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Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification
Journal of Virological Methods 185 (2012) 254–258
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification Jun Peng a,b , Junfang Zhang b , Zihao Xia a , Yongqiang Li a , Junsheng Huang b , Zaifeng Fan a,∗ a
State Key Laboratory of Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, China Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences; Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests; Danzhou 571737, Hainan, China b
a b s t r a c t Article history: Received 3 February 2012 Received in revised form 22 June 2012 Accepted 27 June 2012 Available online 4 July 2012 Keywords: Banana bunchy top virus Virus detection Loop-mediated isothermal amplification (LAMP) Turbidimetry
A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63 ◦ C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/l plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other bananainfected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Banana (Musa nana) is one of the most important fruit crops, and bunchy top disease is a major threat to banana production in many tropical and subtropical regions (Dale, 1987; Dietzgen and Thomas, 1991). The causal virus, Banana bunchy top virus (BBTV) which belongs to the genus Babuvirus, family Nanoviridae (Vetten et al., 2012), spreads between banana plants by the banana aphid Pentalonia nigronervosa and from place to place by transporting propagative materials derived from infected plants (Dale, 1987; Dietzgen and Thomas, 1991). Banana plants infected by BBTV may display distorted bunches, and new growth from infected plants usually become stunted with a bunchy top-like appearance. No effective resistance is known in Musa spp. to this virus, thus control is still based largely on the use of virus-free propagative materials, roguing of infected plants and implementation of quarantine barriers. At present, banana plantlets from tissue culture are used widely, it is necessary, therefore, to develop efficient techniques for BBTV detection to obtain virus-free propagative materials. Published methods for detecting BBTV include virus isolation and purification (Wu and Su, 1990a; Harding et al., 1991;
∗ Corresponding author. Tel.: +86 10 62732771; fax: +86 10 62732771. E-mail address: [email protected] (Z. Fan). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.06.026
Thomas and Dietzgen, 1991), enzyme-linked immunosorbent assay (ELISA) (Wu and Su, 1990b; Thomas and Dietzgen, 1991; Geering and Thomas, 1996), nucleic acid hybridization (Hu et al., 1996; Hafner et al., 1997), PCR (Xie and Hu, 1995; Hu et al., 1996; Hafner et al., 1997; Dietzgen et al., 1999), and immnocapture-PCR (Sharman et al., 2000). Although ELISA and PCR are applied widely, a nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) is more rapid and simpler, using only a water bath or heating block (Notomi et al., 2000). The LAMP assay is performed under isothermal conditions, employing a DNA polymerase with strand-displacing activity and a set of four designed primers which recognize a total of six distinct sequences on the target DNA to be amplified; the amplified products contain singlestranded loops, allowing primers to bind without the need for repeated cycles of thermal denaturation (Notomi et al., 2000). As the LAMP reaction progresses, the by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate, and an increase in the turbidity with the production of white precipitate correlates with the amount of DNA synthesized, monitoring of the LAMP reaction was achieved by realtime measurement of turbidity (Mori et al., 2001, 2004). The objective of this research was to develop a LAMP method for the diagnosis of BBTV infection of banana plants. Additionally, an improved visual closed-tube procedure was developed for highthroughput detection. The application of LAMP for the detection of BBTV in field-grown banana plants is described.
J. Peng et al. / Journal of Virological Methods 185 (2012) 254–258
255
Fig. 1. Nucleic acid sequence of target fragment on DNA-R (containing the replication initiator protein gene) of the BBTV genome used for designing inner and outer primers. The specific sequences used for primer design and their relative positions in the viral genome are indicated by arrows.
