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>

The detection and clinical significance of hepatic fibrosis index / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the relationship between levels of serum HA, LN, IV-C, PC III of chronic hepatitis and indexes of hepatic fibrosis.</p><p><b>METHODS</b>The levels of serum HA, LN, IV-C and PC III of chronic hepatitis of 124 cases and health 18 cases were measured by radio immunoassay, combined with clinical characteristics and 33 cases pathologic slice etc. The diagnostic of the indexes of serum was analyzed with statistics.</p><p><b>RESULTS</b>HA and IV-C are parallel in chronic hepatitis periods. LN and PC III are concert in the same pathologic periods. In G4 period PC III is nearly closed with comparative group. The value of HA, LN, NV-C and PC III in the chronic hepatitis group was significantly higher than that in the normal comparative group. Conclusion The levels of serum HA LN IV-C and PC III are in concert with the degree of hepatic fibrosis, and these indexes are valuable for chronic hepatitis diagnoses combined with the clinic. LN and PC III are coincidence with hepatic fibrosis degree before G4 period.</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.

Research of open situation x-ray of cervical vertebra in healthy people / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the incidence rate of the different width in two sides of vertebra and C2 spinal process tilt, and analyze the different incidence rate between the unusual change of open situation x-ray and migraine.</p><p><b>METHODS</b>Among the 350 investigators, testing the open situation x-ray of cervical vertebra and the questionnaire of migraine situation (163 male and 187 female with an average age of 39.6 years ranging from 26 to 54 years). They were divided into several groups according to the age, including 94 people from 26 to 34 years, 103 people from 35 to 44 years, 153 people from 45 to 54 years. The method of contrasting the incidence rate of open situation x-ray with the rate of migraine was used.</p><p><b>RESULTS</b>Among the 350 investigated people, the incidence rate of the different width in two sides of vertebra and C2 spinal process tilt was 32.28%. The relationship between the different width in two sides of vertebra and the rate of migraine is not obvious (P > 0.05). However,the relationship between C2 spinal process tilt and the migraine was obvious (0.05 > P > 0.01).</p><p><b>CONCLUSION</b>The difference between the different width in two sides of vertebra and the rate of migraine is not obvious in the investigated people, so it is not reliable to diagnose the illness as cervical vertebra disease. While the difference between the C2 spinal process tilt and the migraine is obvious, it is one of the reasons to diagnose the migraine.</p>