project report for the food standards agency fsa project...

Project Report

for

The Food Standards Agency

FSA Project Code Q01114 LFI Project Code 121150

by

Mary Scott MSc Angus Knight PhD

Evaluation of DNA based methods for fruit juice authenticity using lab-on-a-chip capillary electrophoresis endpoint detection

November 2008 Leatherhead Food International, Randalls Road, Leatherhead, Surrey KT22 7RY Tel +44 (0)1372 376761; Fax +44 (0)1372 386228; www.leatherheadfood.com

EXECUTIVE SUMMARY

DNA based analysis for the authentication of fruit juices was evaluated using the polymerase chain reaction (PCR) and Lab on a Chip capillary electrophoresis (LOC). A PCR restriction fragment length polymorphism (RFLP) assay for the detection of grapefruit juice demonstrated the detection of grapefruit juice in orange juice although the assay was relatively insensitive with a limit of detection of 10% v/v. A PCR heteroduplex assay for detecting mandarin juice in orange juice was successfully applied to the LOC system and demonstrated greater sensitivity with a limit of detection of 2.5% v/v. Results for both assays using authentic juice mixtures were consistent with that expected following the random re-annealing of PCR amplified DNA at PCR plateau according to the principles of Hardy-Weinberg law. Calculations of theoretical and expected yields of homoduplex and heteroduplexes indicated that the heteroduplexes were under-estimated by 1.5 fold on the LOC. Although the LOC can provide good quantitative end-point analytical data from PCR methods great care must be taken in data interpretation since results are dependent on the attainment of the PCR plateau. Standard Operating Procedures (SOPs) were prepared for both the detection of mandarin juice in orange juice and for the detection of grapefruit juice in orange juice. Universal PCR reactions for the identification of polymorphic DNA sequences within the plant ribulose biphosphate carboxylase gene (rbcL) were also developed and demonstrated to amplify DNA from processed concentrate derived pomegranate juice.

1. INTRODUCTION The ability to authenticate food products provides a means of monitoring products for the protection of consumers and regulatory compliance (particularly with respect to article 8 of the General Food Law regulation 178/2002 and articles 14 &15 of the Food Safety Act 1990)1,2. The consumer rightly expects that product labelling represents the true identity of the product. However in some cases either accidental or fraudulent substitution occurs. Accidental substitution may occur as a result of inadequate cleaning following the changeover of products during manufacture and may be symptomatic of poor manufacturing practices. Fraudulent substitution is also a serious matter, representing deliberate extension of products with cheaper additives. High profile examples of fraud include water addition, meat and fish species substitution, milk substitution in dairy products, use of non-basmati rice varieties in basmati rice products and adulteration of fruit juices. Fraudulent substitution not only attempts to deceive the consumer but also may go hand in hand with dangerous practices. The ability to detect misrepresentation and deliberate adulteration is therefore essential in order to protect the safety and well being of the consumer. Over the last 10 years, the UK Food Standards Agency (FSA) has supported the development of DNA based approaches to detect and quantify food ingredients. DNA based approaches have been utilised to address a number of food authenticity issues related to processed food 3,4. These include the detection of plant ingredients derived from genetically modified plants 5, plant speciation 6, e.g. rice7, non-durum wheat in durum wheat 8, mandarin juice in orange juice 9,10,11, and authentication of Locust Bean and Guar gums in processed foods 12. The techniques have also been applied extensively to issues in meat and fish adulteration 13-26. Although generally successful the uptake by government enforcement laboratories has been limited due to the nature of the equipment and skill base needed to perform the analyses. However, more convenient technologies are bringing opportunities for routine DNA analyses. Lab on a Chip capillary electrophoresis (LOC) technology represents a simple analytical platform for analysis of DNA based assays. LOC capillary electrophoresis may offer some advantages over standard gel electrophoresis for DNA and protein analysis. The relatively low cost and ease of use of the system combined with reportedly accurate sizing and quantification of fragments can be readily exploited for species profiling and quantification. This has led to significant interest in the system with the result that the approach has been adopted by a number of government enforcement and private laboratories during the last 12 months. In a new research requirement, Q01R0011, the FSA has asked for further extension and validation of methods incorporating lab-on-a-chip capillary electrophoresis. Previous studies carried out by Leatherhead Food International have resulted in the development of molecular assays for the differentiation of citrus species. This has included the development of a quantitative, heteroduplex based assay for determining the extension of poor quality orange juice with mandarin juice; and a restriction fragment length polymorphism (RFLP) based method for determining grapefruit juice in orange juice9,10,11. The aim of this project was to determine if these two assays could be transferred to a LOC format and to develop standard operating procedures for use by public analysts. The first task was to evaluate the LOC system for the detection of grapefruit juice in orange juice using an Eco R1 based RFLP that recognizes amplified grapefruit DNA and not amplified orange DNA. In this assay, PCR primers based on conserved sequences of the chloroplast DNA accD – psaI inter-genic spacer region are used to amplify a 177 bp sequence. Amplified grapefruit DNA can be cleaved by Eco R1 to yield a 125 bp and 52 bp fragments. Amplified orange DNA cannot be cleaved. This RFLP has been previously validated using amplified DNA from authentic juice samples and conventional non-denaturing polyacrylamide gel electrophoresis (PAGE) using 43 grapefruit and 27 orange different authentic samples 11. In any PCR that results in co-amplification of similar targets or alleles then heteroduplex is the predominant product following re-annealing of denatured duplexes when the PCR plateau phase has been reached. As we have described previously 9,10 this re-annealing is

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analogous to the assortment of gametes at fertilisation within a randomly mating population at equilibrium according to Hardy-Weinberg law (H-W law) originally described one hundred years ago 29,30,31. For a population at equilibrium the ratio of homozygous dominant: heterozygotes: homozygous recessive, is given by the ratio p2:2pq:q2 where p and q represent the proportion of dominant (p) and recessive (q) alleles within the population respectively. The sum total of p+q is always equal to one since there are only two alleles. For diploid DNA or mixtures of two haploid genomes e.g. chloroplast or mitochondrial genomes, random re-assortment of PCR products at PCR plateau is expected to distribute heteroduplexes and homoduplexes according to the same formula p2:2pq:q2 where p and q represent the concentrations of authentic and adulterant homoduplexes and pq that of each heteroduplex. The application of H-W law to PCR RFLP analysis requires four key assumptions:

1. That the PCR reaction reaches plateau such that random re-assortment results in heteroduplex formation.

2. The PCR amplification efficiency is equal for the two alleles 3. That there is no heteroduplex RFLP cleavage 4. That homoduplex and heteroduplex are bound by the fluorescent intercalator used for

quantification with equal efficiency.

Quantification of grapefruit derived RFLPs at PCR plateau results in the measurement of q2 and therefore underestimates the actual concentration of grapefruit polymorphisms within the amplified DNA that is represented by %q. Consideration of heteroduplex formation is extremely important in PCR - RFLP analysis since adulterant homoduplex is a minor product at PCR plateau compared with heteroduplex. The ability of restriction endonucleases to cleave mispaired heteroduplexes has been reported. Although a number of restriction enzymes can cleave mismatches, they can to do so at both slower rates and faster rates than homoduplex substrates, often cleaving single strands resulting in partial cleavage. Most type II enzymes such as Eco R1 used in this study, without redundant recognition sites, do not cleave heteroduplexes. However, changes in assay conditions e.g. high glycerol concentrations can result in relaxed specificity or “star” activity allowing heteroduplex cleavage. It should not be assumed that any restriction enzyme either does or does not cleave mismatched target sequences without experimental data 32,33. One approach to overcoming the problem of heteroduplex cleavage is the use of mutation screening enzymes such as CEL I to cleave mis-matches allowing the recognition of heteroduplex molecules 34 and we therefore evaluated the use of this approach during the course of this project. The second task of this project was to evaluate the LOC system for the detection of heteroduplexes between amplified orange and mandarin DNA using a number of approaches. Measurement of the % heteroduplex formed (% 2pq) allows calculation of the relative concentrations of the authentic orange (%p) and adulterant mandarin (%q) species. Although conformational based separations and quantitative heteroduplex analysis can be achieved using conventional non-denaturing PAGE 8no data was available demonstrating that the LOC could be used for conformational separation. Indeed, given the reported sizing accuracy of the LOC system it was considered that heteroduplex analysis was unlikely to be successful and therefore alternative approaches based on heteroduplex cleavage by the CEL I were also considered. A third task for the project was agreed with the Food Standards Agency following the start of the project. This required a determination of the feasibility of applying PCR technology for the authentication of pomegranate juice. This task firstly required the identification of relevant polymorphic DNA sequences from databases allowing the differentiation of pomegranate from other fruit genera and potential adulterants such as elderberry juice. Secondly, this task required the development and optimisation of procedures for the extraction of DNA from processed pomegranate juices and thirdly to investigate the potential of PCR and heteroduplex analysis for determining the adulteration of processed pomegranate juice.

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2. METHODS 2.1 Fruit juice samples Authentic fruit juice samples used in this study are shown in table 1 35. Equal quantities of juices from mandarin samples 92, 93, 94,131, and 133 were prepared by conventional domestic juice pressing by hand and mixed to form a mandarin juice validation mix and likewise samples 144, 278, 290, 352, and 355 were prepared and pooled to form the orange validation mix. Validation mixes for orange and grapefruit by PCR RFLP were prepared from 5 samples each of processed concentrate derived fruit juice. 2.2 DNA extraction from fruit juices DNA was extracted from 100 µl fruit juice following neutralisation with 10 µl 2M Tris-HCl pH 8.0 using a commercially available kit (Phytopure, GE healthcare UK) in accordance with manufacturers instructions and as previously described 9,10,11. Extracted DNA (2µl) was used for PCR. The efficiency of DNA extraction was measured with reference to spiking experiments. Typically 20 µl (2 µg) of a “Precision Molecular Mass Standard” (BioRad, Herts, UK) was spiked into 100 µl of juice sample and the sample neutralised and extracted as above. Precipitated DNA was resuspended in 20 µl 10mM Tris 1mM EDTA buffer (TE) pH 8.0 and 1ul subjected to LOC capillary electrophoresis. Recovered bands were quantified using the LOC in order to determine the % recovery. 2.3 PCR PCR used “Ready To Go” PCR beads (GE healthcare, Little Chalfont UK) or hi fidelity “ Maximase” (Transgenomics, Paris, France) for heteroduplex studies. Reactions were carried out in thin-walled PCR tubes in a final volume of 25 µl in accordance with manufacturer’s instructions.) PCR primers were synthesised by Applied Biosystems, Warrington, UK. Mandarin and orange and grapefruit PCR primers were as previously described 9,10. PCR conditions were further optimised for magnesium ion concentration and annealing temperature for use using a BioRad I – cycler. Grapefruit PCR conditions were optimised for the I - cycler and were 95◦C for 4 mins, followed by 35 cycles of 95 ◦C for 30s, 55◦C for 30s, 72◦C for 30s and 1 cycle of 72◦C for 1 min before holding at 4◦C. Mandarin PCR conditions were also optimised for the I-cycler and the use of Maximase and were, 95◦C for 2 mins, followed by 40 cycles of 94 ◦C for 30s, 55◦C for 30s, 68◦C for 30s and 1 cycle of 68◦C for 5 min before holding at 4◦C. PCRs for the large sub unit of the chloroplast ribulose biphosphate carboxylase gene (rbcL) were rbc104 and rbc83. PCR conditions were 95◦C for 5 mins, followed by 40 cycles of 95 ◦C for 15s, 50◦C for 30s, 72◦C for 30s and 1 cycle of 72◦C for 10 min before holding at 4◦C. 2.4 RFLP Analysis Eco R1 was obtained from Roche UK and used in accordance with manufacturers instructions. PCR product (5 µl) was digested in a total volume of 10 µl at 37◦C for 1 hour and 1 µl analysed using the LOC. 2.5 Mis-Match Analysis Mis-Match analysis based on CEL1 nuclease digestion was carried out in accordance with manufacturer’s instructions using a commercially available kit (Surveyor™, Transgenomics, Paris, France) 2.6 LOC Capillary Electrophoresis All analysis was carried out using the Agilent LOC capillary electrophoresis 2100 Bioanalyzer and reagents obtained from Agilent UK. Estimates of DNA fragment size and quantity were based on the LOC supplied standard within the Agilent DNA 1000 kit. For reference purposes DNA size and quantity were also compared with commercial standards using a 100 bp ladder (“Superladder-low”, Thermo Scientific, Egham, UK)

