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For Peer ReviewHighly sensitive Real-Time PCR method to identify species
origin in heparinoids
Journal: Analytical and Bioanalytical Chemistry
Manuscript ID ABC-01707-2019.R1
Type of Paper: Research Paper
Date Submitted by the Author: 17-Oct-2019
Complete List of Authors: Pecorini, Simone; Biofer, Quality ControlTorrini, Lucia; Biofer, Research and DevelopmentCamurri, Giulio; Biofer, Quality ControlFerraresi, Roberta; Biofer, Quality Control
Keywords: Heparinoid, PCR, Origin, Glycogen, Purification
Analytical & Bioanalytical Chemistry
For Peer Review
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Highly sensitive real-time PCR method to identify species origin in heparinoids
Simone Pecorini1, Giulio Camurri1, Lucia Torrini2, Roberta Ferraresi1,*,
1 Microbiology Quality Control Laboratory, Biofer S.p.A., via Canina, 2, 41036 Medolla (MO), Italy
2 Research and Development Laboratory, Biofer S.p.A., via Canina, 2, 41036 Medolla (MO), Italy
*Corresponding author, email: [email protected]
ABSTRACT
Heparinoids are the starting material for sulodexide production, drug used as intravenous anti-
coagulant, as an alternative to heparin. The origin determination in the starting material for
sulodexide, heparin and derivatives, is crucial for the safety (including the impact related to Bovine
Spongiform Encephalopathy) and efficacy of the final products. Therefore, European countries
have decided to approve the production of heparin only from porcine intestinal mucosa.
PCR (Polymerase Chain Reaction) methods are available to evaluate the origin species of crude
heparin, during heparin production process, while they lack for the same analysis in heparinoids
during sulodexide manufacturing processes.
Notably, two main critical issues occur during the origin determination by using PCR for heparinoid
analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA
amounts are very low in these samples. To overcome these critical issues, our proposed method is
based on two fundamental steps, the DNA concentration by glycogen treatment and DNA
purification, which occur before and after DNA extraction, respectively. Finally, by applying Real-
Time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine and
caprine), to assess possible contamination, and one from swine, to confirm the origin species. To
date, such method is the only one that determines origin species by PCR for heparinoids, that
guarantees quality, safety and traceability of heparin-derived pharmaceutical products. In
conclusion, our proposed method is alternative to Nuclear Magnetic Resonance and ELISA
methods, because Real-Time PCR offers significant advantages in sensitivity, specificity and
robustness.
Keywords Heparinoid, PCR, glycogen, purification, origin, glycosaminoglycans
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INTRODUCTION
After the advent of Bovine Spongiform Encephalopathy (BSE), European countries have decided
to approve the production of the heparin only from porcine intestinal mucosa [1, 2]. To ensure the
safety of these anti-coagulant agents, pharmacopoeias, institutions and monitoring organizations in
medical and pharmaceutical fields declared that the origin of the crude heparin (raw material) must
be known and certificated [2-4].
Chemically, the significant differences among porcine and ruminant (bovine, ovine and caprine)
heparins are represented by the greater abundance of sulfated groups in the first, the differences
in chemical structures, the affinity to bind the anti-thrombin III and the molecular weight distribution.
Notably, concerning anti-coagulant activity, porcine and ovine heparin are quite similar, while
bovine heparin biological activity is weaker [5].
Sulodexide is a key pharmaceutical heparin-like product widely used in thrombosis field, as a pro-
fibrinolytic agent. It is a glycosaminoglycan derived from porcine intestinal mucosa, constituted by
chains of polysaccharides, with different grades and types of sulfation sites [6]. The major
constituents of sulodexide are heparan sulfate and dermatan sulfate, which counts, respectively,
for about 80% and 20% of the total [7, 8]. While crude heparin represents the raw material for
heparin production, heparinoids (HPDs), heparin-derived glycosaminoglycans, constitute the
starting material of sulodexide manufacturing process. Heparin, differently from sulodexide and
HPD, is the only product with a dedicated monograph in European and United States
pharmacopeia.
Pharmacopeia monographs suggest adopting Polymerase Chain Reaction (PCR) as the elective
method to determine origin species. In literature, the detection of ruminant contamination has been
described with different methods, based on immunology, microscopy or spectrometry. While
microscopic and spectrometric methods lack sensitivity, ELISA is more accurate, but a matrix
interference and false positive results may occur [9, 10]. Real-Time PCR represents a valid control
method for the evaluation of the origin of heparin-derived samples, compared to Nuclear Magnetic
Resonance (NMR) [11]. Moreover, Real-Time PCR offers significant advantages in sensitivity,
specificity, accuracy and robustness [12]. PCR methods are known for origin determination in
heparin, but a gap in knowledge exists concerning the determination of origin species in HPD [1].
Generally, the main critical issue, encountered during the development of PCR method for heparin-
containing samples, is the presence of heparin, inhibitor of DNA polymerase [13-15].
The aim of our study is the identification and validation of a Real-Time PCR method to determine
origin species In HPD samples. Therefore, it will be possible to ensure the quality, safety and
accuracy controls during the manufacturing process of sulodexide, as it happens for heparin.
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MATERIALS AND METHODS
No animals were used for this study. The study did not violate human rights.
DNA assay by optical density at 260 nm
Total DNA concentration was assayed by ultraviolet (UV) absorption at 260 nm, by using the UV-
VIS spectrometry (Perkin Elmer, Waltham, USA) with three replicates. Sample with absorbance
more than 1.0 AU (Absorbance Units) was diluted 1:10 or 1:100, from an initial concentration of 4
mg of powder per mL of water. Blank measurements were performed by using molecular biology
grade water.
Nucleotidic impurities
HPD and crude heparin samples were evaluated by using High Performance Liquid
Chromatography (HPLC), applying the method “Nucleotidic impurities” described in the monograph
section of heparin sodium from United States Pharmacopoeia (USP), by using USP Adenosine
reference standard (U.S. Pharmaceutical Convention, Rockville, USA, Cat. No. 1012123) [3].
