The Establishment Of A Real-time RT-PCR Assay For Detection Of Hepatitis E Virus (HEV) And Its Primary Application

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic hepatitis. So far, the HEV infection has been diagnosed mainly by detection of anti-HEV antibodies using ELISA method. However, the limitation of HEV antibody detection in window period of HEV infection, in patients with deficient immunity or even by using EIAs with different sensitivity and specificity may make the diagnosis difficult. Detection of HEV RNA is one of criteria for HEV infection. Conventional RT-PCR assays including nested RT-PCR have been used. However, the sensitivity is not enough and test procedure is time consuming. They are also prone to contamination when performed as nested RT-PCR procedures. To overcome the disadvantage of conventional RT-PCR assays, a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA was developed in this study.26 primers and 4 probes were designed based on a multiple sequence alignment of HEV ORF3 conserved region available in GenBank. By screening primers and probes, the best combination which includes forward primer (401U), the reverse primer (402R) and the probe (301F) had been determined. By optimizing the concentration of primer and probe, the concentration for forward primer, reverse primer and probe is .10pmol/μl, 0.20pmol/μl and 0.20pmol/μl, respectively. Then, the reactive parameters and condition including Taq DNA polymerase, AMV reverse transcriptase, the temperature of reverse transcription and PCR cycling had also been optimized. The concentration for Taq DNA polymerase and AMV reverse...