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Table 1. Authentic Citrus samples.

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3. RESULTS AND DISCUSSION 3.1 PCR and RFLP analysis for detecting grapefruit juice in orange juice 3.1.1 LOC PCR-RFLP for detecting grapefruit juice in orange juice Figure 1 shows the typical RFLP pattern resulting from amplification and cleavage of DNA from orange and grapefruit samples. The theoretical amplicon size was 177 bp and the theoretical Eco R1 cleavage products were 125 bp and 52 bp compared with LOC measured values that ranged from 179-183, 128-135 and 61-69 bp respectively. A minor band was frequently visualised migrating below the 177 bp product in both orange and grapefruit PCR products. This band demonstrated similar Eco R1 cleavage to the major product suggesting that it was not a non-specific amplification product and most likely results from chloroplast heteroplasmy or PCR stutter. The reported sizing accuracy on the chip system is +/- 10 % and the values obtained were in reasonable agreement. In order to evaluate the accuracy of size estimations obtained on the chip system we compared the migration of a 100bp DNA size ladder. On average size estimates were approximately 6% less when run in lane 12 compared with lane 1. In addition, marker sizes were compared with the mean marker size obtained from lane 1 and lane 12. In all cases the LOC overestimated the size by approximately 5-10% with greater accuracy associated with lane 12 than lane 1 presumably resulting from compensation resulting from the observed gel drift and chip to chip variation.

EcoR1 RFLP analysis of orange and grapefruitchloroplast DNA PCR products on the Chip System

Grapefruit Orange

Figure.1 Eco R1 RFLP analysis of authentic grapefruit and orange samples. Lane 1 Authentic grapefruit (88) uncut; Lanes 2-6, Eco R1 digests of amplified DNA from different authentic grapefruit samples (88,96,222,243 & 393). Lanes 7-11, Eco R1 digests of amplified DNA from different authentic orange samples (142,144,278,290 & 352). Lane 12 Negative control.

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3.1.2 Theoretical and practical considerations for quantitative analysis The reported limit of detection of the LOC system is 0.10 ng. In order to determine the theoretical yields of PCR products at PCR plateau a self-calculating spreadsheet based on H-W law was constructed (Table 2). The spreadsheet allows calculation of anticipated levels of heteroduplex and homoduplex at different chip loadings and therefore the theoretical limit of detection for different % DNA mixtures in relation to PCR product yield. For the grapefruit RFLP assay the 125 bp Eco R1 fragment represents 125/177 or 70% of the total PCR product. Since the minimum quantity of DNA for a detectable band using LOC is 0.10 ng then this must represent 70% w/w of the grapefruit PCR product at the limit of detection. Therefore a minimum of 0.14 ng grapefruit homoduplex is required for the detection of 0.10 ng of the 125bp grapefruit Eco R1 RFLP band. Calculations showed that in order to reach a detection limit of approximately 5% w/w DNA by Eco R1 RFLP then a lane loading of 50 ng must be applied to the LOC. In practice, this means that the PCR product yield must reach 50 ng/µl so that the maximum gel loading (1µl) can be applied. However, this can be difficult to achieve in practice (without DNA precipitation) since PCR yields are frequently 10-25 ng/µl resulting in a theoretical RFLP based detection limit of 12.0 – 7.6% v/v juice. Conventional PAGE is approximately twice as sensitive owing to the higher gel loadings achievable. The small proportion of cleavable homoduplex resulting from re-annealing at PCR plateau means that conventional RFLP analysis is an inherently insensitive approach for determining adulteration.

Table 2. Calculation of the theoretical detection limits at different chip loadings for the detection of grapefruit juice (q) in orange juice (p) and the distribution of different duplexes according to H-W law. Data assumes a limit of detection of 0.1 ng (Agilent data) using LOC Eco R1 RFLP analysis or 0.54 ng in PAGE analysis. 3.1.3 Use of Surveyor™ technology for heteroduplex cleavage Since adulterant homoduplex is a minor PCR product at PCR plateau compared with heteroduplex we evaluated the use of the commercially available Cel I nuclease (Surveyor™) for heteroduplex cleavage. Although control experiments using the control template supplied were successful no cleavage of the accD – psaI amplicon from 50% (v/v) orange/grapefruit mixtures was obtained. The assay was also applied to mandarin/orange heteroduplexes without success.

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3.1.4 Validation of the grapefruit assay on the LOC using authentic juices Different mixtures and combinations of authentic orange and grapefruit samples were analysed using the Eco R1 RFLP LOC method. Results obtained (%w/w DNA) were analysed in comparison to the original juice mixture concentrations (%(v/v) juice) using two different approaches. Firstly the 125 bp Eco R1 RFLP grapefruit homoduplex present was quantified using the LOC standard and software. Since the 125 bp band represents 70% of the total homoduplex that must be present the total grapefruit homoduplex present was calculated by multiplying the measured value of the 125 bp band by 1.43. This value was then added to the value for the 177 bp band to give the total PCR product yield. This was more accurate than simply adding the value of the 52 bp band since this band was quantified less efficiently (data not shown). The quantity of grapefruit homoduplex present was expressed as % w/w homoduplex/total PCR product (%G RFLP) this is equivalent to % q2 This represents the conventional approach for calculating adulteration that does not consider heteroduplex formation. Secondly, using the same method of homoduplex quantification, the predicted levels of homoduplex were calculated assuming heteroduplex formation and H-W law (p2:2pq:q2) where p2 and q2 represent the measured quantities of orange and grapefruit homoduplex bands respectively and 2pq the quantity of heteroduplex. In this case the quantity of adulterant (q) is represented by the square root of the calculated value for the fraction of adulterant homoduplex present. Since p + q =1 the % adulteration (%q) = q /p + q X100 In simple terms, according to H-W law, the % adulteration is equal to the square root of the fraction of total adulterant homoduplex (q2) in the total DNA present X 100. The data obtained from fresh authentic juices is shown in Table 3 and Fig. 2. The data showed that the predicted values for adulteration (%q) obtained based on calculation using H-W were in good agreement with the actual juice values and close to the anticipated values assuming equal contributions of DNA from both orange and grapefruit juices. The measured % grapefruit RFLP (%G RFLP) i.e. %q2 was also in good agreement with the theoretical H-W values (Fig.2) The variances were large probably resulting from natural variation or sample preparation. Not unexpectedly, variance was most evident at the 10% adulteration (CV 89.3%) which is close to the theoretical limit of detection based on the chip loading applied.

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0 5 10 15 20 25 30

% Grapefruit (G) juice in Orange juice (v/v)

% P

CR

Pro

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(w/w

) %G - theoretical %q

% G - measured %q

% G RFLP-measured %q2

% G RFLP-theoretical %q2

Figure 2. Comparison of the measured % G RFLP or % q2 compared with the theoretical % G RFLP or % q2 predicted from Hardy-Weinberg law. Data is also shown comparing the calculated juice content (%q) compared with the theoretical values. Theoretical values assume an equal contribution of PCR product from both orange and grapefruit juice DNA and random reassortment of single stranded PCR products at PCR plateau. Error bars represent the standard deviation.

Table 3. Comparison of actual, measured and theoretical values for grapefruit adulteration using PCR and Eco R1 RFLP analysis from authentic juice mixtures. Calculations of adulteration were based on Hardy-Weinberg equilibrium where q represents the concentration of the grapefruit adulterant species and q2 represents the fraction of grapefruit homoduplex (G homoduplex ) within the total amplified DNA. Theoretical values assume an equal contribution of PCR product from both orange and grapefruit juice DNA and random reassortment of single stranded PCR products at PCR plateau. Total DNA values were calculated from the 125bp RFLP.

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Table 4. Comparison of actual, measured and theoretical values for grapefruit adulteration based on duplicate validation data from two separate experiments (Series A and B) using processed juice mixtures. Calculations of adulteration were based on Hardy-Weinberg equilibrium where q represents the concentration of the grapefruit adulterant species and q2 represents the fraction of grapefruit homoduplex (G homoduplex ) within the total amplified DNA. Theoretical values assume an equal contribution of PCR product from both orange and grapefruit juice DNA and random reassortment of single stranded PCR products at PCR plateau. Total DNA values were calculated from the 125bp RFLP.

