[Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis A virus].

OBJECTIVE

To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.

METHODS

According to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients.

RESULTS

The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients.

CONCLUSION

The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.