Before running the HPLC analysis, we treated samples by using 10 µL benzonase (Sigma-Aldrich,
Saint Louis, USA, Cat. No. E1014), 222 U alkaline phosphatase (Sigma-Aldrich, Cat. No P4978)
and 125 µL phosphodiesterase I solution (0.1U/µL, Worthington, Lakewood, USA, Cat. No. VPH),
as clearly described in [3]. We performed the HPLC analysis by using the Agilent Infinity 1260
(Agilent Technologies, Santa Clara, USA), with LC Mode, 4,6 mm x 15 cm column (4-µm packing
L1, from Sigma-Aldrich,), 260 nm UV detector and the following temperatures: 37±1°C for the
autosampler and 18°C for the column. The flow rate is fixed as 1 mL/min, with an injection volume
of 10 µL and the runtime fixed to 25 min. Adenosine USP standard was injected six times to control
reproducibility. Then, other retention times were set up by using the table of relative retention time
from USP [3].
Glycogen treatment, DNA extraction and purification
Glycogen treatment of 1 h was performed, by using glycogen from Mytilus edulis (Sigma-Aldrich),
following manufacturer’s procedure. The starting amount of HPD was 25 mg, dissolved in 100 µL
of molecular biology grade water. Finally, pellets were resuspended in molecular biology grade
water.
DNA extraction was performed by using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany),
according to manufacturer’s protocols. 4 µL internal control DNA (Primer DesignTM Ltd.,
Southampton, UK) were added to HPD and crude heparin samples before the DNA extraction,
together with the addition of proteinase K, as indicated by manufacturer.
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After extraction, DNA was purified by using DNeasy PowerClean Pro Cleanup kit (Qiagen),
according to manufacturer’s instructions, with a 50 µL final elution with molecular biology grade
water.
DNA amplification
Multiplex Real-Time PCR was performed by using the Light Cycler 480 thermal cycler (Roche
Diagnostics, Basel, Switzerland), LightCycler 480 Probe Master (Roche Diagnostics) and
Speciation standard kit of Bos taurus, Capra hircus, Ovis aries and Sus scrofa (Primer DesignTM
Ltd.), according to manufacturer’s instructions. The Real-Time PCR mixture of 20 µL included: 10
µL Probe Master, 1 µL species-specific primer and probe (FAM, 465-510 nm) mix, 1 µL internal
control DNA primer and probe (VIC, 533-580 nm) mix, 3 µL molecular biology grade water and,
finally, 5 µL DNA from treated samples, positive control or negative control (molecular biology
grade water). Amplified sequences are patented and minimum information for publication from the
supplier is summarized in Table S1 (see Electronic Supplementary Material, ESM). The thermal
protocol was set up as follows: 10 min at 95°C for initial denaturation, 50 cycles of denaturation (10
s at 95°C) and annealing/extension (1 min at 60°C, with fluorescence detection) and, extension (15
s at 78°C), and, finally, 30 s at 40°C to cool the plate.
Positive control DNA is different for each tested species and it is constituted by known copy
numbers of the amplified sequence for each species. Reaction conditions worked correctly only if
species-specific positive control DNA crossing point (Cp) is included in the range 16-23
amplification cycles (cs) at the end of the run, as mentioned in the manufacturer’s protocol. Internal
control DNA works as a positive control for the extraction and purification processes, to assess
whether DNA polymerase inhibitors have been correctly removed. Kits from Primer Design
contains a separate mix with specific primers and probe for the detection of such exogenous DNA,
which is not species-specific. The different emission ranges of the two probes, FAM and VIC, allow
performing a multiplex reaction. As already mentioned, such internal control DNA was spiked into
heparinoid sample before the DNA extraction process. Extraction protocol works correctly and
inhibitors are removed when internal control DNA has a Cp within the range 25-31 cs. The
Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler
480 II software database was applied during analysis.
Statistical analysis
Quantitative variables between groups were analyzed by different statistical analyses, depending
on the sample types (performed statistical test is indicated in the caption of each figure). P values
<0.05 were considered statistically significant. Data shown in the graphs are represented as the
mean±SEM (Standard Error of the Mean). Statistical analysis were performed using Prism 8.1
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(Graphpad Software Inc., La Jolla, USA). Normality analysis was performed before statistical
comparisons.
RESULTS
Crude heparin displays higher DNA amount than HPD
Samples of crude heparin and HPD were analyzed by optical density at 260 nm. Results are
shown in table of Fig. 1, reported as absorbance values (ABS, expressed as AU). Sample analysis
to estimate the DNA amount reported high quantity of nucleotides in crude heparins (mean ABS =
1.19±0.07 AU), and, on the other side, lower amounts in HPDs (mean ABS = 0.33±0.01 AU), with
a statistically significant variation (P < 0.0001, Mann-Whitney test), as reported in Fig. 1.
Absorbance values of HPD samples from different suppliers (HPD-X and HPD-Y) are reported in
the table of Fig. 1.
Moreover, different DNA amounts in one sample of crude heparin and one HPD (from Y supplier)
sample were evaluated with a chromatographic analysis of nucleotidic impurities. We observed
several peaks, identified as specific nucleotides, in the crude heparin sample (Fig. 2, orange line),
while in the HPD (Y) sample (Fig. 2, black line) observed peaks presented lower intensity.
Retention times of nucleotidic impurities are reported in table of Fig. 2. Other peaks were not
considered since they were also observed in blank samples. Overall, nucleotidic impurity
percentage in crude heparin was equal to 2.15 % m/m, while HPD (Y) sample presented 0.14 %
m/m.
As displayed in Fig. 3, crude heparin (B) showed higher DNA amount than HPD samples, also
after Real-Time PCR, since the mean Cp for Sus scrofa was 18.81±0.1 cs, similar to the porcine
positive control (A, mean Cp = 19.83±0.04 cs). On the other side, three HPD samples (C) reported
mean Cp = 32.84±0.30 cs (33.44, 33.19 and 31.90 cs, respectively) after DNA extraction and
purification. Negative control (D) was compliant.
DNA purification is crucial to remove inhibitory effect of the heparin on DNA polymerase
Fig. S1 (see ESM) reports the amplification curves of the HPD-Y sample that underwent DNA
extraction and purification (B), and DNA extraction only (C), besides positive control (A, mean Cp =
17.94±0.13 cs). While purified DNA reported positive mean Cp = 29.72±0.01 cs, the extracted-only
DNA showed no Cp, as the negative control (D).
The analysis of internal control DNA, reported in Fig. S2 (see ESM), was performed on the same
samples displayed in Fig. S1 (see ESM). Here, we show that DNA amplification occurs only in
extracted-and-purified samples (A, mean Cp = 29.63±0.03 cs), while extracted-only DNA (B)
presents an amplification curve similar to the negative control (C).
