Protocols

Detection of Plant Genes, Gene Expression andViral RNA from Tissue Prints on FTA® Cards

YVETTE ROY and ANNETTE NASSUTH*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1,Canada

Abstract. A procedure was established for easy and convenient detection of plant DNA,plant RNA and viral RNA from plant tissue prints on FTA® (Flinders Technology Associ-ates, Moscoso et al., 2005) PlantSaver Cards, while avoiding the cross-contamination thatcommonly occurs with prints from plant tissues. Detection was successful by adding 2 mmdiscs of the prints directly to (RT)-PCR reactions. DNA was detected in leaves fromtobacco, tomato and grapes, and in fruit of tomato and grape. Rubisco and malate dehydro-genase RNA were detected in tomato fruit and leaf, tobacco leaf and grape leaf. RNA fromPepino mosaic virus was detected in tomato leaf and fruit and from Rupestis stem pittingassociated virus in grape leaf. Detection was still possible from the tissue prints on FTA®

Cards after storage for many months at room temperature, making this a procedure suit-able for sample collection and storage off site, prior to further processing in the laboratory.

Key words: FTA® PlantSaver Card, plant nucleic acid detection, plant virus detection, RT-PCR, tissue print

Abbreviations: DTT, dithiothreitol; FTA, Flinders Technology Associates; PepMV, pepinomosaic virus; RSPaV, Rupestis stem pitting-associated virus; RT-PCR, reverse transcription-polymerase chain reaction; RubiscoL, large subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase; SSR, simple sequence repeat.

Tissue print detection of DNA,RNA and virus Roy and NassuthIntroduction

Detecting nucleic acids from plant tissues can often pose difficulties due to thecomplex methods currently used to obtain, store and purify the samples. FTA®

Cards (Whatman®) provide an alternate method. A sample can be pressed or spot-ted on the FTA paper and the nucleic acids within the tissue will be bound to itsmatrix, after which it can be archived until further use. The paper is impregnatedwith chelators, denaturants and free-radical traps, which inhibit enzymes, mi-crobes and chemicals that may degrade the DNA or RNA in the sample (Rogersand Burgoyne, 1997; Salvador et al., 2003; Whatman, 2003). Therefore, nucleicacids on FTA® Cards can be stored for long periods of time at ambient tempera-tures, in contrast to the traditional storage in liquid at -20oC or -80oC.

Plant Molecular Biology Reporter 23: 383–395, December 2005© 2005 International Society for Plant Molecular Biology. Printed in Canada.

*Author for correspondence. e-mail: anassuth@uoguelph.ca; fax: 519-837-2075;ph: 519-824-4120 ext 58787.

The FTA® Card was initially developed to detect excess phenylalanine inblood samples as a method to screen newborn infants for Phenylketourea (PKU)(Guthrie and Susi, 1963). The membrane was subsequently used for DNA detec-tion by PCR, in forensic science (Vanek et al., 2001; Krenke et al., 2002; Nelsonet al., 2002; Seah et al., 2003) and human medicine (Devost, 2000; Roberts, 2000;Taback, 2003). The scientific community then recognized the potential of FTA®

Cards to allow for sample collection in the field and later analysis in a laboratory(Gutierrez-Corchero et al., 2002; Crabbe, 2003). It was also discovered that hu-man RNA can be detected by RT-PCR with RNA eluted from prints on FTA®

Cards (Natarajan et al., 2000; Bhattacharya et al., 2004; Moscoso et al., 2005).More recently, scientists have expanded the application of the FTA® Card to de-tect nucleic acids in plant tissue and found it rapid and effective. Detection ofDNA has been reported for FTA prints from leaf tissue of a variety of plants, in-cluding soybean (Lin et al., 2000), Arabidopsis, marijuana, coca, orchid, papaya,petunia, opium poppy, potato, rice, sugar beat, sugarcane (Lin et al., 2000), cas-sava, tobacco, corn (Lin et al., 2000; Ndunguru et al. 2005), tomato (Lin et al.,2000; Bendezu, 2004; Ndunguru et al., 2005), barley (Drescher and Graner, 2002)and impatiens (Tsukaya, 2004; Tsukaya et al., 2005). Natarajan et al. (2000) alsosuccessfully detected RNA in eluants from potato leaf prints.

