from single to multiplex detection of plant …ptfit1/pdf/pp35/pp_35_04.pdf · immunofluorescence...

Plant Research International, Wageningen, The Netherlands

FROM SINGLE TO MULTIPLEX DETECTION OF PLANTPATHOGENS: pUMA, A NEW CONCEPT OF MULTIPLEX

DETECTION USING MICROARRAYS1

P.J.M. Bonants, C.D. Schoen, M. Szemes, A. Speksnijder, M.M. Klerks,P.H.J.F. van den Boogert, C. Waalwijk, J.M. Van der Wolf and C. Zijlstra

Abstract

An overview is given of rapid methods for the detection of plant-related organ-isms in plant material, soil, compost, water, etc. Protein-based detection assayssuch as isozyme analysis, ELISA, immunofluorescence colony-staining and futureapplications of immunological tests are described as well as nucleic acid-basedtests. Examples of RNA amplification tests, such as RT-PCR, NASBA andAmpliDet RNA are given, and numerous DNA-based tests using PCR, either withor without the use of probes, are illustrated. Most tests described are directed to-wards the detection of plant pathogens such as viruses, bacteria, fungi and nema-todes. A test for the detection of mRNA of a mycotoxin producing fungus is alsoshown in this review. This illustrates that the technology of many of the tests de-scribed can equally well be used for the development of assays to detect harmfulorganisms in food, feed, water, air or any other environment in the agrofood pro-duction chain. The latest development in detection are in the field of multiplex de-tection using microarrays is presented as the pUMA technology.

Key words: DNA, PCR, ELISA, AmpliDet RNA, diagnostics, detection, patho-gens, microarrays, bacteria, viruses, nematodes, fungi, mycotoxins,multiplex, NASBA, DGGE, pUMA, TaqMan, Molecular Beacon

Introduction

Quality control and monitoring in the agrofood chain is necessary to ensuresafe agriculture, horticulture and agrofood processing. Since growers, producers,

Phytopathol. Pol. 35: 29–47© The Polish Phytopathological Society, Poznań 2005ISSN 1230-0462

1The work was partially funded by Productschap Tuinbouw and Ministry of Agriculture, Fisheriesand Nature Management (DWK 397III).

processing industry and consumers increasingly demand higher quality for start-ing material and agrofood products, there is a rising interest for rapid, sensitive,specific and user-friendly diagnostic methods. Nucleic acid diagnostic technolo-gies can be of great help nowadays as they have undergone huge innovations overthe last two decades. While enzyme-linked immunosorbent assays (ELISA) werestate of the art in the seventies and mid-eighties for the detection of human, ani-mal and plant pathogens, nowadays molecular methods are able to offer better sen-sitivity, specificity and/or speed. Due to the elucidation of the genomic compositionof organisms, insights in gene expression, genetics and host-pathogen interac-tions, molecular knowledge has explosively expanded. Consequently, DNA andRNA amplification methods can enable an accurate diagnosis, leading to an easierapproach of detection, characterisation and identification of pathogens, genes anddisease suppressing microorganisms or antagonists. These advancements also in-creasingly fulfil the needs required for microbiological quality control and moni-toring in the agrofood chain.

This paper describes some techniques that have been developed at Plant Re-search International (PRI, Wageningen, The Netherlands) for detection of plantpathogens and other plant-related organisms, and that can be used in agrosystems.During culture and production of various agroproducts, such as potatoes, corn, to-matoes, sugar beet, but also flowers and flower bulbs, many plant pathogens causeenormous damage. Methods to detect these plant pathogens enable to check theproduct quality and to monitor the presence of pathogens during processing proce-dures in the whole production chain. Many of these techniques are currently alsounder evaluation for the detection of human pathogens in agrofood processing sys-tems, as recent disease outbreaks are associated to the consumption of plant-re-lated products.

Plant-related organisms in agrosystems

Plant pathogens

Because agrosystems are becoming less dependent on chemical control, there isan increasing demand for fast, reliable methods capable of detecting plant-relatedorganisms. Such methods can be used for:

• Quality monitoring of substrates. The compost and potting soil industryrequires guarantees that the product is free of plant parasitic organisms as well asmeeting the manufacturer’s claims that it contains plant health-promoting organ-isms.

