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Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid
Journal of Virological Methods 121 (2004) 25–29
Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid E. Ragozzino, F. Faggioli∗ , M. Barba Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22–00156 Rome, Italy Received 18 February 2004; received in revised form 14 May 2004; accepted 17 May 2004 Available online 7 July 2004
Abstract A one tube-one step RT-PCR was developed for the detection of seven viroids (Apple scar skin viroid, Apple dimple fruit viroid, Pear blister canker viroid, Hop stunt viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid) in four genera that infect eight plant species. The efficiency and specificity of this method were optimized by the use of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase and HotStarTaq® DNA polymerase which allowed increase sensitivity of viroid detection. The method was assessed with 56 viroid-infected field plants. The multiplex one tube-one step RT-PCR has the advantage of requiring less hands-on time to set up an assay than standard multiplex one, it also reduces the possibility of false positive tests because all steps are performed in the same tube thus, avoiding cross-contamination. The method may be used routinely for viroid detection in sanitary and certification programmes. © 2004 Elsevier B.V. All rights reserved. Keywords: Viroids diagnosis; One tube-one step RT-PCR; RNA extraction method; Multiplex RT-PCR
1. Introduction The reverse transcription-polymerase chain reaction (RT-PCR) technique is a rapid and reliable method for detecting viroids from infected plants (Hadidi and Candresse, 2003). Currently, the RT-PCR is the most sensitive technique for determining the presence or absence of specific RNA templates in mixtures. Avoiding contamination and achieving viroid full-length transcripts are among the goals of this technique. The approach used most frequently for a RT-PCR assay is to run the RT reaction under optimum conditions for reverse-transcriptase (Rtase) activity and then to add a fraction of the RT reaction to a standard PCR reaction (RT-PCR two tubes-two steps). Since additional steps are undesirable, as they increase the risk of contamination, an alternative way is to add the PCR reagents to the RT reaction itself. In this case, the technique is referred to as one tube-two steps
∗ Corresponding author. Tel.: +39 06 820 70207; fax: +39 06 868 02296. E-mail address: [email protected] (F. Faggioli).
0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.05.012
RT-PCR, the reaction conditions are sequentially adapted to the enzymes used and the risk of contamination decreases. Another possibility is to use all the components of the RT and PCR reactions together, thus allowing the simultaneous activities of both the Rtase and Taq DNA polymerase. This form of RT-PCR is called one tube-one step RT-PCR which provides convenience, sensitivity and decreases significantly probable contamination. It is difficult, however, to optimise the conditions of both RT and PCR because all the reagents are mixed before starting the RT reaction. The RT-PCR systems in current use for the detection of most viroids include: a two-step RT-PCR using separate reactions for each viroid (Hadidi and Yang, 1990; Hadidi et al., 1992; Shamloul et al., 1995; Loreti et al., 1997); a polyvalent detection by a mixture of specific primer sets in multiplex standard (Levy et al., 1992; Ito et al., 2002; Ragozzino et al., 2003); fluorescent (Di Serio et al., 2002); standard and multiplex ELISA PCR (Shamloul et al., 2002; Shamloul and Hadidi, 1999); and the use of a common primer set (Faggioli and Ragozzino, 2002). In contrast to the above RT-PCR systems, one-step RT-PCR has been established only for a limited number of viroids (Rezaian et al., 1992; Shamloul et al., 1997; Mumford et al., 2000).
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E. Ragozzino et al. / Journal of Virological Methods 121 (2004) 25–29
Table 1 One tube-one step RT-PCR assays of 56 viroid-infected field samples from eight plant species Samples
Number of tested plants
Viroid
Origin
RT-PCRa
Quince Apple ‘virginia crab’ Pear ‘coscia’ Apple ‘stark red delicious’ Citrus ‘etrog’ Chrysanthemum Peach ‘may grand’ Peach calico isolate Apricot ‘vitillo’ Plum ‘florentia’ Peach GF 305 GF 305 healthy seedling Quince healthy seedling Chrysantemum healthy seedling Citrus healthy seedling
4 4 8 3 4 8 3 2 10 8 2 2 2 2 2
ASSVd ASSVd PBCVd ADFVd CEVd CSVd PLMVd PLMVd HSVd HSVd HSVd – – – –
ISPaVe (Italy) CTIFL (France) ISPaVe (Italy) University of Portici (Italy) University of Bari (Italy) ISPaVe (Italy) ISPaVe (Italy) University of Portici (Italy) University of Portici (Italy) University of Portici (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy)
4/4 4/4 8/8 3/3 4/4 8/8 3/3 2/2 10/10 8/8 2/2 0/2 0/2 0/2 0/2
ASSVd, apple scar skin viroid; CSVd, chrysanthemum stunt viroid; PBCVd, pear blister canker viroid; PLMVd, peach latent mosaic viroid; ADFVd, apple dimple fruit viroid; HSVd, hop stunt viroid; CEVd, citrus exocortis viroid. a Number of positive plants/number of analysed plants.
