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Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants
Journal of Virological Methods 174 (2011) 123–127
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants Dongmei Jiang a,b , Zujian Wu a , Lianhui Xie a , Teruo Sano c , Shifang Li b,∗ a
Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, West 2 Yuangmingyuan Road, Beijing 100193, China c Department of Plant Pathology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan b
a b s t r a c t Article history: Received 19 January 2011 Received in revised form 3 March 2011 Accepted 15 March 2011 Available online 2 April 2011 Keywords: Sap RT-PCR Coleus CbVd Pipettor
A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Viroids are small (246–475 nucleotides), covalently closed single-stranded RNAs. Like viruses, viroids replicate in their host plants and may act as phytopathogenic agents; however, they do not encode any proteins (Fauquet et al., 2005). Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide, and both its stem and leaf are known to be succulent. Coleus is susceptible to infection by several coleus blumei viroid (CbVd) species of the genus Coleviroid, family Pospiviroidae. Up to now, 6 main viroids that infect coleus have been reported: CbVd 1–6 (Fonseca et al., 1989; Spieker, 1996a,b; Spieker et al., 1996; Hou et al., 2009a,b) (Fig. 1a). CbVd-1 was first reported in a commercial yellow coleus in Brazil in 1996 (Fonseca et al., 1989), and then was reported successively in many other countries including Germany, Japan, Canada, Korea and China (Spieker et al., 1990; Ishiguro et al., 1996; Singh and Boucher, 1991; Chung and Choi, 2008; Li et al., 2006). To date, CbVd-2, CbVd-3, and CbVd-4 have only been reported in Germany (Spieker, 1996a,b; Spieker et al., 1996). CbVd-5 and CbVd-6 have only been reported in China until now (Hou et al., 2009a,b). All of the known coleus viroids share a common central conserved region (CCR), which is known to be an important structural element for Pospiviroid replication, i.e., processing and ligation (Diener, 1986; Gas et al., 2007). Recombination is common in CbVds, for example, CbVd-
∗ Corresponding author. Tel.: +86 10 62890875; fax: +86 10 62890875. E-mail addresses: sfl[email protected], [email protected] (S. Li).
2 and CbVd-6 are natural viroid chimeras and CbVd-4 is an in vitro-generated inverse chimera (Spieker, 1996b; Hou et al., 2009b) (Fig. 1a). Until now, CbVd-1 has been detected in many countries while the distribution of the other 5 coleus viroids seems to be limited. Therefore, it is necessary to establish a simple and fast method to further investigate the distribution of CbVds. Many methods for viroid detection have been reported including hybridization using DIG-labeled probes, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and Return-PAGE (Welnicki and Hiruki, 1992; Schumacher et al., 1983, 1986); however, they are laborious and time consuming. Besides, relatively pure coleus nucleic acids are vital for these methods. RT-PCR has proved to be a rapid and sensitive technique for the detection of both RNA viruses (Henson and French, 1993) and viroids (Hadidi and Yang, 1990; Yang et al., 1992; Levy et al., 1994). But a limitation of this procedure is the time consuming preparation of RNAs from plant samples. There are many methods for preparing viroid nucleic acids, such as the LiCl method (Li et al., 1995); however, large amounts of sample (at least 1–2 leaves) are required for template preparation in these conventional methods, and these methods require laborious steps to eliminate polyphenol and polysaccharide (Hadidi and Candresse, 2003). In addition, these procedures are expensive. In plant science, some improvements have been made in the methods for the extraction and purification of viroid RNA templates without tissue grinding; however, special tools are required (Hosokawa et al., 2005).
0166-0934/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.03.018
124 D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127 Fig. 1. (a) Schematic for the proposed secondary structures of coleus blumei viroid 1–6, CbVd-2 and CbVd-6 are natural viroid chimeras and CbVd-4 is an in vitro-generated inverse chimera. CbVd-2 includes the left half of CbVd-3 and the right half of CbVd-1, and CbVd-4 is opposite. CbVd-6 includes the left half of CbVd-3 and the right half of CbVd-5. (b) Primers designed for RT-PCR. Upper nucleotides in the box were designed for primers used in RT-PCR.
D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127
125
Fig. 2. Results of sap-direct RT-PCR on coleus plants. M: marker; PC: positive control (the sample infected by CbVd-1, CbVd-5, and CbVd-6, and the template RNA of RT-PCR was extracted by LiCl method); NC: negative control (healthy sample). (a) Detection results of viroids from sap at different dilutions (1*: undiluted template; 10*: template diluted 10-fold; 100*: template diluted 100-fold; 1000*: template diluted 1000-fold). (b) Detection results of CbVds from different tissues—stem, old leaf, and new leaf. (c) Results of sap-direct RT-PCR method and dot-blot hybridization in different samples (1: sample infected by CbVd-1, CbVd-5, and CbVd-6; 2: sample infected by CbVd-1; 3: sample infected by CbVd-5 and CbVd-6).