• Quality control of products. “Tracing and tracking” of plant-related organ-isms in products throughout the chain is becoming more and more important toassure quality for the consumer (Klerks and Leone 2002). This concerns the moni-toring of crop pests or diseases that may cause problems during the post-harvestphase (e.g. formation of mycotoxins). This topic requires special attention due to

30 P.J.M. Bonants, C.D. Schoen, M. Szemes, A. Speksnijder, M.M. Klerks et al.

changes currently taking place in crop systems (less generic control of crop pestsand diseases).

• Compliance with export regulations. The export of plant products is con-trolled by strict regulations. The presence of quarantine organisms is absolutelyunacceptable. Tests currently in use to show the presence of quarantine organismsare generally very time consuming, keeping shipments that are tested under quar-antine for weeks. Rapid tests could solve this problem.

• Prevention. Preventing contamination is important from the viewpoint ofusing healthy starting material (seeds, bulbs, cuttings). Diseased starting mate-rial in a crop production system is often the source of infection. Tests for detect-ing crop disease organisms have been used for years to select healthy startingmaterial. Another application is the detection of crop pests and diseases in sub-strates. If the pests and diseases present in the soil of a specific plot of land areknown beforehand, an appropriate cultivation plan can be drawn up, and the bestcrops and cultivars can be selected. Detection can also play an important role ingreenhouse horticulture by testing recirculating irrigation water. Such testingcan prevent an entire crop from becoming infected due to recirculated disease or-ganisms.

Many detection tests that have been developed, can be used for the above aims.They can be used on soil, compost, water, plant material, inocula, etc. In our labo-ratory tests have been developed that are directed towards different kinds of plantpathogens:

viruses and viroids (e.g. viruses/viroids of vegetables, fruits, ornamentals,potato),

bacteria: Ralstonia (brown rot), Clavibacter (ring rot), Agrobacterium (rootknot), Erwinia (syn. Pectobacterium) (soft rot), Xanthomonas spp.,

phytoplasmas: Stolbur,fungi: Phytophthora spp., Fusarium oxysporum Schlecht., other Fusarium sp.,

Rhizoctonia solani Kühn, Synchytrium, Olpidium, Phoma, Alternaria, Nectria, Guignardia),nematodes: Meloidogyne chitwoodi Golden, O’Bannon, Santo & Finley, M. fallax

Karssen, M. hapla Chitwood, M. minor Karssen, M. naasi Franklin, M. incognita(Kofoid & White) Chitwood, M. javanica (Treub) Chitwood, M. arenaria (Neal)Chitwood, Globodera rostochiensis Wollenweber, G. pallida Stone,

insects (e.g. Thrips species).In addition, tests are developed for the detection of mRNAs of certain organ-

isms. This enables the detection of metabolically active pathogens. This is particu-larly interesting when it concerns toxin-producing plant pathogens, e.g.mycotoxin-producing Fusarium strains.

From single to multiplex detection of plant pathogens... 31

Method requirements for detection and identification

In the agrosector the need for reliable identification and sensitive detection ofplant pathogens, beneficial microorganisms and, more recently, plant-related hu-man pathogens is increasing. Important developments are nowadays:

quantitative detection to quantify inoculum densities of pathogenic variantsto estimate risks for economic damage,

multiplex detection to detect multiple plant pathogens (targets) in the samesample in a single test.

Different aspects have to be considered and certain criteria have to be fulfilledfor developing effective detection methods:

specificity – the test should be very specific and should only detect the targetpathogen and not closely related species; no false positives or false negativesshould be observed,

sensitivity – the test should be sensitive in detecting, for example, one zoo-spore; this goes especially for quarantine-organisms requiring a nil-tolerance,

costs – for routine testing the test should be cost effective,expertise – the presence of sufficient technical and personal expertise to per-

form the test should be considered,robustness – the test should be robust, including a high repeatability and

reproducibility,high throughput – sometimes the test should be suitable for high throughput

screening,speed – the results obtained should be available within a short time.

Methods for detection and identification

Previously, identification took place on the basis of physiological (Phot. 1), bio-logical and morphological (Phot. 2) characteristics requiring specialised expertise.The procedure was usually very time-consuming and the results were often not un-equivocal.