The development of a one tube-one step RT-PCR technique is described for the detection of seven viroids belong to the two known families of viroids (Pospiviroidae and Avsunviroidae, Flores et al., 2003) and to four genera, namely: Apscaviroid (Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Pear blister canker viroid (PBCVd)), Hostuviroid (Hop stunt viroid (HSVd)), Pospiviroid (Chrysanthemum stunt viroid (CSVd) and Citrus exocortis viroid (CEVd)) and Pelamoviroid (Peach latent mosaic viroid (PLMVd)). The method has been assessed for the detection of these seven viroids from at least eight plant species (apple, pear, quince, citrus, peach, apricot, plum, chrysanthemum), thus making it universal and useful for different species of viroids and/or host plants.
2. Materials and methods 2.1. Viroid isolates ASSVd isolate PK13 (supplied by J.C. Desvignes, Centre Technique Interprofessionel des Fruits et Legumes-CTIFL, Bergerac, France), PBCVd isolate P8 (Istituto Sperimentale per la Patologia Vegetale di Roma (ISPaVe) collection) and ADFVd isolate 1 (provided by A. Ragozzino, University of Portici, Naples, Italy) were maintained in pome fruit plants by graft-inoculation. Citrus isolates of CEVd and HSVd (supplied by V. Savino, University of Bari, Italy) were maintained on graft-inoculated citrus ‘Etrog’ plants. HSVd isolate Tajo (supplied by J.C. Desvignes, CTIFL) and PLMVd isolate MG (ISPaVe collection) were maintained on peach GF 305 chip-inoculated plants. A CSVd isolate (supplied by L. Tomassoli, ISPaVe collection) was maintained on naturally infected chrysanthemum plants. Uninfected peach GF 305, quince, chrysanthemum and citrus ‘Etrog’ plants were used as healthy controls. Moreover, a total of 56 field plants
(apple, pear, quince, citrus, peach, apricot, plum, chrysanthemum), infected by one of the seven viroids, were used to test the efficiency of one tube-one step RT-PCR method developed in this investigation to field material (Table 1). 2.2. Preparation of plant nucleic acids containing viroid RNA targets Two methods were evaluated to select a reliable procedure to prepare viroid RNA targets: (1) Total nucleic acids were extracted according to Faggioli et al. (2001) using 0.2 g of plant material. Briefly, leaves, bark or fruit skin from each sample were powdered in liquid nitrogen and homogenised with 900 l of 0.2 M Tris–HCl pH 8.2, 17.5 l of 5M NaCl, 8 l of 10% Triton X-100 and 2 l of 2-mercaptoethanol. After centrifugation at 9,000 × g for 20 min, the pellet was discarded and the supernatant was mixed with 500 l of water-saturated phenol pH 7.0, 100 l of 5% sodium dodecyl sulfate (SDS) and 100 l of 0.1 mM ethylene diamine tetra acetic acid (EDTA) pH 7.The nucleic acids present in the aqueous phase, obtained after further centrifugation at 9,000 × g for 20 min, were recovered by ethanol precipitation and then resuspended in 500 l of distilled sterile water. (2) Viroid RNA isolation was carried out using the RNeasy Plant Mini Kit, according to the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany). RNA was finally eluted with 50 l of RNase-free water. 2.3. One tube-one step RT-PCR Several parameters such as pre-denaturation of nucleic acids in the presence of complementary primers, use of Taq DNA polymerase or hot start Taq DNA polymerase, use of
E. Ragozzino et al. / Journal of Virological Methods 121 (2004) 25–29
reverse-transcriptases from Moloney-murine leukemia virus (M-MLV) or from Avian myeloblastis virus (AMV) were examined to establish the best conditions for the amplification of viroid RNA targets. The optimum one tube-one step protocol consisted of 1.5 l of total nucleic acids or total RNA containing targeted viroid RNA added to 58.5 l of RT-PCR mixture containing: 6 l buffer 10X of HotStarTaq® DNA polymerase (Qiagen); 3.6 l of 25 mM MgCl2 ; 4 l of 2.5 mM dNTPs; 0.4 l of reverse-transcriptase M-MLV (200U/l) (Invitrogen Corporation, Paisley, Scotland, UK); 0.4 l of HotStarTaq® DNA polymerase (5U/l) (Qiagen); 1 l of Rnase OUTTM inhibitor (40U/l) (Invitrogen), 1.5 l of forward primer (0.1 g/l); 1 l of complementary primer (1 g/l); 40.6 l of depc-sterile water. Reverse transcription was carried out at 42 ◦ C for 45 min followed by 15 min at 95 ◦ C to activate hot start Taq polymerase, and by PCR cycling parameter as follows: denaturation at 95 ◦ C for 45 s, annealing at 60 ◦ C for 45 sec, extension at 72 ◦ C for 1 min, for total of 35 cycles, followed by final extension for 7 min at 72 ◦ C. Amplified products were analysed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide. For each viroid specific primers that amplify the full-length of viroid, were used in particular for PLMVd the primers of Loreti et al. (1999); for PBCVd the primers of Loreti et al. (1997); for HSVd the primers of Astruc et al. (1996); for ADFVd and ASSVd, respectively the primers of Di Serio et al. (1996) and Hadidi and Yang (1990); for CEVd and CSVd the primers of Levy and Hadidi (1992).
