Rapid and sensitive detection of Laem-Singh virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

• Published in: Journal of virological methods 2011-10, Vol.177 (1), p.71-74
• Main Authors: Arunrut, Narong, Seetang-Nun, Yortyot, Phromjai, Jurairat, Panphut, Wattana, Kiatpathomchai, Wansika
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; chromatography; DNA; electrophoresis; Fundamental and applied biological sciences. Psychology; Genes, Viral - genetics; Laem-Singh virus; LAMP; Lateral flow dipstick; Loop-mediated isothermal amplification; LSNV; Microbiology; Nucleic Acid Hybridization; oligonucleotides; PCR; Penaeidae - virology; Penaeus monodon; Reproducibility of Results; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; reverse transcription; reverse transcription loop-mediated isothermal amplification; RNA; RNA Viruses - genetics; RNA Viruses - isolation & purification; Sensitivity and Specificity; Techniques used in virology; Temperature; Virology; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.06.020

► We have developed a rapid RT-LAMP-LFD test for detecting LSNV infections in shrimp. ► The RT-LAMP also provided ∼1000-fold greater LSNV RNA detection sensitivity compared to the RT-PCR, and it did not cross-detect other RNA viruses commonly found in shrimp farmed in Southeast Asia. ► The use of a LFD to detect LSNV DNA amplified by the RT-LAMP afforded additional simplicity, reduced the time needed to generate a result and negated the need for agarose electrophoresis and DNA detection equipment. Laem-Singh virus (LSNV) was discovered recently in Thailand in farmed Giant Tiger shrimp (Penaeus monodon) displaying signs of slow growth syndrome. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here a reverse transcription (RT)-LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect LSNV RNA rapidly and specifically. The reaction was optimized at 65°C for 30min and amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5min was detected at LFD test line 5min after application. Including 10min for rapid RNA extraction, test results could be generated within 1h and did not require electrophoresis. Compared to an existing RT-PCR method, the RT-LAMP-LFD was also ∼1000-fold more sensitive in detecting LSNV RNA.