Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Hepatitis A virus mimotope mapping by phage display peptide library / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach.</p><p><b>METHODS</b>Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV.</p><p><b>CONCLUSION</b>The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.</p>

Characterization of capsid protein VP3-VP1 genes of hepatitis A virus prevalent strains circulated in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China.</p><p><b>METHODS</b>The nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region.</p><p><b>RESULTS</b>The nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains.</p><p><b>CONCLUSION</b>All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.</p>