Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick.
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.
Jaroenram
Wansadaj
W
CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Ratchathewi, Bangkok 10400, Thailand.
Kiatpathomchai
Wansika
W
Flegel
Timothy W
TW
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000821:virology*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D046848:White spot syndrome virus 1 / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D015342:DNA Probes