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DNA detection increases after glycogen treatment
Fig. 4 shows Mean Cp values of 1-h glycogen-treated (+) or untreated (-) HPD samples after DNA
amplification with Sus scrofa specific primers on 11 HPD samples, 4 HPD-X and 7 HPD-Y. The
glycogen treatment allows gaining 1 cycle when we performed Real-Time PCR (P= 0.0066, paired
t-test): mean Cpuntreated was 32.09±0.60 cs, while mean Cptreated was 31.04±0.55 cs. Similarly, table
in this figure reports the mean Cp values of amplified swine DNA from 11 HPD samples, with or
without 1-h glycogen treatment, calculated from two replicates for each sample.
Amplification curves (reported in ESM Fig. S3) show an increase in detected DNA amounts,
comparing samples treated with glycogen for 1 hour, before extracting and purifying DNA (C, mean
Cp = 29.72±0.01 cs), and untreated ones (D, extraction and purification only), with mean Cp =
32.15±0.31 cs. Extending the glycogen treatment to 16 hours (B, overnight), we reported mean Cp
= 28.69±0.03 cs, confirming the increase of DNA amounts. Positive control (A, Cp = 17.94±0.13
cs) and negative control (E) complied.
Real-Time PCR validation method
According to European Pharmacopoeia (EP) [16], we validated our Real-Time PCR-based
qualitative method for the specificity, robustness and detection limit. Method validation was
performed by using positive control DNA supplied from Primer Design, as template for specificity,
robustness and detection limit. After, ten batches of HPDs from the two suppliers were analyzed to
validate this method on HPDs.
Specificity
We tested the specificity of our method, by evaluating whether primers and probes can only detect
the species expected to be present. For each primer kit of the analyzed species, three replicates
were loaded with positive control DNA from four species in the same well. Swine-specific primer kit
amplified Sus scrofa positive control (Cp =18.39±0.05 cs), while other species-specific positive
controls were not amplified (Fig. 5A). Bos taurus positive control presented Cp = 17.95±0.04 cs,
after amplification with bovine-specific primers. They also amplified other species-specific positive
control: Cp Sus scrofa = 42.14 cs; Cp Ovis aries = 40.56±1.29 cs; Cp Capra hircus = 39.42 cs. For
Sus scrofa and Capra hircus, only one replicate out of three was detectable, with a positive
crossing point, while Ovis aries DNA amplification occurred in two samples out of three, as
displayed in Fig. 5B. Amplification of Ovis aries positive control by using ovine-specific primers
reported Cp = 16.11±0.03 cs, while positive control from other species did not display any
amplification (Fig. 5C). Similarly, Capra hircus positive control was amplified by using caprine-
specific primer kit, with Cp = 17.18±0.05 cs (Fig. 5D). Amplification of such positive controls from
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other species with these primers did not show significant amplification. Specificity test results are
summarized in Table S2 (see ESM).
Robustness
EP defined the robustness as “the capacity of an analytical procedure not to be affected by
deliberate, although small, variations in parameters” [16]. This indicates the reliability of such
analytical procedure during normal usage. Our test was set as follows: Probe Master volume was
changed by 5% more and less than the correct volume (5.25 and 4.75 µL, respectively), to verify
whether such changes affect the reliability of the analysis. We chose to change Probe Master
volume, since this is the only reagent outside the standardized primer kits from Primer Design, as
shown in Fig. S4 (see ESM), after the amplification of sample DNA, we reported 32.68±0.02 cs as
mean Cp with 5 µL volume of Probe Master (B), 32.92±0.06 cs when volume was reduced to 4.75
µL (C), and, finally, 32.55±0.07 cs with 5.25 µL volume of Probe Master (D). Analysis of positive
control DNA complies (A). The percentage variation of mean Cp was 0.73% and 0.40% with 5%
volume decrease and 5% volume increase, respectively.
Moreover, cross-contamination must be evaluated, to demonstrate robustness: it is defined as a
test in which 20 alternate samples of negative samples and positive samples were loaded side-by-
side (as reported in the table in ESM Fig. S5). All negative samples revealed as negative, while all
positive controls displayed a Cp included in the range 16-23 cs, as reported in ESM Fig. S5.
Detection Limit
Detection Limit is defined as the lowest detectable amount of nucleic acid in an analyzed sample.
Such amount could be not necessarily accurately quantitated. Although the manufacturer of the
primer kits provided a fixed Detection Limit of 35 cs, we set up our Detection Limit, as the DNA
concentration that reported significant amplification in the 95% of analyzed samples with Real-
Time PCR, as requested by pharmacopeia. After the amplification of species-specific positive
controls, Sus scrofa, Bos taurus and Ovis aries primer kits reported the Detection Limit of 1.2 DNA
copies/µL (respectively, mean Cp was 38.55±0.36, 35.46±0.21 and 36.55±0.16 cs), while the
Detection Limit of Capra hircus primers was 0.6 DNA copies/µL, with 37.21±0.25 as mean Cp.
Data reported in Table 1 refer to the fixed detection limits for each species-specific kit. Other
concentrations, higher or lower, do not satisfy the aforementioned criteria.
The bovine specificity test results reported a critical issue, concerning the amplification of DNA
from other species by using Bos taurus primers, as reported in Fig. 5. Fixed detection limits for Sus
scrofa, Ovis aries and Capra hircus DNA were lower than the observed values (Cp = 42.14 cs, Cp
= 40.56±1.29 cs and Cp = 39.42 cs, respectively). Therefore, these values were considered as
nonspecific amplifications.
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HPD samples from different suppliers display different DNA amounts, after Real-Time PCR
We analyzed HPD samples from two different suppliers, by using UV spectrometry and Real-Time
PCR. On one hand, as displayed in Fig. 6, ten HPDs from the first supplier (arbitrarily named HPD-
X) show an increasing trend in absorbance values (mean absorbance = 0.35±0.01) compared to
ten samples from the second supplier (arbitrarily named HPD-Y, mean absorbance = 0.31±0.02),
without statistical significance. On the other hand, Real-Time PCR analysis reported a statistically
significant variation of crossing point, between the two suppliers: mean Cp of HPD-X samples was
28.30±1.16 cs, while HPD-Y samples reported 31.14±0.61 cs as mean Cp (P = 0.0475, Welch’s t-
test). Notably, for routine PCR analysis positive, negative and internal control were loaded on each
plate and they always complied.
DISCUSSION
Heparin and sulodexide (heparin-like product largely used as anti-coagulant drug) are
heterogeneous mixtures that cannot be entirely characterized [1, 6, 11]. Therefore, great attention
must be paid to their traceability and quality [1]. Besides BSE sanitary risk, bovine heparin has also
been associated with a higher risk of heparin-induced thrombocytopenia than porcine heparin [1,
17]. Differences in chemical structures occur mostly among porcine and bovine heparins, by using
NMR technique, since they slightly differ in the chemical structure. From a therapeutic point of
view, these slight differences result in clearly distinct drug effects [18].
Crude heparin and HPD respectively represent the raw material for heparin and sulodexide
manufacturing processes. In pharmacopeia, a monograph is dedicated only to heparin, but not for
sulodexide nor HPDs [2-4]. Monographs indicate the PCR as the elective technique to determine
origin species. However, Hydrogen Nuclear Magnetic Resonance (H-NMR) is the widely used
method to discriminate the porcine or the bovine origin of heparin, HPDs and sulodexide. However,
by using this technique, a part of the possible contaminants could be hidden by the heparin spectra
[11]. Moreover, standards for the origin determination of other ruminant species, e.g. ovine and
caprine, are not commercially available so far. Finally, such method does not ensure high
sensitivity, since declared purity of swine origin for H-NMR method for origin determination is 94%
in our experience.
In this study, we showed a qualitative method for the detection of species-specific DNA, by using
Real-Time PCR, to evaluate possible contamination from three of the most common ruminant
species in heparinoids. Notably, through this technique, which detects the presence of DNA rather
than the differences in chemical structures, we overcome the critical issue encountered with H-
NMR analysis, as previously mentioned.
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First, we performed UV spectrometry analysis to evaluate differences in DNA amounts in crude
heparin and HPD with low grade of sensitivity: crude heparin displays higher nucleic acid levels
compared to HPD (P < 0.0001). Indeed, HPDs manufacturing process requires more steps than
crude heparin one: this determines, in the end, a substantial reduction of DNA amounts, as
confirmed by our results. After, we performed HPLC analysis to better investigate the relative
abundance of DNA amounts, detected as nucleotidic impurities, in crude heparin compared to
HPDs, with higher levels of sensitivity and reliability. By using HPLC, we confirmed the data
observed with UV spectrometry: crude heparin contains higher DNA amounts respect to HPD.
Finally, also Real-Time PCR analysis revealed very higher DNA amounts in crude heparin if
compared to heparinoid samples, with a difference of 14 amplification cycles among those two
sample types.
DNA detection in HPD samples was possible only after the introduction of different steps, before
and after the DNA extraction, because it alone does not remove all the heparin residues and does
not allow detecting significant DNA amounts by using Real-Time PCR and, therefore, we inserted
DNA purification after its extraction. Notably, the presence of heparin residues heavily interferes
with DNA polymerase [15, 19]. Although we performed DNA purification, we inserted internal
control DNA in the PCR plate, to ascertain possible heparin interference. Moreover, DNA
extraction method is optimized by using proteinase K, which reduces the viscosity of the dissolved
HPD in water, because of its action on the proteins from the intestinal mucosa. As previously
demonstrated, our HPD samples characterize for low DNA amounts. Therefore, we evaluated the
positive impact of glycogen treatment before the DNA extraction on HPDs. Notably, glycogen is an
inert carrier that traps nucleic acids and, by using it, it is possible to recover oligonucleotides as
short as eight base pairs and low amounts of DNA/RNA. Glycogen forms a clearly visible pellet
during ethanol precipitation, since it is insoluble in this solvent. Compared to other nucleic acid
carriers, such as tRNA, linear polyacrylamide, or other methods, e.g. DNA sonication, glycogen
allows better efficiency, it is easy-to-use and it does not require particular safety precautions [20-
22]. Comparing samples treated with or without glycogen, we reported a significant difference in
mean Cp (P = 0.0066), confirming that glycogen treatment allows better detection of low DNA
amounts. Notably, enhancing the gap between crossing point of HPD samples and the threshold of
35 cycles by using glycogen treatment (as specified by kit’s manufacturer), allows a better
detection and analysis, since such threshold represents a first detection limit, as reported in the
manufacturer’s protocol of the species-specific primers. Additionally, manufacturer’s protocol
suggests incubating samples with glycogen for 1 hour or overnight. In both cases, we reported
greater detection in glycogen-treated samples, compared to untreated ones. Although overnight
treatment allows us to gain 1 cycle respect to 1-hour treatment, we chose the latter, since it is
timesaving. Thus, final sample preparation protocol, before the DNA amplification was set up as
follows: 1-h glycogen treatment, DNA extraction, DNA purification. Glycogen treatment
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concentrates nucleic acids and this reveals crucial for the correct method execution in highly
processed HPD samples.
As requested by pharmacopeia, the selected method needs to be validated. To validate qualitative
Real-Time PCR methods, EP and the US Food and Drug Administration guidance for industry
strongly recommends the evaluation of the specificity, detection limit and robustness [16, 23]. Our
method meets all these requirements, since it reveals specific and robust and with low detection
limit. Primer kits are specific, since only species-specific DNA was amplified by these primers.
Even if Bos taurus primers amplified DNA from other three species, registered crossing points
were over the detection limit and, therefore, considered as negative. Robustness is guaranteed
since no significant variations in Cp were reported, although the variation of ± 5% volume of Probe
Master. Moreover, no cross-contamination must be reported in all samples. Detection limit for all
species-specific primer kits was fixed as 1.2 DNA copies/µL, except for Capra hircus one, whose
detection limit was set up to 0.6 DNA copies/µL. For both cases, this allows highly sensitive and
efficient DNA detection. Since our Detection Limits for each species are lower than the one
declared from Primer Design, with higher grade of sensitivity and specificity for HPD.
Lastly, we aimed to characterize different suppliers of HPD, by using UV spectrometry, as well as
Real-Time PCR. Non-statistically significant differences in absorbance values among the two
suppliers, 0.35 rather than 0.31, transformed in significant variations when we performed Real-
Time PCR analysis (higher crossing point means lower DNA starting quantity). Both suppliers were
validated since the quality and the safety of their samples met the criteria requested by
pharmacopeia. We hypothesize that greater DNA fragmentation could occur in samples from the
second supplier (HPD-Y), due to possible differences in manufacturing process determining a
decrease in mean Cp, while absorbance values do not significantly change. This will be
investigated during the setup of the quantitative method.