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3. Results and discussion 3.1. Parameters that Affect the Efficiency of the Assay 3.1.1. Nucleic acid extraction method The two methods used in this investigation for nucleic acid extraction from viroid-infected and uninfected tissues were compared in several respects. Total nucleic acids extraction with phenol and total RNA extraction with RNeasy Plant Mini Kit were efficient in obtaining sufficient amounts of targeted viroid-RNA from all infected tissues, eliminated RT-PCR inhibitors and provided good sensitivity and reliability in the subsequent one tube-one step RT-PCR. Both methods allowed processing large number of samples. The sensitivity of each extraction method was determined for all viroids on samples that were prepared by serial dilutions of extracted nucleic acid and then a 1.5 l aliquot of each sample was tested by one tube-one-step RT-PCR as described. Fig. 1 shows the sensitivity of detection of CSVd and ASSVd was at least 100-fold more sensitive when viroids were RT-amplified from total RNA isolated from infected tissue to RNeasy Plant Mini Kit as compared to RT-amplification from total nucleic acids extracted by phenol. Similar results were obtained for other viroids. The total RNA extraction method is not laborious, fast, and safe as it avoids the use of organic solvents. These results confirmed and extended those reported previously (Faggioli et al., 2002; Shamloul et al., 2002) about the reliability of viroid extraction from infected tissue using non-organic solvents.
Fig. 1. Sensitivity of the one tube-one step RT-PCR technique in detecting Chrysanthemum stunt viroid (CSVd, left) and Apple scar skin viroid (ASSVd, right) in nucleic acid extracts of infected tissue obtained by the two different extraction methods described in the text. Total RNA obtained by a commercial kit (A) or total nucleic acids obtained by phenol extraction (B) were diluited 100 , 10−1 , 10−2 and 10−3 . DNA size markers are shown in lane M (Gene RulerTM 100 bpDNA Ladder, Fermentas, Vilnius, Lithuania).
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E. Ragozzino et al. / Journal of Virological Methods 121 (2004) 25–29
Fig. 2. Effect of Avian myeloblastosis virus (AMV, A) and Moloney-murine leukemia virus (M-MLV, B) reverse-transcriptases on the amplification of Hop stunt viroid (HSVd, 300 bp, lane 1), Apple dimple fruit viroid (ADFVd, 306 bp, lane 2), Pear blister canker viroid (PBCVd, 315 bp, lane 3), Apple scar skin viroid (ASSVd, 330 bp, lane 4), Peach latent mosaic viroid (PLMVd, 337 bp, lane 5), Chrysanthemum stunt viroid (CSVd, 354 bp, lane 6) and Citrus exocortis viroid (CEVd, 370 bp, lane 7). DNA size markers are shown in lane M (Gene RulerTM 100 bpDNA Ladder, Fermentas, Vilnius, Lithuania).
