[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.
College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China.
Li
L
Zhao
Baoan
B
Cui
Hongying
H
Chen
Zhanyong
Z
Wei
Lanlan
L
Zheng
Xiaoli
X
Lü
Yanyan
Y
Jia
Xuyong
X
Zhao
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