We were interested in improving the FTA-based procedure to enable us torapidly detect any type of nucleic acids. Several variations of the procedure weretried to determine the most simple, accurate and cost-effective method. The proce-dure was tested on different plants, such as tomato, grape and tobacco, and differ-ent target sequences, such as malate dehydrogenase (MDH), rubiscoL and greenfluorescence protein (GFP) genes, MDH RNA and Pepino mosaic virus (PepMV)and Rupestris stem pitting associated virus (RSPaV) We report here for the firsttime that detection of DNA, RNA and plant virus is possible by the same FTA®-based procedure.

Materials and Methods

Plant material

Tobacco (N. tabacum and N. benthamiana), tomato (L. esculentum) and grape (V.vinifera) were all grown under standard greenhouse conditions, except that somegrape plants were grown in the field or in tissue culture (supplied by Alois Bilavik)as indicated in text. Some greenhouse and field-grown tomato fruits were bought ina local grocery store. V. vinifera fruits were obtained from field-grown plants (sup-plied by Judy Strommer). N. benthamiana GFP16c, a transgenic plant containingone copy of mGFP5, was obtained from David Baulcombe (Riuz et al., 1998).

Preparation of FTA® tissue print

• Place leaf or cut fruit tissue directly on the FTA® PlantSaver Card (Whatman®).• For leaf tissue, apply pressure with a pestle briefly until plant material transfers

to the card.• For fruit tissue, press the cut side gently on the card.

384 Roy and Nassuth

• Allow the prints to dry for one hour and brush off any excess plant materialwith tissue paper.

• Store at ambient temperature in a dry location, preferably with a desiccant.

Preparation of samples for (RT)-PCR analysis

• Clean the Harris® Micro Punch (Whatman®) with a tissue dampened with 70%ethanol and by taking a disc from a blank, sample-less FTA® Card. Repeat. Dothis every time before punching the next sample.

• Remove a disc from the dried FTA® tissue print using the micro punch andplace the disc directly into a PCR tube.

• Note: For tissues with high amounts of inhibitors, such as grape leaf, wash thedisc twice with 200 µl of 70% ethanol, incubating for 5 min for each wash. Dryat room temperature for 30 min before proceeding.

• Wash with FTA® Reagent (Whatman®): Add 200 µl FTA® Reagent to eachPCR tube, incubate for 3 min at room temperature and discard liquid. Duringthis time pipette the liquid up and down twice in each tube.

• Repeat wash with FTA® Reagent.• Note: For tomato fruit samples it is possible to eliminate the washes with the

FTA® Reagent.• Wash twice with 200 µl 0.1 TE (10 mM Tris, 0.1 mM EDTA, pH 8) as de-

scribed for FTA® Reagent.• Air dry samples for 1 hr at room temperature, cover samples with a lid to avoid

contamination while still allowing space for evaporation.

(RT)-PCR analysis (adapted from Nassuth et al. 2000)

• (RT)-PCR mixture (25 µl final volume) contained, in addition to the FTA®

disc containing the nucleic acid sample, a final concentration of 10 mM Tris-HCl, 50 mM or 100 mM KCl, 2% sucrose, 0.1 mM cresol red, 1.5 mM MgCl2,5 mM DTT, 0.5 µM of each primer (Table 1), 200 µM of Mg2+ balanced dNTPs,2 U Taq DNA polymerase (Fermentas) and 0.1 U avian myeloblastosis virus re-verse transcriptase (AMV RT, Roche).

• (RT)-PCR reactions were incubated at 54oC for 45 min (reverse transcription),followed by 35 cycles of 94oC for 30 s, 54oC for 45 s, and 72oC for 60 s, with afinal elongation step at 72oC for 5 min. For PCR a similar procedure was fol-lowed but without the addition of reverse transcriptase and/or without the45 min incubation at 54oC.