3.1.2. Denaturation With the exception of PLMVd, a denaturation step of viroid RNA and its complementary primer did not increase significantly the yield of viroid amplified cDNA as indicated by gel electrophoresis analysis of amplified viroid cDNAs. The difference observed between PLMVd (family Avsunviroidae, containing a hammerhead structure) and the other six viroids (family Pospiviroidae with a quasi rod-like structure) may be due to the fact that PLMVd has a different molecular structure. 3.1.3. Reverse-transcriptase Comparison of AMV reverse-transcriptase with M-MLV reverse-transcriptase in the RT-PCR reaction mixture indicated that AMV enzyme is less efficient than M-MLV reverse-transcriptase in the one tube-one step RT-PCR (Fig. 2). This may be due to the lack of endonuclease activity and the much lower Rnase H activity of M-MLV reverse-transcriptase which make this enzyme more suitable than the other enzyme in our RT-PCR reaction conditions. These conditions may include viroid concentration, temperature of RT step, and salt concentration of the buffer.
3.2. Taq DNA polymerase The use of hot start Taq DNA polymerase was more specific and efficient than standard Taq DNA polymerase in our protocol. This may be due to inactivation of hot start Taq DNA polymerase during the RT process. This polymerase is activated only when the critical temperature is reached (step at 95 ◦ C for 15 min after the RT), thus decreasing the probability of amplifying non-specific products and also increasing the possibility of amplifying very low copy number of viroid targets. 3.3. Assay of viroid-infected field samples When 56 viroid-infected field samples were tested by one tube-one step RT-PCR, products of the expected sizes were obtained from all viroid isolates assayed (Table 1). Seven viroid bands of different sizes (330 bp for ASSVd; 315 bp for PBCVd; 306 bp for ADFVd; 300 bp for HSVd; 354 bp for CSVd; 370 bp for CEVd and 336-342 bp for PLMVd) were observed only in infected samples but not in negative controls. These findings showed the successful use of the protocol with field samples.
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In conclusion, this study demonstrates the feasibility of one tube-one step RT-PCR for the identification of several viroids from different plant species using a unique and universal protocol. The efficiency and specificity of this method were optimized with the use of a commercial kit for the targeted-viroid RNA extraction, of a M-MLV reverse-transcriptase and a hot start Taq DNA polymerase that allowed increase the sensitivity of detection. This method also has the advantage of requiring less hands-on time to set up an assay and also to reduce the possibility of false positive, since all steps are performed in the same tube avoiding cross-contamination of the specific amplified products. The RT-PCR one tube-one-step technique resulted comparable to other RT-PCR methods in use concerning the reability and sensitivity but more rapid, universal and easily-handled (Hadidi and Candresse, 2003). This method has the potential to be used routinely in sanitary and certification programmes.
References Astruc, N., Marcos, J.F., Macquaire, G., Candresse, T., Pallàs, V., 1996. Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur. J. Plant Pathol. 102, 837– 846. Di Serio, F., Aparicio, F., Alioto, D., Ragozzino, A., Flores, R., 1996. Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease. J. Gen. Virol. 77, 2833– 2837. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., Flores, R., 2002. Apple dimple fruit viroid: sequence variability and its specific detection by multiplex fluorescent RT-PCR in the presence of Apple scar skin viroid. J. Plant Pathol. 84, 27–34. Faggioli, F., Ragozzino, E., Barba, M., 2001. Simultaneous detection of stone or pome fruit viroid by single tube-RT-PCR. Acta Hort. 550, 59–63. Faggioli, F., Ragozzino, E., 2002. Detection of pome fruit viroids by RT-PCR using a single primer pair. J. Plant Pathol. 84 (2), 125–128. Faggioli, F., Ferretti, L., Pasquini, G., Barba, M., 2002. Detection of strawberry latent ring spot virus in leaves of olive trees in Italy using a one-step RT-PCR. J. Phytopathol. 150, 636–639. Flores, R., Randles, J.W., Owens, R.A., 2003. Classification. In: Hadidi, A., Flores, R., Randles, J.W., Semancik, J. (Eds), Viroids. CSIRO, Collingwood, Australia, pp. 71–75. Hadidi, A., Candresse, T., 2003. Polymerase chain reaction. In: Hadidi, A., Flores, R., Randles, J.W., Semancik, J. (Eds), Viroids. CSIRO, Collingwood, Australia, pp. 115–122.
