Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide.
Kiatpathomchai
Wansika
W
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand. wansika@biotec.or.th
Jaroenram
Wansadaj
W
Arunrut
Narong
N
Jitrapakdee
Sarawut
S
Flegel
T W
TW
D000818:Animals; D002845:Chromatography / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral