Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.
Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.
GeneReach Technology Corporation, Taichung City 407, Taiwan. pin@genereachbiotech.com
Pin-Hsing
PH
Chou
Yu-Chan
YC
Lin
Ping-Hua
PH
Teng
Chu-Liang
CL
Chen
Pei-Yu
PY
Lee
D000818:Animals; D003429:Cross Reactions; D019223:Densovirinae / Q000302:isolation & purification; D031541:Fluorescence Resonance Energy Transfer; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D033561:Penaeidae / Q000821:virology*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D046848:White spot syndrome virus 1 / Q000302:isolation & purification*
D015345:Oligonucleotide Probes