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Hadidi, A., Yang, X., 1990. Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261–270. Hadidi, A., Terai, Y., Powell, C.A., Scott, S.W., Desvignes, J.C., Ibrahim, L.M., Levy, L., 1992. Enzymatic cDNA amplification of hop stunt viroid variants from naturally infected fruit crops. Acta Hort. 309, 339–344. Ito, T., Ieki, H., Ozaki, K., 2002. Simultaneous detection of six citrus viroids and apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. J. Virol. Methods 106, 235–239. Levy, L., Hadidi, A., Garsney, S.M., 1992. Reverse transcriptionpolymerase chain reaction assays for the rapid detection of citrus viroids using multiplex primer sets. Proc. Int. Soc. Citricult. 106, 800–803. Levy, R., Hadidi, A., 1992. Direct nucleotide sequencing of PCR-amplified DNAs of the closey related citrus viroids IIa and IIb (cachexia). In: Moreno, P., da Graca, J.V., Timmer, L.W. (Eds.), Proceedings of the 13th International Conference on Organic Citrus Virology (IOCV). Riverside, pp. 180–186. Loreti, S., Faggioli, F., Barba, M., 1997. Identification and characterization of an Italian isolate of pear blister canker viroid. J. Phytopathol. 145, 541–544. Loreti, S., Faggioli, F., Cardoni, M., Mordenti, G., Babini, A.R., Poggi Pollini, C., Barba, M., 1999. Comparison of different diagnostic methods for detection of peach latent mosaic viroid. Bull. OEPP/EPPO Bull. 29, 433–438. Mumford, R.A., Walsh, K., Boonham, N., 2000. A comparison of molecular methods for the routine detection of viroids. Bull. OEPP/EPPO Bull. 30, 431–435. Ragozzino, E., Faggioli, F., Alito, D., Barba, M., 2003. Detection and differentiation of peach latent mosaic viroid and hop stunt viroid in stone fruit trees in Italy using multiplex RT-PCR. Phytopathol. Medit. 42, 79–84. Rezaian, M.A., Krake, L.R., Golino, D.A., 1992. Common identity of grapevine viroids from U.S.A. and Australia revealed by PCR analysis. Intervirol. 34, 38–43. Shamloul, A.M., Faggioli, F., Keith, J.M., Hadidi, A., 2002. A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid. J. Virol. Methods 105, 115–121. Shamloul, A.M., Hadidi, A., 1999. Sensitive detection of potato spinder tuber and temperature fruit tree viroids by reverse transcriptionpolymerase chain reaction-probe capture hybridization. J. Virol. Methods 80, 145–155. Shamloul, A.M., Hadidi, A., Zhu, S.F., Singh, R.P., Sagredo, B., 1997. Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants. Can. J. Plant Pathol. 19, 89–96. Shamloul, A.M., Minafra, A., Hadidi, A., Giunchedi, L., Waterworth, H.E., Allam, E.K., 1995. Peach latent mosaic viroid: nucleotide sequence of an Italian isolate, sensitive detection using RT-PCR and geographic distribution. Acta Hort. 386, 522–530.
Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid E. Ragozzino, F. Faggioli∗ , M. Barba Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22–00156 Rome, Italy Received 18 February 2004; received in revised form 14 May 2004; accepted 17 May 2004 Available online 7 July 2004
Abstract A one tube-one step RT-PCR was developed for the detection of seven viroids (Apple scar skin viroid, Apple dimple fruit viroid, Pear blister canker viroid, Hop stunt viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid) in four genera that infect eight plant species. The efficiency and specificity of this method were optimized by the use of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase and HotStarTaq® DNA polymerase which allowed increase sensitivity of viroid detection. The method was assessed with 56 viroid-infected field plants. The multiplex one tube-one step RT-PCR has the advantage of requiring less hands-on time to set up an assay than standard multiplex one, it also reduces the possibility of false positive tests because all steps are performed in the same tube thus, avoiding cross-contamination. The method may be used routinely for viroid detection in sanitary and certification programmes. © 2004 Elsevier B.V. All rights reserved. Keywords: Viroids diagnosis; One tube-one step RT-PCR; RNA extraction method; Multiplex RT-PCR
1. Introduction The reverse transcription-polymerase chain reaction (RT-PCR) technique is a rapid and reliable method for detecting viroids from infected plants (Hadidi and Candresse, 2003). Currently, the RT-PCR is the most sensitive technique for determining the presence or absence of specific RNA templates in mixtures. Avoiding contamination and achieving viroid full-length transcripts are among the goals of this technique. The approach used most frequently for a RT-PCR assay is to run the RT reaction under optimum conditions for reverse-transcriptase (Rtase) activity and then to add a fraction of the RT reaction to a standard PCR reaction (RT-PCR two tubes-two steps). Since additional steps are undesirable, as they increase the risk of contamination, an alternative way is to add the PCR reagents to the RT reaction itself. In this case, the technique is referred to as one tube-two steps
∗ Corresponding author. Tel.: +39 06 820 70207; fax: +39 06 868 02296. E-mail address: [email protected] (F. Faggioli).