Protein-based methods

Protein-based methods are much more accessible and can be carried out rou-tinely. Isozyme analysis is a relatively fast way to identify species of the nematodegenus Meloidogyne (Esbenshade and Triantaphyllou 1985, 1990). The comparisonof esterase and malate dehydrogenase patterns shows great consistency in theidentification of the species. However, for clear and reliable results the isozymeanalysis can only be done with females at a specific developmental stage.

The introduction of immunological techniques in the 1960s was a break-through. At present, the business community in The Netherlands and abroad con-ducts approximately 10 million ELISA tests each year to detect the presence of


bacteria and viruses in agroproducts such as potatoes, ornamentals and flowerbulbs. Therefore high throughput (HTP) ELISA systems (Phot. 3) were developedwhich can handle these large amounts of tests.

For the detection of certain bacteria immunofluorescence colony-staining(IFC) can be used (Fig. 1). After incubation of the bacteria on agar fluorescently la-belled antibodies are added. After washing unbound antibodies fluorescent colo-nies can be seen under a UV microscope and isolated for further studies, e.g. forconfirmation by PCR (Van der Wolf et al. 1998, 2000, Van der Wolf and VanBeckhoven 2004). In The Netherlands most of the required antisera are suppliedby PRI. They still comprise largely polyclonal antibodies. The disadvantage of suchantibodies is that they are unsuitable for the detection of economically importantfungi, which have many dominant cell wall antigens in common resulting inaspecific reactions.

Nucleic acid-based methods

Since the introduction of amplification methods for DNA and RNA, and the in-creasing availability of sequence data many methods have been developed for usein detection and identification:

RNA level: RT-PCR, NASBA or AmpliDet RNA, using primers and probes(e.g. Molecular Beacons, TaqMan probes),


Phot. 1. Growth of bacteria on selective media(photo by J.M. Van der Wolf)

DNA level: PCR (e.g. ITS-RFLP, SCAR-PCR, AFLP, multiplex PCR, (nested)PCR, RAPD, DGGE) using primers and probes (e.g. Molecular Beacons, TaqManprobes).

The amplicons can be detected by traditional gel-electrophoresis or by real-timedetection with fluorescent probes. An example of such a probe is the TaqManprobe (Oberst et al. 1998, Schoen et al. 1996, Kalinina et al. 1997, Brandt et al.1998, Böhm et al. 1999, Zhang et al. 1999, Bonants et al. in press). This probe con-sists of two groups: a fluorescent reporter and a quencher. The fluorescence isquenched due to proximity of the two groups. During the annealing step of thepolymerase chain reaction the probe is bound on the target and during the elonga-tion step the probe is degraded by the Taq-polymerase resulting in release of thereporter from the probe into solution and increase of fluorescence. The fluores-cence can be measured real time during the polymerase chain reaction in areal-time PCR machine. Amplification plots with different amount of template


Phot. 2. TEM (Transmission Electron Microscopy) picture of viruses (photo by C.D. Schoen)


Phot. 3. High throughput ELISA system(photo by J.M. Van der Wolf)

Fig. 1. Immunofluorescence colony-staining (IFC) of bacteria. A mixture of bacteria ora complex substrate containing bacteria, is grown in a solid medium (A) until colony forming

appears, and is then incubated with a FITC-conjugated specific antibody (B). The antibodybinds to the target bacteria (C), which results in a bright green fluorescent staining of the

colonies containing target bacteria (photograph). The fluorescent colonies can be picked fromthe medium (D) and can be used for reisolation, detection with molecular detection methods

or further characterisation (photo by J.M. Van der Wolf)

show differences in the number of cycles before an increase in fluorescence abovebackground, the so-called Ct-value. In this way quantification of target DNA ispossible. An example of multiplex analysis with TaqMan probes for two potato vi-ruses is shown in Figure 2. PVY and PLRV can be detected simultaneously by usingtwo TaqMan probes labelled with two different fluorescent groups in one amplifi-cation reaction. Other applications using these TaqMan probes have been devel-oped, e.g. for several Fusarium species in wheat (Waalwijk et al. 2004).