Analysis of (RT)-PCR products

• 10 µl of the final PCR product was electrophoresed on a 1.5% agarose gel in 1×TBE buffer (45 mM Tris-Borate, 1 mM EDTA).

Re-amplification from discs

• Remove remaining liquid from the PCR tubes and discard.• Wash each disc with 200 µl 0.1 TE for 2 min.• Repeat wash.

Tissue print detection of DNA, RNA and virus 385

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Results and Discussion

Detection of DNA in fruit and leaf discs: plant genes and transgenes

FTA® Cards were used to successfully amplify DNA in tomato fruit, leaf and peti-ole sap (Figures 1 and 2; petiole sap not shown). To demonstrate the possibility ofdetecting single copy (trans)genes using FTA® Cards, transgenic tobacco leafsamples were used to amplify not only endogenous RbcL and MDH genes butalso the GFP transgene (Figure 3).

Detecting nucleic acids from plant tissue prints presented challenges thatwere not a concern with tissue from other organisms. First, DNA amplificationusing grape leaf and fruit tissues was inconsistent. To determine if the difficultiesamplifying the nucleic acids were due to inhibitors in the plant, purified amplifiabletomato nucleic acid extracts were added to grape leaf discs prior to PCR. As posi-tive controls, tomato extract was also added to tomato leaf discs and to blankdiscs (Figure 4a). Tomato extract DNA in control reactions amplified successfullyusing RbcL primers, while the tomato extract added to a grape leaf disc wasunable to amplify, indicating that the grape leaf prints contained inhibitors thatwere interfering with the PCR reaction. The fact that grape tissue prints containedinhibitors was not surprising because plant tissues from woody plants are knownto contain large amounts of polysaccharides and phenols that interfere with (RT)-PCR amplification (Newbury and Possingham, 1977; Rezaian and Krake, 1987;Demeke and Adams, 1992; John, 1992), and this has been a problem with RT-PCR reactions on grape RNA extracts, especially from older tissues (MacKenzieet al., 1997; Nassuth et al., 2000). Traditional RNA extraction methods can bemodified to include PVP-40 or mercaptoethanol, which reduce the amount ofinhibitors (Monette and James, 1990; John, 1992; Henson and French, 1993;Rowhani et al., 1993; Minafra and Hadidi, 1994; MacKenzie et al., 1997; Zhanget al., 1998; Nassuth et al., 2000). The FTA® method does not allow for thesetypes of modifications, although PVP-40 can be added directly to the (RT)-PCRmixture in order to reduce inhibition of the (RT)-PCR components. Inhibition canalso be reduced by pre-washing the sample discs twice, for 5 min with 200 µl of70% ethanol, as recommended by Ndunguru et al. (2005). Amplification of DNAin grape leaf prints was possible after including this washing procedure and 2%PVP-40 in the PCR reaction mixture (Compare leaf print from greenhouse-growngrape in Figure 4a (G) with those in Figure 4b (g)). Amplification was better fromDNA in leaf prints of young tissue culture plants compared to those from plantsgrown in the greenhouse or field (Figure 4b). For less problematic tissues, such astomato leaves and fruits, a variety of washing buffer types and volumes weretested and it was determined that elimination of the wash with FTA® Reagent, tominimize time and cost, was possible although no amplification was obtainedwith some discs (Figure 1). Therefore we routinely wash leaf tissues twice with200 µl of FTA® Reagent followed by two washes of 200 µl 0.1 TE, as recom-mended by the manufacturer.

Tissue print detection of DNA, RNA and virus 387

Another problem was cross-contamination between samples, due to tissuetransferring via the micro-punch. This was tested by removing a disc from a tissueprint with the micro-punch, then removing a blank disc as a negative control.Since cross-contamination was occurring, the blank disc provided an amplifiedPCR product (Figure 2, lanes labeled B; Figure 4a, B and - lanes). Blood samplesapparently provide no cross-contamination of the micro-punch (Whatman, 2003).This is likely because blood easily absorbs within an FTA® Card, while plantmaterial is more liable to leave tissue on top of the card, readily available toattach to the micro-punch and contaminate the next sample. Testing for cross-contamination was not reported by other researchers who used plant tissue on theFTA® Cards, so we assume that it wasn’t considered. Whatman® suggests cleaningthe micro-punch with ethanol or taking a blank disc between samples (Whatman,

