Australasian Plant Pathology

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Australasian Plant Pathology Volume 30, 2001 © Australasian Plant Pathology Society 2001 A journal for the publication of original research in all branches of plant pathology

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Australasian Plant Pathology, 2001, 30, 227–230

TMV RT-PCR primers for pea germplasm assay V. A. Torok and J. W. Randles

Tobacco mosaic virus RNA as an internal control for duplex RT-PCR assay of pea germplasm V. A. Torok and J. W. RandlesA

AP01028

Department of Applied and Molecular Ecology, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia. A Corresponding author; email: [email protected]

Abstract. Tobacco mosaic virus (TMV) is an easily prepared, plentiful and stable source of homogenous RNA suitable for addition to reverse transcription polymerase chain reaction (RT-PCR) assays as an internal control. We describe primers and conditions used in a duplex assay for Pea seedborne mosaic virus (PSbMV)-infection of pea seed as an example of the utility of the system for RT-PCR screening of plant germplasm for the presence or absence of a virus. Additional keywords: Pisum sativum, Potyvirus, PSbMV diagnostic RT-PCR primers, TMV RT-PCR primers. Introduction The reverse transcription polymerase chain reaction (RTPCR) is being adopted widely for the detection of RNA viruses due to its high sensitivity and specificity, and applications to plant viruses have been recently described (Raj et al. 1998; Jacobi et al. 1998; Grieco and Gallitelli 1999; Eun et al. 2000; Hailstones et al. 2000). However, none of these authors describe the use of internal controls. Assays usually include a positive (with added control template) and negative (no sample added to the reaction mix) reaction. For quality assurance purposes, it is important that diagnostic RT-PCR assays also have an internal standard for detecting false negative reactions. This is because negative RT-PCR results may either be real, due to the absence of the target template, or false due to the failure of the RT-PCR. For medical diagnostics with PCR and RT-PCR, the issue of quality assurance has been addressed. Several internal control methods have been developed including: co-amplification with a second set of primers targeted to an endogenously expressed ‘housekeeping’ gene (Denis and Lustenberger 1995); amplification with the same set of primers targeting an exogenous internal control which has been structurally modified from the target (Pallen et al. 1992; Brightwell et al. 1998); amplification with the same set of primers targeting an exogenous internal control sequence which has homology only with the target at the terminal primer recognition sites (Sachadyn and Kur 1998); and supplementing samples with a known amount of a control sequence prior to extraction (Willhauck et al. 1998). A disadvantage of using endogenously expressed ‘housekeeping’ genes as internal controls is that their level of expression can vary so that amplification conditions for the control may not be suitable for detection of the pathogen. The construction of internal controls by altering the size or sequence of PCR products by cloning techniques is laborious © Australasian Plant Pathology Society 2001

and system specific. Other complications can arise from the formation of heteroduplexes during PCR due to sequence similarity between the target and control templates, or variable efficiency of extraction of added control sequence. We have developed an internal Tobacco mosaic virus RNA (TMV-RNA) control for RT-PCR which avoids the above difficulties. The internal TMV-RNA control appears to have wide applicability and we report here that it can be used as an internal RT-PCR control for Pea seedborne mosaic virus (PSbMV) assays in pea seed. Methods Purification of TMV TMV strain U1 was partially purified from systemically infected tobacco leaves. Leaves were crushed in an equal volume of 0.2 M phosphate buffer (pH 7.0) containing 0.1% monothioglycerol, fibre was removed and the extract was clarified by heating at 60°C for 10 min and centrifugation at 10 000 g for 10 min, followed by a single chloroform extraction. Virus was concentrated by precipitation with 0.5 volumes of saturated ammonium sulphate, resuspension in 20 m M phosphate buffer and dialysis against water. This cycle was repeated twice. Virus was then clarified by centrifugation at 10 000 g and sedimented by ultracentrifugation, resuspended in 10 mM sodium borate buffer (pH 8.2) at a concentration of 10 mg mL –1, and stored at 4°C. TMV-RNA extraction TMV (1 mg mL–1) was incubated with 1 mg mL–1 Proteinase K (Amresco) in Proteinase K buffer (10 mM Tris-HCl (pH 7.8), 5 mM EDTA, 0.5% SDS) for 3–5 h at 37°C. The aqueous phase was extracted once with an equal volume of phenol saturated with 100 mM Tris-HCl (pH 7.0) and once with an equal volume of chloroform saturated with water. RNA was recovered by precipitation with three volumes of ethanol (biotechnology grade; Amresco) in the presence of 0.3 M sodium acetate (pH 5.2). The pellet was washed once with 70% ethanol and dried. The TMV-RNA was resuspended in 30 µL of water, the concentration determined by spectrophotometry and quality assessed by agarose gel electrophoresis. PSbMV-RNA was extracted, as above, from PSbMV that had been purified by the method of Wang et al. (1992).

10.1071/AP01028

0815-3191/01/030227

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V. A. Torok and J. W. Randles

Extraction of total nucleic acid from pea seeds Seed infected with PSbMV at a rate of 1% was supplied by D. Graetz (Plant Pathology Unit, South Australian Research and Development Institute, Australia). Sub-samples for testing by RT-PCR comprised lots of ten dry seeds. Seed lots were crushed in an ICARDA mill (Syria) and the seed coats removed in an Aspirator (SK Engineering and Allied Works). Batches of seed were ground into a course flour (Analysen Mühle; Janke and Kunkel IRA Labortechnik) and 1 g lots incubated in 10 mL of CTAB buffer (1.4 M NaCl, 0.1 M Tris-HCl (pH 8.0) 2% N-cetyl-N,N,Ntrimethylammonium bromide, 20 mM EDTA, 0.2% mercaptoethanol at 60°C for 30 min in a plastic bag. Samples were briefly crushed in the bag with a roller before transfer to a tube. They were extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), pH 7.0, then with one volume of chloroform:isoamyl alcohol (24:1). Total nucleic acids were precipitated by addition of 0.9 volumes of isopropanol in the presence of 0.3 M sodium acetate (pH 5.2) and incubation at –20°C. The pellet was washed with 70% ethanol, dried and resuspended in 200 µL of water. TMV primers The TMV-RNA sequence (variant 1) (GenBank accession numbers V01408; J02415; Goelet et al. 1982) was used to design the TMV RTPCR primers TMV-R (5′ TTC CAA TGA ACG TCG TGA CGT C 3′ located at position 4428-4407) and TMV-F (5′ TAC CCG GCT TTG CAG ACG ATT GTG TAC CA 3′ located at position 4023-4051). Primer design was tested with the Oligo 4.01 primer analysis software (National Biosystems Inc., Plymouth, USA) and Amplify 1.0 for analysing PCR experiments (University of Wisconsin, Genetics, Madison, USA). Conditions for combined ‘duplex’ RT-PCR RT-PCR was done in a two-step reaction. Reverse transcription was done in a 15 µL reaction volume containing 2 µL total nucleic acid from pea seed, 0.4 µM PSbMV reverse primer (5′ CTC CAA AAC CAT GCT TCA CTC TTG A 3′), 0.2 µM TMV-R, 10 pg TMV-RNA, 25 mM TrisHCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.8 mM dNTPs, 1 unit µL–1 RNase Inhibitor (Ambion) and 5 units of AMV reverse transcriptase (GeneWorks). RT was done at 45°C for 40 min. PCR was done in a 20 µL reaction volume containing 2 µL cDNA from the RT step, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 0.8 mM dNTPs, 0.4 µM each of PSbMV specific forward (5′ GTG TTG GAG GAA TCA CAC CAG AAG AAT GTG 3′) and reverse (5′ GCA GTT GCT ACA TCC ATC ATT GTT GGC CAT 3′) primers, 0.2 µM each of the TMV primers TMV-F/TMV-R and 1 unit Taq DNA polymerase (Promega). PCR cycling conditions RT-PCR was done either in a PTC-225 DNA Engine Tetrad (MJ Research Inc.) or GeneAmp PCR System 2400 (Perkin Elmer). PCR mixtures were initially incubated at 94°C for 3 min followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s and extension at 72°C for 30 s, with a final extension step of 72°C for 2 min. RT-PCR products were analysed by electrophoresis in 1.5% agarose gels buffered in Tris-acetate–EDTA (pH 8.0) containing 0.5 µg mL–1 ethidium bromide.