In this study, we finally reached our proposed aim to set up a qualitative Real-Time PCR method to
certify the origin determination of HPD samples. We combined three different sequential steps:
glycogen treatment, DNA extraction and, finally, DNA purification, to ensure the greatest recovery
of DNA amount, on one hand, and the elimination of heparin from the final eluted sample, on the
other hand, in highly processed HPD samples.
In conclusion, our Real-Time PCR-based method revealed efficient and highly sensitive to assess
species origin in HPDs, which undergo long and complex manufacturing process and constitute the
starting materials of the sulodexide manufacturing process. The introduction of glycogen treatment
together with the DNA purification step after the DNA extraction, ensure DNA detection grade and
the elimination of heparin residues. Therefore, this innovative method contributes to improve
quality, safety and traceability of highly processed raw materials, as heparinoid, allowing us to add
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a checkpoint for the control of origin species during the manufacturing process of sulodexide. In
the end, this method is also useful to characterize HPDs from different suppliers.
ACKNOWLEDGEMENTS
The research was performed in the Quality Control and R&D laboratories of Biofer S.p.A., Medolla,
Italy. The authors would like to thank Senior Management of the company, and, particularly, Dr.
Alessandro Lapini Sacchetti and Dr. Gianmaria Ristori for their support, endorsement and
extensive knowledge in the field of this class of active substances.
CONFLICT OF INTEREST
The authors declare and disclose that no direct relationships or interests can potentially influence
or impart bias on the work.
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3. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine, Center for Devices and Radiological Health. Guidance for Industry. Heparin for Drug and Medical Device Use: Monitoring Crude Heparin for Quality. 2013. https://www.fda.gov/media/82924/download. Accessed May 2019.
4. European Directorate for the Quality of Medicine & HealthCare. Heparin sodium. In: European Pharmacopoeia 9.0. Strasbourg; 2017. pp: 2644-2646; Volume II.
5. Bouchard O, Hoppensteadt D, Maia P, de Castro AS, Kumar E, Guler N, Jeske W, Kahn D, Walenga JM, Coyne E, Yao Y, Fareed J. Porcine and ovine mucosal heparins and their depolymerized derivatives are comparable in contrast to their bovine equivalents. Blood. 2016. 2016;128(22):5027.
6. Carroll BJ, Piazza G, Goldhaber SZ. Sulodexide in venous disease. J Thromb Haemost. 2019;17(1):31-8. doi:10.1111/jth.14324.
7. Veraldi N, Guerrini M, Urso E, Risi G, Bertini S, Bensi D et al. Fine structural characterization of sulodexide. J Pharm Biomed Anal. 2018;156:67-79. doi:10.1016/j.jpba.2018.04.012.
8. Coccheri S, Mannello F. Development and use of sulodexide in vascular diseases: implications for treatment. Drug Des Devel Ther. 2013;8:49-65. doi:10.2147/DDDT.S6762.
9. Concannon SP, Wimberley PB, Workman WE. A quantitative PCR method to quantify ruminant DNA in porcine crude heparin. Anal Bioanal Chem. 2011;399(2):757-62. doi:10.1007/s00216-010-4362-8.
10. Tanabe S, Hase M, Yano T, Sato M, Fujimura T, Akiyama H. A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Biosci Biotechnol Biochem. 2007;71(12):3131-5. doi:10.1271/bbb.70683.
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11. Auguste C, Dereux S, Martinez C, Anger P. New developments in quantitative polymerase chain reaction applied to control the quality of heparins. Anal Bioanal Chem. 2011;399(2):747-55. doi:10.1007/s00216-010-4232-4.
12. Sakai Y, Kotoura S, Yano T, Kurihara T, Uchida K, Miake K et al. Quantification of pork, chicken and beef by using a novel reference molecule. Biosci Biotechnol Biochem. 2011;75(9):1639-43. doi:10.1271/bbb.110024.
13. Ding M, Bullotta A, Caruso L, Gupta P, Rinaldo CR, Chen Y. An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma. J Virol Methods. 2011;176(1-2):1-8. doi:10.1016/j.jviromet.2011.05.012.
14. Huang Q, Yao CY, Chen B, Wang F, Huang JF, Zhang X et al. Species-specific identification by inhibitor-controlled PCR of ruminant components contaminating industrial crude porcine heparin. Mol Cell Probes. 2006;20(3-4):250-8. doi:10.1016/j.mcp.2006.01.005.
15. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal. 1999;13(3):133-40.
16. European Directorate for the Quality of Medicine & HealthCare. Nucleic acid amplification techniques. In: European Pharmacopoeia 9.0. Strasbourg; 2017. pp: 214-219; Volume I.
17. Houiste C, Auguste C, Macrez C, Dereux S, Derouet A, Anger P. Quantitative PCR and disaccharide profiling to characterize the animal origin of low-molecular-weight heparins. Clin Appl Thromb Hemost. 2009;15(1):50-8. doi:10.1177/1076029608320831.
18. Tovar AM, Santos GR, Capille NV, Piquet AA, Glauser BF, Pereira MS et al. Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs. Sci Rep. 2016;6:35619. doi:10.1038/srep35619.
19. Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques. 1990;9(2):166.
20. Helms C, Graham MY, Dutchik JE, Olson MV. A new method for purifying lambda DNA from phage lysates. DNA. 1985;4(1):39-49. doi:10.1089/dna.1985.4.39.
21. Hengen PN. Carriers for precipitating nucleic acids. Trends Biochem Sci. 1996;21(6):224-5.
22. Tracy S. Improved rapid methodology for the isolation of nucleic acids from agarose gels. Prep Biochem. 1981;11(3):251-68. doi:10.1080/00327488108061767.
23. Ekins J, Peters SM, Jones YL, Swaim H, Ha T, La Neve F et al. Development of a multiplex real-time PCR assay for the detection of ruminant DNA. J Food Prot. 2012;75(6):1107-12. doi:10.4315/0362-028X.JFP-11-415.