In present study, a simple and rapid sap-direct RT-PCR method was established to detect 3 viroids of the genus Coleviroid (CbVd-1, CbVd-5, and CbVd-6) simultaneously. A pipette with a tip, common in a molecular biology laboratory, was used to prepare a very small amount of sap containing viroid RNA as templates for RT-PCR. A pair of universal primers, which can be used to detect 3 coleus viroids simultaneously, was designed based on the CCR sequences of CbVds. As a result, the presence of CbVd1, CbVd-5, and CbVd-6 can be determined in a short time using only a simple RT-PCR without special tools or cumbersome procedures. A pipettor (0.5–10 L) with short tips (0.5–10 L) was used to obtain sap directly from stems and leaves of coleus plants for template preparation, and the sap was then placed in a 1.5-mL Eppendorf (EP) tube directly. Notably, while sampling from a leaf, it should be kept between plastic papers and pressed; thus, the sap is soaked up easily. As soon as the sap was placed in the EP tubes, it was capped until the sap was put into the RT mix; so that the cross-contamination among different samples was reduced to a minimum. After it was diluted to an appropriate concentration, 1 L of the template was used for RT-PCR. A pair of universal primers (CbVds-P1 and CbVds-P2) was designed based on the CCR sequences of the genus Coleviroid (Fig. 1b). To generate cDNA from viroid RNA, 1 L of sap sample was mixed with 1 L (20 pmol) of CbVds-P2 primer (5 -GCAGCGCTGCCAGGGAACCCAGGT-3 ), 1 L of dNTPs (2.5 mM each, Takara, Dalian, China), 1 L of M-MLV (Promega, Madison, USA), 2 L of 5× M-MLV Reaction Buffer, and 0.25 L of recombinant RNasin ribonuclease inhibitor (40 U, Takara, Dalian, China). Distilled water was then added to a final volume of 10 L. The resulting mixture was incubated at 42 ◦ C for 60 min and 98 ◦ C for 5 min. After the RT reaction, 1 L of the RT solution was mixed with 0.2 L (20 pmol) of each primer (CbVds-P1 (5 -GCAGCGCTGCAACGGAAT-3 ) and CbVds-P2), 2 L of KOD-PlusNeo polymerase reaction buffer (Toyobo, Shanghai, China), 2 L dNTPs (2 mM each, Toyobo, Shanghai, China), 1 L MgSO4 (2.5 mM, Toyobo, Shanghai, China), 0.4 L of KOD-Plus-Neo polymerase (Toyobo, Shanghai, China), and 13.2 L distilled water. The cycling parameters for PCR amplification consisted of one cycle of heat denaturation at 94 ◦ C for 5 min, followed by 30 amplification cycles at 94 ◦ C for 30 s, 63 ◦ C for 30 s, and 72 ◦ C for 30 s. The final elongation step was at 72 ◦ C for 5 min. After RT-PCR, 10 L of the RT-PCR products were separated by electrophoresis on a 1.5% agarose gel and
visualized under UV light after staining with DNAgreen (Tiandz, Beijing, China). The products were purified using a PCR purification kit (Tiangen, Beijing, China). The purified fragments were then cloned into a pMD18-T vector (Takara, Dalian, China) and then transformed into Escherichia coli DH5␣. Clones containing inserts were sequenced using an automated DNA sequencer (ABI PRISM 3730XL DNA Analyzer) and analyzed by DNAMAN Version 5.2.2. To compare the detection sensitivity and accuracy of the sap-direct RT-PCR method with conventional methods, low molecular-weight RNAs of 30 coleus samples collected from outside in different seasons were extracted according to the procedure described by Li et al. (1995). After denatured using formaldehyde, RNA extracts were applied to nylon membranes (Hybond-N+, Amersham Biosciences), and hybridization using specific DIG-labeled cRNA probes for CbVd1, CbVd-5, or CbVd-6 was performed according to instructions of DIG RNA Labeling Kit and Detection Starter Kit (Roche, Basel, Switzerland). The sap collected from the stem of coleus plants infected by CbVd-1, CbVd-5, and CbVd-6 was used as a template for RT-PCR after it was diluted 10, 100, or 1000-fold. Bands that corresponded to CbVd-1, CbVd-5, and CbVd-6 were obtained simultaneously (Fig. 2a) The results of repeated experiments demonstrated that a 100-fold dilution is optimal. All the templates from the stem and leaf of the coleus plants infected by CbVds, irrespective of whether they were old or new, were suitable for the detection of CbVds as long as the sap was under proper consistency. RT-PCR results revealed that CbVd-1, CbVd-5, and CbVd-6 bands amplified from the stem were the clearest, followed by old leaf and new leaf. That is, sap from stem is the optimal template due to its higher detection sensitivity (Fig. 2b). CbVds were not detected from viroid-free plants by the sap-direct RT-PCR method, and CbVd-1 was only detected in coleus plants infected by CbVd-1. CbVd-5 and CbVd-6 were present together in the infected samples used in this study, and the amplified fragments could be distinguished clearly on the gel (Fig. 2c). There are many RT-PCR inhibitors in the sap of plants, such as phenols and polysaccharides, and they could contribute to false-negative results; therefore, the templates should be diluted. In this way, the fragments would be amplified clearly when the concentration of RNA and inhibitor reach a balance. In this study, although the samples were collected in different seasons and viroids concentrations in these samples were not the same; however, a 100-fold dilution was found to be superior, and can be
