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Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick
Journal of Virological Methods 156 (2009) 27–31
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick Teeranart Puthawibool a,b , Saengchan Senapin a,c , Wansika Kiatpathomchai a,c,∗ , Timothy W. Flegel a,b a
CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Dept. Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand c National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand b
a b s t r a c t Article history: Received 18 August 2008 Received in revised form 15 October 2008 Accepted 20 October 2008 Available online 6 December 2008 Keywords: IMNV Infectious myonecrosis virus Penaeus vannamei PCR Loop-mediated isothermal amplification (LAMP)
Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75 min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP–LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV). © 2008 Elsevier B.V. All rights reserved.
1. Introduction The Pacific whiteleg shrimp Penaeus (Litopenaeus) vannamei is a non-native (exotic) penaeid shrimp species to Brazil. It was imported for commercial shrimp culture beginning in 1983 but was not cultivated there on a large scale until after 1995 (Briggs et al., 2004). Disease outbreaks due to infectious myonecrosis (IMN) were first reported in farmed Pacific whiteleg shrimp from Brazil in 2004. In P. vannamei, striated muscles are the principal target organ, but gills and lymphoid organ can also be affected (Lightner et al., 2004). Gross signs of IMN-infected shrimp included necrosis of striated
∗ Corresponding author at: CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: +66 2 2015878; fax: +66 2 3547344. E-mail address: [email protected] (W. Kiatpathomchai). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.018
muscles, primarily in the distal abdominal segments and the tail fan, and the appearance of white discoloration of the affected muscle similar to the color of cooked shrimp. Hatchery brood, larvae and all sizes of shrimp could be affected. The causative agent of IMN (infectious myonecrosis virus or IMNV) was described subsequently in 2006 (Poulos et al., 2006). It is a non-enveloped, icosahedral virus of 40 nm in diameter. The genome consists of double-stranded RNA of 7560 nucleotides containing two open reading frames, designated ORF1 and ORF2 (Lightner et al., 2004; Poulos et al., 2006). ORF1 encodes a putative RNA-binding protein and a capsid protein and ORF2 codes for a putative RNA dependent RNA polymerase (RdRp). The virus has been classified tentatively in the family Totiviridae. Apart from P. vannamei infected naturally, experimental infections have been achieved with Penaeus (Farfantepenaeus) subtiltis, Penaeus monodon and Penaeus (Litopenaeus) stylirostris (Lightner et al., 2004; Tang et al., 2005).
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T. Puthawibool et al. / Journal of Virological Methods 156 (2009) 27–31
Fig. 1. Nucleotide sequence of IMNV RdRp gene (GenBank accession number: AY570982; Poulos et al., 2006). The sequences used to design primers F3 and B3 are shown by underlined sequences. The FIP (F1c/TTTT/F2) and BIP (B1c/TTTT/B2) inner primers are shown by shaded boxes and arrows. The FITC-labeled probe sequence is shown in italic and underlined sequence.
Loop-mediated isothermal amplification (LAMP) allows amplification of DNA with high specificity, sensitivity and rapidity under isothermal conditions. LAMP, described originally by Notomi et al. (2000), can amplify target nucleic acid to 109 copies at 60–65 ◦ C within 1 h. The method relies on autocycling strand displacement DNA synthesis by the Bst DNA polymerase large fragment, a DNA polymerase with high strand displacement activity, and a set of two inner primers and two outer primers. LAMP is highly specific for the target sequence because of the recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences in the later stages of the LAMP reaction. As the reaction is conducted under isothermal conditions, it can be carried out with a simple and inexpensive water bath so that a thermal cycler is not required. As there is no time-loss for thermal changes, amplification efficiency is extremely high (Parida et al., 2004; Savan et al., 2005). LAMP for detection of the shrimp DNA virus white spot syndrome virus (WSSV) has been described by Kono