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Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers
Journal of Virological Methods 159 (2009) 206–210
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers Chia-Hsuan Sung a , Jenn-Kan Lu b,∗ a
Animal Health Inspection Division, Bureau of Animal and Plant Health Inspection and Quarantine, 3F, No. 51, Sec. 2, Chungcing S. Road, Taipei City 100, Taiwan Department of Aquaculture, National Taiwan Ocean University, No. 2, Beining Road, Keelung City 202, Taiwan
b
a b s t r a c t Article history: Received 14 November 2008 Received in revised form 23 March 2009 Accepted 24 March 2009 Available online 2 April 2009 Keywords: Nervous necrosis virus NNV Reverse transcription loop-mediated isothermal amplification RT-LAMP Epinephelus coioides Epinephelus lanceolatus
The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 ◦ C for 60 min. The detection limit of RT-LAMP is 10−6 NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2 h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction Nervous necrosis virus (NNV) is a member of the genus Betanodavirus, within the family Nodaviridae. It is a major cause of mortality in larvae and juveniles of farmed marine fish throughout the world (Shieh and Chi, 2005). NNV infection causes viral nervous necrosis or viral encephalopathy and retinopathy, which is characterized by necrosis and vacuolation of the brain and retina, abnormal swimming behavior and dark body colouring (Mori et al., 1992; Comps et al., 1994; Chi et al., 1997). Nodaviruses are small, non-enveloped viruses with a genome comprising two single stranded, positive-sense RNA molecules (RNA1 and RNA2). RNA1 encodes an RNA-dependent RNA polymerase, while the smaller RNA2 encodes the capsid protein (Shieh and Chi, 2005). Several methods have been developed for detection of piscine nodavirus, including immunohistochemistry (Le Breton et al., 1997), indirect enzyme-linked immunosorbent assay (ELISA) (Arimoto et al., 1992), in situ hybridization (Comps et al., 1996), reverse transcription-polymerase chain reaction (RT-PCR) (Nishizawa et al., 1995), fish cell line (Chi et al., 1999), the microfluidic chip method, nucleic acid sequence amplification (NASBA)
∗ Corresponding author. Tel.: +886 2 24622192x5236; fax: +886 2 24631663. E-mail address: [email protected] (J.-K. Lu).
(Starkey et al., 2004), antigen capture ELISA (Shieh and Chi, 2005) and real-time PCR (Dalla Valle et al., 2005). Most of these methods are time-consuming and require expensive instruments and reagents. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and specific nucleic acid amplification method that can synthesize rapidly large amounts of DNA under isothermal conditions (Notomi et al., 2000). The LAMP reaction uses four primers that recognize six distinct sequences in the target gene and utilize the auto-cycling strand displacement activity of Bst DNA polymerase to synthesize DNA (Notomi et al., 2000). There are several reports of using the LAMP method for fish and shellfish pathogens (Savan et al., 2005). RNA templates can also be detected using reverse transcription-LAMP (RT-LAMP), in which the avian myeloblastosis virus (AMV) reverse transcriptase is added, allowing RT and DNA amplification to occur in one step under isothermal conditions (Tomita et al., 2008). The RT-LAMP has been used to detect RNA virus pathogens: viral hemorrhagic septicaemia virus (VHS) (Soliman and El-Matbouli, 2006), infectious haematopoietic necrosis virus and infectious salmon anaemia virus (Millard et al., 2006) and Taura syndrome virus (TSV) (Kiatpathomchai et al., 2007). This report describes the development of an RT-LAMP assay to detect NNV in groupers. The sensitivity and specificity of this assay and of conventional one-step RT-PCR and nested RT-PCR detection
0166-0934/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.03.022
C.-H. Sung, J.-K. Lu / Journal of Virological Methods 159 (2009) 206–210
methods were compared. RNA was isolated by the non-centrifugal method also used for the RT-LAMP assay. This study represents the first report of NNV detection in groupers using the RT-LAMP method. 2. Materials and methods 2.1. Groupers
207
2.5. Sensitivity of the RT-LAMP in detection of NNV Ten-fold serial dilutions of total RNA extracted from a grouper infected by NNV were used as template in the RT-LAMP the reaction program was 65 ◦ C for 60 min and 80 ◦ C for 2 min. The detection limit of RT-LAMP was compared with that of the conventional onestep RT-PCR and nested RT-PCR. 2.6. One-step RT-PCR for NNV detection