388 Roy and Nassuth

Figure 1. PCR products from tomato fruit and leaf tissue prints on FTA® PlantSaver Cards (a, b, c).Discs taken from prints were washed with TE buffer only and then used to amplify nucleic acids withRubiscoL primers producing products of different sizes (The expected sizes are noted on the right).Discs from (a) were washed with TE buffer and used in a second PCR reaction (c). A 100 bp DNAladder (M) was used to confirm fragment size; nucleic acid extracts were used as a positive control(+) and water was used as a negative control (-). These amplification reactions were repeated on atleast two different tissue prints.

Figure 2. PCR products from tomato (T) leaf prints and blank (B) FTA® PlantSaver Cards wereamplified with RubiscoL primers. A 100 bp DNA ladder (M) was used to confirm fragment size.Tomato nucleic acid extract was used as a positive control (+) and water was used as a negativecontrol (-). This test was repeated on at least two different tissue prints.

2003), but we found that both treatments were necessary twice to prevent cross-contamination. We recommend that a consecutively sampled blank disc be testedfor each (RT)-PCR reaction as a negative control.

Detection of plant RNA

RNA in tomato fruit, leaf and petiole sap, grape leaf and fruit and tobacco leaf waseasily detected from discs placed directly in the (RT)-PCR reaction (Figures 4 and 5;grape fruit not shown). This is simpler than the procedure applied by Natarajan et al.(2000) for human blood, human cells, and plant leaf tissue, as recommended by themanufacturer (Whatman®), which involves eluting RNA from the disc and using theeluant in the RT-PCR reaction (Whatman, 2003). All malate dehydrogenase primers(Table 1) were designed around an intron so that amplification on an RNA templatewould yield a different size RT-PCR product than its DNA counterpart. For example,grape malate dehydrogenase primers amplified multiple fragments between 600 and900 base pairs long due to presence of intron(s) in the DNA (Nassuth et al., 2000),whereas the fragment produced by these primers on RNA template is only 196 basepairs and easily distinguishable by agarose gel electrophoresis (Figure 4b). A simi-lar situation exists for tomato and tobacco (Figure 5a, b).

Detection of viral RNA

Viral RNA was readily amplified directly from discs. Tomato fruit and leaf tissuetested positive for the presence of PepMV in both asymptomatic and symptomaticinfected plants (Figure 6a, b). RSPaV was successfully detected in infected grapeleaf tissue (Figure 6c). An additional advantage of FTA® Cards is that they appar-ently inactivate viral pathogens (Moscoso et al., 2005) allowing for safe shipmentand storage of viral samples.

Tissue print detection of DNA, RNA and virus 389

Figure 3. PCR products from non-transgenic (a) and transgenic (b) tobacco leaf prints on FTA®

PlantSaver Cards were amplified using tobacco malate dehydrogenase (MDH), RubiscoL or GFPprimers. A 100 bp DNA ladder (M) was used to confirm fragment size; nucleic acid extracts wereused as a positive control (+), and water was used as a negative control (-). These amplificationreactions were repeated on at least two different tissue prints.

While this manuscript was in preparation, Ndunguru et al. (2005) reportedthe detection of the RNA viruses Tobacco mosaic virus, Potato virus Y andTobacco etch virus, as well as the DNA geminiviruses, from leaf tissue pressedupon FTA® Cards. However, these researchers eluted the viral RNA and DNAbefore (RT)-PCR, as did others for the detection of animal viruses (Bhattacharyaet al., 2004; Moscoso et al., 2005).

We chose to use PepMV for this study because it is extremely stable (Aguilar,2002) and appears to be well distributed throughout the plant in sufficiently hightiter. Small aliquots of tomato fruit sap (1 µl) and small grape buds were sufficientto detect PepMV (Nassuth and Gu, 2005) and RSPaV (Stewart and Nassuth,2001) respectively by RT-PCR. However, not all viruses are evenly distributedthroughout the plants they infect, which can be a problem for the FTA® methodsince it samples only a small area of the plant. Therefore, we recommend that theFTA® detection procedure be compared with RT-PCR on extracts for each virus ina known infected plant before it is applied to large scale studies.

