Title of article :
Development of rapid, simple and sensitive real-time reverse transcriptase loop-mediated isothermal amplification method (RT-LAMP) to detect viral diseases (PRDV, YHV, IHHNV and TSV) of penaeid shrimp
Author/Authors :
Sudhakaran, Raja University of Miyazaki - Faculty of Agriculture, Japan , Mekata, Tohru University of Miyazaki - Interdisciplinary Graduate School of Agriculture and Engineering, Japan , Mekata, Tohru Fisheries Research Agency - National Research Institute of Aquaculture - Aquatic Animal Health Division, Japan , Inada, Mari University of Miyazaki - Graduate School of Agriculture, Japan , Okugawa, Shogo University of Miyazaki - Graduate School of Agriculture, Japan , Kono, Tomoya University of Miyazaki - Faculty of Agriculture, Japan , Supamattaya, Kidchakan Prince of Songkla University - Faculty of Natural Resources, Thailand , Yoshida, Terutoyo University of Miyazaki - Faculty of Agriculture, Japan , Sakai, Masahiro University of Miyazaki - Faculty of Agriculture, Japan , Itami, Toshiaki University of Miyazaki - Faculty of Agriculture, Japan
Abstract :
A one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV), hypodermal and haematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR, RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid, where specific amplification is obtained for PRDV, IHHNV, TSV and YHV in 60 min under isothermal conditions at 63 oC employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA, rendering it ten-fold more sensitive than conventional LAMP assay.
