Title Development and Evaluation of Real-Time Loop-Mediated Isothermal Amplification Methods for the Rapid Detection of Penaeid Viruses
Authers Tohru MEKATA, Raja SUDHAKARAN, and Toshiaki ITAMI
Keywords RT-LAMP, WSSV, YHV, IHHNV, TSV, Penaeid shrimp, Japan
Citation Bull. Fish. Res. Agen. No.35, 39-50, 2012
Abstract
Shrimp viral diseases are a major impediment to commercial shrimp farming. The diseases affecting shrimp culture are white spot syndrome virus (WSSV), yellow head virus (YHV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Taura syndrome virus (TSV). Viral diseases are particularly difficult to control after the onset of infection. Therefore, prophylaxes to prevent or reduce the losses through vertical and horizontal transmission are most important. Various diagnostic methods have been developed to detect shrimp viral diseases, including bioassays, histopathology, polymerase chain reaction (PCR), and quantitative real-time PCR. Although the PCR-based methods are sensitive and highly specific, they require expensive equipment, costly reagents, and are time consuming. Therefore, a simple, quick and sensitive detection method is urgently needed to prevent the invasion of shrimp viral diseases into Japan from other countries. The loop-mediated isothermal amplification (LAMP) assay is a novel approach to amplify nucleic acid with high specificity, sensitivity, and rapidity under isothermal conditions, thereby obviating the need for a thermal cycler. Further, during the LAMP reaction, an insoluble byproduct, magnesium pyrophosphate, is produced in proportion to the large amounts of the target DNA amplified. Hence, real-time quantification can be achieved by measuring the turbidity of the magnesium pyrophosphate using an inexpensive photometer. This real-time LAMP method allows quantitative analysis of nucleic acid templates (real-time LAMP). In the present study, a comparatively less expensive quantitative real-time LAMP assay was successfully applied for detection of shrimp viral diseases and proven to have high sensitivity and specificity.
URI http://www.fra.affrc.go.jp/bulletin/bull/bull35/35-6.pdf