3.1.5 Validation of the grapefruit assay on the LOC using commercially available juices In contrast to the data obtained from fresh juices attempts to validate the assay using mixtures of processed orange and grapefruit juices provided different results. The data (Table 4) shows much higher levels of adulteration calculated from the measured values using H-W (H-W - RFLP) than that of the measured value (RFLP). Both H-W RFLP and the RFLP values are much greater than the theoretical values based on H-W predicted values assuming equal concentrations of DNA in orange and grapefruit juice. The reasons for this are at present unclear however this may reflect a relatively low PCR product yield (approximately half that of the fresh juices) and a failure to reach PCR plateau. A failure to reach plateau prevents segregation according to H-W. If this occurs then the resulting PCR product will only contain homoduplexes and thus results of RFLP analysis appear more similar to the original juice mixtures. 3.2 Heteroduplex analysis for detecting mandarin juice in orange juice. 3.2.1 Resolution of DNA heteroduplexes on the LOC Figure 3 shows the results obtained from LOC analysis of PCR amplified DNA extracted from authentic juice samples of orange juice and mandarin juice and 50% (v/v) mixtures. The LOC size estimates of the homoduplex bands of 198 bp and 206 bp size were within the published LOC sizing errors. The two heteroduplexes showed conformational based migration, typical of electrophoresis under non-denaturing conditions and migrated at approximately 251 bp and 269 bp. The resolution of the heteroduplexes on the chip system was unexpected and demonstrates that the chip system is a non-denaturing electrophoresis system. A faint band of 246 bp was often present migrating below or co-migrating with the 251 bp band. The origin of this band is unknown but might result from co-migration of an alternative heteroduplex or triplex molecule. When present this band was included as a heteroduplex band in our analysis since its concentration increased with increasing mandarin content (Band A, Table 6.)) A final step of heating to 94◦C for 2 mins followed by slow re-annealing (2.2 ◦C /s) was also applied to the PCR to ensure re-annealing and heteroduplex formation however this did not change the yield of heteroduplex indicating that the reaction had plateaued after 40 cycles. The LOC was capable of partially resolving the 8 bp size difference distinguishing orange and mandarin homoduplexes. This partial resolution is shown in the electropherogram in Fig. 4 and is also just visible in Fig. 3. Although this resolution was small in comparison to the heteroduplex bands it enabled quantitative analysis of all four bands obtained from 50% (v/v) orange and mandarin juice mixtures. The resolution obtained allowed comparison of the observed and predicted band yields according to H-W law for 50% (v/v) mixtures. Analysis of homoduplexes at lower values (<50% (v/v)) was not possible owing to the dilution of the adulterant homoduplex band by the authentic homoduplex resulting in heteroduplexes. Measured heteroduplex values were always less than theoretical values in agreement with previous data8.

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Heteroduplex analysis on the chip system

Figure. 3. Heteroduplex analysis on the LOC. Lanes 1-3 authentic mandarin samples,181,184 and 189; lanes 4-6 authentic orange samples 164, 290 and 355, lanes 7-9 50% mandarin in orange mixes, 181/164; 181/290 and 184/355 . Lane 10 negative control.

Figure 4. An example electropherogram obtained following LOC analysis of PCR product obtained from a 50% mandarin in orange juice mixture. The discrepancy between the quantities of observed and predicted total heteroduplex according to H-W law was investigated by repeated (X9) analysis of PCR products from 50% v/v juice mixtures in order to confirm the observed discrepancy and determine a correction factor. The mean value for predicted heteroduplex based on measured homoduplex was 49.03% (S.D. 1.27%) however the mean measured heteroduplex was only 32.95% (S.D. 5.68%). Statistical analysis using the Student’s unpaired t-test confirmed that the predicted and measured values for heteroduplex were significantly different (P<0.0001) and that measured heteroduplex values were 1.49X less than expected values based on the measured homoduplexes. Measured values for heteroduplex were therefore multiplied by a

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correction factor of 1.49 in order to correct for the observed differences. The data suggests that the mandarin/orange DNA heteroduplexes exhibit less affinity for the fluorescent intercalator used for LOC band quantification than the corresponding homoduplexes. Although this seems a plausible explanation we have not as yet excluded other possible explanations related to the calculation of peak area using the Agilent software. 3.2.2 Reproducibility of LOC analysis between chips Table 5 shows a comparison of data obtained from different chips using the same DNA extracted and amplified from different % (v/v) mixtures of retail freshly squeezed pasteurised mandarin and orange juice mixtures. Results were compared with actual, measured, theoretical and corrected values by quantifying heteroduplex bands and for the predicted values at 50% v/v mixtures by quantifying the homoduplex bands. Results showed excellent reproducibility of the analysis between chips. The application of the correction factor is also shown, the corrected values showed a closer fit with the theoretical values than the measured values based on heteroduplex quantification alone.

Table 5. Comparison of the measured, theoretical, predicted and corrected values for % heteroduplex (2 pq%) obtained between chips using PCR and DNA extracted from mixtures of retail freshly squeezed pasteurised mandarin and orange juices. Theoretical values assume an equal contribution of PCR product from both orange and mandarin juice DNA and random re-assortment of single stranded PCR products at PCR plateau. 3.2.3 Validation of the mandarin assay using freshly squeezed authentic samples The assay was validated using mixtures of freshly squeezed authentic samples. Attempts to validate the assay using concentrate derived juices obtained from retailers were compromised by the detection of heteroduplex in the PCR product indicating the presence of mandarin or mandarin hybrid juice in the retail samples (data not shown). We therefore prepared pooled authentic mandarin and orange juices mixtures and mixed these at different concentrations (% v/v). The DNA was extracted and PCR amplified in duplicate for 40 cycles using maximase PCR (Table 6). Duplicate data for complete repetition of juice mixing, DNA extraction and PCRs is shown for extractions 1 and 2. The results of PCR repetition are also shown for extraction 2 using the same extracted DNA. Typical results are shown in Fig. 5.

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Figure 5. Example data for the detection of mandarin juice in orange juice following heteroduplex analysis using PCR and the LOC. Lane 1-6, 0, 2.5, 5, 10, 15, and 50% v/v mandarin in orange juice.

Heteroduplexes

Homoduplexes

Table 6. Validation data obtained using % v/v mixtures of blended authentic mandarin in orange mixtures (%q) (Valmix M and Valmix O). Results are shown for duplicate PCR analysis at different % mandarin juice between two separate extractions (1 and 2) and separate PCR (PCR 1 and 2).

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The assay readily detected 5% v/v mandarin juice in orange juice on the LOC. Data analysis was either based on the measured % (w/w) heteroduplex/total PCR product ((2pq/ p2+2pq+q2)X100) or correction of the measured value using the 1.49 X correction factor described above. The results were compared with the theoretical % (w/w) heteroduplex values predicted using H-W law from the original juice mixtures (assuming equal concentrations of DNA are obtained from each species). Corrected values for % (w/w) heteroduplex were in much closer agreement with predicted H-W values than those based on the measured values alone. H-W calculated values for measured adulteration based on the quantification of the homoduplex bands in the 50% (v/v) mixtures showed that authentic orange was 100% authentic and mandarin 97% authentic. The discrepancy from 100% in mandarin results from the measured background heteroduplex that presumably results from chloroplast heteroplasmy. Summary results are shown in Table 7 for variation between PCR reactions, between DNA extractions and PCR reactions, and as variation for the pooled data set. The results are also shown in comparison to previous data obtained by PAGE analysis (Fig. 7.). The results obtained between the two assay formats were similar. However comparison of the two data sets is complicated since we do not know if the intercalator used for PAGE (ethidium bromide) demonstrates a lesser affinity for heteroduplex than the proprietary intercalator used for LOC (for which a correction factor was applied). Some of the assay variance is derived from LOC analysis alone and PCR repetition as shown from both Table 5 and Table 7. Not unexpectedly variance is greatest at the limit of detection. These data are in agreement with the Agilent published data for quantification on the chip system that shows a 20% error in the accuracy of quantification and a 15% error in the reproducibility of quantification (Agilent DNA 1000 kit guide). Data for variance showed that approximately 8% of the variance was attributable to the LOC analysis alone. The remaining variance was largely attributed to variance in the PCR. Other analytical errors in LOC and PAGE analysis are probably associated with natural variation. Overall, variance in the pooled LOC data was actually very good for this type of analysis and less than the PAGE data (although this may be attributable to the use of less authentic samples). Both theoretical H-W values and LOC H-W values approximate to a linear fit over the 0 - 15% v/v juice range (r2 = 0.9961 and 0.9933 respectively) The LOC mandarin heteroduplex assay can reliably detect mandarin juice in processed orange juice at 5% v/v adulteration provided sufficient PCR product is analysed. Overall, and similar to our previous data based on PAGE and ethidium bromide staining, mandarin juice appeared to have less DNA/unit volume than orange juice and may account for the deviance from H-W law. Of some concern was the finding that the authentic mandarin juice mixtures possessed a higher level of background heteroduplex than that previously observed (5% w/w measured heteroduplex). We therefore examined the levels of heteroduplex in each of the five mandarin juices used to make the authentic composite orange and mandarin juices. The average heteroduplex was 1.78%. However one of these mandarin 92 (Nova) exhibited 4.75% (w/w) heteroduplex however this cannot account for all of the observed heteroduplex since M92 represents only 1/5 of the mixture and possibly this represents occasional bias in the amplification for heteroplasmic alleles. Such high levels of background were not seen in previous studies. In order to confirm that similar background did not occur in orange samples we next tested authentic samples in PCR using DNA extracted previously and DNA extracted during the course of this work. Of the eight authentic orange samples tested (271,273,275,277,350,351,353 and 357) only one (273) produced any detectable heteroduplex bands (1.85% (w/w)). Of the ten authentic hybrid samples tested (126,135,136,137,160,175,183,215,282,284, and 336) none produced detectable heteroduplex bands. The data was consistent with previous studies based on the analysis of 40 authentic orange species and 18 authentic mandarin and mandarin hybrid samples when analysed either separately or as mixtures and suggests that mandarin sample 92 is not typical.

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LOCy = 1.0865x + 0.7783

R2 = 0.9933

Theoreticaly = 1.6693x + 0.6095

R2 = 0.9961

PAGEy = 1.158x + 1.34

R2 = 0.97230.00

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Actual mandarin in orange (% v/v)

% 2pq

%2pq - theoretical%2pq LOC%2pq PAGELinear (%2pq LOC)Linear (%2pq - theoretical)Linear (%2pq PAGE)

Figure 7. Expected yield of heteroduplex (%2pq) according to Hardy Weinberg law plotted against % mandarin in orange juice. Data is shown for analysis using LOC (this work) and for PAGE analysis 8 . Theoretical data assumes an equal contribution of PCR product from both orange and mandarin juice DNA and random re-assortment of single stranded PCR products at PCR plateau. Error bars represent the standard deviation.

Table 7. Analytical variation associated with LOC heteroduplex analysis of mandarin in orange mixtures.