The exquisite sensitivity of this technique is putting a high demand on mea-sures to prevent false positive reactions due to contamination of the laboratorywith nucleic acid. Therefore, monitoring of false-positives by the inclusion of nega-tive controls is required. In addition, the reaction performance must be measured.The reliability of the test is increased by inclusion of a general internal amplifica-tion control in the reaction mix (Klerks et al. in press). When no signals are mea-sured for amplification of the internal control, the test conditions did not allowpolymerase chain reaction during the assay and the test results cannot be trusted.This prevents the scoring of false negative results.

NASBA (Nucleic Acid Sequence Based Amplification) is another amplificationreaction (Compton 1991). NASBA is an isothermal amplification method suitablefor the detection of RNA, which uses three enzymes (reverse transcriptase, RNaseH and T7-polymerase) and results in millions of copies of antisense RNA. Within90 min at 41°C a billion amplification of a specific RNA sequence can be obtained.Because NASBA detects RNA, viability test can be performed (Bentsink et al.2002). An example with NASBA on Ralstonia solanacearum (Smith) Yabuuchi et al.is shown in Figure 3. Bacteria are incubated on metal strips for various hours andthe results with NASBA, PCR and plating on medium are shown in Figure 3. Inlanes 5, 6 and 7 non-viable bacteria can be detected by PCR, but not with NASBAdemonstrating that after 24 h bacteria are dead. To allow real-time amplification


Fig. 2. A. Real-time multiplex RT-PCR with TaqMan probes for the detection of Potato virus Y(PVY) and Potato leaf roll virus (PLRV). Six samples were tested consisting of PVY (1 and 2),PVY and PLRV (3 and 6) or PLRV (4 and 5). Fluorescence is measured using PVY-specific

TaqMan probe (left) or PLRV-specific TaqMan probe (right). B. Gel electrophoretic analysisof the amplification products; lanes 1–6 correspond to samples 1–6

and detection, NASBA was combined with Molecular Beacon (MB) probes, calledAmpliDet RNA (Leone et al. 1998). MB is another fluorescent probe with a re-porter and a quencher, which can be used to detect the amplicon (Tyagi andKramer 1996). MBs are oligonucleotides with a loop and a stem structure. Theloop sequence is complementary to the target sequence. The fluorescent reporterand the quencher are located at the end of the stem sequence. Without target theMB is closed and no fluorescence is recorded. When target is present, the MB isbound to the target and stretched. The reporter and quencher are now distant fromeach other and fluorescence will be recorded. Several AmpliDet RNA methodshave been developed for detection of ribosomal RNA (Van Beckhoven et al. 2002,Van der Wolf et al. 2004), messenger RNA (Klerks et al. 2000) and viral RNA(Klerks et al. 2001 a, b, 2004, Vaskova et al. 2004, Szemes et al. 2002). MB has alsobeen used in PCR to detect DNA (Bonants et al. in press).

The availability of an AmpliDetRNA method for the detection ofmRNA of a toxin producing Fusariumsp. (Klerks et al. 2000) is of particularinterest, since this plant pathogen is re-cognised as a food safety hazard. Espe-cially on wheat this fungus causesmuch concern (Waalwijk et al. 1997,2003, 2004). Figure 4 shows detectionof Fusarium sp. in different seed lots us-ing the AmpliDet RNA method. AnAmpliDet RNA method has also beendeveloped for the detection of two po-tato viruses (PVY/PLRV) (Klerks et al.2001 a). Both viruses occur as single or


Fig. 3. Viability test with NASBA for Ralstonia solanacearum. Metal strips were inoculated withR. solanacearum and airdried for 0 to 120 h. The bacteria were then washed from the strips,plated on a solid medium and tested in NASBA and PCR. The plating results indicate the

presence of live, viable cells. NASBA detects 16s RNA and acts as an indicator for viability.PCR detects the presence of genomic target DNA of live and dead cells. Lanes 1–7 indicate the

different incubation times of R. solanacearum on metal strips, lanes 8 and 9 are controls,+ and – indicate bacterial growth on the medium

Fig. 4. AmpliDet RNA method for the detectionof mRNA of a toxin producing Fusarium sp.