Reusing FTA® discs for multiple experiments for DNA detection

After using samples for PCR or RT-PCR, we reused the washed discs in a secondreaction. The primers chosen for the second reaction were such that they couldnot use the first amplification product as template, thereby ensuring that thevisualized amplification product was truly from a second amplification on theoriginal DNA template (Figure 1). For plants containing large amounts of inhibitors,

390 Roy and Nassuth

Figure 4. PCR (a) and RT-PCR (b) products from grape leaf prints on FTA® PlantSaver Cards.(a) Discs were taken from blank (B), tomato (T), and grape (G) prints. Tomato nucleic acid extractwas added (+) or not (-), and the resulting samples were PCR amplified using RubiscoL primerswhich are known to amplify DNA from both tomato and grape (Myslik and Nassuth, 2001). (b) Discswere taken from leaf prints of grape plants grown in tissue culture (t), the greenhouse (g), or the field(f). For (b) the discs were washed twice with 200 µl of 70% ethanol prior to the normal washingprocedure, and 2% PVP-40 was added to the RT-PCR mixture. A 100 bp DNA ladder (M) was usedto confirm fragment size; nucleic acid extracts were used as a positive control (+), and water wasused as a negative control (-). These amplification reactions were repeated on at least two differenttissue prints.

such as grape, a second (RT)-PCR reaction was often more successful than thefirst (results not shown), presumably due to reduction of inhibitors through thewashes and RT-PCR procedure. However, reusing plant tissue discs for (RT)-PCR was generally not reliable (Figure 1c), in contrast to what had been reportedfor tissues from other organisms. Del Rio et al. (1995) found that blood staineddiscs may be used repeatedly for up to 3 separate PCR reactions.

Storage and integrity of samples

To determine if nucleic acid templates degrade over time to the extent that theycan no longer be amplified, we tested prints that had been stored for variousamounts of time. All tests turned out positive and showed that plant DNA largerthan 800 nucleotides could be detected after eight months, plant RNA up to243 nucleotides in length could be detected after five and a half months and viralRNA up to 719 nucleotides could be detected after six months of storage at ambi-ent temperatures. Viral RNA was detectable after even longer periods of time(8 months), although this was tested only for shorter PCR fragments (312 bp).Drescher and Graner (2002) amplified an 1800 bp fragment from barley leafDNA. Natarajan et al. (2000) amplified a 1.05 kb fragment on RNA eluted frompotato leaf prints, and Ndunguru et al. (2005) also used eluant to amplify a 2.8 kbfragment from a DNA virus and a 2.1 kb fragment from an RNA virus.

Tissue print detection of DNA, RNA and virus 391

Figure 5. RT-PCR products from prints of (a) tomato leaf, fruit and petiole sap and (b) tobacco leaffrom N. benthamiana and N. tabacum, on FTA® PlantSaver Cards, produced with plant species-specific malate dehydrogenase (MDH) primers. A 100 bp DNA ladder (M) was used to confirmfragment size; nucleic acid extracts were used as a positive control (+), and water was used as anegative control (-). These amplification reactions were repeated on at least two different tissueprints. Note that amplification yield from the DNA template is lower because the primers have beendepleted from amplifying the RNA template (Nassuth et al., 2000).

Lin et al. (2000) stored plant tissue prints for up to one month at room tem-perature, while others stored their plant prints at temperatures of -20oC or lower(Natarajan et al., 2000). There was no information on how long plant tissue couldbe stored on FTA® Cards at freezing temperatures, but Lange et al. (1998) used asimilar system, the Generation DNA Purification System (Gentra Systems Inc.),and were able to store their plant samples for up to 6 months at -20oC. Whatman®

has reported that genomic DNA can be stored on FTA® Cards for up to 14 yearsat room temperature (Whatman, 2003). To increase the integrity of the sample ithas been recommended to store samples at freezing temperatures, but this in-creases the chance of moisture uptake by the FTA® Card, possibly spoiling thesample. By storing the samples at ambient temperatures for long periods of timewe have demonstrated the reliability of the FTA® paper to collect samples in afield or greenhouse and to mail samples to laboratory facilities, without compro-mising amplification abilities.

