[Diagonsis establishment of fluorescen quantitative PCR assay for pseudorabies wild-type virus and vaccine virus].
We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.
Zhao
Li
L
College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China.
Cui
Baoan
B
Chen
Hongying
H
Wei
Zhanyong
Z
Zheng
Lanlan
L
Lü
Xiaoli
X
Jia
Yanyan
Y
Zhao
Xuyong
X
D000818:Animals; D005456:Fluorescent Dyes; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011557:Pseudorabies / Q000175:diagnosis / Q000517:prevention & control / Q000821:virology*; D022421:Pseudorabies Vaccines / Q000276:immunology / Q000302:isolation & purification*; D013552:Swine
D005456:Fluorescent Dyes; D022421:Pseudorabies Vaccines