et al. (2004). However, LAMP is also useful for RNA template detection by the use of reverse transcriptase together with DNA polymerase (Notomi et al., 2000; Whiting and Champoux, 1998). Analysis of LAMP products (amplicons) is carried out usually by agarose gel electrophoresis, followed by ethidium bromide staining. As a result, non-specific amplification products may cause false positive results. To help overcome this problem, the identity of LAMP products can be confirmed by restriction endonuclease digestion (Notomi et al., 2000) and hybridization with specific probes (Mori et al., 2006). In order to further simplify and speed up the total time for the LAMP-based assay, amplicon detection by a chromatographic, lateral flow dipstick (LFD) rather electrophoresis was successfully applied for Taura syndrome virus (TSV) (Kiatpathomchai et al., 2008). In that process, generic LFD strips (Milenia® GenLine HybriDetect) were used to detect biotinlabeled amplicons hybridized with an FITC-labeled DNA probe linked, in turn, to a gold-labeled anti-FITC antibody. The total assay interval was approximately 75 min, excluding RNA extraction time, and detection sensitivity was equivalent to other methods used
commonly for nested RT-PCR detection of TSV. The purpose of this study was to develop a similar RT-LAMP–LFD method for detection of IMNV. 2. Materials and methods 2.1. Shrimp samples Pacific whiteleg shrimp (P. vannamei) with opaque, whitish abdomens were obtained from a shrimp farm in the Situbondo District of East Java Province in Indonesia mid-June 2006. They were positive for IMNV using a newly developed RT-PCR detection method (Senapin et al., 2007) and the IQ2000 IMNV Detection and Prevention System kit (Farming IntelliGene Tech. Corp., Taiwan). Normal whiteleg shrimp were obtained from Samutsakorn province of Thailand. 2.2. RNA extraction Shrimp muscle tissue from the 6th abdominal segment was homogenized in TRIzol reagent (Invitrogen) and RNA was extracted following the manufacturer’s instructions. RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. 2.3. Primers for RT-LAMP RT-LAMP primers for IMNV were designed according to the published sequence of the RNA-dependent RNA polymerase (RdRp) gene of IMNV genome (Gen-Bank accession no. AY570982; Poulos et al., 2006) using Primer Explorer version 3 (http://primerexplorer.jp/lamp3.0.0/index.html). The details of the primers are given in Fig. 1 and Table 1. The normal primers and biotin-labeled FIP primer were synthesized by Bio Basic Inc., Canada.
T. Puthawibool et al. / Journal of Virological Methods 156 (2009) 27–31
29
Table 1 Primers and probe used for RT-LAMP of the RdRp gene of IMNV. Primer name
Genome position
Sequences 5 -3
IMNV-F3 IMNV-B3 IMNV-FIP
682-706 878-856 774-750/TTTT/707-724
IMNV-BIP
780-804/TTTT/854-835
FITC-Probe
727-747
GGT TTT AAA GAA TAC TTT GGA GTGA ACT TGA CTA ACT ACA ATT TCT CC TCC AGC TTG TAG TTT CTC AAT CAT TTT TTC GCT TGA CGA AAA ACC AG TGG ATT ACC ATT TGA CTA TGC ATC ATT TTA CTC TTC TCA CCA TTG TCTT CAA CAG CAT CAG AGA GAA ATT
2.4. Optimization of temperature for RT-LAMP To determine the optimum temperature for amplification, the RT-LAMP reactions were carried out at 60, 63 and 65 ◦ C for 1 h, followed by the analysis of the LAMP products by gel electrophoresis. The reaction mixture contained 2 M each of inner primers IMNV-FIP and IMNV-BIP, 0.2 M each of outer primers IMNV-F3 and IMNV-B3, 1.4 mM of dNTP mix (Promega, Madison, WI, USA), 0.4 M betaine (Sigma–Aldrich, St. Louis, MO, USA), 6 mM MgSO4 , 8 U of Bst DNA polymerase (large fragment; New England Biolabs Inc., Beverly, MA, USA), 1× of the supplied buffer, 0.25 U of AMV Reverse transcriptase (Promega) and 2 ng of RNA extracted from IMNV infected shrimp in a final volume of 25 l. 2.5. Biotin-labeling RT-LAMP conditions The biotin-labeling RT-LAMP reactions were carried out at 63 ◦ C for 1 h. The reaction mixture contained 2 M of the biotin-labeled primer FIP, 2 M of inner primer IMNV-BIP, 0.2 M each of outer primers IMNV-F3 and IMNV-B3, 1.4 mM of dNTP mix (Promega, Madison, WI, USA), 0.4 M betaine (Sigma–Aldrich, St. Louis, MO, USA), 6 mM MgSO4 , 8 U of Bst DNA polymerase (large fragment; New England Biolabs Inc., Beverly, MA, USA), 1× of the supplied buffer, 0.25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega) and 2 l of varying amounts of RNA template in a final volume of 25 l.