Conclusions

We show here for the first time that it is possible to detect plant RNA and viralRNA by the same easy, convenient method that can detect plant DNA, by (RT)-PCR amplification directly from a disc taken from FTA® tissue prints. We

392 Roy and Nassuth

Figure 6. RT-PCR products from tomato leaf and fruit prints on FTA® PlantSaver Cards. Viral RNAin tomato was amplified using PepMV RdRp primers (a) and PepMV CP primers (b), and in grapeusing RSPaV primers (c). Tomato leaf samples were taken from tissues of both symptomatic (s) andasymptomatic (n) plants. Asymptomatic tomato fruits were bought at the grocery store and camefrom either greenhouse (n) or field (f-n). Grape leaf discs (c) were washed twice with 200 µl of 70%ethanol prior to the normal washing procedure and addition to an RT-PCR mixture containing 2%PVP-40. A 100 bp DNA ladder (M) was used to confirm fragment size; nucleic acid extracts wereused as a positive control (+), and water was used as a negative control (-). These amplificationreactions were repeated on at least two different tissue prints.

detected DNA in leaves from tobacco, tomato and grapes, and in fruit fromtomato and grape, while avoiding cross-contamination, which often occurs whenusing plant tissue prints. Plant RNA was detected in tomato fruit and leaf, tobaccoleaf and grape leaf, and the RNA viruses PepMV and RSVaP were detected intomato leaf and fruit and grape leaf, respectively.

This procedure could be used to test the presence of a transgene, do SSRanalysis (Merdinoglu et al., 2005), marker genotyping (Lange et al., 1998; Drescherand Graner, 2002) or simple and rapid plant virus detection. The ability to obtainand store the prints at ambient temperatures means that these tests could be em-ployed for wide-scale studies in the field. Restriction digestion and/or cloning andsequencing of the (RT)-PCR products would expand the application to additionalstudies, such as differentiating between virus (Martinez-Culebras et al., 2002) orplant species by, for example, DNA barcoding (Kress et al., 2005).

Acknowledgments

We thank Alois Bilavik and Judy Strommer for grape material and David C.Baulcombe for transgenic tobacco. The constructive criticism of the manuscriptby Mahbuba Siddiqua and Fariba Shahmir is acknowledged. This work was sup-ported by grants from AMCO Farms Inc., IRAP and OMAF to A.N., as well asthe Work Study Program and an URA through the University of Guelph to Y.R.

References

Aguilar JM, Hernandez-Gallardo MD, Cenis JL, Lacasa A, and Aranda MA (2002) Com-plete sequence of the Pepino mosaic virus RNA genome. Arch Virol 147: 2009-2015.

Bendezu IF (2004) Detection of the tomato Mi 1.2 gene by PCR using non-organic DNApurification. Nematropica 34: 23-30.

Bhattacharya SS, Kulka M, Lampel KA, Cebula TA, and Goswami BB (2004) Use of re-verse transcription and PCR to discriminate between infectious and non-infectioushepatitis A virus. J Virol Methods 116: 181-187.

Crabbe MJ (2003) A novel method for the transport and analysis of genetic material frompolyps and zooxanthellae of scleractinian corals. J Biochem Biophys Meth 57: 171-176.

Del Rio SA, Marino MA, and Belgrader P (1995) Reusing the same blood-stained punchfor sequential DNA amplifications and typing. Biotechniques 20: 970-974.

Demeke T and Adams RP (1992) The effects of plant polysaccharides and buffer additiveson PCR. Biotechniques 12: 332-334.

Devost NC and Choy FY (2000) Mutation analysis of Gaucher Disease using dot-bloodsamples on FTA filter paper. Am J Med Genet 94: 417-420.

