Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).</p><p><b>METHODS</b>Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.</p><p><b>RESULTS</b>The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.</p><p><b>CONCLUSION</b>The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.</p>

Establishment and application of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte).</p><p><b>METHODS</b>Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated.</p><p><b>RESULTS</b>We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.</p><p><b>CONCLUSIONS</b>The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.</p>

Analysis on Hantaan virus in hemorrhagic fever patients with renal syndrome in Heilongjiang / 中国地方病学杂志

Objective To separate and amplify Hantaan virus(HV)in serum of hemorrhagic fever patients with renal syndrome(HFRS)in Heilongjiang,and look for its difference from intemational standard type strain(76-118strains).Methods HVs of different phase in the 8erum of 50 HFRS patients were separated and amplified by RTnested-PCR,its products were analyzed the amplified by sequencing.Results Detectable rate of HV in the patients serum was 36.36%(8/22)in 7 days after onset,it Was 13.04%(3/23)in patients having an onset 8 days to 14 days earlier,5 cases were not detectable 15 days after onset.Comparing the sequence of HV S gene fragment,sample 1,9,18,31,37,38,44 strain had a homology of 90.24%,86.72%,89.97%,89.16%,86.45%,87.26%and 89.43%to 76-118 strains,respectively.Conclusions The positive rate is the highset in 7 days after onset.Nucleotide sequence difference exists between pathogenic strain of Heilongjiang's HV and international standard strain,indicating that not only hosts but also locations can affect HV.