Fig. 2. Result of triplicate optimization for RT-LAMP conditions at various temperatures using 2 ng of RNA extracted from IMNV-infected shrimp. Lane M = molecular marker; lane N = no-template control.
odon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV), gill associated virus (GAV). Healthy shrimp DNA was used as a control template. The biotin-labeled RT-LAMP products were analyzed by 2% agarose gel electrophoresis and by LFD. 2.9. Nested RT-PCR for IMNV detection Ten-fold serial dilutions of 100 ng/l of total RNA extracted from IMNV-infected shrimp were used as the template (2 l) for nested RT-PCR detection of IMNV using the IQ2000TM Detection and Prevention System (Farming IntelliGene Technology Corporation) according to the manufacturer’s protocol and using a method that targeted the RNA-dependent RNA polymerase (RdRp) gene of IMNV (Senapin et al., 2007). The nested PCR products were detected by 2% agarose gel electrophoresis followed by ethidium bromide staining and visualization on a UV transluminator.
2.6. Lateral flow dipstick assay 3. Results A DNA probe was designed from the IMNV sequence between F2 and F1c regions (Fig. 1 and Table 1). The DNA probe labeled with FITC at 5 end was synthesized by Bio Basic Inc., Canada. Twenty picomole of the FITC labeled probe were added to the RT-LAMP products as recommended by previous reports (Kiatpathomchai et al., 2008; Nimitphak et al., 2008). After hybridization at 63 ◦ C for 5 min, 8 l of the hybridized product were added to 150 l of the assay buffer in a new tube. Finally, the LFD strip was dipped into the mixture and waited for 5 min. 2.7. Sensitivity of RT-LAMP by gel electrophoresis and LFD Ten-fold serial dilutions (10−2 to 10−6 ) of 100 ng/l of total RNA extracted from IMNV-infected shrimp were used as the template (2 l) for biotin labeling RT-LAMP following optimized conditions. The products were analyzed by 2% agarose gel electrophoresis and by LFD as described above. 2.8. Specificity of RT-LAMP detection The specificity of RT-LAMP primers was examined using 200 ng of total RNA/DNA extracted from shrimp infected with other viruses commonly found in Southeast Asia. These included infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. mon-
3.1. Optimization of reaction temperature for IMNV detection When the RT-LAMP was carried out at 60, 63 and 65 ◦ C for 60 min with 2 ng of RNA template extracted from IMNV infected samples (Fig. 2), all three temperatures tested gave amplicons, but the clearest and strongest bands were obtained at 63 ◦ C. Thus, 63 ◦ C was chosen as the standard for all subsequent tests. 3.2. Comparison of RT-LAMP and nested RT-PCR sensitivity with gel electrophoresis Using equivalent quantities of RNA extracted from IMNVinfected shrimp as templates (2 l) at various dilutions, detection limits for RT-LAMP and nested RT-PCR were found to be comparable at 10−4 dilution for nested RT-PCR based on the RdRp region (Senapin et al., 2007) (Fig. 3(A)), nested RT-PCR using the IQ2000TM Detection and Prevention System (Fig. 3(B)) and RT-LAMP using the optimized temperature (Fig. 3(C)). 3.3. Sensitivity of the combined RT-LAMP and LFD The RT-LAMP–LFD combination for IMNV detection revealed a limit of detection at 10−4 dilution of total RNA template from IMNV-infected shrimp (Fig. 3(D)). This was equivalent to the
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T. Puthawibool et al. / Journal of Virological Methods 156 (2009) 27–31
Fig. 4. Specificity test results of the RT-LAMP method for IMNV using 200 ng each of RNA/DNA templates and detection by gel electrophoresis (A) or by LFD (B). Lane M = molecular marker; lane N = no-template control; lane 1 = RNA template from IMNV-infected P. vannamei; lane 2 = RNA template from MrNV-infected M. rosenbergii; lane 3 = RNA template from TSV-infected P. vannamei; lane 4 = RNA template from GAV-infected P. monodon; lane 5 = RNA template from YHV-infected P. monodon; lane 6: DNA template from HPV-infected P. monodon; lane 7 = DNA template from MBV-infected P. monodon; lane 8 = DNA template from WSSV-infected P. monodon; lane 9 = DNA template from IHHNV-infected P. monodon; lane 10 = RNA template from healthy P. vannamei.
Fig. 3. Sensitivity test results for detection of IMNV using 100 ng (lane U) and 10−1 to 10−6 dilutions of template RNA extracted from an IMNV-infected shrimp sample by RdRp-based nested RT-PCR (A), nested RT-PCR using the IQ2000TM IMNV Detection and Prevention System (B), RT-LAMP (C) and RT-LAMP–LFD (D). Lane M = molecular marker; lane P = kit positive control; lane N1 = 100 ng RNA template from normal shrimp; lane N2 = no-template control.