mixed infections in potato tubers and are responsible for major economic lossesworldwide. Moreover, detection of PLRV and PVY directly on tubers with the cur-rently used enzyme-linked immunosorbent assays (ELISA) is not reliable (Hill andJackson 1984, Spiegel and Martin 1993). The AmpliDet RNA method enabled thedetection of PLRV and PVY directly from tubers in one reaction, already within 2 hfrom sampling. Healthy tubers remained negative. The multiplex AmpliDet RNAdetected and discriminated the viruses also in mix-infected tubers (results notshown). An AmpliDet RNA method was also developed for several PVY strainswith different sequences (Szemes et al. 2002). Different MBs were used to detectthe different strains simultaneously in the NASBA reaction (data not shown). Thedifference between a linear and a MB probe has been studied. Two probes havebeen developed for Ralstonia solanacearum: a linear and a MB with the same se-quence. Cross-reaction can be seen with the linear probe, but not with the MB. Theclosely related bacterial species Pseudomonas cepacia (ex Burkholder) Palleroni &Holmes 1702 and 1708 are being detected with the linear probe, but not with theMB (Van der Wolf et al. 1999).

Denaturant Gradient Gel Electrophoreses (DGGE) is a separation method fora mixture of PCR products of equal length. Fragment separation is based on melt-ing behaviour and fingerprints can be generated from samples with genetic vari-ability (Muyzer 1999). PCR products are generated with rRNA gene directedprimers to profile complex microbial communities and to infer the phylogenetic af-filiation of community members by sequencing or probing the bands within the

profile. Primers can also be used to am-plify and to analyse variability of specificgroups within microbial communities(Kowalchuk et al. 1997). PCR-DGGE isa reliable method for determining thediversity of microbes in environmentalsamples and goes beyond the small per-centage of culturability of these micro-organisms. Evaluation of microbial com-munities has been applied to soilswhere disease suppression of soilbornepathogenic fungi is caused by antibiot-ics producing bacteria (Garbeva et al.2004). In Photograph 4 an example of aDGGE profile is shown.

AFLP (Amplified Fragment LengthPolymorphism) is a powerful technique(Vos et al. 1995) for identification andhas often been used to study geneticvariation in fungi (Mayer et al. 1997,Bonants et al. 1999, 2000, Baayen et al.2000, Eikemo et al. 2004, Ivors et al.2004). Rhizoctonia solani is a fungal


Phot. 4. DGGE profiling of fungal (18S) andbacterial (16S) communities of six soil samples

(photo by A. Speksnijder)

pathogen on sugar beet. Isolates from different fields were collected and analysedby AFLP DNA fingerprinting to study genetic variation. Photograph 5 shows thatthe genetic variation for isolates from one field is zero, while there is some varia-tion between fields.

For the detection of fungi the Internal Transcribed Spacer (ITS) region of rDNAhas often been used. Universal primers: ITS1/ITS4 (White et al. 1990) and specificprimers were developed to detect a wide range of fungi (Waalwijk et al. 1996,Ristaino et al. 1998, Schubert et al. 1999, Stark et al. 1998, Bonants et al. 2003). Anexample is the pathogen Phytophthora fragariae Hickman, a quarantine organism,for which a sensitive and specific detection method was needed. This Oomycete ispathogenic to strawberry and until now a bait test has been used to detect the fun-gus in infected material (Duncan 1980). Sampled strawberry roots are incubated insoil or water with a bait plant for several weeks at 14°C, after which typical oo-spores are visible in infected roots of the bait plant. Based on sequence analysis ofthe ITS regions (Cooke and Duncan 1997, Cooke et al. 2000) specific primers andprobes were developed in PRI together with Scottish Crop Research Institute(SCRI, Dundee, Scotland) and used for the detection of P. fragariae and P. cactorumLebert & Cohn, J. Schröter in roots, water and soil (Lacourt et al. 1997, Bonants et


Phot. 5. AFLP DNA fingerprinting patterns of Rhizoctonia solani isolates from different sugarbeet fields (photo by P.J.M. Bonants)

al. 1997, 2001, Duncan et al. 2001, Cooke et al. 2001). Sensitivity was increased byusing nested PCR (Lacourt et al. 1997, Bonants et al. 1997, Niepold andSchober-Butin 1997, Niessen et al. 1997, Stark et al. 1998).