Results and Discussion Specificity of reaction in duplex RT-PCR format The RT-PCR of TMV-RNA with the TMV-F/TMV-R primers produced a 406 bp amplicon from the 3′ region of the 183 KDa polymerase gene of TMV. The RT-PCR of PSbMVRNA with our specific diagnostic PSbMV primers produced a 1.1 kb amplicon. PSbMV-RNA with all four TMV/PSbMV primers amplified a 1.1 kb product only (Fig. 1, lane 1) and

Fig. 1. Duplex RT-PCR with PSbMV-RNA and TMV-RNA template. Lane M = marker (1 kb ladder GIBCO BRL); lane 1, RT-PCR with PSbMVRNA template under duplex RT-PCR conditions; lane 2, RT-PCR with TMV-RNA template under duplex RT-PCR conditions; lane 3, RT-PCR on PSbMV-RNA and TMV-RNA templates under duplex RT-PCR conditions.

TMV-RNA template with all four TMV/PSbMV primers amplified a 406 bp product only (Fig. 1, lane 2). In the duplex PSbMV/TMV RT-PCR, both the PSbMV template RNA and the TMV control RNA were amplified without apparent interference from each other (Fig. 1, lane 3). We concluded, therefore, that the TMV primers did not amplify PSbMV RNA and the PSbMV primers did not amplify TMV RNA. Application of duplex RT-PCR to seed testing Fig. 2 shows the use of the TMV internal standard in batch testing of pea seed for PSbMV infection. The results represent a test of 250 pea seeds by RT-PCR for PSbMV. Each of the 25 RT-PCR reactions contained a sample from ten pea seeds and the TMV internal control. The figure shows that 24 of the 25 duplex RT-PCR reactions contained a 406 bp TMV amplicon indicating that these RT-PCR reactions had been successful. Lane 5 lacked the 406 bp TMV amplicon indicating that this RT-PCR reaction had failed. A 1.1 kb PSbMV-specific amplicon was present in lanes 7, 10, 14, 17, 21 (Fig. 2) indicating that these samples were positive for PSbMV. These results show the ability of the assay to distinguish between false negative reactions (failed RT-PCR) and true negative reactions (absence of pathogen template). This is important when using RT-PCR as a diagnostic method for certifying freedom from a specific seedborne pathogen. Optimisation of the RT-PCR The internal TMV-RNA standard was designed to be amplified under the same conditions as the PSbMV-RNA specific RT-PCR assay but to produce a specific amplicon

TMV RT-PCR primers for pea germplasm assay

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Fig. 2. Batch testing of pea seed for PSbMV by duplex RT-PCR. Lane M, marker (1 kb plus ladder, GIBCO BRL); lanes 1–25, duplex RT-PCR with total nucleic extracts from batches of dry pea seed; lane (+), positive control containing PSbMV RNA and TMV-RNA; lane (–), negative control containing TMV-RNA only. PSbMV positive samples are indicated by the presence of two amplicons, negatives are indicated by a single TMV specific amplicon, and a failed reaction is indicated by absence of the TMV specific amplicon (see lane 5).

differing in size from the PSbMV amplicon. The optimum duplex RT-PCR conditions which did not show interference with the PSbMV amplification were a TMV-RNA template concentration ranging from 1–10 pg per 15 µL RT reaction; TMV primer concentrations of 0.2 µM for each primer and PSbMV primer concentrations of 0.4 µM for each primer, and a MgCl2 concentration of 1.5 µM. The TMV RT-PCR reaction alone was reliable under a broad range of conditions; TMVRNA concentrations ranging from 1 pg to 1 ng per 15 µL RT reaction; primer concentrations ranging from 0.2 µM to 0.4 µM for each primer; MgCl2 concentrations ranging from 1.5 µM to 2.5 µM, and annealing temperatures ranging from 60–68°C. TMV RT-PCR was also successful when partially purified intact TMV was added at 1 µg per 15 µL RT as the template rather than RNA. Limiting factors When the concentration of the TMV-RNA template was 10 pg per 15 µL RT reaction, the sensitivity of the PSbMV assay was not affected. Under these conditions the reliable limit of PSbMV detection was 100 pg PSbMV-RNA per 15 µL RT reaction (results not shown). This level of sensitivity was the same as for the PSbMV RT-PCR assay alone. When the TMV-RNA control template was increased to 1 ng per 15 µL RT reaction, the level of sensitivity for PSbMV was reduced to 500 pg PSbMV-RNA per 15 µL RT reaction. This result indicated that TMV-RNA template could compete with the PSbMV-RNA template and limit the sensitivity of the test if its concentration is not accurately determined. Modification of the size of the TMV amplicon was tested because it has been suggested that smaller PCR amplicons are amplified more efficiently than larger ones (Pallen et al. 1992;