0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.05.012
RT-PCR, the reaction conditions are sequentially adapted to the enzymes used and the risk of contamination decreases. Another possibility is to use all the components of the RT and PCR reactions together, thus allowing the simultaneous activities of both the Rtase and Taq DNA polymerase. This form of RT-PCR is called one tube-one step RT-PCR which provides convenience, sensitivity and decreases significantly probable contamination. It is difficult, however, to optimise the conditions of both RT and PCR because all the reagents are mixed before starting the RT reaction. The RT-PCR systems in current use for the detection of most viroids include: a two-step RT-PCR using separate reactions for each viroid (Hadidi and Yang, 1990; Hadidi et al., 1992; Shamloul et al., 1995; Loreti et al., 1997); a polyvalent detection by a mixture of specific primer sets in multiplex standard (Levy et al., 1992; Ito et al., 2002; Ragozzino et al., 2003); fluorescent (Di Serio et al., 2002); standard and multiplex ELISA PCR (Shamloul et al., 2002; Shamloul and Hadidi, 1999); and the use of a common primer set (Faggioli and Ragozzino, 2002). In contrast to the above RT-PCR systems, one-step RT-PCR has been established only for a limited number of viroids (Rezaian et al., 1992; Shamloul et al., 1997; Mumford et al., 2000).
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E. Ragozzino et al. / Journal of Virological Methods 121 (2004) 25–29
Table 1 One tube-one step RT-PCR assays of 56 viroid-infected field samples from eight plant species Samples
Number of tested plants
Viroid
Origin
RT-PCRa
Quince Apple ‘virginia crab’ Pear ‘coscia’ Apple ‘stark red delicious’ Citrus ‘etrog’ Chrysanthemum Peach ‘may grand’ Peach calico isolate Apricot ‘vitillo’ Plum ‘florentia’ Peach GF 305 GF 305 healthy seedling Quince healthy seedling Chrysantemum healthy seedling Citrus healthy seedling
4 4 8 3 4 8 3 2 10 8 2 2 2 2 2
ASSVd ASSVd PBCVd ADFVd CEVd CSVd PLMVd PLMVd HSVd HSVd HSVd – – – –
ISPaVe (Italy) CTIFL (France) ISPaVe (Italy) University of Portici (Italy) University of Bari (Italy) ISPaVe (Italy) ISPaVe (Italy) University of Portici (Italy) University of Portici (Italy) University of Portici (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy) ISPaVe (Italy)
4/4 4/4 8/8 3/3 4/4 8/8 3/3 2/2 10/10 8/8 2/2 0/2 0/2 0/2 0/2
ASSVd, apple scar skin viroid; CSVd, chrysanthemum stunt viroid; PBCVd, pear blister canker viroid; PLMVd, peach latent mosaic viroid; ADFVd, apple dimple fruit viroid; HSVd, hop stunt viroid; CEVd, citrus exocortis viroid. a Number of positive plants/number of analysed plants.
The development of a one tube-one step RT-PCR technique is described for the detection of seven viroids belong to the two known families of viroids (Pospiviroidae and Avsunviroidae, Flores et al., 2003) and to four genera, namely: Apscaviroid (Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Pear blister canker viroid (PBCVd)), Hostuviroid (Hop stunt viroid (HSVd)), Pospiviroid (Chrysanthemum stunt viroid (CSVd) and Citrus exocortis viroid (CEVd)) and Pelamoviroid (Peach latent mosaic viroid (PLMVd)). The method has been assessed for the detection of these seven viroids from at least eight plant species (apple, pear, quince, citrus, peach, apricot, plum, chrysanthemum), thus making it universal and useful for different species of viroids and/or host plants.