Drescher A and Graner A (2002) PCR-genotyping of barley seedlings using DNA samplesfrom tissue prints. Plant Breeding 121: 228-231.

French CJ, Bouthillier M, Bernardy M, Ferguson G, Sabourin M, Johnson RC, Masters C,Godkin S, and Mumford R (2001) First report of pepino mosaic virus in Canada, andthe United States. Plant Dis 85: 1121.

Guthrie R and Susi A (1963) A simple phenylalanine method for detecting phenyl-ketonuria in large populations of newborn infants. Am Acad Pediatrics 32: 338-343.

Tissue print detection of DNA, RNA and virus 393

Gutierrez-Corchero F, Arruga MV, Sanz L, Garcia C, Hernandez MA, and Campos F(2002) Using FTA registered cards to store avian blood samples for genetic studies.Their application in sex determination. Mol Ecol Notes 2: 75-77.

Henson JM and French R (1993) The polymerase chain reaction and plant disease diagno-sis. Annu Rev Phytopathol 31: 81-109.

John ME (1992) An efficient method for isolation of RNA and DNA from plants contain-ing polyphenolics. Nucleic Acids Res 20: 2381.

Krenke BE, Tereba A, Anderson SJ, Buel E, Culhane S, Finis CJ, Tomsey CS, Zachetti JM,and Sprecher CJ (2002) Validation of a 16-locus fluorescent multiplex system. J Fo-rensic Sci 47: 773-785.

Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, and Janzen DH (2005) Use of DNAbarcodes to identify flowering plants. Proc Natl Acad Sci USA 102: 8369-8374.

Lange DA, Penuela S, Denny RL, Mudge J, Concibido VC, Orf H, and Young ND (1998)A Plant DNA isolation protocol suitable for polymerase chain reaction based marker-assisted breeding. Crop Sci 38: 217-220.

Lin J-J, Fleming R, Kuo J, Matthews BF, and Saunders JA (2000) Detection of plant genesusing a rapid, nonorganic DNA purification method. Biotechniques 28: 346-350.

MacKenzie DJ, McLean M, Mukerji S, and Green M (1997) Improved RNA extractionfrom woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Dis 81: 222-226.

Martinez-Culebras P, Lazaro A, Abad Campos P, and Jorda C (2002) A RT-PCR assaycombined with RFLP analysis for detection and differentiation of isolates of PepinoMosaic Virus (PepMV) from tomato. Eur J Plant Pathol 108: 887-892.

Meng B, Credi R, Petrovic N, Tomazic I, and Gonsalves D (2003) Antiserum to recombi-nant virus coat protein detects rupestris stem pitting associated virus in grapevines.Plant Dis 87: 515-522.

Merdinoglu D, Butterlin G, Bevilacqua L, Chiquet V, Adam-Blondon A, and Decroocq S(2005) Development and characterization of a large set of microsattellite markers ingrapevine (Vitis Vinifera L.) suitable for multiplex PCR. Mol Breeding 15: 349-366.

Minafra A and Hadidi A (1994) Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification.J Virol Methods 47: 175-188.

Monette PL and James D (1990) Detection of two strains of grapevine virus A. Plant Dis74: 898-900.

Moscoso H, Raybon EO, Thayer SG, and Hofacre CL (2005) Molecular detection andserotyping of infectious bronchitis virus from FTA filter paper. Avian Dis 49: 24-29.

Mumford RA and Metcalfe EJ (2001) The partial sequencing of the genomic RNA of aUK isolate of Pepino Mosaic Virus and the comparison of the coat protein sequencewith other isolates from Europe and Peru. Arch Virol 146: 2455-2460.

Myslik JT and Nassuth A (2001) Rapid detection of viruses, transgenes and mRNAs insmall plant leaf samples. Plant Mol Biol Rep 19: 329-340.

Nassuth A and Gu C (2005) Pepino Mosaic Virus. In: Molecular Diagnosis of PlantViruses. Studium Press, Huston, Texas. Submitted.