Identification and detection methods have also been developed for nematodes.An ITS-RFLP approach has been shown to be a reliable way to distinguish theroot-knot nematodes Meloidogyne chitwoodi, M. fallax, M. hapla and the speciesclosely related to M. incognita (Zijlstra et al. 1995, 1997). Multiplex ITS-PCR isfaster and offers a good alternative to estimate species composition in mixtures(Zijlstra 1997). More recently, species-specific pairs of PCR-primers were devel-oped that amplify Sequence Characterised Amplified Regions (SCARs). Based onsequences of species-specific RAPD (Random Amplified Polymorphic DNA) frag-ments, pairs of SCAR primers were developed that specifically amplify M.chitwoodi, M. fallax, M. hapla (Zijlstra 2000), M. incognita, M. javanica and M. arenaria(Zijlstra et al. 2000). Other specific PCR test assays have been developed for thedetection of M. minor (Karssen et al. in press) and M. naasi (Zijlstra et al. in press).More recently, some progress has been made in developing a multiplex TaqMan forthe detection of M. chitwoodi and/or M. fallax.

Multiplex amplification

The newest development in analysis of nucleic acids is the microarray technol-ogy, in which different oligonucleotides can be spotted on little more than 1 mm2.Microarray technology provides the next generation of DNA diagnostics to measuredifferent pathogens on a single chip. For each pathogen many different target DNAsequences can be detected in parallel. This will improve specificity, allowing detec-tion of pathogenic variants of the target pathogen and avoiding laborious confir-mation procedures.

Several microarray systems are available. PamGene has developed a revolution-ary three-dimensional microarray with a higher capacity to bind oligonucleotidesthan a two-dimensional glass array, which results in a higher sensitivity. Therate-limiting step in a two-dimensional array planar format is the diffusion of thesample molecules (the target) toward the attached probe during hybridisation. Be-cause diffusion is slow, incubation usually takes place overnight. To bypass thislimitation, PamGene has developed a porous aluminium oxide substrate as solidsupport (Van Beuningen et al. 2001). The substrate, with a thickness of 60 m, haslong branched capillaries, which are interconnected inside (Rigby et al. 1990). Thediameter of the individual pores is 200 nm. The reactive surface of this material isincreased 500-fold as compared with a flat two-dimensional surface. In addition,the permeable nature of the microarray facilitates the pressurised movement offluid, such as the sample solution, through its structure. The flow-throughmicroarray substantially reduces hybridisation times and increases signal and sig-nal-to-noise ratios (Schoen et al. in press). An additional advantage of the systemis that the temperature can be regulated and controlled during the hybridisationprocess. Photograph 6 shows the PamGene system. Hybridisation signals can beobtained within 5 min. An example is shown in Photograph 7. ITS amplicons ofseveral Phytophthora species labelled with a fluorescent primer are hybridised at


37°C to a microarray on which severaloligos (20–25 nt) are spotted. The oligoswere selected based on sequence differ-ence of the ITS regions of rDNA of sev-eral Phytophthora species. After hybridisa-tion the temperature can be raised andfluorescence can be monitored for eachindividual spot. A melting curve (Fig. 5)can be determined and specific andaspecific hybridisation signals can be de-termined.

Amplification of the target DNA ofthe pathogen to be detected is a prerequi-site for sensitive detection. In a simplexPCR assay where one set of PCR primersis used, small samples of DNA can pro-duce sufficient copies to be visualised onmicroarrays. To amplify multiple targetsin one PCR assay, multiple primer sets


Phot. 7. Picture of hybridisation signal of fluorescently labelled ITS-PCR amplicons of severalPhytophthora species to a microarray on which species-specific oligos were spotted

(photo by P.J.M. Bonants)