2. Materials and methods 2.1. Viroid isolates ASSVd isolate PK13 (supplied by J.C. Desvignes, Centre Technique Interprofessionel des Fruits et Legumes-CTIFL, Bergerac, France), PBCVd isolate P8 (Istituto Sperimentale per la Patologia Vegetale di Roma (ISPaVe) collection) and ADFVd isolate 1 (provided by A. Ragozzino, University of Portici, Naples, Italy) were maintained in pome fruit plants by graft-inoculation. Citrus isolates of CEVd and HSVd (supplied by V. Savino, University of Bari, Italy) were maintained on graft-inoculated citrus ‘Etrog’ plants. HSVd isolate Tajo (supplied by J.C. Desvignes, CTIFL) and PLMVd isolate MG (ISPaVe collection) were maintained on peach GF 305 chip-inoculated plants. A CSVd isolate (supplied by L. Tomassoli, ISPaVe collection) was maintained on naturally infected chrysanthemum plants. Uninfected peach GF 305, quince, chrysanthemum and citrus ‘Etrog’ plants were used as healthy controls. Moreover, a total of 56 field plants
(apple, pear, quince, citrus, peach, apricot, plum, chrysanthemum), infected by one of the seven viroids, were used to test the efficiency of one tube-one step RT-PCR method developed in this investigation to field material (Table 1). 2.2. Preparation of plant nucleic acids containing viroid RNA targets Two methods were evaluated to select a reliable procedure to prepare viroid RNA targets: (1) Total nucleic acids were extracted according to Faggioli et al. (2001) using 0.2 g of plant material. Briefly, leaves, bark or fruit skin from each sample were powdered in liquid nitrogen and homogenised with 900 l of 0.2 M Tris–HCl pH 8.2, 17.5 l of 5M NaCl, 8 l of 10% Triton X-100 and 2 l of 2-mercaptoethanol. After centrifugation at 9,000 × g for 20 min, the pellet was discarded and the supernatant was mixed with 500 l of water-saturated phenol pH 7.0, 100 l of 5% sodium dodecyl sulfate (SDS) and 100 l of 0.1 mM ethylene diamine tetra acetic acid (EDTA) pH 7.The nucleic acids present in the aqueous phase, obtained after further centrifugation at 9,000 × g for 20 min, were recovered by ethanol precipitation and then resuspended in 500 l of distilled sterile water. (2) Viroid RNA isolation was carried out using the RNeasy Plant Mini Kit, according to the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany). RNA was finally eluted with 50 l of RNase-free water. 2.3. One tube-one step RT-PCR Several parameters such as pre-denaturation of nucleic acids in the presence of complementary primers, use of Taq DNA polymerase or hot start Taq DNA polymerase, use of
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reverse-transcriptases from Moloney-murine leukemia virus (M-MLV) or from Avian myeloblastis virus (AMV) were examined to establish the best conditions for the amplification of viroid RNA targets. The optimum one tube-one step protocol consisted of 1.5 l of total nucleic acids or total RNA containing targeted viroid RNA added to 58.5 l of RT-PCR mixture containing: 6 l buffer 10X of HotStarTaq® DNA polymerase (Qiagen); 3.6 l of 25 mM MgCl2 ; 4 l of 2.5 mM dNTPs; 0.4 l of reverse-transcriptase M-MLV (200U/l) (Invitrogen Corporation, Paisley, Scotland, UK); 0.4 l of HotStarTaq® DNA polymerase (5U/l) (Qiagen); 1 l of Rnase OUTTM inhibitor (40U/l) (Invitrogen), 1.5 l of forward primer (0.1 g/l); 1 l of complementary primer (1 g/l); 40.6 l of depc-sterile water. Reverse transcription was carried out at 42 ◦ C for 45 min followed by 15 min at 95 ◦ C to activate hot start Taq polymerase, and by PCR cycling parameter as follows: denaturation at 95 ◦ C for 45 s, annealing at 60 ◦ C for 45 sec, extension at 72 ◦ C for 1 min, for total of 35 cycles, followed by final extension for 7 min at 72 ◦ C. Amplified products were analysed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide. For each viroid specific primers that amplify the full-length of viroid, were used in particular for PLMVd the primers of Loreti et al. (1999); for PBCVd the primers of Loreti et al. (1997); for HSVd the primers of Astruc et al. (1996); for ADFVd and ASSVd, respectively the primers of Di Serio et al. (1996) and Hadidi and Yang (1990); for CEVd and CSVd the primers of Levy and Hadidi (1992).
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3. Results and discussion 3.1. Parameters that Affect the Efficiency of the Assay 3.1.1. Nucleic acid extraction method The two methods used in this investigation for nucleic acid extraction from viroid-infected and uninfected tissues were compared in several respects. Total nucleic acids extraction with phenol and total RNA extraction with RNeasy Plant Mini Kit were efficient in obtaining sufficient amounts of targeted viroid-RNA from all infected tissues, eliminated RT-PCR inhibitors and provided good sensitivity and reliability in the subsequent one tube-one step RT-PCR. Both methods allowed processing large number of samples. The sensitivity of each extraction method was determined for all viroids on samples that were prepared by serial dilutions of extracted nucleic acid and then a 1.5 l aliquot of each sample was tested by one tube-one-step RT-PCR as described. Fig. 1 shows the sensitivity of detection of CSVd and ASSVd was at least 100-fold more sensitive when viroids were RT-amplified from total RNA isolated from infected tissue to RNeasy Plant Mini Kit as compared to RT-amplification from total nucleic acids extracted by phenol. Similar results were obtained for other viroids. The total RNA extraction method is not laborious, fast, and safe as it avoids the use of organic solvents. These results confirmed and extended those reported previously (Faggioli et al., 2002; Shamloul et al., 2002) about the reliability of viroid extraction from infected tissue using non-organic solvents.