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D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127
Table 1 Detection results of CbVd-1, CbVd-5, and CbVd-6 using Dot-blot hybridization and Sap-direct RT-PCR. The number shown in the table is the number of samples (out of 30) that tested positive for the viroids. Numbers with * indicate the different detection results for CbVd-6 using two different methods. Assay
Dot-blot
Sap-direct RT-PCR
CbVd-1 CbVd-5 CbVd-6
29 16 15*
29 16 16*
applied to almost all coleus samples. Only few samples were needed to be diluted 10-fold or 1000-fold to obtain a clearer picture. The results of hybridization demonstrated that 29 of the 30 samples were positive for CbVd-1, 16 were positive for CbVd-5, and 15 were positive for CbVd-6. The result of sap-direct RT-PCR showed that 29 of the 30 samples were positive for CbVd-1, and CbVd-5 and CbVd-6 were positive in 16 samples (Table 1). Therefore, the results of the sap-direct RT-PCR corresponded to results of hybridization except for one sample, which was positive for CbVd-6 in sap-direct RT-PCR but negative in hybridization. That is, sap-direct RT-PCR method was more sensitive than dot-blot hybridization. Maybe the concentration of CbVd-6 in this sample was inadequate for hybridization, but sufficient for detection by RT-PCR. After cloning, 3 clones containing inserts of each viroid were sequenced. The results of sequence analysis revealed that the 3 bands from bottom to top on the gel (Fig. 2a) represented CbVd-1, CbVd-5, and CbVd-6, respectively. The sap-direct RT-PCR method is convenient because templates can be obtained from the stem or leaf without tissue grinding step. Viroids possess a thermodynamically stable secondary structure (Steger and Riesner, 2003), and viroid molecules are not destroyed by the high amount of RNase in plant materials. Thus, RNA in an EP tube can be maintained for a long period by capping. In our study, the sap without dilution in EP tubes could be available at −20 ◦ C for a long time, even more than 1 year; while they could not be stored at room temperature for more than 2 h, or 4 ◦ C (or 0 ◦ C) for more than 6 h. Notably, the sap from coleus plants at different dilution can be used only once. Therefore, to be on the safe side, it is better to draw sap from coleus plants just before RT-PCR. If the collection location is far from the laboratory, it is preferable to collect the stalk or leaf from the plant, and obtain the sap before they were dry. Many DNA polymerases can be used in this PCR method, and KODPlus-Neo is better due to its high amplification efficiency. Many ornamental plants are succulent and the sap can be drawn easily by the pipettor. The pipettor is a common tool in the molecular biology laboratory, and is considered to be more convenient than syringe needles, razors, or other tools that could be used to collect sap from plants (Hosokawa et al., 2005). In addition, pipettor tips can be taken out of the tubes more easily than other tools. The results of batch-detection revealed that the sap-direct RTPCR method can distinguish the three viroids from different coleus different viroids accurately (Fig. 2c), no matter the concentration of viroids were high or low in the plants. Compared to other methods, this method is not only rapid and accurate based on a simple RT-PCR, but also requires a lesser amount template (without purification). The method is especially feasible for mass detection. One microliter of sap is enough for the RT-PCR template, and the RT-PCR products are sufficient to be cloned conveniently. CbVd-5 and CbVd-6 have been reported in China for nearly 2 years; however, there has been no report about the two viroids in other countries except Japan. This rapid method could be very useful to clarify the geographic distribution of these two viroids quickly. Although the pathogenicity of CbVds is not clear and in most cases, CbVds induce no apparent symptoms in the coleus, the quarantine and control of CbVds cannot be ignored for the reason that