The orange-spotted grouper (Epinephelus coioides) and king grouper (Epinephelus lanceolatus) with or without NNV clinical signs were collected from cultivation farms in Kaohsiung, Taiwan. Grouper brain and other tissues were collected for RNA extraction. 2.2. RNA extraction Total RNA was extracted from the brain using Trizol® reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s recommendation. Homogenization of grouper tissue with 1000 l Trizol reagent and 200 l chloroform mixed well, incubated 5 min at room temperature and centrifuged at 12,000 rpm for 15 min at 4 ◦ C. The aqueous phase was transferred to a fresh tube and added 500 l of isopropyl alcohol for RNA precipitation. After washing with 100% and 75% ethanol, RNA was dissolved in DEPC-treated ddH2 O and stored at −80 ◦ C. For simple RNA isolation, we used the QuickExtractTM FFPE RNA extraction kit (EPICENTRE Biotechnologies, Madison, USA). 10–50 mg of tissue was used for 100 l of the QuickExtract FFPE RNA extraction solution. Heat the tube for 30 min at 56 ◦ C, and then for 2 min at 98 ◦ C. 2 ml of buffer 1 and 2 ml of RNase-free DNase I to the extracted RNA. The sample was mixed and incubated the tube at 37 ◦ C for 10 min. 2 ml of stop solution to the tube and incubated at 65 ◦ C for 10 min. RNA was stored at −80 ◦ C. 2.3. Design of primers for RT-LAMP NNV RT-LAMP primers targeting the NNV coat protein mRNA (GenBank accession no. AY744705) were designed using Primer Explorer Software (http://primerexplorer.jp/elamp4.0.0/index. html). Primer sequences are shown in Table 1.
Ten-fold serial dilutions of total RNA extracted from NNVinfected grouper were then amplified by one-step RT-PCR, specific primers according to the OIE (World organisation for animal health) manual of diagnostic tests for aquatic animals 2006. The RT-PCR was carried out in a total volume of 50 l reaction mixture with a QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The program was: 50 ◦ C for 30 min, 15 min initial denaturation at 95 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 30 s, and elongation at 72 ◦ C for 50 s, followed by a 7 min extension at 72 ◦ C and cooling to 4 ◦ C. The products were electrophoresed and analyzed on a 1.6% agarose gel. 2.7. Nested RT-PCR for NNV detection The first step RT-PCR was carried out in a total volume of 50 l reaction mixture with F1 (5 -GGATTTGGACGTGCGACCAA-3 )/R3 (5 -CGAGTCAACA CGGGTGAAGA-3 ) primer and QIAGEN OneStep RT-PCR Kit (QIAGEN, USA) according to the manufacturer’s instructions. The program was: 5 min initial denaturation at 94 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 30 s, and elongation at 72 ◦ C for 50 s, followed by a 7 min extension at 72 ◦ C and cooling to 4 ◦ C. Take 100 times dilution of first step RTPCR product 1 l as template for the second step PCR. Total 50 l hat contains 5 l of 10× PCR buffer with 200 mM Tris–HCl (pH 8.4), 500 mM KCl, 1.5 l of 50 mM MgCl2, 1 l of 10 mM dNTP mix, 1 l of 10 M F2 (5 -CGTGTCAGT GCTGTGTCGCT-3 ) and R3 primer, 36.3 l of sterile deionized water and 0.2 l of Taq DNA Polymerase. The PCR reaction program was followed by 30 cycles of 94 ◦ C for 30 s, 58 ◦ C for 30 s and 72 ◦ C for 30 s F2 and R3 primer for second step PCR, the final product was 427 bp of NNV coat protein gene as described in Nishizawa et al. (1995). The PCR products were electrophoresed and analyzed on a 1.6% agarose gel.
2.4. Determination of RT-LAMP reaction conditions 2.8. Specificity of RT-LAMP detection The RT-LAMP was performed in a 25 l reaction mixture consisting of 5 l of target RNA, 40 pmol each of the NNV-FIP and NNV-BIP primers, 5 pmol each of the NNV-F3 and NNV-B3 primers, 12.5 l of 2× reaction mix (supplied in the Loopamp RNA Amplification Kit, Eiken Chemical Co. Ltd., Japan) and 1 l enzyme mix containing Bst DNA polymerase and AMV reverse transcriptase. Amplification was carried out at 65 ◦ C for 10, 15, 20, 25, 30, 35, 40, 50 or 60 min. The reaction was then terminated by heating at 80 ◦ C for 2 min. Various reaction temperatures (55, 57.5, 60, 62.5 and 65 ◦ C) were also tested. The RT-LAMP products were then analyzed by electrophoresis on 1.6% agarose gel. The optimal reaction conditions were determined and used in all further experiments.