Brightwell et al. 1998) and may compete with the target template. Thus, by increasing the size of the TMV internal control amplicon to more closely match that of the PSbMV amplicon, it would be expected that the drive of the reaction kinetics towards the smaller PCR product would be reduced. Alternative TMV primers were designed to produce larger TMV amplicons, but these primers produced spurious bands in the TMV/PSbMV duplex RT-PCR. We concluded that by limiting the amount of TMV-RNA control added to our duplex RT-PCR, TMV would not be amplified in preference to PSbMV, overcoming the expected difficulties associated with the co-amplification of differently sized products. This method allows the concentration of control template to be optimised, unlike methods which use an endogenous control. Scope of this method We have developed the TMV internal control for testing pea germplasm for PSbMV, and particularly for plant breeding and quarantine purposes. The system has been tested successfully with other RNA plant viruses (V. A. Torok, N. Thompson and J. W. Randles, unpublished data) and we consider that the conditions described here offer a reliable internal control for general application to the diagnosis of RNA viruses by RT-PCR. The method should be applicable to a wide range of other RT-PCR assays. TMV does not present risks to the user, is easily purified in large quantity and the virus can be stored for years at 4•C in the presence of trace amounts of the preservative sodium azide. Acknowledgements We thank D. Graetz for supplying PSbMV-infected pea seed and the Grains Research and Development Corporation for provision of a postgraduate research scholarship to V. A. T.

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References Brightwell G, Pearce M, Leslie D (1998) Development of internal controls for PCR detection of Bacillus anthracis. Molecular and Cellular Probes 12, 367–377. Denis MG, Lustenberger P (1995) Qualitative low-level internal control for nested RT-PCR. BioTechniques 19, 906–908. Eun AJC, Seoh ML, Wong SM (2000) Simultaneous quantitation of two orchid viruses by the TaqMan® real-time RT-PCR. Journal of Virological Methods 87, 151–160. Goelet P, Lomonossoff GP, Butler PJ, Akam ME, Gait MJ, Karn J (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proceedings of the National Academy of Sciences USA 79, 5818–5822. Grieco F, Gallitelli D (1999) Multiplex reverse transcriptionpolymerase chain reaction applied to virus detection in globe artichoke. Journal of Phytopathology 147, 183–185. Hailstones DL, Bryant KL, Broadbent P, Zhou C (2000) Detection of Citrus tatter leaf virus with reverse transcription-polymerase chain reaction (RT-PCR). Australasian Plant Pathology 29, 240–248. Jacobi V, Bachand GD, Hamelin JD, Castello JD (1998) Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses. Journal of Virological Methods 74, 167–178.

Pallen MJ, Puckey LH, Wren BW (1992) A rapid, simple method for detecting PCR failure. PCR Methods and Applications 1, 91–92. Raj SK, Saxena S, Hallen V, Singh BP (1998) Reverse transcriptionpolymerase chain reaction (RT-PCR) for direct detection of cucumber mosaic virus (CMV) in gladiolus. Biochemistry and Molecular Biology International 44, 89–95. Sachadyn P, Kur J (1998) The construction and use of a PCR internal control. Molecular and Cellular Probes 12, 259–262. Wang D, Hayes IM, Maule AJ (1992) Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA. Journal of Virological Methods 36, 223–230. Willhauck M, Vogel S, Keilholz U (1998) Internal control for quality assurance of diagnostic RT-PCR. BioTechniques 25, 656–659.

Received 18 December 2000, accepted 26 March 2001

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