Kfj. Tang et Dv. Lightner, Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction, AQUACULTURE, 189(1-2), 2000, pp. 11-21
A competitive polymerase chain reaction (PCR) method for quantification of
white spot syndrome virus (WSSV) genome was developed. A pair of WSSV prime
rs, designated WSSV341F/R, was selected to amplify a 341-bp DNA fragment fr
om the WSSV genome. For a competitive internal standard, we constructed and
cloned a 289-bp DNA fragment, the result of a 52-bp deletion from the 341-
bp amplicon. In a competitive PCR reaction, we co-amplified the target WSSV
DNA with known concentrations of the internal standard using WSSV341F/R pr
imers. The amplicons from WSSV DNA and from internal standard DNA differed
in size and could be distinguished after gel electrophoresis. The concentra
tion of WSSV genomes was determined from its relation to the concentration
of the internal standard. The log-log plot of the ratio of the amplicons (i
nternal standard: WSSV) on the internal standard concentration was linear.
Using this competitive PCR procedure, we quantified WSSV DNA in the samples
of hemolymph and tissues of the cephalothorax of individual WSSV-infected
shrimp. The number of WSSV genomes in both hemolymph and tissues correspond
ed to the severity of infection determined by histological evaluation. In a
ddition, the changes in number of WSSV genomes in the hemolymph during the
course of the infection were determined. (C) 2000 Elsevier Science B.V. All
rights reserved.