2. Materials and methods 2.1. Virus sources and DNA extraction Samples of plants showing BBTV or virus-like symptoms were obtained from different banana plantations on Hainan Island, China. The samples of leaves and suckers were collected, and genomic DNA was extracted using a modified CTAB DNA extraction method according to Gawel and Jarret (1991).
products were detected directly by visual observation of the solution color by mixing the pre-added 1 l of SYBR Green I to the reaction solution by gentle centrifugation (for about 5 s, up to 2000 × g). Green fluorescence was observed clearly with the naked eye with a positive reaction, whereas the color remained orange with the negative reactions. The LAMP products (5 l) were analyzed by electrophoresis on a 2% (w/v) agarose gel and stained subsequently with ethidium bromide. 2.5. LAMP specificity
2.2. Primer design LAMP primers were designed according to sequence of DNAR (containing the replication initiator protein gene) of BBTV genome (accession number U97525) using PrimerExplorer V4 software (http://primerexplorer.jp/e/). A forward inner primer FIP (5 -CTTTCGTTTCTCAAGGTGTGCGTCGAGATGAAGAGAAGAAG 3 ) consisted of F1c (the complementary sequence of F1, 5 -CTTTCGTTTCTCAAGGTGTGC-3 , nt 339–319 in the BBTV genome) and F2 (5 -GTCGAGATGAAGAGAAGAAG3 , nt 265–284), and a backward inner primer BIP (5 -GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ) consisted of B1c (the complementary sequence of B1, 5 GGGAGCCAAGAAGAAGCA-3 , nt 340–357 in the BBTV genome) and B2 (5 -GGACCTTCGATTCTTGTATC-3 , nt 398–379). The outer primers F3 (5 -TGATGCGGGATGAGTTTA-3 , nt 188–205) and B3 (5 -CACGCATATCCTGTATGAC-3 , nt 466–448) were used for the initiation of LAMP reaction. The primer sequences and their respective binding sites were indicated in Fig. 1. 2.3. Reaction mixtures and optimal conditions The LAMP reaction mix contained 1.6 M each of the primers BBTV-FIP and BBTV-BIP, 0.2 M each of the primers BBTV-F3 and BBTV-B3, 1.6 mM of dNTPs, 1 M of betaine (Solarbio, Beijing, China), 4 mM of MgSO4 , 10× ThermoPol reaction buffer (20 mM Tris–HCl, 10 mM KCl, 10 mM of (NH4 )2 SO4 , and 0.1% TritonX-100), 8 U of Bst DNA polymerase, and 1 l of BBTV DNA template, and doubledistilled water to a final volume of 25 l. Then, an equal volume of paraffin oil was added to the tube to prevent evaporation, followed by adding 1 l of 1:10-diluted SYBR Green I (Invitrogen, Carlsbad, CA) to the interior of the lid prior to amplification. The LAMP reaction was carried out in a Loopamp real-time turbidimeter (LA-320C; Teramecs, Kyoto, Japan) at 63 ◦ C for 90 min and terminated at 80 ◦ C for 10 min.
To confirm the specificity of the LAMP assay, the RNA of Cucumber mosaic virus (CMV), and the DNAs of Banana streak OL virus (genus Badnavirus, family Caulimoviridae), Ralstonia solanacearum, Fusarium oxysporum f. sp. cubense race 1 (Foc1) and race 4 (Foc4) were used in the analyses. For CMV RNA template, an additional 0.2 l (8 U) of reverse transcriptase (Takara, Dalian, China) was added to LAMP reaction solution. 2.6. LAMP sensitivity To determine the sensitivity of the BBTV LAMP assay, a DNA fragment of 748 bp containing the LAMP target region of the BBTV genome was amplified by PCR using primers BBTV-RP1 (5 -ATGTGG TATGCTGGATGTTC-3 ) and BBTV-RP2r (5 GGTTCATATTTCCCGCTTTGA-3 ). The concentration of primers was adjusted to 10 M for subsequent usage. The PCR mix was 50 l in volume and contained 5 l of 10× EX Taq buffer (Mg2+ Plus), 1 l of dNTP mixture (10 mM each), 5 U of EX Taq DNA polymerase (Takara, Dalian, China) and 2 l each of the primers. The thermal cycling program was as follows: an initial denaturation step at 94 ◦ C for 3 min, followed by 35 cycles of at 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 45 s, with a final extension at 72 ◦ C for 7 min. PCR product was purified and cloned into the pMD18-T vector (Takara, Dalian, China) following the manufacturer’s instructions. The recombinant plasmid, designated pMD18-T-BBTV, was adjusted to the concentration of 100 ng/l, and diluted into a 10-fold serials (1 × 100 to 1 × 107 copies) before mixed with extracted DNA from healthy banana used as a reference to assess the detection limit of the LAMP assay. To evaluate the feasibility of LAMP method for diagnosis of samples collected in the field, all samples collected from different geographic locations in Hainan Island were detected with PCR and LAMP assay, respectively. 3. Results
