Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

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 lab...

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Bibliographic Details
Published in:Journal of virological methods 2013-03, Vol.188 (1-2), p.51-56
Main Authors: Khunthong, Sasiwarat, Jaroenram, Wansadaj, Arunrut, Narong, Suebsing, Rungkarn, Mungsantisuk, Idsada, Kiatpathomchai, Wansika
Format: Article
Language:English
Subjects:
Animals
Aquaculture
chromatography
detection limit
disease control
DNA
Fluorescent Dyes
gel electrophoresis
Gill-associated virus
Immunochromatography - methods
Lateral flow dipstick
Litopenaeus vannamei
Loop-mediated isothermal amplification (LAMP)
Metapenaeus brevicornis
Molecular Diagnostic Techniques - methods
mortality
Nucleic Acid Amplification Techniques - methods
Oligonucleotide Probes - genetics
pathogens
Penaeidae - virology
Penaeus (Litopenaeus) vannamei
Penaeus monodon
reverse transcriptase polymerase chain reaction
reverse transcription loop-mediated isothermal amplification
RNA
Roniviridae - isolation & purification
Sensitivity and Specificity
shrimp
shrimp culture
Temperature
Thailand
Veterinary Medicine - methods
virulence
Yellow head virus (YHV) detection
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Summary: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 45min, followed by hybridization with an FITC-labeled DNA probe for 5min and detected by LFD within 5min (time required approximately 55min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1pg 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2012.11.041