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3.3 PCR analysis of Pomegranate juice 3.3.1 Identification of polymorphic rbcL sequences DNA sequences for the large sub-unit of the plant chloroplast ribulose biphosphate carboxylase gene (rbcL) from pomegranate and different genera requested by the FSA were obtained from the National Centre for Biotechnology Information USA www.ncbi.nlm.nih.gov and aligned using commercially available software. In some cases the FSA specified genera or species were not available in which case a more closely related genus was used. Full length sequences were not always available. The sequence alignment is shown in Fig. 8. The sequence alignment demonstrates a number of polymorphisms differentiating plant genera including unique pomegranate SNPs and RFLPs distinguishing other plant genera from pomegranate (e.g. a unique elderberry family RFLP creating an Eco R1 site (GAATTC) at position 112). 3.3.2 Selection and evaluation of PCR primers Two sets of PCR primers were designed (Fig.8) based on the sequence alignment and polymorphic sequences. It was anticipated that design of primers amplifying a 176bp amplicon (rbc176) could also allow resolution of different heteroduplexes resulting from admixtures on the chip system. Unfortunately we were unable to obtain authentic pomegranate juices from industrial contacts and we therefore used squeezed pomegranate juice and processed juices obtained from retailers in order to test the PCR reactions. The primer set rbc176 performed poorly in PCR and was not investigated further however rbc83 did amplify control DNA efficiently and was selected for further study. Although the corresponding pomegranate sequence data was not available previous studies had shown that the PCR primer pair rbc104 were capable of amplifying a polymorphic plant DNA sequence from processed jams and yoghurts. It was therefore was also considered that these primers may also be useful for determining pomegranate adulteration. 3.3.3 Optimisation of DNA extraction from processed pomegranate juice Unlike most orange and grapefruit juices that contain particulates, pomegranate juice in most processed concentrate derived samples is usually clarified. DNA extractions were therefore performed on either 100 µl juice or 1 ml samples that were microfuged for ten minutes, followed by removal of 900 µl supernatant and resuspension in the remaining juice. Processed concentrate derived pomegranate juice was spiked prior to extraction with low molecular weight DNA quantification standards in order to measure spike recovery. Overall spike recovery in control TE samples was 39.7% (SD 16.8%) and in juice samples 34.7% (SD 19.73%). The efficiency of spike recovery from the pomegranate juice was 87.4% compared with recovery from TE. The recovery of the smallest spiked fragment (200 bp) was 20.7% when spiked in juice compared with 18.5% in TE. Although recovery of low molecular weight DNA was poor this was considered typical of most DNA extraction methods and therefore further optimisation of extraction procedures was not investigated. 3.3.4 PCR amplification of DNA extracted from processed fruit juices Following optimisation of the annealing temperature and magnesium ion concentration the rbc104 and rbc83 PCRs were applied to DNAs extracted from pasteurised pressed fruit juices obtained from retail outlets. Fig. 9 demonstrates successful amplification of extracted DNA from retail samples and demonstrates the general utility of the PCR primers. The PCR reactions were further applied to a number of different processed juices (Figs. 9 & 10). Both primer sets produced PCR amplicons from processed fruit juices including a number of pomegranate samples. rbc104 primers appeared to amplify more efficiently than rbc83 although scope exists for further optimisation of both the DNA extraction and PCR.

http://www.ncbi.nlm.nih.gov/

http://www.ncbi.nlm.nih.gov/

18

Figure 8. Partial nucleotide sequence alignment based on available rbc L sequences.

10 20 30 40 50 60 70 80 90Pomegranate T T AT CCCT T AGACCT T T TT GA AGAAGGT T CTGTT ACT AAT AT GT T T ACTT CCAT TGAGGGT AATGT A T T TGGGT T CAAAGCCCTGNNNGCT CT ACGElderberry family L14066 T T ACCCAT T AGACCT T T TT GA AGAAGGT T CTGTT ACT AACAT GT T T ACTT CCAT TGT GGGT AATGT A T T TGGGT T CAAAGCCCTGCGCGCT CT ACGGrapefrui t AJ238407 T T ACCCGT T AGACCT T T TT GA AGAAGGT T CTGTT ACT AACAT GT T T ACTT CCAT TGT GGGT AATGT A T T TGGT T T CAAAGCA CTGCGCGCT CT ACGgrape rbcL T T ACCCT T T AGACCT T T TT GA AGAAGGCT CTGTT ACT AACAT GT T T ACTT CCAT TGT GGGT AATGT GT T TGGGT T CAAAGCT CTGCGCGCT CT ACGCranberry L12625 T T AT CCT T T AGACCT T T TT GA AGAAGGT T CAGTT ACT AACAT GT T T ACTT CCAT TGT GGGT AATGT T T T TGGGT T CAAAGCT CTGCGGGCT CT ACGCurrant L11204 T T AT CCCT T AGACCT T T TT GA AGAAGGT T CTGTT ACT AAT AT GT T T ACTT CCAT TGT GGGT AATGT A T T TGGGT T CAAAGCCCTGCGCGCT CT ACGRaspberry U06825 T T ACCCCT T AGACCT T T TT GA AGAAGGT T CGGTT ACT AACAT GT T T ACTT CCAT TGT AGGT AATGT GT T TGGGT T CAAGGCCT TGCGCGCT CT ACGStrawberry U06805 T T ACCCCT T AGACCT T T TT GA AGAGGGT T CGGTT ACT AACAT GT T T ACTT CGAT TGT AGGT AATGT GT T TGGGT T CAAGGCCT TGCGCGCT CT ACGPeach AF206813 T T ACCCCT T AGACCT T T TT GA AGAGGGT T CTGTT ACT AACAT GT T T ACTT CCAT TGT AGGT AATGT GT T TGGGT T CAAGGCCCTGCGCGCT CT ACGPlum L01947 T T ACCCCT T AGACCT T T TT GA AGAGGGT T CTGTT ACT AACAT GT T T ACTT CCAT TGT AGGT AATGT GT T TGGGT T CAAGGCCCTGCGCGCT CT ACGApricot AF411489 T T ACCCCT T AGACCT T T TT GA AGAGGGT T CTGTT ACT AACAT GT T T ACTCCAT T TGT AGGGAATGT GT T TGGGGT CAAGGCCCTGCGCGCCCACCGTomato L14403 T T ACCCT T T AGACCT T T TT GA AGAAGGT T CCGTT ACCAAT AT GT T T ACTT CCAT TGT AGGT AACGT A T T TGGGT T CAAAGCCCTGCGCGCT CT ACGBananna AF378770 T T AT CCT T T AGACCT T T TT GA AGAAGGT T CTGTT ACT AACAT GT T T ACTT CCAT TGT GGGT AATGT A T T TGGT T T CAAAGCCT T ACGAGCT CT ACGRhubarb M77702 T T ACCCAT T AGACCT T T TT GA AGAAGGT T CTGTT ACT AAT AT GT T T ACTT CCAT TGT GGGT AATGT A T T TGGGT T CAAAGCCCTGCGT GCT CT ACG

Consensus T T Ac CCy T T AGACCT T T TT GA AGA aGG t T C t GTT AC t AA c AT GT T T ACT t C c a T TGt g GG t AA tGT r T T TGGg t T CAA aGC c c T g c gc GC t C t a CG

100 110 120 130 140 150 160 170 180 190

Pomegranate T CTGGAGGATCT GAGAATCCCT ACTGCA T AT ACT AA AACT TT CCAAGGCCCGCCT CAT GGT AT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGElderberry family L14066 T CTGGAAGATCT GCGAATT CCTGGTGCT T ATGTT AA AACNTT CCAAGGCCCGCCT TAT GGT AT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGGrapefrui t AJ238407 T CTAGAGGATCT A CGAATCCCT CCTGAGT AT ACT AA AACT TT CCAAGGCCCGCCT CACGGCAT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGgrape rbcL T CTAGAGGATCT GCGAATCCCCCCTGCT T AT T CT AA AACT TT CCAAGGCCCGCCT CAT GGCAT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGCranberry L12625 T CTGGAAGATCT A CGAATCCCTGT TGCGT ATGTT AA AACT TT CCAAGGACCT CCT CACGGCAT CCAAGT TGAAAGAGATAAA T TGAACAAGT ATGGCurrant L11204 T CTGGAGGATCT GCGAATCCCCGT TGCT T ATGTGAA AACT TT CCAAGGCCCGCCT CACGGCAT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGRaspberry U06825 T CTGGAGGATT T A CGAATCCCT CCTGCT T ATGTT AA AACT TT CCAAGGCCCGCCT CACGGGAT CCAAGT TGAAAGAGATAAA T TGAACAAGT ATGGStrawberry U06805 T CTGGAGGATT T A CGAATCCCT ACTGCT T ATGTT AA AACT TT CCAAGGCCCGCCT CACGGGAT CCAAGT TGAAAGAGATAAA T TGAACAAGT ATGGPeach AF206813 T CTGGAGGATT T GCGAATCCCT ACTGCT T ATGTT AA AACT TT CCAAGGCCCGCCT CAT GGGAT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGPlum L01947 T CTGGAGGATT T GCGAATCCCT ACTGCT T ATGTT AA AACT TT CCAAGGCCCGCCT CAT GGGAT CCAAGT TGAGAGAGATAAA T TGAACAAGT ATGGApricot AF411489 CCTGGAGGATT T CCGAATCCCT ACT CCT T ATGTT AA AACCTT CCAAGCCCCCCCT CAT GGGGT CCAAGT TGAGAGAGATAAA T T T AACAA AT ATGGTomato L14403 T CTGGARGATCT GCGAATCCCT CCTGCT T ATGTT AA AACT TT CCAAGGTCCGCCT CAT GGGAT CCAAGT TGAAAGAGATAAA T TGAACAAGT ATGGBananna AF378770 T CTGGAGGATCT GCGAATT CCCACT TCT T AT T CCAA AACT TT CCAAGGCCCGCCT CACGGCAT TCAGGT TGAAAGAGATAAGT TGAACAAGT ATGGRhubarb M77702 T T TGGAGGATT T GCGAATCCCT CCTGCT T AT T CGAA AACT TT CCAAGGCCCGCCT CACGGT AT CCAAGT TGAAAGAGATAAA T TGAACAA AT ATGG

Consensus t c Tg GAg GATc T g c GAATc CC t mc T gc t T AT g t t AA AAC t TT CCAAGg cCCg CCT cA y GG s a T cCA a GT TGAg AGAGATAA a T T g AACAA g T ATGG

200

rbc176 rbc83

rbc176rbc83

Pomegranate Apple Guava Mixed

rbc104

rbc83

Figure 9. rbc104 and rbc83 PCR products obtained from retail pressed pasteurised fruit juices and a mixed sample of the three juices.. Lanes 1 & 2 pomegranate juice; lanes 3 & 4 apple juice; lanes 5&6 guava juice, lanes 7 & 8 Mixed pomegranate, apple and guava juices.