2.4. Analysis of LAMP products Real-time turbidity readings at 650 nm were obtained and a turbidity threshold value of 0.1 was used. After reaction, LAMP
For the specificity test, no cross-reactivity with templates extracted, or from other banana-infecting pathogens was found. Only amplified DNA products from banana infected with BBTV
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Fig. 2. Specificity test of the LAMP assay for the detection of BBTV. (A) Agarose gel electrophoresis analysis of LAMP amplicon showing the specificity of LAMP assay for detection of BBTV. Lane 1, genomic DNA of Banana bunchy top virus (BBTV); lane 2, healthy plant control; lane 3, the cDNA of Cucumber mosaic virus (CMV); lane 4, the genomic DNA of Banana streak OL virus; lanes 5–7, DNAs of Ralstonia solanacearum, Fusarium oxysporum f. sp. cubense race 1 (Foc1) and Fusarium oxysporum f. sp. cubense race 4 (Foc4), respectively; and lane 8, water control; lane M, Trans2K plus II DNA marker. (B) Visual detection of the LAMP amplification product. The original orange color of SYBR green turned green in the positive reaction mixture (sample 1). (C) A turbidity vs. time graph plotted by the built-in software from the data obtained for LAMP specificity assay with a real-time turbidimeter.
showed ladder-like bands, while no amplicons were detected for other banana pathogens and control (Fig. 2A). The color of LAMP products changed from orange to green when BBTV was detected with SYBR Green I, while the color remained orange for the healthy plant and water controls (Fig. 2B). The turbidity vs. time curve obtained by using the real-time turbidimetry to monitor the DNA synthesis reaction indicated that the primer set was able to amplify the target DNA sequence (Fig. 2C). It is difficult to quantify precisely BBTV genomic DNA in infected banana plants, thus, the plasmid DNA mixed with total DNA
extracted from banana leaf tissues was used as a reference for both LAMP and PCR to evaluate the sensitivity of BBTV LAMP and PCR, respectively. Since some inhibitory compounds usually exist in banana tissues, ten-fold serial dilution of BBTV plasmid DNA mixed with extracted total DNA from healthy banana leaves used as a reference was demonstrated to be a convenient approach to test the detection limit of both LAMP and PCR. The sensitivity test showed that this LAMP assay could detect as low as 1 pg/l of DNA, while detection limit of PCR was about 100 pg/l plasmid DNA (Fig. 3). Thus, the detection sensitivity of the LAMP assay was
Fig. 3. Comparison of the sensitivity of LAMP with that of PCR assay. (A) Sensitivity of LAMP assay for the detection of the pMD18-T-BBTV plasmid DNA. Lane M, Trans2K Plus II DNA marker, lanes 1–8 correspond to serial 10-fold dilutions of BBTV pMD18-T-BBTV plasmid ranging from 1 × 100 to 1 × 107 copies when mixed with extracted DNA from healthy banana; (B) sensitivity of the PCR assay for the detection of the pMD18-T-BBTV plasmid DNA using the BBTV-RP1/BBTV-RP2r primer set; samples in the lanes 1–8 correspond to those in the lanes with the same number in panel A. (C) Visual inspection of the LAMP assay. The original orange color of SYBR green turned green in the positive reaction mixture. (D) A turbidity vs. time graph plotted from the data obtained for LAMP assay with a real-time turbidimeter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
J. Peng et al. / Journal of Virological Methods 185 (2012) 254–258
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Fig. 4. Detection of field-grown banana samples using LAMP and PCR methods. (A) PCR detection of field-grown banana samples. Lanes 1 and 2, the water and healthy controls; lanes 3–8, positive samples detected by PCR; lane M, Trans2K plus II DNA marker; (B) visual observation of the LAMP detection of field-grown banana samples; samples in the lanes 1–8 correspond to those with the same lane number in panel (A); and (C) the turbidity vs. time graph plotted automatically with the data obtained from LAMP assay with a real-time turbidimeter.