Nassuth A, Pollari E, Helmeczy K, Stewart S, and Kofalvi SA (2000) Improved RNAextraction and one-tube RT-PCR assay for simultaneous detection of control plantRNA plus several viruses in plant extracts. J Virol Methods 90: 37-49.

Natarajan P, Trinh T, Mertz L, Goldsborough M, and Fox DK (2000) Paper-based archiv-ing of mammalian and plant samples for RNA analysis. Biotechniques 29: 1328-1333.

Ndunguru J, Taylor NJ, Yadav J, Aly H, Legg JP, Aveling T, Thompson G, and FauquetCM (2005) Application of FTA technology for sampling, recovery and molecular char-

394 Roy and Nassuth

acterization of viral pathogens and virus-derived transgenes from plant tissues. Virol J2: 45.

Nelson MS, Levedakou EN, Matthews JR, Early BE, Freeman DA, Kuhn CA, SprecherCJ, Amin AS, McElfresh KC, and Schumm JW (2002) Detection of a primer-bindingsite polymorphism for the STR locus D16S539 using the powerplex 1.1 system andvalidation of a degenerate primer to correct for the polymorphism. J Forensic Sci 47:345-349.

Newbury HJ and Possingham JV (1977) Factors affecting the extraction of intact ribonu-cleic acid from plant tissue containing interfering phenolic compounds. Plant Physiol60: 543-547.

Rezaian MA and Krake LR (1987) Nucleic acid extraction and virus detection in grape-vine. J Virol Methods 17: 277-285.

Roberts R, Sullivan P, Joyce P, and Kennedy MA (2000) Rapid and comprehensive deter-mination of cytochrome P450 CYP2D6 poor metabolizer genotypes by multiplex poly-merase chain reaction. Human mutation 16: 77-85.

Rogers C and Burgoyne L (1997) Bacterial typing: storing and processing of stabilizedreference bacteria for polymerase chain reaction without preparing DNA- an exampleof an automatable procedure. Analytical Biochem 247: 223-227.

Rowhani A, Chay C, Golino DA, and Falk BW (1993) Development of a polymerase chainreaction technique for the detection of grapevine fanleaf virus in grapevine tissue.Phytopathology 83: 749-758.

Ruiz MT, Voinnet O, and Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937-946.

Salvador JM and De Ungria MCA (2003) Isolation of DNA from saliva of betel quidchewers using treated cards. J Forensic Sci 48: 794-797.

Seah LH, Jeevan NH, Othman MI, Jaya P, Ooi YS, Wong PC, and Kee SS (2003) STRdata for the AmpF/STR identifiler loci in three ethnic groups (Malay, Chinese, Indian)of the malaysian population. Forensic Sci Int 138: 134-137.

Stewart S and Nassuth (2001) RT-PCR based detection of rupestris stem pitting associatedvirus within field-grown grapevines throughout the year. Plant Dis 85: 617-620.

Taback B, Giuliano AE, Hansen NM, Singer FR, Shu S, and Hoon DSB (2003) Detectionof tumor-specific genetic alterations in bone marrow from early-stage breast cancerpatients. Cancer Res 63: 1884-1887.

Tsukaya H (2004) Gene flow between Impatiens radicans and I. javensis (Balsaminaceae)in Gunung Pangrango, central Java, Indonesia. Am J Bot 91: 2119-2123.

Tsukaya H, Iokawa Y, Kondo M, and Ohba H (2005) Large-scale general collection ofwild-plant DNA in Mustang, Nepal. J Plant Res 118: 57-60.

Vanek D, Hradil R, and Budowle B (2001) Czech population data on 10 short tandem re-peat loci of SGM plus STR system kit using DNA purified in FTA cards. Forensic SciInt 119: 107-108.

Whatman (2003) Nucleic acid sample preparation. Avaibable at: http://www.whatman.com/products/?pageID=7.31. Accessed August 4, 2005.

Zhang Y-P, Uyemoto JK, Golino DA, and Rowhani A (1998) Nucleotide sequence and RT-PCR detection of a virus associated with grapevine rupestris stem-pitting disease.Phytopathology 88: 1231-1237.

Tissue print detection of DNA, RNA and virus 395