The specificity of NNV RT-LAMP primers was tested using 200 ng of total RNA/DNA extracted from the orange-spotted grouper iridovirus infected grouper, NNV-infected grouper, and healthy grouper brain tissue were used as the template. 3. Results 3.1. Optimization of the RT-LAMP reaction conditions The RT-LAMP method was carried out using NNV-infected grouper RNA as the template to determine the optimal temperature
Table 1 Detail of RT-LAMP primers designed for detection of NNV. Primer name
Position of coat protein gene
Length
Sequence
NNV-F3 NNV-B3 NNV-FIP NNV-BIP
467–485 662–681 494–513/TTTT/536–555 578–598/TTTT/634–651
19 nt 20 nt 44 nt 43 nt
5 -AGTCGTTGCCAAATGGTGG-3 5 -TGGTCTCTTCAGGTGTCTCA-3 5 -GACGCTGCTCCTTTCCCGACTTTTAACAGTCCGACCTCAGTACA-3 5 -ACTCCTGTGTGTCGGCAACAATTTTCGCTCAGTCGAACACTCC-3
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Fig. 1. The optimal condition of RT-LAMP method. (A) Reaction temperature. Lanes 1–5: RT-LAMP carried out at 55, 57.5, 60, 62.5 and 65 ◦ C, respectively. Lane M: 100 bp DNA ladder. (B) Reaction time. Lanes 1–9: RT-LAMP carried out for 10, 15, 20, 25, 30, 35, 40, 50 and 60 min, respectively. Lane M: 100 bp DNA ladder.
and reaction time. Fig. 1 shows that the RT-LAMP product was observed at 55, 57.5, 60, 62.5 and 65 ◦ C (Fig. 1A). However, 65 ◦ C was chosen as the optimal temperature as specificity of the reaction increases at higher temperatures. On the other hand, the RT-LAMP product detected the NNV-RNA as early as 25–30 min at 65 ◦ C (Fig. 1B). Although the RT-LAMP product could be detected at 25–30 min, the optimal reaction time was 60 min to ensure detection of a lower concentration of target template.
the manufacturer’s recommendation for the FFPE RNA extraction kit, the first 56 ◦ C incubation step was shortened from 30 min to 10 and 20 min, while the results of the RT-LAMP were the same (Fig. 5A). The RT-LAMP detection limit could reach 10−3 dilution of FFPE extracted RNA (Fig. 5B).
3.2. Comparison of sensitivity of the RT-LAMP and RT-PCR The detection limit of the RT-LAMP reaction was tested using 10fold serial dilutions of NNV-infected grouper RNA and compared with results from the RT-PCR method. The detection limit of RTLMAP was 10−6 (Fig. 2). The detection limit of the one-step RT-PCR was 10−2 to 10−3 and the detection limit of nested RT-PCR was 10−5 (Fig. 2). The sensitivity of RT-LAMP for the detection of NNV was about 1000 times and 10 times more sensitive than the one-step RT-PCR and the nested RT-PCR respectively. 3.3. Specificity of the RT-LAMP detection The RT-LAMP amplified product was only detected in the NNVinfected orange-spotted grouper and in the king grouper when NNV-RNA was present (Fig. 3). There was no amplification of orange-spotted grouper iridovirus infected grouper DNA or healthy grouper genomic DNA and RNA. No cross-reaction was observed in this study. 3.4. Detection of NNV in grouper tissues by LAMP Total RNA was isolated from various tissues, including brain, eye, gill, heart, liver, spleen and muscle, and the NNV RNA was detected in the brain, eye, gill, heart, liver and muscle by RT-LAMP (Fig. 4). 3.5. Rapid extraction of RNA for RT-LAMP Brain tissue RNA was extracted by an FFPE RNA extraction kit; the RNA was also used for the RT-LAMP assay. In comparison with
Fig. 2. Sensitivity of RT-LAMP, one-step RT-PCR and nested PCR. (A) RT-LAMP products; (B) one-step RT-PCR; (C) nested RT-PCR. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–8: 10−1 to 10−7 dilutions of RNA from NNV-infected grouper brain tissue.