approximately 100 times higher than that of PCR. All the experiments were performed independently for three times, and nearly identical results were obtained. To evaluate the feasibility of the LAMP method for detecting BBTV in the field-grown bananas, a total of 66 samples were tested by both PCR and LAMP. Results from the LAMP assay showed that 60 samples were tested positive, 6 samples negative by LAMP, which were conformed with the PCR results; however, 2 samples tested positive by LAMP were tested negative by PCR. The 2 samples were then propagated by tissue culture and the plantlets were re-tested to be positive by both LAMP and PCR. The detection rate of PCR and LAMP for the samples collected in the field in this study were 87.8% (58/66) and 90.9% (60/66), respectively (Fig. 4).
dye compared with gel electrophoresis. The results in this work suggests that the established BBTV LAMP method should be useful for both disease monitoring in banana plantations and large-scale propagation of virus-free banana plantlets by tissue culture. Acknowledgements This research was supported by the Special Fund for AgroScientific Research in the Public Interest (200903049), A grant from the Ministry of Education (IRT1042), Research Fund for the Doctoral Program of Higher Education of China (20110008110020), and the Chinese National Non-profit Institute Research Grant of CATAS-EPI (2009hzs1J07). We thank Dr Tao Zhou in our laboratory and the Guangzhou Deaou Biotechnology Co. Ltd. for technical assistance.
4. Discussion References In this study, a method for rapid diagnosis of BBTV in banana planting materials using LAMP assay was developed and this approach has the potential to become a valuable diagnostic tool in banana production. The advantages of LAMP over conventional molecular methods are more sensitive and rapid. The LAMP does not require a thermal cycler because reactions can be performed using a heating block or in a water bath under isothermal conditions. SYBR Green I is one of the most sensitive nucleic acid fluorescence dyes available (Matsui et al., 2004), however, the LAMP reaction would be inhibited if SYBR green I were added directly to the reaction solution at a concentration required for visualization (Goto et al., 2009; Tao et al., 2011). Thus, the fluorescent dye is often added after the reaction, which likely causes aerosol pollution. In this study, an improvement was made by adding fluorescent dye to the interior of the lid prior to amplification. After reaction, the SYBR Green I would be mixed with LAMP reaction solution by brief gentle centrifugation (for about 5 s, up to 2000 × g), forming different colors to aid in the judgment of the results. The risk of cross-contamination is minimal using the improved closed-tube detection system, which facilitates the screening of samples and would be helpful for high-throughput application. In conclusion, this LAMP assay is a rapid, cost-effective, sensitive and specific method for the detection of BBTV. In addition, the closed-tube visual detection system is suitable for general laboratories and rapid for obtaining results using the SYBR Green I
Dale, J.L., 1987. Banana bunchy top: an economically important tropical plant virus disease. Advances in Virus Research 33, 325–333. Dietzgen, R.G., Thomas, J.E., 1991. Properties of virus-like particles associated with banana bunchy top disease in Hawaii, Indonesia and Tonga. Australasian Plant Pathology 20, 161–165. Dietzgen, R.G., Thomas, J.E., Smith, G.R., Maclean, D.L., 1999. PCR-based detection of viruses in banana and sugarcane. Current Topics in Virology 1, 105–118. Gawel, N.J., Jarret, R.L., 1991. A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Molecular Biology Reporter 9, 262–266. Geering, A.D.W., Thomas, J.E., 1996. A comparison of four serological tests for the detection of banana bunchy top virus in banana. Australian Journal of Agricultural Research 47, 403–412. Goto, M., Honda, E., Ogura, A., Nomoto, A., Hanaki, K., 2009. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy napthol blue. Biotechniques 46, 167–172. Hafner, G.J., Harding, R.M., Dale, J.L., 1997. A DNA primer associated with banana bunchy top virus. Journal of General Virology 78, 479–486. Harding, R.M., Burns, T.M., Dale, J.L., 1991. Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA. Journal of General Virology 72, 225–230. Hu, J.S., Wang, M., Sether, D., Xie, W., Leonhardt, K.W., 1996. Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa). Annals of Applied Biology 128, 55–64. Matsui, K., Ishii, N., Honjo, M., Kawabata, Z., 2004. Use of the SYBR Green I fluorescent dye and a centrifugal filter device for rapid determination of dissolved DNA concentration in fresh water. Aquatic Microbial Ecology 36, 99–105. Mori, Y., Nagamine, K., Tomita, N., Notomi, T., 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications 289, 150–154. Mori, Y., Kitao, M., Tomita, N., Notomi, T., 2004. Real-time turbidimetry of LAMP reaction for quantifying template DNA. Journal of Biochemical and Biophysical Methods 59, 145–157.