they may cause damage to other plants, just as hop stunt viroid can cause damage to hop even though it infects grapevines latently (Sano and Shikata, 1985; Sasaki and Shikata, 1977). Rapid and simple methods could play a considerable role in the quarantine and control of CbVds, and this study can also provide a reference for the detection of other viroids, even viruses. Acknowledgements This work was supported by grants from the Special Fund for Agro-scientific Research in the Public Interest (nyhyzx-200903004 and 200903034), National Basic Research and Development Program of China (973 Program) (No. 2009CB119200), the National Natural Science Foundation of China (Nos. 30771403, 30970134 and 31071765). This work was also supported by NSFC-JSPS Joint Research Project between China and Japan (30811140157), Key Project of the Fujian Provincial Department of Science and Technology (2007L2002). References Chung, B.M., Choi, G.S., 2008. Incidence of coleus blumei viroid 1 in seeds of commercial coleus in Korea. Plant Pathol. J. 24 (3), 305–308. Diener, T.O., 1986. Viroid processing: a model involving the central conserved region and hairpin I. In: Proc. Natl. Acad. Sci. U.S.A. , 83, pp. 58–62. Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., 2005. Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, Amsterdam, Netherlands, pp. 1145–1160. Fonseca, M.E.N., Boiteux, L.S., Singh, R.P., Kitajima, E.W., 1989. A small viroid in coleus species from Brazil. Fitopatol. Bras. 14, 94–96. Gas, M.E., Hernandez, C., Flores, R., Daròs, J.A., 2007. Processing of nuclear viroids in vivo: an interplay between RNA conformations. PLoS Pathog. 3 (11), 1813–1826, e182. Hadidi, A., Candresse, T., 2003. Polymerase chain reaction. In: Hadidi, A., Flores, R., Randles, J.W., Semanick, J.S. (Eds.), Viroids. CSIRO, Collingwood, Australia, pp. 115–122. Hadidi, A., Yang, X., 1990. Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261–270. Henson, J.M., French, R., 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31, 81–100. Hosokawa, W., Matsushita, Y., Uchida, H., Yazawa, S., 2005. Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue. J. Virol. Methods 131, 28–33. Hou, W.Y., Li, S.F., Wu, Z.J., Jiang, D.M., Sano, T., 2009b. Coleus blumei viroid 6: a new tentative member of the genus Coleviroid derived from natural genome shuffling. Arch. Virol. 154, 993–997. Hou, W.Y., Sano, T., Li, S.F., Li, F., Li, L., Wu, Z.J., 2009a. Identification and characterization of a new Coleviroid (CbVd-5). Arch. Virol. 154, 315–320. Ishiguro, A., Sano, T., Harada, Y., 1996. Nucleotide sequence and host range of a coleus viroid isolated from coleus (coleus blumei Benth) in Japan. Ann. Phytopathol. Soc. Jpn. 62, 84–86. Levy, L., Lee, I.M., Hadidi, A., 1994. Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid and MLO nucleic acids. J. Virol. Methods 49, 295–304. Li, S.F., Onodera, S., Sano, T., Yoshida, K., Wang, G.P., Shikata, E., 1995. Gene diagnosis of viroids: comparisons of return-PAGE and hybridization using dig labelled DNA and RNA probes for practical diagnosis of hop stunt, citrus exocortis and apple scar skin viroids in their natural host plants. Ann. Phytopathol. Soc. Jpn. 61, 381–390. Li, S.F., Su, Q., Guo, R., 2006. First report of coleus blumei viroid from coleus in China. Plant Pathol. 55, 565. Sano, T., Shikata, E., 1985. A viroid isolated from grapevine. Shokubutsu Boueki 39, 361–364. Sasaki, M., Shikata, E., 1977. On some properties of hop stunt disease agent, a viroid. Proc. Jpn. Acad. Ser. B 53, 109–112. Schumacher, J., Meyer, N., Riesner, D., Weidemann, H.L., 1986. Diagnostic procedure for detection of viroids and viruses with circular RNAs by “Return”-gel electrophoresis. J. Phytopathol. 115 (4), 332–343. Schumacher, J., Randles, J.W., Riesner, D., 1983. A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Anal. Biochem. 135, 288–295. Singh, R.P., Boucher, A., 1991. High incidence of transmission and occurrence of a viroid in commercial seeds of coleus in Canada. Plant Dis. 75 (2), 184–187. Spieker, R.L., 1996a. A new sequence variant of coleus blumei viroid 1 from the coleus blumei cultivar “Rainbow Gold”. Arch. Virol. 141, 2153–2161. Spieker, R.L., 1996b. In vitro-generated ‘inverse’ chimeric coleus blumei viroids evolve in vivo into infectious RNA replicons. J. Gen. Virol. 77, 2839–2846. Spieker, R.L., Haas, B., Charng, Y., Freimuller, K., Sanger, H.L., 1990. Primary and secondary structure of a new viroid ‘species’ (CbVd 1) present in the coleus blumei cultivar ‘Bienvenue’. Nucleic Acids Res. 18, 3998.
D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127 Spieker, R.L., Marinkovic, S., Sanger, H.L., 1996. A new sequence variant of coleus blumei viroid 3 from the coleus blumei cultivar ‘Fairway Ruby’. Arch. Virol. 141, 1377–1386. Steger, G., Riesner, D., 2003. Molecular characteristics. In: Hadidi, A., Flores, R., Randles, J.W., Semancik, J.S. (Eds.), Viroids. CSIRO, Collingwood, Australia, pp. 15– 29.
