A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus.
A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments.
Yuan
Li
L
Institute of Hydrobiology, Chinese Academy of Sciences, East Lake Southern Road #7, Wuhan 430072, Hubei, PR China.
Zhang
Xiaohua
X
Chang
Mingxian
M
Jia
Chensong
C
Hemmingsen
Sean M
SM
Dai
Heping
H
D000818:Animals; D003400:Astacoidea / Q000821:virology*; D009500:Neutralization Tests / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D046848:White spot syndrome virus 1 / Q000302:isolation & purification*