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Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63. Sharman, M., Thomas, J.E., Dietzgen, R.G., 2000. Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. Journal of Virological Methods 89, 75–88. Tao, Z.Y., Zhou, H.Y., Xia, H., Xu, S., Zhu, H.W., Culleton, R.L., Han, E.T., Lu, F., Fang, Q., Gu, Y.P., Liu, Y.B., Zhu, G.D., Wang, W.M., Li, J.L., Cao, J., Gao, Q., 2011. Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivas infection. Parasites and Vectors 4, 115. Thomas, J.E., Dietzgen, R.G., 1991. Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia. Journal of Virological Methods 72, 217–224.
Vetten, H.J., Dale, J.L., Grigoras, I., Gronenborn, B., Harding, R., Randles, J.W., Sano, Y., Thomas, J.E., Timchenko, T., Yeh, H.H., 2012. Family Nanoviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, San Diego, CA, USA, pp. 395–404. Wu, R.Y., Su, H.J., 1990a. Purification and characterization of Banana bunchy top virus. Journal of Phytopathology 128, 153–160. Wu, R.Y., Su, H.J., 1990b. Production of monoclonal antibodies against banana bunchy top virus and their use in enzyme-linked immunosorbent assay. Journal of Phytopathology 128, 203–208. Xie, W.S., Hu, J.S., 1995. Molecular cloning, sequence analysis, and detection of banana bunchy top virus in Hawaii. Phytopathology 85, 339–347.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification Jun Peng a,b , Junfang Zhang b , Zihao Xia a , Yongqiang Li a , Junsheng Huang b , Zaifeng Fan a,∗ a
State Key Laboratory of Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, China Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences; Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests; Danzhou 571737, Hainan, China b
a b s t r a c t Article history: Received 3 February 2012 Received in revised form 22 June 2012 Accepted 27 June 2012 Available online 4 July 2012 Keywords: Banana bunchy top virus Virus detection Loop-mediated isothermal amplification (LAMP) Turbidimetry
A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63 ◦ C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/l plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other bananainfected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Banana (Musa nana) is one of the most important fruit crops, and bunchy top disease is a major threat to banana production in many tropical and subtropical regions (Dale, 1987; Dietzgen and Thomas, 1991). The causal virus, Banana bunchy top virus (BBTV) which belongs to the genus Babuvirus, family Nanoviridae (Vetten et al., 2012), spreads between banana plants by the banana aphid Pentalonia nigronervosa and from place to place by transporting propagative materials derived from infected plants (Dale, 1987; Dietzgen and Thomas, 1991). Banana plants infected by BBTV may display distorted bunches, and new growth from infected plants usually become stunted with a bunchy top-like appearance. No effective resistance is known in Musa spp. to this virus, thus control is still based largely on the use of virus-free propagative materials, roguing of infected plants and implementation of quarantine barriers. At present, banana plantlets from tissue culture are used widely, it is necessary, therefore, to develop efficient techniques for BBTV detection to obtain virus-free propagative materials. Published methods for detecting BBTV include virus isolation and purification (Wu and Su, 1990a; Harding et al., 1991;
∗ Corresponding author. Tel.: +86 10 62732771; fax: +86 10 62732771. E-mail address: [email protected] (Z. Fan). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.06.026
Thomas and Dietzgen, 1991), enzyme-linked immunosorbent assay (ELISA) (Wu and Su, 1990b; Thomas and Dietzgen, 1991; Geering and Thomas, 1996), nucleic acid hybridization (Hu et al., 1996; Hafner et al., 1997), PCR (Xie and Hu, 1995; Hu et al., 1996; Hafner et al., 1997; Dietzgen et al., 1999), and immnocapture-PCR (Sharman et al., 2000). Although ELISA and PCR are applied widely, a nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) is more rapid and simpler, using only a water bath or heating block (Notomi et al., 2000). The LAMP assay is performed under isothermal conditions, employing a DNA polymerase with strand-displacing activity and a set of four designed primers which recognize a total of six distinct sequences on the target DNA to be amplified; the amplified products contain singlestranded loops, allowing primers to bind without the need for repeated cycles of thermal denaturation (Notomi et al., 2000). As the LAMP reaction progresses, the by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate, and an increase in the turbidity with the production of white precipitate correlates with the amount of DNA synthesized, monitoring of the LAMP reaction was achieved by realtime measurement of turbidity (Mori et al., 2001, 2004). The objective of this research was to develop a LAMP method for the diagnosis of BBTV infection of banana plants. Additionally, an improved visual closed-tube procedure was developed for highthroughput detection. The application of LAMP for the detection of BBTV in field-grown banana plants is described.