Fig. 5. Melting curve analysis ofPhytophthora nicotianae probe NIC1

Phot. 6. PamGene microarray system consisting of hybridisationunit connected to peristaltic pump and waterbath, fluorescence

microscope and CCD camera (photo by P.J.M. Bonants)

can be used. However, the drawback of such multiplex PCR assays is the enormousdecrease in sensitivity and the limitation in the number of different targets to beamplified in one assay. Moreover, the dynamic range of the targets present in thesample to be tested is not always reflected in the outcome of the test. Most of thetime targets present in very low amounts will not be amplified in contrast to thoseabundantly present. To overcome some of these problems PRI has recently devel-oped a principle for multiplex detection based on padlock probe technology(pUMA) which offer a means of introducing a universal amplification step into de-tection by microarrays, thereby increasing sensitivity. pUMA stands for “Pad-lock-based Universal Multiplex detection Array”. A schematic representation ofthe technique is given in Figure 6. Padlock probes (PLPs) are oligonucleotides car-rying the target complementary regions at their 5’ and 3’ ends, which recognise ad-jacent sequences on the target DNA or RNA molecule. Thus, upon hybridisation,the ends of the probes get into adjacent position, and they can be joined by enzy-matic ligation. Ligation occurs and the probes are circularised only when both endsegments recognise correctly the target sequences. Non-circularised probes are re-moved by exonuclease treatment, while the circularised ones may be amplified byusing universal primers. Subsequently, the target-specific products are detectedvia the microarray. Beside the target-specific recognition sequences and the uni-versal primer binding sites, the PLPs contain a unique identifier sequence, theso-called ZipCode, which is actually the region recognised by the oligonucleotidesbound to the array. Thus the targeted pathogens and the recognition molecules ofthe array are independent, making the assay easily modifiable, extendable andadaptable in other fields, such as in detection of soil organisms.

DNA extracted from a sample under investigation is mixed with specific pad-lock probes that bind to the targets to be detected. These specific padlock probes


Fig. 6. Schematic view for padlock probe hybridisation, ligation and PCR amplificationof circularised probes followed by hybridisation to a microarray system. Padlock probe consistsof T1, T2, Zip-Code, F and R. T1 – target complementary sequence, T2 – target complementary

sequence, Zip-Code – unique identifier sequence, F – universal forward primer sequence,R – universal reverse primer sequence

are subsequently ligated, amplified and detected on a microarray. Amplificationoccurs using one generic PCR primer set.

Results with the prototype pUMA enabling simultaneous detection of sixpathogens, look very promising (Fig. 7). Combination of microarrays with 96-wellplates will enable the analysis of 96 samples at the same time. Application of thepUMA technique is universal and opens possibilities to monitor health hazardproblems: in a single test numerous harmful organisms can be detected in a sensi-tive, reliable and fast manner.

Conclusions

In the agrosector methods for detection of plant pathogens have been used formany years. Techniques used for these purposes have rapidly developed during thepast years from techniques based on morphological characteristics to innovativebiochemical and molecular tests. Some of the latter techniques are described inthis review and are implemented in the plant diagnostic field. Much experience hasbeen gained with extraction of pathogens and/or nucleic acids from complex sub-strates, complex nucleic acid amplification strategies, application of multiplex de-tection using microarrays or other platforms, production of recombinant antibodies,etc. The expertise gained by researchers in the agrosector can also be of great valuefor the development of tests for quality monitoring in agrofood processing sys-tems. Food safety is a relevant issue in society and likewise plant health in generalis an important link in the food production chain. An integrated approach to thedevelopment of diagnostic tests would be favourable both for the agrosector andthe agrofood processing sector.


Fig. 7. Representation of the prototype pUMA for six different targets. Targets were mixed withsix PLPs followed by padlock probe hybridisation, ligation and PCR amplification of circularised

probes and finally by hybridisation to an array system (nylon membranes) on which sixZipCode oligos were spotted four times

Acknowledgements

The authors wish to thank José Van Beckhoven, Dorine Donkers-Venne,Maudie Egberts, Marga van Gent-Pelzer, Richard van Hoof, Anita Luttikholt,Marjon Krijger, Greet de Raaij, Nari Anderson, Dianne van der Wal and Marjannede Weerdt for their contribution to the work presented. The collaboration withPamGene International BV, especially Riet Hilhorst, Lars Vahlkamp and PietBoender is highly appreciated.

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Authors’ address: Dr Peter J.M. Bonants,Plant Research International B.V.,Droevendaalsesteweg 1,6708 PB Wageningen,The Netherlandse-mail: [email protected]

Accepted for publication: 9.03.2005


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