detection limit for LAMP or nested RT-PCR methods followed by electrophoresis, as described above. This equivalence of methods was also similar to results previously reported for the combination of LAMP–LFD for hepatopancreatic parvovirus (HPV) detection in the black tiger shrimp Penaeus monodon (Nimitphak et al., 2008). 3.4. Specificity of LAMP detection by gel electrophoresis and LFD Cross-amplification tests using 200 ng each of nucleic acid extracts from healthy P. vannamei and from shrimp infected with other viruses (i.e., DNA viruses IHHNV, MBV, PmDNV, WSSV and RNA viruses YHV, TSV, MrNV, GAV) all gave negative results with our RT-LAMP method followed by either electrophoresis (Fig. 4(A)) or LFD (Fig. 4(B)). 4. Discussion An RT-LAMP diagnostic method that targeted a 197 bp sequence of the RdRp gene was successfully developed for the detection of IMNV in shrimp. The RT-LAMP carried out at 63 ◦ C for 60 min was
faster than typical nested RT-PCR methods that require 30–45 min for an RT reaction and 2–3 h for PCR cycling. Sensitivity of the method was comparable to that of common nested RT-PCR methods for IMNV, similar to the report of Kono et al. (2004) showing that LAMP detection sensitivity was comparable or superior to that of nested PCR. For specificity test, there was no cross-reaction with other shrimp viruses commonly found in Southeast Asia. In addition, no positive result was obtained with DNA extracts from healthy shrimp. This indicates that the RT-LAMP method was specific for IMNV. Combining the RT-LAMP protocol with LFD detection of amplicons reduced the time and complication associated with usual detection by electrophoresis, and it resulted in a total analysis time (excluding the RNA extraction step) to less than 75 min. The high sensitivity and specificity, the relatively short analysis time and the use of relatively inexpensive equipment are key advantages of the RT-LAMP–LFD method. In addition, the LFD detection step confirms the identity of the specific amplicon by hybridization and avoids the use carcinogens such as ethidium bromide. The test platform can be adapted easily for rapid detection of other shrimp infectious agents simply by designing appropriate sets of LAMP primers and specific FITC probes to be used with the generic LFD employed. Since the cost for RT-LAMP–LFD detection is comparable with that for standard nested RT-PCR followed by electrophoresis, it constitutes a highly sensitive, safe and rapid alternative for IMNV detection. Furthermore, field samples from Indonesia were examined by RT-LAMP–LFD using RNA extracts by one-step guanidinium thiocyanate (GuSCN) homogenization method described by Teng et al. (2007). It was found that the GuSCN RNA extraction combined with RT-LAMP–LFD gave the same results when compared with that of TRIzol extracts (data not shown). The equal sensitivity we found supports the idea of previous report that the GuSCN RNA extraction combined with RT-LAMP protocol has the potential for further development for diagnosis of diseases in the field.
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Acknowledgements This study was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Teeranart Puthawibool has been supported by The Higher Education Development Project-Agricultural Biotechnology Consortium (ADB). References Briggs, M.R.P., Funge-Smith, S.J., Subasinghe, R., Phillips, M., 2004. Introductions and Movement of Penaeus vannamei and Penaeus stylirostris in Asia and the Pacific. RAP Publication 2004/10. FAO Regional Office for Asia and the Pacific, Bangkok, Thailand. Kiatpathomchai, W., Jaroenram, W., Arunrut, N., Jitrapakdee, S., Flegel, T.W., 2008. Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J. Virol. Methods 153, 214–217. Kono, T., Savan, R., Sakai, M., Itami, T., 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 115, 59–65. Lightner, D.V., Pantoja, C.R., Poulos, B.T., Tang, K.F.J., Redman, R.M, Andrade, T.P.D., Bonami, J.R., 2004. Infectious Myonecrosis: New Disease in Pacific White Shrimp. The Advocate. Global Aquaculture Alliance. October 2004. Mori, Y., Hirano, T., Notomi, T., 2006. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol. 6, 3. Nimitphak, T., Kiatpathomchai, W., Flegel, T.W., 2008. Shrimp hepatopancreatic parvovirus (HPV) detection by combining loop-mediated isothermal amplification with a lateral flow dipstick (LAMP-LFD). J. Virol. Methods 154, 56–60.
