[Establishing a real-time PCR assay for anatid herpesvirus 1].
OBJECTIVE
To develop a real-time quantitative PCR assay to detect duck plague virus (DPV) for the rapid diagnosis of DPV infection, the investigation of its nosogenesis and the screening of effective antiviral drugs.


METHODS
The primer and probe were designed according to the gene sequence of DPV DNA polymerase gene. To establish a standard curve, a plasmid containing 125 bp PCR product was constructed and severed as a positive control. The TaqMan based real-time PCR method was adopted and compared with the traditional PCR approach in sensitivity, reliability and specificity.


RESULTS
A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 2.3 x 10 to 2.3 x 10(5) copies. A minimum of 23 positive plasmids could be detected, indicating a good sensitivity of the assay. The coefficients of variance (CVs) were 1.22-6.69 and 2.09-8.84 for the intra-assay and inter-assay tests respectively, indicating a good reliability. No amplification products were found for DNA from other pathogens, indicating a good specificity. The comparative study proved that the TaqMan technology was much more sensitive than traditional PCR assay.


CONCLUSIONS
The real-time quantitative PCR assay for DPV DNA has good sensitivity, specificity and reliability.
Yang
Fa-long
FL
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Jia
Wen-xiang
WX
Xie
Yi
Y
Zeng
Wei
W
Yang
Wei-qing
WQ
Yue
Hua
H
D018138:Alphaherpesvirinae / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D004372:Ducks; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004279:DNA, Viral