Diagonsis establishment of fluorescen quantitative PCR assay for pseudorabies wild-type virus and vaccine virus.
- Author:
Li ZHAO
1
;
Baoan CUI
;
Hongying CHEN
;
Zhanyong WEI
;
Lanlan ZHENG
;
Xiaoli LÜ
;
Yanyan JIA
;
Xuyong ZHAO
Author Information
1. College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Fluorescent Dyes;
Herpesvirus 1, Suid;
genetics;
isolation & purification;
Polymerase Chain Reaction;
methods;
Pseudorabies;
diagnosis;
prevention & control;
virology;
Pseudorabies Vaccines;
immunology;
isolation & purification;
Swine
- From:
Chinese Journal of Biotechnology
2008;24(7):1149-1154
- CountryChina
- Language:Chinese
-
Abstract:
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.