19

rbc104rbc83

Pomegranate(fresh) 35% Pomegranate

5% Aronia(concentrate)

Pomegranate and grape(fresh)

Pomegranate and grape(concentrate) Pomegranate and grape

(concentrate)

Figure 10. PCR amplification of DNA extracted from freshly squeezed and concentrate derived pomegranate juices obtained from retail outlets using PCR primers rbc104 and rbc83.conditions. Heteroduplexes appeared to be present in some of the samples as shown by fainter band migrating above the PCR product obtained from the mixed samples however. It is well known that successful PCR from processed products requires the amplification of small amplicons owing to DNA degradation during processing. The rbc83 and rbc104 PCRs described here fulfil this criterion. The detection of adulteration by LOC RFLP using small fragments is more difficult owing to the reduced levels of fluorescent dye intercalation obtained with small fragments and the lack of availability of suitable polymorphic restriction sites. Although some evidence for heteroduplex formation, indicative of adulteration, was present in the co-amplification of mixed juice samples (Fig. 9) and in the retail pomegranate juices (Fig. 10) no gross differences in LOC migration were evident presumably owing to the low molecular weight product The approach could be improved by using Pyrosequencing technology to directly sequence the PCR products. Pyrosequencing technology has a number of advantages compared with other methods. Firstly reactions are internally controlled using the authentic species as control and allows the simultaneous detection of multiple adulterants. The method is definitive, relying on sequence characterisation instead of indirect characterisation with separate or multiple probe(s). The method is quick and simple with minimal operator intervention, leading to greater accuracy. Pyrosequencing™ technology is capable of multiple sequence determinations within 10-15 minutes. Heteroduplex formation does not influence results compared with RFLP data and is much more sensitive. Results reported here (and previously reported findings demonstrate that the approach is quantitative (subject to the usual PCR caveats that may result from differences in amplification rates of different alleles). The approach can be applied to other areas of food authenticity issues where DNA is present and single nucleotide variations can provide identification. This includes poultry, fish, and meat speciation including variable positions resulting from the detection of specific methylated DNA

20

sequences present in different animal tissues and the authentication of herbal products, medicines, and nut containing products. A comparison of different technologies for allele frequency determination including, RFLP, real-time Pyrosequencing™ analysis, single base extension (SBE), Taq man TM and MALDI-TOF based primer extension revealed that all of these methods (if used appropriately) can provide reasonably accurate allele frequency estimation of SNPs in DNA pools36. However, only Pyrosequencing™ allows the multiple detection of different variable positions in a simple format. The sequence alignments and regions of rbcL identified in this study are ideally suited for the application of this technology.

21

4. CONCLUSIONS The data has shown that two different PCR assays for fruit juice authentication can be transferred to the chip system and that PCR end-point quantification can be achieved using the LOC provided that the analysis is performed and interpreted correctly. Standard operating procedures have been drafted for these two methods on the LOC platform (see appendix). Interpretation of data according to H-W law provided a much closer relationship with the authentic juice mixtures than that obtained using current analytical approaches. The failure to consider H-W in this type of PCR analysis may lead to significant errors in quantification. Accurate end-point quantification of PCR must either ensure that PCR plateau is reached so that duplexes are re-assorted according to H-W law or that PCR plateau is not reached such that only homoduplexes are measured. The ability of the selected endonuclease to cleave heteroduplex must also be carefully assessed. As predicted by H-W law for an RFLP assay without heteroduplex cleavage the grapefruit assay was insensitive with a limit of detection of 10% v/v juice. Similar detection limits are likely for microsatellite or indel based markers where measurement occurs at PCR plateau and heteroduplexes are not measured. Although increased sensitivity could be achieved by measurement before plateau (where H-W law does not apply) this would be difficult to ensure in practice. Consideration of pre and post plateau measurement is very important for end-point quantification and should be considered carefully in LOC analysis performed under non-denaturing conditions. Alternatively, accurate analysis could be obtained using denaturing capillary electrophoresis as demonstrated for Basmati rice 37,38 , under these conditions data interpretation according to H-W law does not apply since all PCR products are examined as single stranded DNA. However, this is currently more expensive requiring fluorescently labelled primers and not technically feasible owing to a lack of denaturing LOC equipment. Assay validation of the grapefruit assay was successful for freshly squeezed juices. Validation with processed juices provided different quantitative data and probably results from the failure of the PCR reactions to reach plateau such that only homoduplex PCR product is formed. This may result from the smaller number of PCR cycles used (35X) in this assay compared with the mandarin assay (40X). This needs to be considered in any ring-trial. Sensitivity may be improved by examining isoschizomers for mis-match cleavage or directly detecting adulterant by other approaches e.g. Pyrosequencing. The mandarin assay was highly effective at quantitatively detecting mandarin juice in orange juice in both freshly squeezed and processed juices and readily detects 5% v/v adulteration. The Agilent LOC system is a non-denaturing electrophoresis system. This means that the LOC cannot provide unequivocal size determinations and it will always be necessary to run alternative standards to ensure accurate band identification. The LOC can resolve DNA heteroduplexes, but does not quantify heteroduplex bands as efficiently as homoduplex bands. LOC analysis under estimates the quantity of heteroduplex by 1.49X. Two PCR reactions based on separate regions of the rbcL gene demonstrated that small fragments of DNA can be extracted and amplified from processed pomegranate juice. Further characterisation of these sequences requires DNA sequencing. Part of the work described in this project has been accepted for publication: Scott, M., Knight, A. (2009) Quantitative PCR analysis for fruit juice authentication using PCR and Lab-on-a-Chip capillary electrophoresis according to Hardy-Weinberg law. Journal of Agricultural and . Food Chemistry (accepted)

22

5. REFERENCES

1. Regulation (EC) 178/2002 of the European Parliament and Council 2002. Official Journal of the European Communities. L31/1 1.2.2002

2. UK- Food Safety Act (1990) http://www.opsi.gov.uk/si/si2004/uksi_20043279_en.pdf

3. Candrian, U., Meyer, R. (1995) Applications of nucleic acids amplification methods in

food analysis. Current status and future trends in analytical food chemistry. Proceedings of the Eighth European Conference on Food Chemistry. Austrian Chemical Society 1. 192-199

4. Lockley A.K., Bardsley R.G. (2000) DNA-based methods for food

authentication.Trends in Food Science and Technology 11, 67-77

5. Hubner P., Waiblinger H.U., Pietsch K., Brodmann P. (2001) Validation of PCR methods for quantitation of genetically modified plants in food. Journal of the Association of Analytical Chemists International 84 (6), 1855-1864

6. Knight, A.I., (1998) Method for detecting a particular plant species in a product.

Patent WO 98/14607

7. Bligh H.F.J. (2000) Detection of adulteration of Basmati rice with non-premium long-grain rice. International Journal of Food Science and Technology 35, 257-265

8. Bryan G.J., Dixon A., Gale M.D., Wiseman G. (1998) A PCR-based method for the

detection of hexaploid bread wheat adulteration of durum wheat and pasta. Journal of Cereal Science 28, 135-145

9. Mooney, R.; Chappell, L., Knight, A.I., (1999) Validation of Molecular Assays for Fruit

Juice Authentication. MAFF Final Report ANO672

10. Mooney, R, Chappell, L., Knight, A. (2006) Evaluation of a polymerase chain reaction-based heteroduplex assay for detecting the adulteration of processed orange juice with mandarin juice. Journal of the Association of Analytical Chemists International 89, 1052-1060.

11. Ortola-Vidal, A., Eggington, A.J.’ Knight, A.I. (2000) Further development and

validation of assays for fruit authentication. MAFF Final report ANO6118

12. Meyer, K., Rosa, C., Hischenhuber, C., Meyer, R. (2000) Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis. Journal of the Association of Analytical Chemists International, 8, 89-99

13. Abdulmawjood A., Bulte M.(2002) Identification of ostrich meat by restriction fragment

length polymorphism (RFLP) analysis of cytochrome b gene. Journal of Food Science 67, 1688-1691

14. Amicucci A., Guidi C., Zambonelli A., Potenza L., Stocchi V. (2002) Molecular

approaches for the detection of truffle species in processed food products. Journal of the Science of Food and Agriculture 82,, 1391-1397

15. Bania J., Ugorski M., Polanowski A., Adamczyk E. (2001) Application of polymerase

chain reaction for detection of goats' milk adulteration by milk of cow. Journal of Dairy Research 68, 333-336

24

http://www.opsi.gov.uk/si/si2004/uksi_20043279_en.pdf

16. Bottero M.T., Civera T., Nucera D., Rosati S., Sacchi P., Turi R.M. (2003) A multiplex

polymerase chain reaction for the identification of cows', goats' and sheep's milk in dairy products. International Dairy Journal 13,, 277-282

17. Brodmann P.D., Nicholas G., Schaltenbrand P., Ilg E.C. (2001) Identifying unknown

game species: experience with nucleotide sequencing of the mitochondrial cytochrome b gene and a subsequent basic local alignment search tool search. European Food Research Technology 212, 491-496

18. Lockley A.K., Bardsley R.G. (2002) Intron variability in an actin gene can be used to

discriminate between chicken and turkey DNA. Meat Science 61, 163-168

19. Meyer R., Candrian U., Luthy J., (1994) Detection of pork in heated heat products by the polymerase Chain Reaction. Journal of the Association of Analytical Chemists International 77, 617-622

20. Meyer, R., Hofelein, C., Luthy, J., Candrian, U. (1995) Polymerase Chain Reaction-

Restriction Fragment Length Polymorphism Analysis: A simple method for species identification in food. Journal of the Association of Analytical Chemists International 78, 1542-1551

21. Piknova L., Krahulcova J., Kuchta T. (2002) Detection of the cow's milk component in

ewe's and goat's cheese using polymerase chain reaction. Bulletin of Food Research 41, 163-167

22. Rehbein H., Horstkotte B. (2002( Knowing what's what: ensuring fish and shellfish

authenticity. Food Science and Technology 16, 40-43

23. Sanjuan A., Comesana A.S. (2002) Molecular identification of nine commercial flatfish species by polymerase chain reaction-restriction fragment length polymorphism analysis of a segment of the cytochrome b region. Journal of Food Protection 65, 1016-1023

24. Tajima K., Enishi O., Amari M., Mitsumori M., Kajikawa H., Kurihara M., Yanai S.,

Matsui H., Yasue H., Mitsuhashi T., Kawashima T., Matsumoto M (2002) PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements. Bioscience, Biotechnology, and Biochemistry 66, 2247-2250

25. Unseld M., Beyerman B., Brandt P., Hiesel R. (1995) Identification of the species

origin of highly processed meat products by mitochondrial DNA sequences. PCR Methods and Applications 4,241-243

26. Zeleny R., Bernreuther A., Schimmel H., Pauwels J. (2002) Evaluation of PCR-based

beef sexing methods.Journal of Agricultural and Food Chemistry 50, 4169-4175

27. Ortola-Vidal A., Knight A. (2003) Improved authentication of jams and Yoghurts using PCR. FSA project Q01073

28. Ortola-Vidal, A., Schnerr, H., Rojmyr, M., Lysholm., F., Knight, A (2007) Quantitative

Identification of Plant Genera in Food Products using PCR and Pyrosequencing™ Technology. Food Control 18, 921-927

29. Hardy, G.H. (1908) Mendelian proportions in a mixed population. Science 28, 49-50

30. Weinberg, W. (1908) Über den Nachweis der Vererbung beim

Menschen.Jahresh.Ver.Vaterl.Naturkd. Wuerttemb. 64: 368-382

31. Stern, C. (1943) The Hardy-Weinberg law. Science 97: 137-138

25

32. Petranovic, M., Petranovic, D., Dohet, C., Brooks, P., Radman, M. (1990) Some

restriction endonucleases tolerate single mismatches of the pyrimidine-purine type. Nucleic Acids Research 18, 2159-2162.

