Available online at www.sciencedirect.com Journal of Virological Methods 146 (2007) 125–128 Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification Wansika Kiatpathomchai a,b,∗ , Wansadaj Jareonram b , Sarawut Jitrapakdee c , T.W. Flegel b a National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand b CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand c Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Received 27 March 2007; received in revised form 7 June 2007; accepted 11 June 2007 Available online 23 July 2007 Abstract Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In this study, using the RT-LAMP method, a protocol for detecting Taura syndrome virus which is a causative agent of Penaeus vannamei was developed. Time and temperature conditions for detection of TSV were optimized for 60 min at 63 ◦ C. The nucleic acids of other shrimp pathogens (yellow head virus; YHV and white spot syndrome; WSSV) were not amplified by this RT-LAMP system. The detection of TSV using RT-LAMP was 10 times more sensitive than the RT-PCR but less sensitive than nested RT-PCR. However this system was more convenient, rapid, and does not require sophisticated PCR machine. © 2007 Elsevier B.V. All rights reserved. Keywords: Taura syndrome virus (TSV); Penaeus vannamei; Shrimp; Loop-mediated isothermal amplification; LAMP; RT-LAMP; RT-PCR 1. Introduction Taura syndrome virus (TSV) was first discovered in Ecuador in 1992 (Jimenez, 1992). It was a serious cause of shrimp mortality for reared Penaeus (Litopenaeus) vannamei (P. van- namei) in the Americas where it spreads principally through the regional and international transfer of live postlarvae and brood- stock (Brock, 1997). TSV is a small, non-enveloped icosahedral virus containing a single-stranded positive-sense RNA genome of 10,205 nucleotides (Bonami et al., 1997; Mari et al., 2002) and was classified in the family Dicistroiridae (Mayo, 2002, 2005). The capsid is comprised of three major proteins, i.e. 55, 40, 24 kDa designated VP1, VP2 and VP3, respectively as well as a 58 kDa minor protein designated V0 (Bonami et al., 1997; Mari et al., 2002). ∗ 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: wansika@biotec.or.th (W. Kiatpathomchai). Loop-mediated isothermal amplification (LAMP) assay is a novel approach that allows amplification of DNA with high specificity, sensitivity and rapidity under isothermal conditions. LAMP, originally described by Notomi et al. (2000), can amplify target nucleic acid to 10 9 copies at 60–65 ◦ C within 1 h. The method relies on autocycling strand displacement DNA syn- thesis by the Bst DNA polymerase large fragment, a DNA polymerase with high strand displacement activity, and a set of two specially designed 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 performed with a sim- ple and inexpensive water bath. Therefore, a thermal cycler is not required. As there is no time loss in thermal changes, the amplification efficiency of the LAMP method is extremely high (Parida et al., 2004; Savan et al., 2005). The development of a loop-mediated isothermal amplifica- tion (LAMP) assay for detection of shrimp white spot syndrome virus (WSSV) DNA was described by Kono et al. (2004). The 0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.06.007