Fig. 1. Sensitivity of the one tube-one step RT-PCR technique in detecting Chrysanthemum stunt viroid (CSVd, left) and Apple scar skin viroid (ASSVd, right) in nucleic acid extracts of infected tissue obtained by the two different extraction methods described in the text. Total RNA obtained by a commercial kit (A) or total nucleic acids obtained by phenol extraction (B) were diluited 100 , 10−1 , 10−2 and 10−3 . DNA size markers are shown in lane M (Gene RulerTM 100 bpDNA Ladder, Fermentas, Vilnius, Lithuania).
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Fig. 2. Effect of Avian myeloblastosis virus (AMV, A) and Moloney-murine leukemia virus (M-MLV, B) reverse-transcriptases on the amplification of Hop stunt viroid (HSVd, 300 bp, lane 1), Apple dimple fruit viroid (ADFVd, 306 bp, lane 2), Pear blister canker viroid (PBCVd, 315 bp, lane 3), Apple scar skin viroid (ASSVd, 330 bp, lane 4), Peach latent mosaic viroid (PLMVd, 337 bp, lane 5), Chrysanthemum stunt viroid (CSVd, 354 bp, lane 6) and Citrus exocortis viroid (CEVd, 370 bp, lane 7). DNA size markers are shown in lane M (Gene RulerTM 100 bpDNA Ladder, Fermentas, Vilnius, Lithuania).
3.1.2. Denaturation With the exception of PLMVd, a denaturation step of viroid RNA and its complementary primer did not increase significantly the yield of viroid amplified cDNA as indicated by gel electrophoresis analysis of amplified viroid cDNAs. The difference observed between PLMVd (family Avsunviroidae, containing a hammerhead structure) and the other six viroids (family Pospiviroidae with a quasi rod-like structure) may be due to the fact that PLMVd has a different molecular structure. 3.1.3. Reverse-transcriptase Comparison of AMV reverse-transcriptase with M-MLV reverse-transcriptase in the RT-PCR reaction mixture indicated that AMV enzyme is less efficient than M-MLV reverse-transcriptase in the one tube-one step RT-PCR (Fig. 2). This may be due to the lack of endonuclease activity and the much lower Rnase H activity of M-MLV reverse-transcriptase which make this enzyme more suitable than the other enzyme in our RT-PCR reaction conditions. These conditions may include viroid concentration, temperature of RT step, and salt concentration of the buffer.
3.2. Taq DNA polymerase The use of hot start Taq DNA polymerase was more specific and efficient than standard Taq DNA polymerase in our protocol. This may be due to inactivation of hot start Taq DNA polymerase during the RT process. This polymerase is activated only when the critical temperature is reached (step at 95 ◦ C for 15 min after the RT), thus decreasing the probability of amplifying non-specific products and also increasing the possibility of amplifying very low copy number of viroid targets. 3.3. Assay of viroid-infected field samples When 56 viroid-infected field samples were tested by one tube-one step RT-PCR, products of the expected sizes were obtained from all viroid isolates assayed (Table 1). Seven viroid bands of different sizes (330 bp for ASSVd; 315 bp for PBCVd; 306 bp for ADFVd; 300 bp for HSVd; 354 bp for CSVd; 370 bp for CEVd and 336-342 bp for PLMVd) were observed only in infected samples but not in negative controls. These findings showed the successful use of the protocol with field samples.
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In conclusion, this study demonstrates the feasibility of one tube-one step RT-PCR for the identification of several viroids from different plant species using a unique and universal protocol. The efficiency and specificity of this method were optimized with the use of a commercial kit for the targeted-viroid RNA extraction, of a M-MLV reverse-transcriptase and a hot start Taq DNA polymerase that allowed increase the sensitivity of detection. This method also has the advantage of requiring less hands-on time to set up an assay and also to reduce the possibility of false positive, since all steps are performed in the same tube avoiding cross-contamination of the specific amplified products. The RT-PCR one tube-one-step technique resulted comparable to other RT-PCR methods in use concerning the reability and sensitivity but more rapid, universal and easily-handled (Hadidi and Candresse, 2003). This method has the potential to be used routinely in sanitary and certification programmes.
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