33. Langhans, M.T., Palladino, M.J. (2008) Cleavage of mispaired heteroduplex DNA

substrates by numerous restriction enzymes. Current issues in Molecular Biology 11, 1-12.

34. Qiu, P., Shandilya, H., D’Alessio, D., O’Connor, K., Durocher, J., Gerard, G.F. (2004)

Mutation detection using SurveyorTM nuclease. Biotechniques 38, 702 – 707.

35. Pegg, A.K., Patel, Evans, M.T.A., Farnell, P.J. (1996) Collection of authentic samples (fruit, leaves and rootstock) from important fruit-producing countries. Project report ANO 630. UK Ministry of Agriculture Fisheries and Food .

36. Shifman, S., Pisante-Shalom, A., Yakir, B., Darvasi, A. (2002) Quantitative

technologies for allele frequency estimation of SNPs in DNA pools. Molecular and . Cellular. Probes 16, 429-434.

37. Vemireddy, L.R., Archak, S., Nagaraju, J. (2007) Capillary electrophoresis is essential

for microsatellite marker based detection and quantification of adulteration of Basmati rice (Oryza sativa). Journal of Agricultural and Food Chemistry 55 , 8112-8117

38. Archak, S., Vemireddy, L.R., Archak, S., Nagaraju, J. (2007) High-throughput

multiplex microsatellite marker assay for detection and quantification of adulteration in Basmati rice (Oryza sativa). Electrophoresis 28

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6.0 APPENDIX 6.1 Draft SOP for detecting mandarin juice in orange juice – pending approval from the Agency Additives and Authenticity Methodology Working Group

FOOD STANDARDS AGENCY STANDARD OPERATING PROCEDURE (SOP) xxx

Version 1.0, Month, 200X

STANDARD OPERATING PROCEDURE FOR DETERMINING MANDARIN JUICE IN ORANGE JUICE

Prepared by: Dr Angus I Knight, Leatherhead Food International

Date December 2008

Approved by _________________________ Date _______________

27

CONTENTS

1. HISTORY / BACKGROUND....................................................................................................12

1.1 BACKGROUND...........................................................................................................................12 1.2 CHANGES IN CURRENT VERSION................................................................................................12

2. PURPOSE....................................................................................................................................12

3. SCOPE .........................................................................................................................................12

4. DEFINITIONS AND ABBREVIATIONS ................................................................................12

5. PRINCIPLE OF THE METHOD..............................................................................................12

6. MATERIALS AND EQUIPMENT ...........................................................................................12

6.1 CHEMICALS...............................................................................................................................12 6.2 WATER .....................................................................................................................................13 6.3 SOLUTIONS, STANDARDS AND REFERENCE MATERIALS .............................................................13 6.4 COMMERCIAL KITS....................................................................................................................13 6.5 PLASTICWARE...........................................................................................................................13 6.6 GLASSWARE..............................................................................................................................13 6.7 EQUIPMENT...............................................................................................................................13

7. PROCEDURES...........................................................................................................................13

7.1 SAMPLE PREPARATION..............................................................................................................13 7.2 QUALITY ASSURANCE...............................................................................................................14

8. CALCULATIONS AND DATA ANALYSIS ...........................................................................14

9. RELATED PROCEDURES.......................................................................................................14

1. HISTORY / BACKGROUND

1.1 Background

Pure orange juice commands premium prices. Under EU legislation pure orange juice can only be labelled as pure orange juice if the juice has been derived solely from cultivars of Citrus sinensis. Poor quality early season orange juice may be adulterated with other juices eg mandarin juice obtained from Citrus reticulata in order to add flavour, colour, or extend the product. Addition of other juices obtained from other Citrus sp. is not permitted in pure orange juice and this practice results in unfair trading and consumer deception. 1.2 Changes in current version. This is the original version

2. PURPOSE

The purpose of this assay is to detect the presence of mandarin juice in orange juice.

28

SOP xxx, Version 1.0

3. SCOPE

This method is applicable to freshly squeezed and concentrate derived processed fruit

juices. The DNA extraction method may be applied to different juices.

4. DEFINITIONS AND ABBREVIATIONS

PCR Polymerase Chain Reaction LOC Lab on a chip

5. PRINCIPLE OF THE METHOD

The method described in this SOP allows the determination of mandarin DNA in the presence of orange DNA, when the DNA is extracted from juice mixtures. The method is based on the fact that differences exist in the chloroplast DNA sequences in oranges compared with mandarins. The method uses the polymerase chain reaction (PCR) to amplify a specific fragment of chloroplast DNA extracted from orange and mandarin juice. A single pair of PCR primers based on conserved orange and mandarin chloroplast DNA sequences is used for amplification. When these PCR products are mixed at the plateau phase of PCR they re-anneal to form heteroduplexes owing to the presence of an 8bp indel. The heteroduplexes can be recognised by their reduced mobility using LOC capillary electrophoresis and quantified in order to measure adulteration. The method consists of the following steps:

1. DNA extraction 2. PCR 3. Analysis of DNA amplicons using the Agilent DNA 1000 Bioanalyser

The following controls must be used in the analysis:

1. DNA extraction control. 2. PCR negative control. 3. Authentic orange/mandarin mixture (50%v/v)

6. MATERIALS AND EQUIPMENT

6.1 Chemicals

Ethanol absolute (>99.8%) Product code : E/0650DF/17 Isopropanol (Propan-2-ol) (>99.5%) Product code : P/7490/08

Page 29 of 53


Chloroform (>99.8%) Product code: C/4960/17 Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG 1M Tris.HCl 0.1M EDTA (100x TE buffer pH8.0) Product code: T-9285 Supplier: Sigma Aldrich Company Ltd, Fancy Road Poole, Dorset BH11 4QH Eco R1 (40 u/ul) Product code: 10200310001 Glycogen Product code: 10901393001 Supplier: Roche Diagnostics Ltd. Charles Avenue, Burgess Hill RH15 9RY

6.2 Water

Ultrapure water Product code: VX10977035 Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG

6.3 Solutions, standards and reference materials

Authentic reference juice samples are obtained by hand squeezing authentic fruit

samples using a domestic juicer using samples collected in MAFF project ANO630

Pegg, A.K., Patel, Evans, M.T.A., Farnell, P.J. (1996) Collection of authentic samples (fruit, leaves and rootstock) from important fruit-producing countries. Project report ANO 630. UK Ministry of Agriculture Fisheries and Food . There are currently no standards for quantitative analysis

DNA molecular mass standard

Precision Molecular Mass Standard. Product code: 170-8207 Supplier: BioRad Laboratories Ltd. Maxted Road, Hemel Hempstead, Herts, HP2 7DX

Page 30 of 53


All other standards for DNA molecular size and mass are supplied by Agilent for the DNA 100 chips (see equipment) Batch numbers for standards and chip reagents must be recorded.

6.4 Commercial kits

DNA Extraction kit Phytopure DNA isolation kit Product code: RPN8510 Supplier: GE Healthcare, Little Chalfont, Bucks HP7 9NA PCR Ready-to-go Pure Taq beads (alternative suppliers of Taq polymerase may be used). Product code: 27-9557-01 Supplier: GE Healthcare, Little Chalfont, Bucks HP7 9NA Custom PCR Primer Synthesis Supplier: Applied Biosystems, 120 Birchwood Boulevard, Warrington, Cheshire, WA3 7Q

Page 31 of 53


6.5 Plasticware

0.5 ml Treff thin-walled PCR tubes Product code: 96.4625.9.06 Eprak refill tube racks Product code: SL-5006 RT10F filter tips for p10 RT20F filter tips for P20 RT200F filter tips for P200 RT1000-F filter tips for P1000 Supplier: Anachem 20 Charles Street Luton Beds LU2 0EB 1.5 ml eppendorf tubes Product code: DIS-800-090Y Universal containers Product code: DIS-800-100Q DNA AWAY wipes Product code: MBP-BIO-007H Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG

6.6 Glassware

None 6.7 Equipment

Micro-centrifuge – MSE Micro Centaur (or equivalent) capable of 10,000g

Capillary electrophoresis system – Agilent 2100 Bio-analyser

DNA 100 chips and reagents Product code: 5067-1504 Supplier: Agilent technologies Ltd, Life Science and Chemical Analysis, Lakeside, Cheadle Royal Business Park Stockport, Cheshire SK8 3GR

Page 32 of 53


7. PROCEDURES

7.1 DNA EXTRACTION FROM FRUIT JUICES DNA samples should be extracted in duplicate for each sample. This method is based on modifications to a commercially available kit (Phytopure) and may be used for different fruit juices. The method consists of three steps: cell lysis, DNA extraction, and DNA precipitation. Control DNA extractions should be performed with 50% v/v authentic orange and mandarin juice samples and also include a negative TE blank extraction. Extractions should be performed in duplicate. If viscous juice samples are aliquoted then wide bore pipette tips should be utilised. If the extraction method is applied to clarified juices juices then 1ml aliquots should be microfuged for 5 min and 900 µl of the supernatant removed prior to cell lysis. Juice concentrates may also be extracted after diluting 1 : 5 with H2O. Avoid cross-contamination between samples by using fresh filter tips. N.B. wear gloves and safety glasses when handling chloroform and note any relevant COSHH assessments for reagents. The efficiency of DNA extraction should be measured – see section 7.5 7.1.1 Cell Lysis 1. Neutralise 100 µl fruit juice with 10 µl 2M Tris-HCl pH 8.0. 2. Add 600 µl of reagent 1 from the Phytopure kit , ensuring that all the reagents are fully dissolved. 3. Add 200 µl of reagent from the Phytopure kit 2. 4. Invert the tube several times until a homogenous mixture is obtained. 5. Incubate the mixture at 65°C in a shaking water bath for 10 minutes . (Alternatively, regular manual agitation during the incubation will suffice.) 6. Place the sample on ice for 20 minutes. 7.1.2 DNA extraction 1. Extract the sample once with 500 µl cold (-20°C) chloroform by vortexing and

microfuging (12,000g 10min). Retain upper aqueous phase after extraction. 2. Add 500 µl cold (-20 °C) chloroform and mix. 3. Add 100 µl DNA extraction silica from the Phytopure kit. Ensure that the silica is

fully resuspended by vortexing briefly. 4. Shake at room temp for 5 min. 5. Microfuge as above. 6. Remove and retain the upper aqueous phase without disturbing the interface and

precipitate the DNA (see next section).