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Welnicki, M., Hiruki, C., 1992. Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid. J. Virol. Methods 39, 91–99. Yang, X., Hadidi, A., Garnsey, S.M., 1992. Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytopathology 82, 279–285.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants Dongmei Jiang a,b , Zujian Wu a , Lianhui Xie a , Teruo Sano c , Shifang Li b,∗ a
Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, West 2 Yuangmingyuan Road, Beijing 100193, China c Department of Plant Pathology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan b
a b s t r a c t Article history: Received 19 January 2011 Received in revised form 3 March 2011 Accepted 15 March 2011 Available online 2 April 2011 Keywords: Sap RT-PCR Coleus CbVd Pipettor
A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Viroids are small (246–475 nucleotides), covalently closed single-stranded RNAs. Like viruses, viroids replicate in their host plants and may act as phytopathogenic agents; however, they do not encode any proteins (Fauquet et al., 2005). Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide, and both its stem and leaf are known to be succulent. Coleus is susceptible to infection by several coleus blumei viroid (CbVd) species of the genus Coleviroid, family Pospiviroidae. Up to now, 6 main viroids that infect coleus have been reported: CbVd 1–6 (Fonseca et al., 1989; Spieker, 1996a,b; Spieker et al., 1996; Hou et al., 2009a,b) (Fig. 1a). CbVd-1 was first reported in a commercial yellow coleus in Brazil in 1996 (Fonseca et al., 1989), and then was reported successively in many other countries including Germany, Japan, Canada, Korea and China (Spieker et al., 1990; Ishiguro et al., 1996; Singh and Boucher, 1991; Chung and Choi, 2008; Li et al., 2006). To date, CbVd-2, CbVd-3, and CbVd-4 have only been reported in Germany (Spieker, 1996a,b; Spieker et al., 1996). CbVd-5 and CbVd-6 have only been reported in China until now (Hou et al., 2009a,b). All of the known coleus viroids share a common central conserved region (CCR), which is known to be an important structural element for Pospiviroid replication, i.e., processing and ligation (Diener, 1986; Gas et al., 2007). Recombination is common in CbVds, for example, CbVd-
∗ Corresponding author. Tel.: +86 10 62890875; fax: +86 10 62890875. E-mail addresses: sfl[email protected], [email protected] (S. Li).
2 and CbVd-6 are natural viroid chimeras and CbVd-4 is an in vitro-generated inverse chimera (Spieker, 1996b; Hou et al., 2009b) (Fig. 1a). Until now, CbVd-1 has been detected in many countries while the distribution of the other 5 coleus viroids seems to be limited. Therefore, it is necessary to establish a simple and fast method to further investigate the distribution of CbVds. Many methods for viroid detection have been reported including hybridization using DIG-labeled probes, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and Return-PAGE (Welnicki and Hiruki, 1992; Schumacher et al., 1983, 1986); however, they are laborious and time consuming. Besides, relatively pure coleus nucleic acids are vital for these methods. RT-PCR has proved to be a rapid and sensitive technique for the detection of both RNA viruses (Henson and French, 1993) and viroids (Hadidi and Yang, 1990; Yang et al., 1992; Levy et al., 1994). But a limitation of this procedure is the time consuming preparation of RNAs from plant samples. There are many methods for preparing viroid nucleic acids, such as the LiCl method (Li et al., 1995); however, large amounts of sample (at least 1–2 leaves) are required for template preparation in these conventional methods, and these methods require laborious steps to eliminate polyphenol and polysaccharide (Hadidi and Candresse, 2003). In addition, these procedures are expensive. In plant science, some improvements have been made in the methods for the extraction and purification of viroid RNA templates without tissue grinding; however, special tools are required (Hosokawa et al., 2005).
0166-0934/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.03.018
124 D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127 Fig. 1. (a) Schematic for the proposed secondary structures of coleus blumei viroid 1–6, CbVd-2 and CbVd-6 are natural viroid chimeras and CbVd-4 is an in vitro-generated inverse chimera. CbVd-2 includes the left half of CbVd-3 and the right half of CbVd-1, and CbVd-4 is opposite. CbVd-6 includes the left half of CbVd-3 and the right half of CbVd-5. (b) Primers designed for RT-PCR. Upper nucleotides in the box were designed for primers used in RT-PCR.
D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127
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Fig. 2. Results of sap-direct RT-PCR on coleus plants. M: marker; PC: positive control (the sample infected by CbVd-1, CbVd-5, and CbVd-6, and the template RNA of RT-PCR was extracted by LiCl method); NC: negative control (healthy sample). (a) Detection results of viroids from sap at different dilutions (1*: undiluted template; 10*: template diluted 10-fold; 100*: template diluted 100-fold; 1000*: template diluted 1000-fold). (b) Detection results of CbVds from different tissues—stem, old leaf, and new leaf. (c) Results of sap-direct RT-PCR method and dot-blot hybridization in different samples (1: sample infected by CbVd-1, CbVd-5, and CbVd-6; 2: sample infected by CbVd-1; 3: sample infected by CbVd-5 and CbVd-6).