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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 Res. 28, e63. Parida, M., Posadas, G., Inoue, S., Hasebe, F., Morita, K., 2004. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J. Clin. Microbiol. 42, 257–263. Poulos, B.T., Tang, K.F.J., Pantoja, C.R., Bonami, J.B., Lightner, D.V., 2006. Purification and characterization of infectious myonecrosis virus of penaeid shrimp. J. Gen. Virol. 87, 987–996. Senapin, S., Phewsaiya, K., Briggs, M., Flegel, T.W., 2007. Outbreaks of infectious myonecrosis virus (IMNV) in Indonesia confirmed by genome sequencing and use of an alternative RT-PCR detection method. Aquaculture 266, 32– 38. Savan, R., Kono, T., Itami, T., Sakai, M., 2005. Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens. J. Fish. Dis. 28, 573–581. Tang, K.F., Pantoja, C.R., Poulos, B.T., Redman, R.M., Lightner, D.V., 2005. In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV). Dis. Aquat. Org. 63, 261–265. Teng, P.H., Chen, C.L., Sung, P.F., Lee, F.C., Ou, B.R., Lee, P.Y., 2007. Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot-blot hybridization. J. Virol. Methods 146, 317–326. Whiting, S.H., Champoux, J.J., 1998. Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase: mechanistic implications. J. Mol. Biol. 8 (278), 559–577.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick Teeranart Puthawibool a,b , Saengchan Senapin a,c , Wansika Kiatpathomchai a,c,∗ , Timothy W. Flegel a,b a
CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Dept. Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand c National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand b
a b s t r a c t Article history: Received 18 August 2008 Received in revised form 15 October 2008 Accepted 20 October 2008 Available online 6 December 2008 Keywords: IMNV Infectious myonecrosis virus Penaeus vannamei PCR Loop-mediated isothermal amplification (LAMP)
Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75 min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP–LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV). © 2008 Elsevier B.V. All rights reserved.
1. Introduction The Pacific whiteleg shrimp Penaeus (Litopenaeus) vannamei is a non-native (exotic) penaeid shrimp species to Brazil. It was imported for commercial shrimp culture beginning in 1983 but was not cultivated there on a large scale until after 1995 (Briggs et al., 2004). Disease outbreaks due to infectious myonecrosis (IMN) were first reported in farmed Pacific whiteleg shrimp from Brazil in 2004. In P. vannamei, striated muscles are the principal target organ, but gills and lymphoid organ can also be affected (Lightner et al., 2004). Gross signs of IMN-infected shrimp included necrosis of striated
∗ Corresponding author at: CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: +66 2 2015878; fax: +66 2 3547344. E-mail address: [email protected] (W. Kiatpathomchai). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.018
muscles, primarily in the distal abdominal segments and the tail fan, and the appearance of white discoloration of the affected muscle similar to the color of cooked shrimp. Hatchery brood, larvae and all sizes of shrimp could be affected. The causative agent of IMN (infectious myonecrosis virus or IMNV) was described subsequently in 2006 (Poulos et al., 2006). It is a non-enveloped, icosahedral virus of 40 nm in diameter. The genome consists of double-stranded RNA of 7560 nucleotides containing two open reading frames, designated ORF1 and ORF2 (Lightner et al., 2004; Poulos et al., 2006). ORF1 encodes a putative RNA-binding protein and a capsid protein and ORF2 codes for a putative RNA dependent RNA polymerase (RdRp). The virus has been classified tentatively in the family Totiviridae. Apart from P. vannamei infected naturally, experimental infections have been achieved with Penaeus (Farfantepenaeus) subtiltis, Penaeus monodon and Penaeus (Litopenaeus) stylirostris (Lightner et al., 2004; Tang et al., 2005).
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T. Puthawibool et al. / Journal of Virological Methods 156 (2009) 27–31
Fig. 1. Nucleotide sequence of IMNV RdRp gene (GenBank accession number: AY570982; Poulos et al., 2006). The sequences used to design primers F3 and B3 are shown by underlined sequences. The FIP (F1c/TTTT/F2) and BIP (B1c/TTTT/B2) inner primers are shown by shaded boxes and arrows. The FITC-labeled probe sequence is shown in italic and underlined sequence.
