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Title: Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick
Authors: Sasiwarat Khunthong
Wansadaj Jaroenram
Narong Arunrut
Rungkarn Suebsing
Idsada Mungsantisuk
Wansika Kiatpathomchai
Mahidol University
Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE)
Thailand National Center for Genetic Engineering and Biotechnology
Keywords: Immunology and Microbiology
Issue Date: 1-Mar-2013
Citation: Journal of Virological Methods. Vol.188, No.1-2 (2013), 51-56
Abstract: Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65. °C for 45. min, followed by hybridization with an FITC-labeled DNA probe for 5. min and detected by LFD within 5. min (time required approximately 55. min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1. pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field. © 2012 Elsevier B.V.
ISSN: 18790984
Appears in Collections:Scopus 2011-2015

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