In present study, a simple and rapid sap-direct RT-PCR method was established to detect 3 viroids of the genus Coleviroid (CbVd-1, CbVd-5, and CbVd-6) simultaneously. A pipette with a tip, common in a molecular biology laboratory, was used to prepare a very small amount of sap containing viroid RNA as templates for RT-PCR. A pair of universal primers, which can be used to detect 3 coleus viroids simultaneously, was designed based on the CCR sequences of CbVds. As a result, the presence of CbVd1, CbVd-5, and CbVd-6 can be determined in a short time using only a simple RT-PCR without special tools or cumbersome procedures. A pipettor (0.5–10 L) with short tips (0.5–10 L) was used to obtain sap directly from stems and leaves of coleus plants for template preparation, and the sap was then placed in a 1.5-mL Eppendorf (EP) tube directly. Notably, while sampling from a leaf, it should be kept between plastic papers and pressed; thus, the sap is soaked up easily. As soon as the sap was placed in the EP tubes, it was capped until the sap was put into the RT mix; so that the cross-contamination among different samples was reduced to a minimum. After it was diluted to an appropriate concentration, 1 L of the template was used for RT-PCR. A pair of universal primers (CbVds-P1 and CbVds-P2) was designed based on the CCR sequences of the genus Coleviroid (Fig. 1b). To generate cDNA from viroid RNA, 1 L of sap sample was mixed with 1 L (20 pmol) of CbVds-P2 primer (5 -GCAGCGCTGCCAGGGAACCCAGGT-3 ), 1 L of dNTPs (2.5 mM each, Takara, Dalian, China), 1 L of M-MLV (Promega, Madison, USA), 2 L of 5× M-MLV Reaction Buffer, and 0.25 L of recombinant RNasin ribonuclease inhibitor (40 U, Takara, Dalian, China). Distilled water was then added to a final volume of 10 L. The resulting mixture was incubated at 42 ◦ C for 60 min and 98 ◦ C for 5 min. After the RT reaction, 1 L of the RT solution was mixed with 0.2 L (20 pmol) of each primer (CbVds-P1 (5 -GCAGCGCTGCAACGGAAT-3 ) and CbVds-P2), 2 L of KOD-PlusNeo polymerase reaction buffer (Toyobo, Shanghai, China), 2 L dNTPs (2 mM each, Toyobo, Shanghai, China), 1 L MgSO4 (2.5 mM, Toyobo, Shanghai, China), 0.4 L of KOD-Plus-Neo polymerase (Toyobo, Shanghai, China), and 13.2 L distilled water. The cycling parameters for PCR amplification consisted of one cycle of heat denaturation at 94 ◦ C for 5 min, followed by 30 amplification cycles at 94 ◦ C for 30 s, 63 ◦ C for 30 s, and 72 ◦ C for 30 s. The final elongation step was at 72 ◦ C for 5 min. After RT-PCR, 10 L of the RT-PCR products were separated by electrophoresis on a 1.5% agarose gel and
visualized under UV light after staining with DNAgreen (Tiandz, Beijing, China). The products were purified using a PCR purification kit (Tiangen, Beijing, China). The purified fragments were then cloned into a pMD18-T vector (Takara, Dalian, China) and then transformed into Escherichia coli DH5␣. Clones containing inserts were sequenced using an automated DNA sequencer (ABI PRISM 3730XL DNA Analyzer) and analyzed by DNAMAN Version 5.2.2. To compare the detection sensitivity and accuracy of the sap-direct RT-PCR method with conventional methods, low molecular-weight RNAs of 30 coleus samples collected from outside in different seasons were extracted according to the procedure described by Li et al. (1995). After denatured using formaldehyde, RNA extracts were applied to nylon membranes (Hybond-N+, Amersham Biosciences), and hybridization using specific DIG-labeled cRNA probes for CbVd1, CbVd-5, or CbVd-6 was performed according to instructions of DIG RNA Labeling Kit and Detection Starter Kit (Roche, Basel, Switzerland). The sap collected from the stem of coleus plants infected by CbVd-1, CbVd-5, and CbVd-6 was used as a template for RT-PCR after it was diluted 10, 100, or 1000-fold. Bands that corresponded to CbVd-1, CbVd-5, and CbVd-6 were obtained simultaneously (Fig. 2a) The results of repeated experiments demonstrated that a 100-fold dilution is optimal. All the templates from the stem and leaf of the coleus plants infected by CbVds, irrespective of whether they were old or new, were suitable for the detection of CbVds as long as the sap was under proper consistency. RT-PCR results revealed that CbVd-1, CbVd-5, and CbVd-6 bands amplified from the stem were the clearest, followed by old leaf and new leaf. That is, sap from stem is the optimal template due to its higher detection sensitivity (Fig. 2b). CbVds were not detected from viroid-free plants by the sap-direct RT-PCR method, and CbVd-1 was only detected in coleus plants infected by CbVd-1. CbVd-5 and CbVd-6 were present together in the infected samples used in this study, and the amplified fragments could be distinguished clearly on the gel (Fig. 2c). There are many RT-PCR inhibitors in the sap of plants, such as phenols and polysaccharides, and they could contribute to false-negative results; therefore, the templates should be diluted. In this way, the fragments would be amplified clearly when the concentration of RNA and inhibitor reach a balance. In this study, although the samples were collected in different seasons and viroids concentrations in these samples were not the same; however, a 100-fold dilution was found to be superior, and can be