Loop-mediated isothermal amplification (LAMP) allows amplification of DNA with high specificity, sensitivity and rapidity under isothermal conditions. LAMP, described originally by Notomi et al. (2000), can amplify target nucleic acid to 109 copies at 60–65 ◦ C within 1 h. The method relies on autocycling strand displacement DNA synthesis by the Bst DNA polymerase large fragment, a DNA polymerase with high strand displacement activity, and a set of two inner primers and two outer primers. LAMP is highly specific for the target sequence because of the recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences in the later stages of the LAMP reaction. As the reaction is conducted under isothermal conditions, it can be carried out with a simple and inexpensive water bath so that a thermal cycler is not required. As there is no time-loss for thermal changes, amplification efficiency is extremely high (Parida et al., 2004; Savan et al., 2005). LAMP for detection of the shrimp DNA virus white spot syndrome virus (WSSV) has been described by Kono et al. (2004). However, LAMP is also useful for RNA template detection by the use of reverse transcriptase together with DNA polymerase (Notomi et al., 2000; Whiting and Champoux, 1998). Analysis of LAMP products (amplicons) is carried out usually by agarose gel electrophoresis, followed by ethidium bromide staining. As a result, non-specific amplification products may cause false positive results. To help overcome this problem, the identity of LAMP products can be confirmed by restriction endonuclease digestion (Notomi et al., 2000) and hybridization with specific probes (Mori et al., 2006). In order to further simplify and speed up the total time for the LAMP-based assay, amplicon detection by a chromatographic, lateral flow dipstick (LFD) rather electrophoresis was successfully applied for Taura syndrome virus (TSV) (Kiatpathomchai et al., 2008). In that process, generic LFD strips (Milenia® GenLine HybriDetect) were used to detect biotinlabeled amplicons hybridized with an FITC-labeled DNA probe linked, in turn, to a gold-labeled anti-FITC antibody. The total assay interval was approximately 75 min, excluding RNA extraction time, and detection sensitivity was equivalent to other methods used
commonly for nested RT-PCR detection of TSV. The purpose of this study was to develop a similar RT-LAMP–LFD method for detection of IMNV. 2. Materials and methods 2.1. Shrimp samples Pacific whiteleg shrimp (P. vannamei) with opaque, whitish abdomens were obtained from a shrimp farm in the Situbondo District of East Java Province in Indonesia mid-June 2006. They were positive for IMNV using a newly developed RT-PCR detection method (Senapin et al., 2007) and the IQ2000 IMNV Detection and Prevention System kit (Farming IntelliGene Tech. Corp., Taiwan). Normal whiteleg shrimp were obtained from Samutsakorn province of Thailand. 2.2. RNA extraction Shrimp muscle tissue from the 6th abdominal segment was homogenized in TRIzol reagent (Invitrogen) and RNA was extracted following the manufacturer’s instructions. RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. 2.3. Primers for RT-LAMP RT-LAMP primers for IMNV were designed according to the published sequence of the RNA-dependent RNA polymerase (RdRp) gene of IMNV genome (Gen-Bank accession no. AY570982; Poulos et al., 2006) using Primer Explorer version 3 (http://primerexplorer.jp/lamp3.0.0/index.html). The details of the primers are given in Fig. 1 and Table 1. The normal primers and biotin-labeled FIP primer were synthesized by Bio Basic Inc., Canada.
T. Puthawibool et al. / Journal of Virological Methods 156 (2009) 27–31
29
Table 1 Primers and probe used for RT-LAMP of the RdRp gene of IMNV. Primer name
Genome position
Sequences 5 -3
IMNV-F3 IMNV-B3 IMNV-FIP
682-706 878-856 774-750/TTTT/707-724
IMNV-BIP
780-804/TTTT/854-835
FITC-Probe
727-747
GGT TTT AAA GAA TAC TTT GGA GTGA ACT TGA CTA ACT ACA ATT TCT CC TCC AGC TTG TAG TTT CTC AAT CAT TTT TTC GCT TGA CGA AAA ACC AG TGG ATT ACC ATT TGA CTA TGC ATC ATT TTA CTC TTC TCA CCA TTG TCTT CAA CAG CAT CAG AGA GAA ATT
2.4. Optimization of temperature for RT-LAMP To determine the optimum temperature for amplification, the RT-LAMP reactions were carried out at 60, 63 and 65 ◦ C for 1 h, followed by the analysis of the LAMP products by gel electrophoresis. The reaction mixture contained 2 M each of inner primers IMNV-FIP and IMNV-BIP, 0.2 M each of outer primers IMNV-F3 and IMNV-B3, 1.4 mM of dNTP mix (Promega, Madison, WI, USA), 0.4 M betaine (Sigma–Aldrich, St. Louis, MO, USA), 6 mM MgSO4 , 8 U of Bst DNA polymerase (large fragment; New England Biolabs Inc., Beverly, MA, USA), 1× of the supplied buffer, 0.25 U of AMV Reverse transcriptase (Promega) and 2 ng of RNA extracted from IMNV infected shrimp in a final volume of 25 l. 2.5. Biotin-labeling RT-LAMP conditions The biotin-labeling RT-LAMP reactions were carried out at 63 ◦ C for 1 h. The reaction mixture contained 2 M of the biotin-labeled primer FIP, 2 M of inner primer IMNV-BIP, 0.2 M each of outer primers IMNV-F3 and IMNV-B3, 1.4 mM of dNTP mix (Promega, Madison, WI, USA), 0.4 M betaine (Sigma–Aldrich, St. Louis, MO, USA), 6 mM MgSO4 , 8 U of Bst DNA polymerase (large fragment; New England Biolabs Inc., Beverly, MA, USA), 1× of the supplied buffer, 0.25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega) and 2 l of varying amounts of RNA template in a final volume of 25 l.