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7.1.3 DNA precipitation 1. Add an equal volume (700 µl) cold (-20 °C) isopropanol. 2. Add 1 µl glycogen (20 mg/ml in H2O). 3. Mix gently by inversion to allow the DNA to precipitate. Leave at room temp 5

minutes. 4. Microfuge as above. 5. Carefully wash the pellet with 1 ml 70% ethanol. 6. Microfuge as above. 7. Discard the supernatant and dry the pellet. 8. Resuspend the pellet in 20 µl 1 X TE and use 2 µl for PCR (see next section) 7.2 PCR PCR reactions should be performed at least in triplicate for each sample i.e. a total of 6 PCR reactions for duplicate extractions of a fruit juice sample PCR reactions should be set up in a class II safety cabinet (if available) to protect reagents from PCR and DNA contamination. The room should be separated from PCR thermal cycling and product analysis. Use separate pipettes designated for PCR work in conjunction with disposable filter tips. DNA should be added as a final step. Following PCR reaction set up and analysis the surfaces and pipettes should be cleaned using DNA AWAY wipes. PCR uses pre-packaged commercially available reagents and represents the preferred method for reaction preparation providing greater convenience. The reactions utilise “Ready To Go” PCR beads containing lyophilised reagents. The reagents required are: “Ready to Go” PCR beads PCR Primers H2O The volumes used are shown below. When brought to a final volume of 25 µl by addition of sample DNA each reaction contains: 200 µM each dNTP, 50 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl (pH 9.0 at room temperature). For 1 reaction H2O 18 µl Primer BFOR (2.5pmol/µl) 2.5 µl Primer BREV (2.5pmol/µl) 2.5 µl _________________________________________ Total 23 µl The primers BFOR (AGAAAGATACAATCCCGCTAAACG) and BREV (GTATCCGCAATTCAATATAGATGGA) and H2O are mixed prior to dispensing 23 µl aliquots into each tube. The beads are allowed to dissolve in the solution for 5 minutes, this may be aided by gentle vortexing or tapping of the side of the tube. Add 2 µl template DNA to each reaction and place in thermal cycler. Add 2 µl of the

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dummy extraction as a negative control. Alternative suppliers of Taq polymerase may be used. 7.2.1 PCR conditions PCR conditions have been optimised using a Perkin Elmer 480 and a BioRad I cycler and are shown below. The use of different PCR machines may require the use of modified cycling parameters. PCR machines should be calibrated every six months. Perform thermal cycling and analysis of PCR products in a separate room. Never allow reagents, pipettes, gloves, laboratory coats etc used to handle PCR products to come into contact with reagents or pipettes used for setting up PCR reactions or DNA sample preparation. 94 oC 3 min 1 cycle 94 oC 30 s 60 oC 15 s 72 oC 30 s 40 cycles 94 oC 30 s 60 oC 15 s 72 oC 10 min 1 cycle 18 oC HOLD Following completion of the PCR step, reactions may be held at 18°C for 24 hours prior to analysis. PCR products may be stored indefinitely at -20°C. 7.3 Lab on a Chip (LOC) analysis 1µl PCR samples are analysed on the chip system in duplicate and in accordance with manufacturers instructions using manufacturer supplied sizing standards and software. Controls using authentic mandarin and orange juice PCR products from a 50% mixture must be analysed to confirm band identity and the migration of heteroduplexes and homoduplexes. The lower heteroduplex band may be visualised as two bands. If this occurs then both should be included in the analysis. An example electropherogram is shown in Figure 1.

1.1

7.4 Sample preparation

Samples may be used directly following purchase or squeezing or stored at -20C prior to use. Where the solution is not homogeneous or lumps exist then the juice solution should be blended in a domestic blender. Wide bore pipettes should be used for aliquoting viscous solutions.

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7.5 Quality Assurance

The efficiency of DNA extraction may be measured by spiking experiments. Typically 20 µl (2 µg) of a “Precision Molecular Mass Standard” (BioRad, Herts, UK) is spiked into 100 µl of juice sample and the sample neutralised and extracted as above. Precipitated DNA is resuspended in 20 µl 10mM Tris 1mM EDTA buffer (TE) pH 8.0 and 1 ul subjected to LOC capillary electrophoresis. Recovered bands are quantified using the LOC in order to determine the % w/w recovery. Recovery of the 200 bp mass standard should be >10% . In order to ensure that contaminating DNA is not present then a dummy (blank) extraction followed by PCR analysis should be carried out using TE alone.

8 CALCULATIONS AND DATA ANALYSIS

Adulteration is calculated assuming that PCR plateau has been reached after 40 cycles and therefore that the principles of Hardy-Weinberg (H-W) Law apply and the total PCR product can be represented by the ratio p2:2pq:q2 where p and q represent the quantities of authentic and adulterant species. 2pq represents the total heteroduplex (Hardy-Weinberg%) and 2pq% the fraction of the total PCR product. The value obtained for 2pq must be multiplied by 1.49 to correct for reduced heteroduplex measurement using the LOC. The 2 pq% is calculated from the total product yield from all bands. For 5% adulteration the theoretical % heteroduplex represents 9.5% of the total PCR product according to H-W law. The LOC underestimates heteroduplex by 1.49X and therefore this is equivalent to the measurement of 6.3% heteroduplex.

The quantity of adulterant may be measured in comparison to the standard LOC curve generated from a validation study using authentic juices (below). In practice actual yields of heteroduplex are 30% less than expected owing to natural variation. Since heteroduplex represents the major product of mixed allele PCR at PCR plateau the assay is sensitive and readily detects 5% v/v adulteration.

The LOC has a limit of detection of 0.149 ng heteroduplex and if there are two heteroduplex bands then the minimal gel loading of heteroduplex is 0.3 ng representing 6.3% of the product heteroduplex. That is, detection at 5% v/v mandarin juice requires a minimum gel loading of 4.76 ng. In practice results should be based on the analysis of 10 ng measured PCR product to avoid analysis close to the limit of detection.

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Heteroduplexes

Homoduplexes

Figure 1. Example electropherogram for the detection of mandarin juice in orange juice following heteroduplex analysis using PCR and the LOC. Lane 1-6, 0, 2.5, 5, 10, 15, and 50% v/v mandarin in orange juice.

Figure 2. Standard curve and slope for calculating % v/v mandarin in orange from

corrected % heteroduplex values.

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9 RELATED PROCEDURES

None

---------- END OF DOCUMENT ----------

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6.2 Draft SOP for detecting grapefruit juice in orange juice pending approval from the Agency Additives and Authenticity Methodology Working Group

FOOD STANDARDS AGENCY

STANDARD OPERATING PROCEDURE (SOP) xxx

Version 1.0, Month, 200X

STANDARD OPERATING PROCEDURE FOR DETERMINING GRAPEFRUIT JUICE IN ORANGE JUICE

Prepared by: Dr Angus I Knight, Leatherhead Food International

Date December 2008

Approved by _________________________ Date _______________

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CONTENTS

1. HISTORY / BACKGROUND....................................................................................................12

1.1 BACKGROUND...........................................................................................................................12 1.2 CHANGES IN CURRENT VERSION................................................................................................12

2. PURPOSE....................................................................................................................................12

3. SCOPE .........................................................................................................................................12

4. DEFINITIONS AND ABBREVIATIONS ................................................................................12

5. PRINCIPLE OF THE METHOD..............................................................................................12

6. MATERIALS AND EQUIPMENT ...........................................................................................12

6.1 CHEMICALS...............................................................................................................................12 6.2 WATER .....................................................................................................................................13 6.3 SOLUTIONS, STANDARDS AND REFERENCE MATERIALS .............................................................13 6.4 COMMERCIAL KITS....................................................................................................................13 6.5 PLASTICWARE...........................................................................................................................13 6.6 GLASSWARE..............................................................................................................................13 6.7 EQUIPMENT...............................................................................................................................13

7. PROCEDURES...........................................................................................................................13

7.1 SAMPLE PREPARATION..............................................................................................................13 7.2 QUALITY ASSURANCE...............................................................................................................14

8. CALCULATIONS AND DATA ANALYSIS ...........................................................................14

9. RELATED PROCEDURES.......................................................................................................14

8. HISTORY / BACKGROUND

8.1 Background

Pure orange juice commands premium prices. Under EU legislation (5) in order to label pure orange juice as pure orange juice the juice must be derived from cultivars of Citrus sinensis. Poor quality early season orange juice may be adulterated with other juices e.g. grapefruit juice obtained from Citrus paradise, in order to add flavour or extend the product. Addition of other juices obtained from other Citrus sp. is not permitted in pure orange juice and this practice results in unfair trading and consumer deception. 8.2 Changes in current version. This is the original version

9. PURPOSE

The purpose of this assay is to detect the presence of grapefruit juice in orange juice.

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10. SCOPE

This method is applicable to freshly squeezed and concentrate derived processed fruit

juices. The DNA extraction method may be applied to other juices

11. DEFINITIONS AND ABBREVIATIONS

PCR Polymerase Chain Reaction LOC Lab on a chip

12. PRINCIPLE OF THE METHOD

The method described in this SOP allows the determination of grapefruit DNA in the presence of orange DNA, when the DNA is extracted from juice mixtures. The method is based on the fact that differences exist in the chloroplast DNA sequences in oranges compared with grapefruit. The method uses the polymerase chain reaction (PCR) to amplify a specific fragment of chloroplast DNA extracted from orange juice. A single pair of PCR primers based on conserved orange and grapefruit chloroplast DNA sequences is used for amplification. The amplified orange and mandarin PCR fragments differ in the presence of an Eco R1 restriction site that is unique to the grapefruit chloroplast amplicon. Amplification of mixed DNA samples allows the recognition of grapefruit amplicons following cleavage of the 177 bp amplicon DNA into grapefruit specific fragments of 125 bp and 52 bp.

41


The method consists of the following steps.

4. DNA extraction 5. PCR 6. Eco R1 restriction enzyme digestion 7. Analysis of DNA fragments using the Agilent DNA 1000 bioanalyser

.The following controls must be used in the analysis:

4. DNA extraction control. 5. PCR negative control. 6. DNA digestion control

13. MATERIALS AND EQUIPMENT

13.1 Chemicals

Ethanol absolute (>99.8%) Product code : E/0650DF/17 Isopropanol (Propan-2-ol) (>99.5%) Product code : P/7490/08 Chloroform (>99.8%) Product code: C/4960/17 Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG 1M Tris.HCl 0.1M EDTA (100x TE buffer pH8.0) Product code: T-9285 Supplier: Sigma Aldrich Company Ltd, Fancy Road Poole, Dorset BH11 4QH Eco R1 (40 u/µl) Product code: 10200310001 Glycogen Product code: 10901393001 Supplier: Roche Diagnostics Ltd. Charles Avenue, Burgess Hill RH15 9RY

42


13.2 Water

Ultrapure water Product code: VX10977035 Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG

6.3 Solutions, standards and reference materials Authentic reference juice samples are obtained by hand squeezing authentic fruit samples using a domestic juicer samples collected in MAFF project ANO630 Pegg, A.K., Patel, Evans, M.T.A., Farnell, P.J. (1996) Collection of authentic samples (fruit, leaves and rootstock) from important fruit-producing countries. Project report ANO 630. UK Ministry of Agriculture Fisheries and Food . There is currently no standard for quantitative analysis DNA molecular mass standard Precision Molecular Mass Standard. Product code: 170-8207 Supplier: BioRad Laboratories Ltd. Maxted Road, Hemel Hempstead, Herts, HP2 7DX All other standards for DNA molecular size and mass are supplied by Agilent for the DNA 100 chips (see equipment) Batch numbers for standards and chip reagents must be recorded.