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190x275mm (96 x 96 DPI)
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FIGURE CAPTIONS
Figure 1 Histograms report the mean absorbance at UV wavelength of 260 nm, expressed as Absorbance Units (AU), of 10 samples of crude heparin (CH, 1.19±0.07 AU) and 20 samples of heparinoids (HPDs, 0.33±0.01 AU). A significant variation in absorbance was observed comparing crude heparin with HPDs (****: P < 0.0001, Mann-Whitney test). Table reports the absorbance values for each sample and mean Absorbance values.
Figure 2 Overlay of chromatograms of crude heparin (orange line) and heparinoid (HPD-Y, black line), after analysis of nucleotide impurities by using HPLC [3]. Table reports the retention times of nucleotide impurities in crude heparin and heparinoid, identified from the relative retention times supplied by United States Pharmacopoeia. We observed several peaks, identified as specific nucleotides, in the crude heparin sample (nucleotide impurities: 2.15% m/m), while in the HPD sample detected peaks presented lower intensity (nucleotide impurities: 0.14% m/m).
Figure 3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 19.83±0.04 cs), crude heparin (B, blue lines, Cp: 18.81±0.1 cs), three different heparinoid samples from one supplier (HPD-Y, C, green, yellow and brown lines, Cp: 32.84±0.30 cs; 33.44, 33.19 and 31.90 cs, each) and negative control (D, black lines). A 14-fold increase in mean Cp was observed comparing crude heparin and HPD-Y samples. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles.
Figure 4 The graph reports the crossing points of amplified swine DNA from 11 heparinoid samples with (+) or without (-) glycogen treatment of 1 hour, 4 from the first supplier (HPD-X) and 7 from the other supplier (HPD-Y). Mean Cp values, expressed as amplification cycles, of Sus scrofa DNA, with or without 1-h glycogen treatment, were calculated from two replicates for each sample. Green lines report a decrease in sample Cp, while red lines show an increase in Cp after glycogen treatment. At the bottom of the table, we report mean Cp±SEM values from all the HPD samples, with or without 1-h glycogen treatment. We reported a significant decrease of Cp in samples treated with glycogen for 1 h, compared to untreated ones (**: P < 0.01, paired t-test ), regardless of the supplier. Data are expressed as mean Cp (cs). DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: cycles; SEM: Standard Error of the Mean.
Figure 5 Specificity test results of each species-specific primer kit: Sus scrofa (A, grey lines, Cp: 18.39±0.05 cs), Bos taurus (B, blue lines, Cp: 17.95±0.04 cs), Ovis aries (C, red lines, Cp: 16.11±0.03 cs) and Capra hircus (D, green lines, Cp: 17.18±0.05 cs). For each test, DNA positive control of each species and negative control were loaded. While porcine, ovine and caprine-specific primers only amplified species-specific DNA positive control, bovine-specific primers (B) reported amplification of porcine (Cp: 42.14 cs, grey line), ovine (Cp: 40.56±1.29 cs, red lines) and caprine (Cp: 39.42 cs, green line) DNA, with Cp>35 cs. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles.
Figure 6 Absorbance at UV wavelength of 260 nm, expressed as AU, and Cp values, expressed as cs, for 20 heparinoid samples, 10 from one supplier (HPD-X) and 10 from the other (HPD-Y). Table in the figure reports the mean crossing point value of each sample. Histograms report the mean absorbance (A) and mean Cp (B) of HPDs from the two suppliers. While we reported no significant variation in absorbance, mean Cp from HPD-Y was significantly higher than the one from HPD-X
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(*: P< 0.05, Welch’s t-test). DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. AU: Absorbance Units; Cp: Crossing point; cs: cycles.
TABLE CAPTIONS
Table 1 Fixed detection limit for all the tested species-specific primer kits, expressed as copies of DNA positive control in 1 µL. Reported data show the Cp, expressed as cs, of three samples treated for 1 h with glycogen, extracted and purified, after three independent dilution series. Mean Cp and SEM are reported on the right of the table. DNA amplification was performed by using the four species-specific primer kits. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles; SEM: Standard Error of the Mean.
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Fig. 1
Absorbance at 260 nm (AU)Crude Heparin (CH)
CH-01 CH-02 CH-03 CH-04 CH-05 CH-06 CH-07 CH-08 CH-09 CH-10 Mean1.2 1.2 1.3 1.2 1.2 1.2 1.3 1.1 1.1 1.1 1.19±0.07
Heparinoid (HPD)
HPD-X01 HPD-X02 HPD-X03 HPD-X04 HPD-X05 HPD-X06 HPD-X07 HPD-X08 HPD-X09 HPD-X10 Mean0.34 0.40 0.41 0.41 0.36 0.36 0.30 0.32 0.28 0.31
0.33±0.01HPD-Y01 HPD-Y02 HPD-Y03 HPD-Y04 HPD-Y05 HPD-Y06 HPD-Y07 HPD-Y08 HPD-Y09 HPD-Y100.33 0.35 0.34 0.25 0.24 0.21 0.31 0.35 0.34 0.40
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Fig. 2
Res
pons
e
Time (min)
CRUDE HEPARIN (Nucleotide impurities: 2.15% m/m)
HEPARINOID (Nucleotide impurities: 0.14% m/m)
C dC
U
mdC
G
dGT
A
dA
Retention time (min)Nucleotide USP relative retention time Crude Heparin Heparinoid
Cytidine (C) 0.28 3.490 3.476Deoxycytidine (dC) 0.38 4.597 4.574
Uridine (U) 0.40 4.818 4.8005-metyl-2-deoxycytidine (mdC) 0.66 9.044 8.977
Guanosine (G) 0.81 10.116 10.0602-deoxyguanosine (dG) 0.89 11.462 11.446
Thymidine (T) 0.92 12.309 12.326Adenosine (A) 1.00 13.161 13.167
2-deoxyadenosine (dA) 1.04 13.473 13.500
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Fig. 3Fl
uore
scen
ce F
AM
cha
nnel
(465
-510
nm
)
Cycles
A
B
C
D
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Fig. 4
1-h glycogen treatment
- + Mean (cs) Mean (cs)
HPD-X07 33.60 32.37HPD-X08 32.91 32.56HPD-X09 32.57 33.14HPD-X10 32.66 31.58HPD-Y05 29.51 29.13HPD-Y06 30.05 29.28HPD-Y07 30.68 28.56HPD-Y08 29.31 29.59HPD-Y09 35.63 33.64HPD-Y10 33.96 31.88HPD-Y11 32.15 29.72
Mean±SEM 32.09±0.60 31.04±0.55
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Fig. 5
Fluo
resc
ence
FA
M c
hann
el (4
65-5
10 n
m)
Cycles
Fluo
resc
ence
FA
M c
hann
el (4