Fig. 2. Result of triplicate optimization for RT-LAMP conditions at various temperatures using 2 ng of RNA extracted from IMNV-infected shrimp. Lane M = molecular marker; lane N = no-template control.
odon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV), gill associated virus (GAV). Healthy shrimp DNA was used as a control template. The biotin-labeled RT-LAMP products were analyzed by 2% agarose gel electrophoresis and by LFD. 2.9. Nested RT-PCR for IMNV detection Ten-fold serial dilutions of 100 ng/l of total RNA extracted from IMNV-infected shrimp were used as the template (2 l) for nested RT-PCR detection of IMNV using the IQ2000TM Detection and Prevention System (Farming IntelliGene Technology Corporation) according to the manufacturer’s protocol and using a method that targeted the RNA-dependent RNA polymerase (RdRp) gene of IMNV (Senapin et al., 2007). The nested PCR products were detected by 2% agarose gel electrophoresis followed by ethidium bromide staining and visualization on a UV transluminator.
2.6. Lateral flow dipstick assay 3. Results A DNA probe was designed from the IMNV sequence between F2 and F1c regions (Fig. 1 and Table 1). The DNA probe labeled with FITC at 5 end was synthesized by Bio Basic Inc., Canada. Twenty picomole of the FITC labeled probe were added to the RT-LAMP products as recommended by previous reports (Kiatpathomchai et al., 2008; Nimitphak et al., 2008). After hybridization at 63 ◦ C for 5 min, 8 l of the hybridized product were added to 150 l of the assay buffer in a new tube. Finally, the LFD strip was dipped into the mixture and waited for 5 min. 2.7. Sensitivity of RT-LAMP by gel electrophoresis and LFD Ten-fold serial dilutions (10−2 to 10−6 ) of 100 ng/l of total RNA extracted from IMNV-infected shrimp were used as the template (2 l) for biotin labeling RT-LAMP following optimized conditions. The products were analyzed by 2% agarose gel electrophoresis and by LFD as described above. 2.8. Specificity of RT-LAMP detection The specificity of RT-LAMP primers was examined using 200 ng of total RNA/DNA extracted from shrimp infected with other viruses commonly found in Southeast Asia. These included infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. mon-
3.1. Optimization of reaction temperature for IMNV detection When the RT-LAMP was carried out at 60, 63 and 65 ◦ C for 60 min with 2 ng of RNA template extracted from IMNV infected samples (Fig. 2), all three temperatures tested gave amplicons, but the clearest and strongest bands were obtained at 63 ◦ C. Thus, 63 ◦ C was chosen as the standard for all subsequent tests. 3.2. Comparison of RT-LAMP and nested RT-PCR sensitivity with gel electrophoresis Using equivalent quantities of RNA extracted from IMNVinfected shrimp as templates (2 l) at various dilutions, detection limits for RT-LAMP and nested RT-PCR were found to be comparable at 10−4 dilution for nested RT-PCR based on the RdRp region (Senapin et al., 2007) (Fig. 3(A)), nested RT-PCR using the IQ2000TM Detection and Prevention System (Fig. 3(B)) and RT-LAMP using the optimized temperature (Fig. 3(C)). 3.3. Sensitivity of the combined RT-LAMP and LFD The RT-LAMP–LFD combination for IMNV detection revealed a limit of detection at 10−4 dilution of total RNA template from IMNV-infected shrimp (Fig. 3(D)). This was equivalent to the
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Fig. 4. Specificity test results of the RT-LAMP method for IMNV using 200 ng each of RNA/DNA templates and detection by gel electrophoresis (A) or by LFD (B). Lane M = molecular marker; lane N = no-template control; lane 1 = RNA template from IMNV-infected P. vannamei; lane 2 = RNA template from MrNV-infected M. rosenbergii; lane 3 = RNA template from TSV-infected P. vannamei; lane 4 = RNA template from GAV-infected P. monodon; lane 5 = RNA template from YHV-infected P. monodon; lane 6: DNA template from HPV-infected P. monodon; lane 7 = DNA template from MBV-infected P. monodon; lane 8 = DNA template from WSSV-infected P. monodon; lane 9 = DNA template from IHHNV-infected P. monodon; lane 10 = RNA template from healthy P. vannamei.