6.4 Commercial kits

DNA Extraction kit Phytopure DNA isolation kit Product code: RPN8510 Supplier: GE Healthcare, Little Chalfont, Bucks HP7 9NA PCR Ready-to-go Pure Taq beads (alternative suppliers of Taq polymerase may be used). Product code: 27-9557-01 Supplier: GE Healthcare, Little Chalfont, Bucks HP7 9NA Custom PCR Primer Synthesis Supplier: Applied Biosystems, 120 Birchwood Boulevard, Warrington, Cheshire, WA3 7Q

43


6.5 Plasticware

0.5ml Treff thin walled PCR tubes Product code: 96.4625.9.06 Eprak refill tube racks Product code: SL-5006 RT10F filter tips for p10 RT20F filter tips for P20 RT200F filter tips for P200 RT1000-F filter tips for P1000 Supplier: Anachem 20 Charles Street Luton Beds LU2 0EB 1.5ml eppendorf tubes Product code: DIS-800-090Y Universal containers Product code: DIS-800-100Q DNA AWAY wipes Product code: MBP-BIO-007H Supplier: Fisher Scientific, Bishop Meadow Road, Loughborough, Leics. LE11 5RG

6.6 Glassware

None 6.7 Equipment

Micro-centrifuge – MSE Micro Centaur (or equivalent) capable of 10,000g

Capillary electrophoresis system – Agilent 2100 Bio-analyser

DNA 100 chips and reagents Product code: 5067-1504 Agilent Technologies Ltd, Life Science and Chemical Analysis, Lakeside, Cheadle Royal Business Park Stockport, Cheshire SK8 3GR

44


7 PROCEDURES

7.1 DNA EXTRACTION FROM FRUIT JUICES DNA samples should be extracted in duplicate for each sample. This method is based on modifications to a commercially available kit (Phytopure) and may be used for different fruit juices. The method consists of three steps: cell lysis, DNA extraction, and DNA precipitation. Control DNA extractions should be performed with 50% v/v authentic orange and grapefruit juice samples and also include a negative TE blank extraction. Extractions should be performed in duplicate. If viscous juice samples are aliquoted then wide bore pipette tips should be utilised. If the extraction method is applied to clarified juices eg pomegranate or grape juices, then 1ml aliquots should be microfuged for 5 min and 900 ul of the supernatant removed prior to cell lysis. Juice concentrates may also be extracted after diluting 1 : 5 with H2O. Avoid cross-contamination between samples by using fresh filter tips. N.B. wear gloves and safety glasses when handling chloroform and note any relevant COSHH assessments for reagents. The efficiency of DNA extraction should be measured (see section 7.5). 7.1.1 Cell Lysis 1. Neutralise 100 µl fruit juice with 10µl 2M Tris-HCl pH 8.0. 2. Add 600 µl of reagent 1 from the Phytopure kit , ensuring that all the reagents are fully dissolved. 3. Add 200 µl of reagent 2 from the Phytopure kit. 4. Invert the tube several times until a homogenous mixture is obtained. 5. Incubate the mixture at 65°C in a shaking water bath for 10 minutes . (Alternatively, regular manual agitation during the incubation will suffice.) 6. Place the sample on ice for 20 minutes. 7.1.2 DNA extraction 7. Extract the sample once with 500 µl cold (-20°C) chloroform by vortexing and

microfuging (12,000g 10min). Retain upper aqueous phase after extraction. 8. Add 500 µl cold (-20 °C) chloroform and mix. 9. Add 100 µl DNA extraction silica from the Phytopure kit. Ensure that the silica is

fully resuspended by vortexing briefly. 10.Shake at room temp for 5 min. 11.Microfuge as above. 12.Remove and retain the upper aqueous phase without disturbing the interface and

precipitate the DNA (see next section).

45


7.1.3 DNA precipitation 9. Add an equal volume (700 µl) cold (-20 °C) isopropanol. 10.Add 1 µl glycogen (20 mg/ml in H2O). 11.Mix gently by inversion to allow the DNA to precipitate. Leave at room temp 5

minutes. 12.Microfuge as above. 13.Carefully wash the pellet with 1ml 70% ethanol. 14.Microfuge as above. 15.Discard the supernatant and dry the pellet. 16.Resuspend the pellet in 20 µl 1 X TE and use 2 µl for PCR (see next section)

7.3 PCR PCR reactions should be performed at least in triplicate for each sample i.e. a total of 6 PCR reactions for duplicate extractions of a fruit juice sample PCR reactions should be set up in a class II safety cabinet (if available) to protect reagents from PCR and DNA contamination. The room should be separated from PCR thermal cycling and product analysis. Use separate pipettes designated for PCR work in conjunction with disposable filter tips. DNA should be added as a final step. Following PCR reaction set up and analysis the surfaces and pipettes should be cleaned using DNA AWAY wipes PCR uses pre-packaged commercially available reagents and represents the preferred method for reaction preparation providing greater convenience. The reactions utilise “Ready To Go” PCR beads containing lyophilised reagents The reagents required are: “Ready to Go” PCR beads Primers H2O When brought to a final volume of 25 µl by addition of sample DNA each reaction contains: 200 µM each dNTP, 50 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl (pH 9.0 at room temperature). For 1 reaction H2O 18 µl Primer Chl A (2.5pmol/µl) 2.5 µl Primer Chl B (2.5pmol/µl) 2.5 µl _________________________________________ Total 23 µl The primers Chl A (GCC GGG CAA ATA AAA TGA ATT TC) and Chl B (GAA AAG AAT TTC TTA CAA ATT CCC ) and H2O are mixed prior to dispensing 23 µl aliquots into each tube. The beads are allowed to dissolve in the solution for 5 minutes, this

46


may be aided by gentle vortexing or tapping of the side of the tube. Add 2 µl template DNA to each reaction and place in a thermal cycler. Add 2 µl of the dummy extraction as a negative control. 7.2.1 PCR conditions PCR conditions have been optimised using a Perkin Elmer 480 and a BioRad I cycler and are shown below. 94 oC 3 minutes 1 cycle 94 oC 30 seconds 50 oC 15 seconds 72 oC 30 seconds 40 cycles 94 oC 30 seconds 50 oC 15 seconds 72 oC 10 minutes 1 cycle 18 oC HOLD Following completion of PCR, reactions may be held at 18oC for 24 hours prior to analysis. PCR products may be stored indefinitely at -20° C. The use of different PCR machines may require the use of modified cycling parameters. PCR machines should be calibrated every six months. Perform thermal cycling and analysis of PCR products in a separate room from that used for reaction set up. Never allow reagents, pipettes, gloves, laboratory coats etc used to handle PCR products to come into contact with reagents or pipettes used for setting up PCR reactions or DNA sample preparation. 7.3 Eco R1 Restriction enzyme digestion Restriction enzyme digestion is carried out in accordance with manufacturers instructions. Typically 5 µl PCR product is digested in a total volume of 10 µl including 1 µl 10X reaction buffer and 1 µl (40 units) of enzyme at 37◦C in a water bath or heating block for 60 minutes. 7.4 Lab on a Chip (LOC) analysis 1µl of undigested and digested samples are analysed on the chip system in duplicate and in accordance with manufacturers instructions using manufacturer supplied sizing standards and software. Controls using authentic grapefruit and orange juice PCR products obtained from authentic juices should be present in the analysis as well as a blank DNA extraction. The theoretical amplicon sizes are 177 bp and the theoretical Eco R1 cleavage products are 125 bp and 52 bp. LOC measured values may range

47


from 179-183, 128-135 and 61-69 bp respectively. A minor band is frequently visualised migrating below the 177 bp product in both orange and grapefruit PCR products. This band demonstrates similar Eco R1 cleavage and may result from chloroplast heteroplasmy or PCR stutter. This band is not included within the analysis. The size and quantity of the 177 bp and 125 bp bands are recorded.

1.2

9.4 Sample preparation

Samples may be used directly following purchase or squeezing or stored at -20C prior to use. If the solution is not homogeneous or lumps exist then the juice solution should be blended in a domestic blender. Wide bore pipettes should be used for aliquoting viscous solutions.

9.5 Quality Assurance

The efficiency of DNA extraction may be measured by spiking experiments. Typically 20 µl (2 µg) of a “Precision Molecular Mass Standard” (BioRad, Herts, UK) is spiked into 100 µl of juice sample and the sample neutralised and extracted as above. Precipitated DNA is resuspended in 20 µl 10mM Tris 1mM EDTA buffer (TE) pH 8.0 and 1 µl subjected to LOC capillary electrophoresis. Recovered bands are quantified using the LOC in order to determine the % recovery. Recovery of the 200 bp mass standard should be >10% . In order to ensure that contaminating DNA is not present then a dummy (blank) extraction followed by PCR analysis should be carried out using TE alone.

10 CALCULATIONS AND DATA ANALYSIS

The level of adulteration is calculated assuming that PCR has gone to completion and

that the PCR plateau phase has been reached after 40 cycles. Under these conditions

the principles of Hardy-Weinberg (H-W) Law apply and the total PCR product can be

represented by the ratio p2:2pq:q2 where p and q represent the quantities of authentic

and adulterant species. q2 represents the homoduplex fraction of the grapefruit PCR

product cleavable by Eco R1. The 177 bp and 125 bp bands are measured using the

Agilent software and recorded – see Figure 1. The quantity of the 125bp band is

multiplied by 1.4 since the 125 bp represents only 70% of q2 within the PCR product.

To find % q2 we must determine the fraction of the product occupied by q2 and

multiply this by 100. The % grapefruit adulteration (% q) is equal to the square root of

48


% q2. The sensitivity of the assay is determined by the concentration of PCR product

following digestion that is loaded on the LOC and the limit of detection of the LOC

(0.1 ng). This may be calculated according to Hardy Weinberg law as shown in Table

1. The assay is not sensitive and >20 ng PCR product are required to detect 10% v/v

grapefruit adulteration.

EcoR1 RFLP analysis of orange and grapefruitchloroplast DNA PCR products on the Chip System

Grapefruit Orange

Figure 1. Example Eco R1 RFLP analysis of authentic grapefruit and orange samples. Lane 1 Authentic grapefruit (88) uncut; Lanes 2-6, Eco R1 digests of amplified DNA from different authentic grapefruit samples (88,96,222,243 & 393). Lanes 7-11, Eco R1 digests of amplified DNA from different authentic orange samples (142,144,278,290 & 352). Lane 12 Negative control.

49


Table 1. Calculation of the theoretical detection limits at different chip loadings for the detection of grapefruit juice (q) in orange juice (p) and the distribution of different duplexes according to H-W law. Data assumes a limit of detection of 0.1 ng (Agilent data) using LOC Eco R1 RFLP analysis.

11 RELATED PROCEDURES

None

---------- END OF DOCUMENT ----------

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