65-5
10 n
m)
Cycles
Fluo
resc
ence
FA
M c
hann
el (4
65-5
10 n
m)
Cycles
Fluo
resc
ence
FA
M c
hann
el (4
65-5
10 n
m)
Cycles
Sus scrofa Bos taurus
Ovis aries Capra hircus
A B
C D
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A
B
Fig. 6
UV spectroscopy Real-Time PCRSample ABS 260 nm (AU) Mean ABS SEM Cp (cs) Mean Cp SEM
HPD-X01 0.34
0.35 0.01
27.46 27.28
28.30 1.16
HPD-X02 0.40 25.13 25.06HPD-X03 0.41 24.89 24.84HPD-X04 0.41 24.26 24.26HPD-X05 0.36 25.98 25.94HPD-X06 0.36 25.78 25.73HPD-X07 0.30 32.48 32.26HPD-X08 0.32 32.57 32.55HPD-X09 0.28 33.41 32.87HPD-X10 0.31 31.55 31.61HPD-Y01 0.33
0.31 0.02
33.46 33.41
31.14 0.61
HPD-Y02 0.35 33.27 33.10HPD-Y03 0.34 32.01 31.79HPD-Y04 0.25 30.75 30.84HPD-Y05 0.24 29.11 29.14HPD-Y06 0.21 29.26 29.29HPD-Y07 0.31 28.59 28.52HPD-Y08 0.35 29.56 29.61HPD-Y09 0.34 33.61 33.67HPD-Y10 0.40 31.94 31.81
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Table 1
SpeciesDetection Limit
(DNA copies/µL)
Crossing point, glycogen-treated DNA positive control (cycles) Mean Cp SEM
Sus scrofa 1.2 38.01 38.93 38.91 40.01 37.95 37.42 37.77 40.44 37.55 38.55 0.36Bos taurus 1.2 35.65 35.31 34.70 35.40 34.95 35.94 35.09 36.81 35.29 35.46 0.21Ovis aries 1.2 36.72 36.12 35.79 36.07 36.98 36.81 36.92 37.22 36.28 36.55 0.16
Capra hircus 0.6 38.01 35.91 37.79 36.78 38.43 36.58 36.85 37.60 36.91 37.21 0.25
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Analytical and Bioanalytical Chemistry
Electronic Supplementary Material
Highly sensitive real-time PCR method to identify species origin
in heparinoids
Simone Pecorini, Giulio Camurri, Lucia Torrini, Roberta Ferraresi
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
A
B
C D
Fig. S1 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoid (HPD-Y, second supplier) after 1-h glycogen treatment, extraction and purification (B, green lines, 29.72±0.01 cs), the same heparinoid after 1-h glycogen treatment and extraction without purification (C, blue lines) and negative control (D, black lines). DNA amplification occurred only in extracted-and-purified samples, while extracted-only DNA presented an amplification curve similar to the negative control. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
VIC
chan
nel (
533-
580
nm)
Cycles
A
B C
Fig. S2 Typical Real-Time PCR profile of the internal control DNA after extraction and purification of the same HPD-Y sample analyzed in Figure 3 and 4 (A, brown lines, Cp: 29.63±0.03 cs), after extraction only (B, green lines) and negative control (C, grey lines). While glycogen-treated, extracted and purified DNA reported positive amplification, the glycogen-treated and extracted DNA showed no Cp. DNA amplification was performed by using the internal control-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
A
BC D
E
Fig. S3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoids after overnight glycogen treatment, extraction and purification (B, green lines, Cp: 28.69±0.03 cs), heparinoids after 1-h glycogen treatment, extraction and purification (C, blue lines, Cp: 29.72±0.01 cs), heparinoids without glycogen treatment (D, yellow lines, Cp: 32.15±0.31 cs) and negative control (E, black lines). Although after the overnight glycogen treatment Cp was lower than the one after 1-h treatment, the latter determined a decrease in Cp, if compared to the extracted-and-purified sample. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
AB
C
D
Probe Master mix volume (µL)4.75 (C) 5.00 (B) 5.25 (D)
Cp (cs) 32.98 32.69 32.6232.86 32.66 32.48
Mean Cp (cs) 32.92 32.68 32.55SEM (cs) 0.06 0.02 0.07
% variation 0.73% 0.40%
Fig. S4 Results of robustness test performed with Sus scrofa primer kit. We performed robustness test by modifying the probe master mix volume: amplification curves of DNA positive control (A, blue lines), samples by using standard volume (B, grey lines, Cp: 32.68±0.02), by using 5%-reduced-volume (C, red lines, Cp: 32.92±0.06 cs) and 5%-increased volume (D, green lines, Cp: 32.05±0.07 cs) of probe master mix. 5%-variation of probe master mix volume reflected in slight variations in mean Cp (0.73% and 0.40%), confirming the robustness of the method. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
Sus scrofaBos taurusOvis ariesCapra hircusNegative control
16-23 cs
1 2 3 4 5 6 7 8 9 10 11 12
ABlank Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus
- - 16.88 - 18.30 - 18.63 - 16.62 - 18.16 -
BPos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra
19.26 - 18.99 - 17.44 - 18.50 - 19.51 - 19.43 -
CPos Ctr Capra Neg Ctr Capra
17.90 -
Fig. S5 Results of cross-contamination test, as Cp. We loaded more than 20 samples, alternating negative and DNA positive control samples in one Real-Time PCR microplate. Reaction conditions worked correctly only if the amplification of species-specific positive control DNA crossing point occurred in the range 16-23 cs, as mentioned in the manufacturer’s protocol. All negative samples revealed as negative, while all positive samples presented Cp included in the correct range. Therefore, cross-contamination was successfully tested. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: amplification cycles
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Table S1 Accession numbers, context sequence lengths and anchor nucleotides for each species-specific primer used in the DNA amplification (Primer DesignTM Ltd.). bp: base pairs
Accession Number Context Sequence Anchor NucleotideSus scrofa AF034253.1 195 bp 14549
Bos taurus NC_006853 208 bp 15931
Ovis aries HM236178.1 106 bp 14872
Capra hircus KM998968.1 123 bp 8037
Table S2 Results of the specificity test for each species-specific primer kit. Data report the mean Cp of DNA positive control from all species, after amplification with each species-specific primer kit, expressed as cs. By using bovine species-specific primers, positive amplification occurred only in one sample of Sus scrofa (Cp: 42.14 cs) and Capra hircus (Cp: 39.42 cs) DNA positive control, instead of two (as displayed for Ovis aries, Cp: 40.56±1.29 cs). This explains why single values were reported for these two species. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles
Mean crossing point, by using species-specific primer kits (±SEM, cycles)
Sus scrofa Bos taurus Ovis aries Capra hircus
Sus scrofa 18.39±0.05 42.14 - -
Bos taurus - 17.95±0.04 - -
Ovis aries - 40.56±1.29 16.11±0.03 -
DNA
posit
ive
cont
rol
Capra hircus - 39.42 - 17.18±0.05
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Analytical and Bioanalytical Chemistry
Electronic Supplementary Material
Highly sensitive real-time PCR method to identify species origin
in heparinoids
Simone Pecorini, Giulio Camurri, Lucia Torrini, Roberta Ferraresi
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
A
B
C D
Fig. S1 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoid (HPD-Y, second supplier) after 1-h glycogen treatment, extraction and purification (B, green lines, 29.72±0.01 cs), the same heparinoid after 1-h glycogen treatment and extraction without purification (C, blue lines) and negative control (D, black lines). DNA amplification occurred only in extracted-and-purified samples, while extracted-only DNA presented an amplification curve similar to the negative control. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
VIC
chan
nel (
533-
580
nm)
Cycles
A
B C
Fig. S2 Typical Real-Time PCR profile of the internal control DNA after extraction and purification of the same HPD-Y sample analyzed in Figure 3 and 4 (A, brown lines, Cp: 29.63±0.03 cs), after extraction only (B, green lines) and negative control (C, grey lines). While glycogen-treated, extracted and purified DNA reported positive amplification, the glycogen-treated and extracted DNA showed no Cp. DNA amplification was performed by using the internal control-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
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FAM
cha
nnel
(465
-510
nm
)
Cycles
A
BC D
E
Fig. S3 Typical Real-Time PCR profile of the Sus scrofa DNA from positive control (A, red lines, Cp: 17.94±0.13 cs), heparinoids after overnight glycogen treatment, extraction and purification (B, green lines, Cp: 28.69±0.03 cs), heparinoids after 1-h glycogen treatment, extraction and purification (C, blue lines, Cp: 29.72±0.01 cs), heparinoids without glycogen treatment (D, yellow lines, Cp: 32.15±0.31 cs) and negative control (E, black lines). Although after the overnight glycogen treatment Cp was lower than the one after 1-h treatment, the latter determined a decrease in Cp, if compared to the extracted-and-purified sample. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
AB
C
D
Probe Master mix volume (µL)4.75 (C) 5.00 (B) 5.25 (D)
Cp (cs) 32.98 32.69 32.6232.86 32.66 32.48
Mean Cp (cs) 32.92 32.68 32.55SEM (cs) 0.06 0.02 0.07
% variation 0.73% 0.40%
Fig. S4 Results of robustness test performed with Sus scrofa primer kit. We performed robustness test by modifying the probe master mix volume: amplification curves of DNA positive control (A, blue lines), samples by using standard volume (B, grey lines, Cp: 32.68±0.02), by using 5%-reduced-volume (C, red lines, Cp: 32.92±0.06 cs) and 5%-increased volume (D, green lines, Cp: 32.05±0.07 cs) of probe master mix. 5%-variation of probe master mix volume reflected in slight variations in mean Cp (0.73% and 0.40%), confirming the robustness of the method. DNA amplification was performed by using the porcine-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: cycles
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Fluo
resc
ence
FAM
cha
nnel
(465
-510
nm
)
Cycles
Sus scrofaBos taurusOvis ariesCapra hircusNegative control
16-23 cs
1 2 3 4 5 6 7 8 9 10 11 12
ABlank Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus
- - 16.88 - 18.30 - 18.63 - 16.62 - 18.16 -
BPos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra Pos Ctr Capra Neg Ctr Bos Pos Ctr Bos Neg Ctr Sus Pos Ctr Sus Neg Ctr Ovis Pos Ctr Ovis Neg Ctr Capra
19.26 - 18.99 - 17.44 - 18.50 - 19.51 - 19.43 -
CPos Ctr Capra Neg Ctr Capra
17.90 -
Fig. S5 Results of cross-contamination test, as Cp. We loaded more than 20 samples, alternating negative and DNA positive control samples in one Real-Time PCR microplate. Reaction conditions worked correctly only if the amplification of species-specific positive control DNA crossing point occurred in the range 16-23 cs, as mentioned in the manufacturer’s protocol. All negative samples revealed as negative, while all positive samples presented Cp included in the correct range. Therefore, cross-contamination was successfully tested. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: crossing point; cs: amplification cycles
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Table S1 Accession numbers, context sequence lengths and anchor nucleotides for each species-specific primer used in the DNA amplification (Primer DesignTM Ltd.). bp: base pairs
Accession Number Context Sequence Anchor Nucleotide Sus scrofa AF034253.1 195 bp 14549 Bos taurus NC_006853 208 bp 15931 Ovis aries HM236178.1 106 bp 14872
Capra hircus KM998968.1 123 bp 8037
Table S2 Results of the specificity test for each species-specific primer kit. Data report the mean Cp of DNA positive control from all species, after amplification with each species-specific primer kit, expressed as cs. By using bovine species-specific primers, positive amplification occurred only in one sample of Sus scrofa (Cp: 42.14 cs) and Capra hircus (Cp: 39.42 cs) DNA positive control, instead of two (as displayed for Ovis aries, Cp: 40.56±1.29 cs). This explains why single values were reported for these two species. DNA amplification was performed by using the four species-specific primers. The Universal CC FAM (510)-VIC (580) [465-510,533-580] color compensation from Roche LightCycler 480 II software database was applied during analysis. Cp: Crossing point; cs: amplification cycles
Mean crossing point, by using species-specific primer kits (±SEM, cycles)
Sus scrofa Bos taurus Ovis aries Capra hircus
DNA
posit
ive
cont
rol Sus scrofa 18.39±0.05 42.14 - -
Bos taurus - 17.95±0.04 - -
Ovis aries - 40.56±1.29 16.11±0.03 -
Capra hircus - 39.42 - 17.18±0.05
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