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Table 1 Detection results of CbVd-1, CbVd-5, and CbVd-6 using Dot-blot hybridization and Sap-direct RT-PCR. The number shown in the table is the number of samples (out of 30) that tested positive for the viroids. Numbers with * indicate the different detection results for CbVd-6 using two different methods. Assay
Dot-blot
Sap-direct RT-PCR
CbVd-1 CbVd-5 CbVd-6
29 16 15*
29 16 16*
applied to almost all coleus samples. Only few samples were needed to be diluted 10-fold or 1000-fold to obtain a clearer picture. The results of hybridization demonstrated that 29 of the 30 samples were positive for CbVd-1, 16 were positive for CbVd-5, and 15 were positive for CbVd-6. The result of sap-direct RT-PCR showed that 29 of the 30 samples were positive for CbVd-1, and CbVd-5 and CbVd-6 were positive in 16 samples (Table 1). Therefore, the results of the sap-direct RT-PCR corresponded to results of hybridization except for one sample, which was positive for CbVd-6 in sap-direct RT-PCR but negative in hybridization. That is, sap-direct RT-PCR method was more sensitive than dot-blot hybridization. Maybe the concentration of CbVd-6 in this sample was inadequate for hybridization, but sufficient for detection by RT-PCR. After cloning, 3 clones containing inserts of each viroid were sequenced. The results of sequence analysis revealed that the 3 bands from bottom to top on the gel (Fig. 2a) represented CbVd-1, CbVd-5, and CbVd-6, respectively. The sap-direct RT-PCR method is convenient because templates can be obtained from the stem or leaf without tissue grinding step. Viroids possess a thermodynamically stable secondary structure (Steger and Riesner, 2003), and viroid molecules are not destroyed by the high amount of RNase in plant materials. Thus, RNA in an EP tube can be maintained for a long period by capping. In our study, the sap without dilution in EP tubes could be available at −20 ◦ C for a long time, even more than 1 year; while they could not be stored at room temperature for more than 2 h, or 4 ◦ C (or 0 ◦ C) for more than 6 h. Notably, the sap from coleus plants at different dilution can be used only once. Therefore, to be on the safe side, it is better to draw sap from coleus plants just before RT-PCR. If the collection location is far from the laboratory, it is preferable to collect the stalk or leaf from the plant, and obtain the sap before they were dry. Many DNA polymerases can be used in this PCR method, and KODPlus-Neo is better due to its high amplification efficiency. Many ornamental plants are succulent and the sap can be drawn easily by the pipettor. The pipettor is a common tool in the molecular biology laboratory, and is considered to be more convenient than syringe needles, razors, or other tools that could be used to collect sap from plants (Hosokawa et al., 2005). In addition, pipettor tips can be taken out of the tubes more easily than other tools. The results of batch-detection revealed that the sap-direct RTPCR method can distinguish the three viroids from different coleus different viroids accurately (Fig. 2c), no matter the concentration of viroids were high or low in the plants. Compared to other methods, this method is not only rapid and accurate based on a simple RT-PCR, but also requires a lesser amount template (without purification). The method is especially feasible for mass detection. One microliter of sap is enough for the RT-PCR template, and the RT-PCR products are sufficient to be cloned conveniently. CbVd-5 and CbVd-6 have been reported in China for nearly 2 years; however, there has been no report about the two viroids in other countries except Japan. This rapid method could be very useful to clarify the geographic distribution of these two viroids quickly. Although the pathogenicity of CbVds is not clear and in most cases, CbVds induce no apparent symptoms in the coleus, the quarantine and control of CbVds cannot be ignored for the reason that