J. Peng et al. / Journal of Virological Methods 185 (2012) 254–258
255
Fig. 1. Nucleic acid sequence of target fragment on DNA-R (containing the replication initiator protein gene) of the BBTV genome used for designing inner and outer primers. The specific sequences used for primer design and their relative positions in the viral genome are indicated by arrows.
2. Materials and methods 2.1. Virus sources and DNA extraction Samples of plants showing BBTV or virus-like symptoms were obtained from different banana plantations on Hainan Island, China. The samples of leaves and suckers were collected, and genomic DNA was extracted using a modified CTAB DNA extraction method according to Gawel and Jarret (1991).
products were detected directly by visual observation of the solution color by mixing the pre-added 1 l of SYBR Green I to the reaction solution by gentle centrifugation (for about 5 s, up to 2000 × g). Green fluorescence was observed clearly with the naked eye with a positive reaction, whereas the color remained orange with the negative reactions. The LAMP products (5 l) were analyzed by electrophoresis on a 2% (w/v) agarose gel and stained subsequently with ethidium bromide. 2.5. LAMP specificity
2.2. Primer design LAMP primers were designed according to sequence of DNAR (containing the replication initiator protein gene) of BBTV genome (accession number U97525) using PrimerExplorer V4 software (http://primerexplorer.jp/e/). A forward inner primer FIP (5 -CTTTCGTTTCTCAAGGTGTGCGTCGAGATGAAGAGAAGAAG 3 ) consisted of F1c (the complementary sequence of F1, 5 -CTTTCGTTTCTCAAGGTGTGC-3 , nt 339–319 in the BBTV genome) and F2 (5 -GTCGAGATGAAGAGAAGAAG3 , nt 265–284), and a backward inner primer BIP (5 -GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ) consisted of B1c (the complementary sequence of B1, 5 GGGAGCCAAGAAGAAGCA-3 , nt 340–357 in the BBTV genome) and B2 (5 -GGACCTTCGATTCTTGTATC-3 , nt 398–379). The outer primers F3 (5 -TGATGCGGGATGAGTTTA-3 , nt 188–205) and B3 (5 -CACGCATATCCTGTATGAC-3 , nt 466–448) were used for the initiation of LAMP reaction. The primer sequences and their respective binding sites were indicated in Fig. 1. 2.3. Reaction mixtures and optimal conditions The LAMP reaction mix contained 1.6 M each of the primers BBTV-FIP and BBTV-BIP, 0.2 M each of the primers BBTV-F3 and BBTV-B3, 1.6 mM of dNTPs, 1 M of betaine (Solarbio, Beijing, China), 4 mM of MgSO4 , 10× ThermoPol reaction buffer (20 mM Tris–HCl, 10 mM KCl, 10 mM of (NH4 )2 SO4 , and 0.1% TritonX-100), 8 U of Bst DNA polymerase, and 1 l of BBTV DNA template, and doubledistilled water to a final volume of 25 l. Then, an equal volume of paraffin oil was added to the tube to prevent evaporation, followed by adding 1 l of 1:10-diluted SYBR Green I (Invitrogen, Carlsbad, CA) to the interior of the lid prior to amplification. The LAMP reaction was carried out in a Loopamp real-time turbidimeter (LA-320C; Teramecs, Kyoto, Japan) at 63 ◦ C for 90 min and terminated at 80 ◦ C for 10 min.