C.-H. Sung, J.-K. Lu / Journal of Virological Methods 159 (2009) 206–210
209
Fig. 5. Rapid extraction RNA for RT-LAMP. (A) RNA from rapid extraction by different incubation time. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–4: 10, 20 and 30 min incubation time; (B) different dilutions of RNA for RT-LAMP. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–4: 10−1 to 10−3 dilutions of RNA. Fig. 3. Specificity of the RT-LAMP reaction. Lane M: 100 bp DNA ladder, lane 1: negative control, lane 2: healthy grouper genomic DNA, lane 3: orange-spotted grouper iridovirus infected grouper genomic DNA, lane 4: RNA from NNV-infected grouper, lane 5: RNA from NNV-infected orange-spotted grouper and lane 6: RNA from NNVinfected king grouper. 200 ng DNA/RNA was used for RT-LAMP.
4. Discussion Viral nervous necrosis is a serious viral disease in the grouper cultivation industry in Taiwan. Many stages in their life cycle can be infected with NNV, especially in hatchery-reared larvae and juve-
Fig. 4. Detection of NNV-RNA from various grouper tissue by RT-LAMP. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–8: RNA from brain, eye, gill, heart, spleen, liver and muscle, respectively.
niles (Chi et al., 2003). Once infected with NNV, the grouper may become a carrier and facilitate further spread of the virus. Thus, an effective diagnostic method is required for disease prevention and control. The current “gold standard method” of detection of NNV uses conventional RT-PCR (Nishizawa et al., 1995) but there are still some disadvantages, such as the need to use a thermal cycler for the reaction, the multiple and complex operation process and low amplificational efficiency (Mori et al., 2001; Tomita et al., 2008). In this study, the RT-LAMP method was carried out for the detection of NNV in groupers. Two sets of primers used were able to amplify a 215 bp sequence from the coat protein gene of NNV. The optimal condition for the detection of NNV RNA was 65 ◦ C for 60 min. The amplified product was, however, detected after 25–30 min and this suggests that the detection of NNV is possible in a very short period of time. However, for complete amplification, 60 min is considered an optimal reaction time (Kiatpathomchai et al., 2007). The RT-LAMP method used for the detection of NNV was shown to be highly sensitive; its detection limit was 10−6 of NNV-RNA. The sensitivity of RT-LAMP was about 1000 times more sensitive than one-step RT-PCR and 10 times more sensitive than nested RT-PCR, detection limits being 10−2 to 10−3 and 10−5 , respectively. One-step RT-PCR can complete all procedures in one step and in one tube, but the sensitivity is lower than the nested RT-PCR and RT-LAMP. Although the nested RT-PCR method can obtain higher sensitivity, it needs a two-step operation and takes another 2–3 h. In comparison with RT-LAMP, RT and DNA amplification can proceed under the same conditions within 1 h, and using only a one-step operation. This shows that RT-LAMP is a more sensitive, rapid and a convenient method. To simplify the operation of RNA extraction, we used the FFPE kit, which can complete all the steps in one tube without centrifugation. The RNA extracted by the FFEP kit could be used for the NNV RT-LAMP. According to the manufacturer’s recommendations, the first incubation time is 56 ◦ C for 30 min. In order to decrease the operation time, the first step incubation can be shortened to 20 or 10 min, and this does not influence the final result of the RT-LAMP assay. Thus, the RNA extraction can be finished within 30 min with-
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out centrifugation. With this combination of the FFEP and RT-LAMP, all operation processes can be completed within 2 h, from sample to result. The RT-LAMP assay developed was shown to be a rapid, sensitive and specific method for detecting NNV in grouper tissues. This method would be a useful tool for routine diagnosis and for large-scale screening in field studies. Acknowledgements This study was supported by Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ), Council of Agriculture, Executive Yuan of Taiwan. References Arimoto, M., Mushiake, K., Mizuta, Y., Nakai, T., Muroga, K., Furusawa, T., 1992. Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish Pathol. 27, 191–195. Chi, S., Lo, C.F., Kou, G., Chang, P., Peng, S., Chen, S., 1997. Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatcheryreared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel). J. Fish Dis. 20, 185–193. Chi, S.C., Lin, S.C., Su, H.M., Hu, W.W., 1999. Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae. Virus Res. 63, 107–114. Chi, S.C., Shieh, J.R., Lin, S.J., 2003. Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis. Aquat. Organ. 55, 221–228. Comps, M., Pepin, J., Bonami, J., 1994. Purification and characterization of two fish encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture 123, 1–10. Comps, M., Trindade, M., Delsert, C., 1996. Investigation of fish encephalitis viruses (FEV)expression in marine fishes using DIG-labelled probes. Aquaculture 143, 113–121. Dalla Valle, L., Toffolo, V., Lamprecht, M., Maltese, C., Bovo, G., Belvedere, P., Colombo, L., 2005. Development of a sensitive and quantitative diagnostic assay for fish
nervous necrosis virus based on two-target real-time PCR. Vet. Microbiol. 110, 167–179. Kiatpathomchai, W., Jareonram, W., Jitrapakdee, S., Flegel, T.W., 2007. Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification. J. Virol. Methods 146, 125–128. Le Breton, A., Grisez, L., Sweetman, J., Ollevier, F., 1997. Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax L. J. Fish Dis. 20, 145–151. Millard, P.J., Bickerstaff, L.E., LaPatra, S.E., Kim, C.H., 2006. Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification. J. Fish Dis. 29, 201–213. Mori, K., Nakai, T., Muroga, K., Arimoto, M., Mushiake, K., Furusawa, I., 1992. Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368–371. Mori, Y., Nagamine, K., Tomita, N., Notomi, T., 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289, 150–154. Nishizawa, T., Mori, K., Furuhashi, M., Nakai, T., Furusawa, I., Muroga, K., 1995. Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish. J. Gen. Virol. 76 (Pt 7), 1563–1569. 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. 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. Shieh, J.R., Chi, S.C., 2005. Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA. Dis. Aquat. Organ. 63, 53–60. Soliman, H., El-Matbouli, M., 2006. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet. Microbiol. 114, 205–213. Starkey, W.G., Millar, R.M., Jenkins, M.E., Ireland, J.H., Muir, K.F., Richards, R.H., 2004. Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA). Dis. Aquat. Organ. 59, 93–100. Tomita, N., Mori, Y., Kanda, H., Notomi, T., 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protoc. 3, 877–882.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers Chia-Hsuan Sung a , Jenn-Kan Lu b,∗ a
Animal Health Inspection Division, Bureau of Animal and Plant Health Inspection and Quarantine, 3F, No. 51, Sec. 2, Chungcing S. Road, Taipei City 100, Taiwan Department of Aquaculture, National Taiwan Ocean University, No. 2, Beining Road, Keelung City 202, Taiwan
b
a b s t r a c t Article history: Received 14 November 2008 Received in revised form 23 March 2009 Accepted 24 March 2009 Available online 2 April 2009 Keywords: Nervous necrosis virus NNV Reverse transcription loop-mediated isothermal amplification RT-LAMP Epinephelus coioides Epinephelus lanceolatus
The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 ◦ C for 60 min. The detection limit of RT-LAMP is 10−6 NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2 h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction Nervous necrosis virus (NNV) is a member of the genus Betanodavirus, within the family Nodaviridae. It is a major cause of mortality in larvae and juveniles of farmed marine fish throughout the world (Shieh and Chi, 2005). NNV infection causes viral nervous necrosis or viral encephalopathy and retinopathy, which is characterized by necrosis and vacuolation of the brain and retina, abnormal swimming behavior and dark body colouring (Mori et al., 1992; Comps et al., 1994; Chi et al., 1997). Nodaviruses are small, non-enveloped viruses with a genome comprising two single stranded, positive-sense RNA molecules (RNA1 and RNA2). RNA1 encodes an RNA-dependent RNA polymerase, while the smaller RNA2 encodes the capsid protein (Shieh and Chi, 2005). Several methods have been developed for detection of piscine nodavirus, including immunohistochemistry (Le Breton et al., 1997), indirect enzyme-linked immunosorbent assay (ELISA) (Arimoto et al., 1992), in situ hybridization (Comps et al., 1996), reverse transcription-polymerase chain reaction (RT-PCR) (Nishizawa et al., 1995), fish cell line (Chi et al., 1999), the microfluidic chip method, nucleic acid sequence amplification (NASBA)
∗ Corresponding author. Tel.: +886 2 24622192x5236; fax: +886 2 24631663. E-mail address: [email protected] (J.-K. Lu).