they may cause damage to other plants, just as hop stunt viroid can cause damage to hop even though it infects grapevines latently (Sano and Shikata, 1985; Sasaki and Shikata, 1977). Rapid and simple methods could play a considerable role in the quarantine and control of CbVds, and this study can also provide a reference for the detection of other viroids, even viruses. Acknowledgements This work was supported by grants from the Special Fund for Agro-scientific Research in the Public Interest (nyhyzx-200903004 and 200903034), National Basic Research and Development Program of China (973 Program) (No. 2009CB119200), the National Natural Science Foundation of China (Nos. 30771403, 30970134 and 31071765). This work was also supported by NSFC-JSPS Joint Research Project between China and Japan (30811140157), Key Project of the Fujian Provincial Department of Science and Technology (2007L2002). References Chung, B.M., Choi, G.S., 2008. Incidence of coleus blumei viroid 1 in seeds of commercial coleus in Korea. Plant Pathol. J. 24 (3), 305–308. Diener, T.O., 1986. Viroid processing: a model involving the central conserved region and hairpin I. In: Proc. Natl. Acad. Sci. U.S.A. , 83, pp. 58–62. Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., 2005. Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, Amsterdam, Netherlands, pp. 1145–1160. Fonseca, M.E.N., Boiteux, L.S., Singh, R.P., Kitajima, E.W., 1989. A small viroid in coleus species from Brazil. Fitopatol. Bras. 14, 94–96. Gas, M.E., Hernandez, C., Flores, R., Daròs, J.A., 2007. Processing of nuclear viroids in vivo: an interplay between RNA conformations. PLoS Pathog. 3 (11), 1813–1826, e182. Hadidi, A., Candresse, T., 2003. Polymerase chain reaction. In: Hadidi, A., Flores, R., Randles, J.W., Semanick, J.S. (Eds.), Viroids. CSIRO, Collingwood, Australia, pp. 115–122. Hadidi, A., Yang, X., 1990. Detection of pome fruit viroids by enzymatic cDNA amplification. J. Virol. Methods 30, 261–270. Henson, J.M., French, R., 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31, 81–100. Hosokawa, W., Matsushita, Y., Uchida, H., Yazawa, S., 2005. Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue. J. Virol. Methods 131, 28–33. Hou, W.Y., Li, S.F., Wu, Z.J., Jiang, D.M., Sano, T., 2009b. Coleus blumei viroid 6: a new tentative member of the genus Coleviroid derived from natural genome shuffling. Arch. Virol. 154, 993–997. Hou, W.Y., Sano, T., Li, S.F., Li, F., Li, L., Wu, Z.J., 2009a. Identification and characterization of a new Coleviroid (CbVd-5). Arch. Virol. 154, 315–320. Ishiguro, A., Sano, T., Harada, Y., 1996. Nucleotide sequence and host range of a coleus viroid isolated from coleus (coleus blumei Benth) in Japan. Ann. Phytopathol. Soc. Jpn. 62, 84–86. Levy, L., Lee, I.M., Hadidi, A., 1994. Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid and MLO nucleic acids. J. Virol. Methods 49, 295–304. Li, S.F., Onodera, S., Sano, T., Yoshida, K., Wang, G.P., Shikata, E., 1995. Gene diagnosis of viroids: comparisons of return-PAGE and hybridization using dig labelled DNA and RNA probes for practical diagnosis of hop stunt, citrus exocortis and apple scar skin viroids in their natural host plants. Ann. Phytopathol. Soc. Jpn. 61, 381–390. Li, S.F., Su, Q., Guo, R., 2006. First report of coleus blumei viroid from coleus in China. Plant Pathol. 55, 565. Sano, T., Shikata, E., 1985. A viroid isolated from grapevine. Shokubutsu Boueki 39, 361–364. Sasaki, M., Shikata, E., 1977. On some properties of hop stunt disease agent, a viroid. Proc. Jpn. Acad. Ser. B 53, 109–112. Schumacher, J., Meyer, N., Riesner, D., Weidemann, H.L., 1986. Diagnostic procedure for detection of viroids and viruses with circular RNAs by “Return”-gel electrophoresis. J. Phytopathol. 115 (4), 332–343. Schumacher, J., Randles, J.W., Riesner, D., 1983. A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Anal. Biochem. 135, 288–295. Singh, R.P., Boucher, A., 1991. High incidence of transmission and occurrence of a viroid in commercial seeds of coleus in Canada. Plant Dis. 75 (2), 184–187. Spieker, R.L., 1996a. A new sequence variant of coleus blumei viroid 1 from the coleus blumei cultivar “Rainbow Gold”. Arch. Virol. 141, 2153–2161. Spieker, R.L., 1996b. In vitro-generated ‘inverse’ chimeric coleus blumei viroids evolve in vivo into infectious RNA replicons. J. Gen. Virol. 77, 2839–2846. Spieker, R.L., Haas, B., Charng, Y., Freimuller, K., Sanger, H.L., 1990. Primary and secondary structure of a new viroid ‘species’ (CbVd 1) present in the coleus blumei cultivar ‘Bienvenue’. Nucleic Acids Res. 18, 3998.
D. Jiang et al. / Journal of Virological Methods 174 (2011) 123–127 Spieker, R.L., Marinkovic, S., Sanger, H.L., 1996. A new sequence variant of coleus blumei viroid 3 from the coleus blumei cultivar ‘Fairway Ruby’. Arch. Virol. 141, 1377–1386. Steger, G., Riesner, D., 2003. Molecular characteristics. In: Hadidi, A., Flores, R., Randles, J.W., Semancik, J.S. (Eds.), Viroids. CSIRO, Collingwood, Australia, pp. 15– 29.
127
Welnicki, M., Hiruki, C., 1992. Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid. J. Virol. Methods 39, 91–99. Yang, X., Hadidi, A., Garnsey, S.M., 1992. Enzymatic cDNA amplification of citrus exocortis and cachexia viroids from infected citrus hosts. Phytopathology 82, 279–285.