Fig. 3. Sensitivity test results for detection of IMNV using 100 ng (lane U) and 10−1 to 10−6 dilutions of template RNA extracted from an IMNV-infected shrimp sample by RdRp-based nested RT-PCR (A), nested RT-PCR using the IQ2000TM IMNV Detection and Prevention System (B), RT-LAMP (C) and RT-LAMP–LFD (D). Lane M = molecular marker; lane P = kit positive control; lane N1 = 100 ng RNA template from normal shrimp; lane N2 = no-template control.
detection limit for LAMP or nested RT-PCR methods followed by electrophoresis, as described above. This equivalence of methods was also similar to results previously reported for the combination of LAMP–LFD for hepatopancreatic parvovirus (HPV) detection in the black tiger shrimp Penaeus monodon (Nimitphak et al., 2008). 3.4. Specificity of LAMP detection by gel electrophoresis and LFD Cross-amplification tests using 200 ng each of nucleic acid extracts from healthy P. vannamei and from shrimp infected with other viruses (i.e., DNA viruses IHHNV, MBV, PmDNV, WSSV and RNA viruses YHV, TSV, MrNV, GAV) all gave negative results with our RT-LAMP method followed by either electrophoresis (Fig. 4(A)) or LFD (Fig. 4(B)). 4. Discussion An RT-LAMP diagnostic method that targeted a 197 bp sequence of the RdRp gene was successfully developed for the detection of IMNV in shrimp. The RT-LAMP carried out at 63 ◦ C for 60 min was
faster than typical nested RT-PCR methods that require 30–45 min for an RT reaction and 2–3 h for PCR cycling. Sensitivity of the method was comparable to that of common nested RT-PCR methods for IMNV, similar to the report of Kono et al. (2004) showing that LAMP detection sensitivity was comparable or superior to that of nested PCR. For specificity test, there was no cross-reaction with other shrimp viruses commonly found in Southeast Asia. In addition, no positive result was obtained with DNA extracts from healthy shrimp. This indicates that the RT-LAMP method was specific for IMNV. Combining the RT-LAMP protocol with LFD detection of amplicons reduced the time and complication associated with usual detection by electrophoresis, and it resulted in a total analysis time (excluding the RNA extraction step) to less than 75 min. The high sensitivity and specificity, the relatively short analysis time and the use of relatively inexpensive equipment are key advantages of the RT-LAMP–LFD method. In addition, the LFD detection step confirms the identity of the specific amplicon by hybridization and avoids the use carcinogens such as ethidium bromide. The test platform can be adapted easily for rapid detection of other shrimp infectious agents simply by designing appropriate sets of LAMP primers and specific FITC probes to be used with the generic LFD employed. Since the cost for RT-LAMP–LFD detection is comparable with that for standard nested RT-PCR followed by electrophoresis, it constitutes a highly sensitive, safe and rapid alternative for IMNV detection. Furthermore, field samples from Indonesia were examined by RT-LAMP–LFD using RNA extracts by one-step guanidinium thiocyanate (GuSCN) homogenization method described by Teng et al. (2007). It was found that the GuSCN RNA extraction combined with RT-LAMP–LFD gave the same results when compared with that of TRIzol extracts (data not shown). The equal sensitivity we found supports the idea of previous report that the GuSCN RNA extraction combined with RT-LAMP protocol has the potential for further development for diagnosis of diseases in the field.
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Acknowledgements This study was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Teeranart Puthawibool has been supported by The Higher Education Development Project-Agricultural Biotechnology Consortium (ADB). References Briggs, M.R.P., Funge-Smith, S.J., Subasinghe, R., Phillips, M., 2004. Introductions and Movement of Penaeus vannamei and Penaeus stylirostris in Asia and the Pacific. RAP Publication 2004/10. FAO Regional Office for Asia and the Pacific, Bangkok, Thailand. Kiatpathomchai, W., Jaroenram, W., Arunrut, N., Jitrapakdee, S., Flegel, T.W., 2008. Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J. Virol. Methods 153, 214–217. Kono, T., Savan, R., Sakai, M., Itami, T., 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 115, 59–65. Lightner, D.V., Pantoja, C.R., Poulos, B.T., Tang, K.F.J., Redman, R.M, Andrade, T.P.D., Bonami, J.R., 2004. Infectious Myonecrosis: New Disease in Pacific White Shrimp. The Advocate. Global Aquaculture Alliance. October 2004. Mori, Y., Hirano, T., Notomi, T., 2006. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol. 6, 3. Nimitphak, T., Kiatpathomchai, W., Flegel, T.W., 2008. Shrimp hepatopancreatic parvovirus (HPV) detection by combining loop-mediated isothermal amplification with a lateral flow dipstick (LAMP-LFD). J. Virol. Methods 154, 56–60.
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