(Starkey et al., 2004), antigen capture ELISA (Shieh and Chi, 2005) and real-time PCR (Dalla Valle et al., 2005). Most of these methods are time-consuming and require expensive instruments and reagents. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and specific nucleic acid amplification method that can synthesize rapidly large amounts of DNA under isothermal conditions (Notomi et al., 2000). The LAMP reaction uses four primers that recognize six distinct sequences in the target gene and utilize the auto-cycling strand displacement activity of Bst DNA polymerase to synthesize DNA (Notomi et al., 2000). There are several reports of using the LAMP method for fish and shellfish pathogens (Savan et al., 2005). RNA templates can also be detected using reverse transcription-LAMP (RT-LAMP), in which the avian myeloblastosis virus (AMV) reverse transcriptase is added, allowing RT and DNA amplification to occur in one step under isothermal conditions (Tomita et al., 2008). The RT-LAMP has been used to detect RNA virus pathogens: viral hemorrhagic septicaemia virus (VHS) (Soliman and El-Matbouli, 2006), infectious haematopoietic necrosis virus and infectious salmon anaemia virus (Millard et al., 2006) and Taura syndrome virus (TSV) (Kiatpathomchai et al., 2007). This report describes the development of an RT-LAMP assay to detect NNV in groupers. The sensitivity and specificity of this assay and of conventional one-step RT-PCR and nested RT-PCR detection
0166-0934/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.03.022
C.-H. Sung, J.-K. Lu / Journal of Virological Methods 159 (2009) 206–210
methods were compared. RNA was isolated by the non-centrifugal method also used for the RT-LAMP assay. This study represents the first report of NNV detection in groupers using the RT-LAMP method. 2. Materials and methods 2.1. Groupers
207
2.5. Sensitivity of the RT-LAMP in detection of NNV Ten-fold serial dilutions of total RNA extracted from a grouper infected by NNV were used as template in the RT-LAMP the reaction program was 65 ◦ C for 60 min and 80 ◦ C for 2 min. The detection limit of RT-LAMP was compared with that of the conventional onestep RT-PCR and nested RT-PCR. 2.6. One-step RT-PCR for NNV detection
The orange-spotted grouper (Epinephelus coioides) and king grouper (Epinephelus lanceolatus) with or without NNV clinical signs were collected from cultivation farms in Kaohsiung, Taiwan. Grouper brain and other tissues were collected for RNA extraction. 2.2. RNA extraction Total RNA was extracted from the brain using Trizol® reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s recommendation. Homogenization of grouper tissue with 1000 l Trizol reagent and 200 l chloroform mixed well, incubated 5 min at room temperature and centrifuged at 12,000 rpm for 15 min at 4 ◦ C. The aqueous phase was transferred to a fresh tube and added 500 l of isopropyl alcohol for RNA precipitation. After washing with 100% and 75% ethanol, RNA was dissolved in DEPC-treated ddH2 O and stored at −80 ◦ C. For simple RNA isolation, we used the QuickExtractTM FFPE RNA extraction kit (EPICENTRE Biotechnologies, Madison, USA). 10–50 mg of tissue was used for 100 l of the QuickExtract FFPE RNA extraction solution. Heat the tube for 30 min at 56 ◦ C, and then for 2 min at 98 ◦ C. 2 ml of buffer 1 and 2 ml of RNase-free DNase I to the extracted RNA. The sample was mixed and incubated the tube at 37 ◦ C for 10 min. 2 ml of stop solution to the tube and incubated at 65 ◦ C for 10 min. RNA was stored at −80 ◦ C. 2.3. Design of primers for RT-LAMP NNV RT-LAMP primers targeting the NNV coat protein mRNA (GenBank accession no. AY744705) were designed using Primer Explorer Software (http://primerexplorer.jp/elamp4.0.0/index. html). Primer sequences are shown in Table 1.
Ten-fold serial dilutions of total RNA extracted from NNVinfected grouper were then amplified by one-step RT-PCR, specific primers according to the OIE (World organisation for animal health) manual of diagnostic tests for aquatic animals 2006. The RT-PCR was carried out in a total volume of 50 l reaction mixture with a QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The program was: 50 ◦ C for 30 min, 15 min initial denaturation at 95 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 30 s, and elongation at 72 ◦ C for 50 s, followed by a 7 min extension at 72 ◦ C and cooling to 4 ◦ C. The products were electrophoresed and analyzed on a 1.6% agarose gel. 2.7. Nested RT-PCR for NNV detection The first step RT-PCR was carried out in a total volume of 50 l reaction mixture with F1 (5 -GGATTTGGACGTGCGACCAA-3 )/R3 (5 -CGAGTCAACA CGGGTGAAGA-3 ) primer and QIAGEN OneStep RT-PCR Kit (QIAGEN, USA) according to the manufacturer’s instructions. The program was: 5 min initial denaturation at 94 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 58 ◦ C for 30 s, and elongation at 72 ◦ C for 50 s, followed by a 7 min extension at 72 ◦ C and cooling to 4 ◦ C. Take 100 times dilution of first step RTPCR product 1 l as template for the second step PCR. Total 50 l hat contains 5 l of 10× PCR buffer with 200 mM Tris–HCl (pH 8.4), 500 mM KCl, 1.5 l of 50 mM MgCl2, 1 l of 10 mM dNTP mix, 1 l of 10 M F2 (5 -CGTGTCAGT GCTGTGTCGCT-3 ) and R3 primer, 36.3 l of sterile deionized water and 0.2 l of Taq DNA Polymerase. The PCR reaction program was followed by 30 cycles of 94 ◦ C for 30 s, 58 ◦ C for 30 s and 72 ◦ C for 30 s F2 and R3 primer for second step PCR, the final product was 427 bp of NNV coat protein gene as described in Nishizawa et al. (1995). The PCR products were electrophoresed and analyzed on a 1.6% agarose gel.