To confirm the specificity of the LAMP assay, the RNA of Cucumber mosaic virus (CMV), and the DNAs of Banana streak OL virus (genus Badnavirus, family Caulimoviridae), Ralstonia solanacearum, Fusarium oxysporum f. sp. cubense race 1 (Foc1) and race 4 (Foc4) were used in the analyses. For CMV RNA template, an additional 0.2 l (8 U) of reverse transcriptase (Takara, Dalian, China) was added to LAMP reaction solution. 2.6. LAMP sensitivity To determine the sensitivity of the BBTV LAMP assay, a DNA fragment of 748 bp containing the LAMP target region of the BBTV genome was amplified by PCR using primers BBTV-RP1 (5 -ATGTGG TATGCTGGATGTTC-3 ) and BBTV-RP2r (5 GGTTCATATTTCCCGCTTTGA-3 ). The concentration of primers was adjusted to 10 M for subsequent usage. The PCR mix was 50 l in volume and contained 5 l of 10× EX Taq buffer (Mg2+ Plus), 1 l of dNTP mixture (10 mM each), 5 U of EX Taq DNA polymerase (Takara, Dalian, China) and 2 l each of the primers. The thermal cycling program was as follows: an initial denaturation step at 94 ◦ C for 3 min, followed by 35 cycles of at 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 45 s, with a final extension at 72 ◦ C for 7 min. PCR product was purified and cloned into the pMD18-T vector (Takara, Dalian, China) following the manufacturer’s instructions. The recombinant plasmid, designated pMD18-T-BBTV, was adjusted to the concentration of 100 ng/l, and diluted into a 10-fold serials (1 × 100 to 1 × 107 copies) before mixed with extracted DNA from healthy banana used as a reference to assess the detection limit of the LAMP assay. To evaluate the feasibility of LAMP method for diagnosis of samples collected in the field, all samples collected from different geographic locations in Hainan Island were detected with PCR and LAMP assay, respectively. 3. Results
2.4. Analysis of LAMP products Real-time turbidity readings at 650 nm were obtained and a turbidity threshold value of 0.1 was used. After reaction, LAMP
For the specificity test, no cross-reactivity with templates extracted, or from other banana-infecting pathogens was found. Only amplified DNA products from banana infected with BBTV
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Fig. 2. Specificity test of the LAMP assay for the detection of BBTV. (A) Agarose gel electrophoresis analysis of LAMP amplicon showing the specificity of LAMP assay for detection of BBTV. Lane 1, genomic DNA of Banana bunchy top virus (BBTV); lane 2, healthy plant control; lane 3, the cDNA of Cucumber mosaic virus (CMV); lane 4, the genomic DNA of Banana streak OL virus; lanes 5–7, DNAs of Ralstonia solanacearum, Fusarium oxysporum f. sp. cubense race 1 (Foc1) and Fusarium oxysporum f. sp. cubense race 4 (Foc4), respectively; and lane 8, water control; lane M, Trans2K plus II DNA marker. (B) Visual detection of the LAMP amplification product. The original orange color of SYBR green turned green in the positive reaction mixture (sample 1). (C) A turbidity vs. time graph plotted by the built-in software from the data obtained for LAMP specificity assay with a real-time turbidimeter.