2.4. Determination of RT-LAMP reaction conditions 2.8. Specificity of RT-LAMP detection The RT-LAMP was performed in a 25 l reaction mixture consisting of 5 l of target RNA, 40 pmol each of the NNV-FIP and NNV-BIP primers, 5 pmol each of the NNV-F3 and NNV-B3 primers, 12.5 l of 2× reaction mix (supplied in the Loopamp RNA Amplification Kit, Eiken Chemical Co. Ltd., Japan) and 1 l enzyme mix containing Bst DNA polymerase and AMV reverse transcriptase. Amplification was carried out at 65 ◦ C for 10, 15, 20, 25, 30, 35, 40, 50 or 60 min. The reaction was then terminated by heating at 80 ◦ C for 2 min. Various reaction temperatures (55, 57.5, 60, 62.5 and 65 ◦ C) were also tested. The RT-LAMP products were then analyzed by electrophoresis on 1.6% agarose gel. The optimal reaction conditions were determined and used in all further experiments.
The specificity of NNV RT-LAMP primers was tested using 200 ng of total RNA/DNA extracted from the orange-spotted grouper iridovirus infected grouper, NNV-infected grouper, and healthy grouper brain tissue were used as the template. 3. Results 3.1. Optimization of the RT-LAMP reaction conditions The RT-LAMP method was carried out using NNV-infected grouper RNA as the template to determine the optimal temperature
Table 1 Detail of RT-LAMP primers designed for detection of NNV. Primer name
Position of coat protein gene
Length
Sequence
NNV-F3 NNV-B3 NNV-FIP NNV-BIP
467–485 662–681 494–513/TTTT/536–555 578–598/TTTT/634–651
19 nt 20 nt 44 nt 43 nt
5 -AGTCGTTGCCAAATGGTGG-3 5 -TGGTCTCTTCAGGTGTCTCA-3 5 -GACGCTGCTCCTTTCCCGACTTTTAACAGTCCGACCTCAGTACA-3 5 -ACTCCTGTGTGTCGGCAACAATTTTCGCTCAGTCGAACACTCC-3
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Fig. 1. The optimal condition of RT-LAMP method. (A) Reaction temperature. Lanes 1–5: RT-LAMP carried out at 55, 57.5, 60, 62.5 and 65 ◦ C, respectively. Lane M: 100 bp DNA ladder. (B) Reaction time. Lanes 1–9: RT-LAMP carried out for 10, 15, 20, 25, 30, 35, 40, 50 and 60 min, respectively. Lane M: 100 bp DNA ladder.
and reaction time. Fig. 1 shows that the RT-LAMP product was observed at 55, 57.5, 60, 62.5 and 65 ◦ C (Fig. 1A). However, 65 ◦ C was chosen as the optimal temperature as specificity of the reaction increases at higher temperatures. On the other hand, the RT-LAMP product detected the NNV-RNA as early as 25–30 min at 65 ◦ C (Fig. 1B). Although the RT-LAMP product could be detected at 25–30 min, the optimal reaction time was 60 min to ensure detection of a lower concentration of target template.
the manufacturer’s recommendation for the FFPE RNA extraction kit, the first 56 ◦ C incubation step was shortened from 30 min to 10 and 20 min, while the results of the RT-LAMP were the same (Fig. 5A). The RT-LAMP detection limit could reach 10−3 dilution of FFPE extracted RNA (Fig. 5B).
3.2. Comparison of sensitivity of the RT-LAMP and RT-PCR The detection limit of the RT-LAMP reaction was tested using 10fold serial dilutions of NNV-infected grouper RNA and compared with results from the RT-PCR method. The detection limit of RTLMAP was 10−6 (Fig. 2). The detection limit of the one-step RT-PCR was 10−2 to 10−3 and the detection limit of nested RT-PCR was 10−5 (Fig. 2). The sensitivity of RT-LAMP for the detection of NNV was about 1000 times and 10 times more sensitive than the one-step RT-PCR and the nested RT-PCR respectively. 3.3. Specificity of the RT-LAMP detection The RT-LAMP amplified product was only detected in the NNVinfected orange-spotted grouper and in the king grouper when NNV-RNA was present (Fig. 3). There was no amplification of orange-spotted grouper iridovirus infected grouper DNA or healthy grouper genomic DNA and RNA. No cross-reaction was observed in this study. 3.4. Detection of NNV in grouper tissues by LAMP Total RNA was isolated from various tissues, including brain, eye, gill, heart, liver, spleen and muscle, and the NNV RNA was detected in the brain, eye, gill, heart, liver and muscle by RT-LAMP (Fig. 4). 3.5. Rapid extraction of RNA for RT-LAMP Brain tissue RNA was extracted by an FFPE RNA extraction kit; the RNA was also used for the RT-LAMP assay. In comparison with
Fig. 2. Sensitivity of RT-LAMP, one-step RT-PCR and nested PCR. (A) RT-LAMP products; (B) one-step RT-PCR; (C) nested RT-PCR. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–8: 10−1 to 10−7 dilutions of RNA from NNV-infected grouper brain tissue.
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Fig. 5. Rapid extraction RNA for RT-LAMP. (A) RNA from rapid extraction by different incubation time. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–4: 10, 20 and 30 min incubation time; (B) different dilutions of RNA for RT-LAMP. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–4: 10−1 to 10−3 dilutions of RNA. Fig. 3. Specificity of the RT-LAMP reaction. Lane M: 100 bp DNA ladder, lane 1: negative control, lane 2: healthy grouper genomic DNA, lane 3: orange-spotted grouper iridovirus infected grouper genomic DNA, lane 4: RNA from NNV-infected grouper, lane 5: RNA from NNV-infected orange-spotted grouper and lane 6: RNA from NNVinfected king grouper. 200 ng DNA/RNA was used for RT-LAMP.
4. Discussion Viral nervous necrosis is a serious viral disease in the grouper cultivation industry in Taiwan. Many stages in their life cycle can be infected with NNV, especially in hatchery-reared larvae and juve-
Fig. 4. Detection of NNV-RNA from various grouper tissue by RT-LAMP. Lane M: 100 bp DNA ladder, lane 1: negative control, lanes 2–8: RNA from brain, eye, gill, heart, spleen, liver and muscle, respectively.
niles (Chi et al., 2003). Once infected with NNV, the grouper may become a carrier and facilitate further spread of the virus. Thus, an effective diagnostic method is required for disease prevention and control. The current “gold standard method” of detection of NNV uses conventional RT-PCR (Nishizawa et al., 1995) but there are still some disadvantages, such as the need to use a thermal cycler for the reaction, the multiple and complex operation process and low amplificational efficiency (Mori et al., 2001; Tomita et al., 2008). In this study, the RT-LAMP method was carried out for the detection of NNV in groupers. Two sets of primers used were able to amplify a 215 bp sequence from the coat protein gene of NNV. The optimal condition for the detection of NNV RNA was 65 ◦ C for 60 min. The amplified product was, however, detected after 25–30 min and this suggests that the detection of NNV is possible in a very short period of time. However, for complete amplification, 60 min is considered an optimal reaction time (Kiatpathomchai et al., 2007). The RT-LAMP method used for the detection of NNV was shown to be highly sensitive; its detection limit was 10−6 of NNV-RNA. The sensitivity of RT-LAMP was about 1000 times more sensitive than one-step RT-PCR and 10 times more sensitive than nested RT-PCR, detection limits being 10−2 to 10−3 and 10−5 , respectively. One-step RT-PCR can complete all procedures in one step and in one tube, but the sensitivity is lower than the nested RT-PCR and RT-LAMP. Although the nested RT-PCR method can obtain higher sensitivity, it needs a two-step operation and takes another 2–3 h. In comparison with RT-LAMP, RT and DNA amplification can proceed under the same conditions within 1 h, and using only a one-step operation. This shows that RT-LAMP is a more sensitive, rapid and a convenient method. To simplify the operation of RNA extraction, we used the FFPE kit, which can complete all the steps in one tube without centrifugation. The RNA extracted by the FFEP kit could be used for the NNV RT-LAMP. According to the manufacturer’s recommendations, the first incubation time is 56 ◦ C for 30 min. In order to decrease the operation time, the first step incubation can be shortened to 20 or 10 min, and this does not influence the final result of the RT-LAMP assay. Thus, the RNA extraction can be finished within 30 min with-
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