showed ladder-like bands, while no amplicons were detected for other banana pathogens and control (Fig. 2A). The color of LAMP products changed from orange to green when BBTV was detected with SYBR Green I, while the color remained orange for the healthy plant and water controls (Fig. 2B). The turbidity vs. time curve obtained by using the real-time turbidimetry to monitor the DNA synthesis reaction indicated that the primer set was able to amplify the target DNA sequence (Fig. 2C). It is difficult to quantify precisely BBTV genomic DNA in infected banana plants, thus, the plasmid DNA mixed with total DNA
extracted from banana leaf tissues was used as a reference for both LAMP and PCR to evaluate the sensitivity of BBTV LAMP and PCR, respectively. Since some inhibitory compounds usually exist in banana tissues, ten-fold serial dilution of BBTV plasmid DNA mixed with extracted total DNA from healthy banana leaves used as a reference was demonstrated to be a convenient approach to test the detection limit of both LAMP and PCR. The sensitivity test showed that this LAMP assay could detect as low as 1 pg/l of DNA, while detection limit of PCR was about 100 pg/l plasmid DNA (Fig. 3). Thus, the detection sensitivity of the LAMP assay was
Fig. 3. Comparison of the sensitivity of LAMP with that of PCR assay. (A) Sensitivity of LAMP assay for the detection of the pMD18-T-BBTV plasmid DNA. Lane M, Trans2K Plus II DNA marker, lanes 1–8 correspond to serial 10-fold dilutions of BBTV pMD18-T-BBTV plasmid ranging from 1 × 100 to 1 × 107 copies when mixed with extracted DNA from healthy banana; (B) sensitivity of the PCR assay for the detection of the pMD18-T-BBTV plasmid DNA using the BBTV-RP1/BBTV-RP2r primer set; samples in the lanes 1–8 correspond to those in the lanes with the same number in panel A. (C) Visual inspection of the LAMP assay. The original orange color of SYBR green turned green in the positive reaction mixture. (D) A turbidity vs. time graph plotted from the data obtained for LAMP assay with a real-time turbidimeter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 4. Detection of field-grown banana samples using LAMP and PCR methods. (A) PCR detection of field-grown banana samples. Lanes 1 and 2, the water and healthy controls; lanes 3–8, positive samples detected by PCR; lane M, Trans2K plus II DNA marker; (B) visual observation of the LAMP detection of field-grown banana samples; samples in the lanes 1–8 correspond to those with the same lane number in panel (A); and (C) the turbidity vs. time graph plotted automatically with the data obtained from LAMP assay with a real-time turbidimeter.
approximately 100 times higher than that of PCR. All the experiments were performed independently for three times, and nearly identical results were obtained. To evaluate the feasibility of the LAMP method for detecting BBTV in the field-grown bananas, a total of 66 samples were tested by both PCR and LAMP. Results from the LAMP assay showed that 60 samples were tested positive, 6 samples negative by LAMP, which were conformed with the PCR results; however, 2 samples tested positive by LAMP were tested negative by PCR. The 2 samples were then propagated by tissue culture and the plantlets were re-tested to be positive by both LAMP and PCR. The detection rate of PCR and LAMP for the samples collected in the field in this study were 87.8% (58/66) and 90.9% (60/66), respectively (Fig. 4).
dye compared with gel electrophoresis. The results in this work suggests that the established BBTV LAMP method should be useful for both disease monitoring in banana plantations and large-scale propagation of virus-free banana plantlets by tissue culture. Acknowledgements This research was supported by the Special Fund for AgroScientific Research in the Public Interest (200903049), A grant from the Ministry of Education (IRT1042), Research Fund for the Doctoral Program of Higher Education of China (20110008110020), and the Chinese National Non-profit Institute Research Grant of CATAS-EPI (2009hzs1J07). We thank Dr Tao Zhou in our laboratory and the Guangzhou Deaou Biotechnology Co. Ltd. for technical assistance.
4. Discussion References In this study, a method for rapid diagnosis of BBTV in banana planting materials using LAMP assay was developed and this approach has the potential to become a valuable diagnostic tool in banana production. The advantages of LAMP over conventional molecular methods are more sensitive and rapid. The LAMP does not require a thermal cycler because reactions can be performed using a heating block or in a water bath under isothermal conditions. SYBR Green I is one of the most sensitive nucleic acid fluorescence dyes available (Matsui et al., 2004), however, the LAMP reaction would be inhibited if SYBR green I were added directly to the reaction solution at a concentration required for visualization (Goto et al., 2009; Tao et al., 2011). Thus, the fluorescent dye is often added after the reaction, which likely causes aerosol pollution. In this study, an improvement was made by adding fluorescent dye to the interior of the lid prior to amplification. After reaction, the SYBR Green I would be mixed with LAMP reaction solution by brief gentle centrifugation (for about 5 s, up to 2000 × g), forming different colors to aid in the judgment of the results. The risk of cross-contamination is minimal using the improved closed-tube detection system, which facilitates the screening of samples and would be helpful for high-throughput application. In conclusion, this LAMP assay is a rapid, cost-effective, sensitive and specific method for the detection of BBTV. In addition, the closed-tube visual detection system is suitable for general laboratories and rapid for obtaining results using the SYBR Green I
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