A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patient / 中华流行病学杂志

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

A study on seroprevalence of anti-HAV IgG in adults of 4 cities in China / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the seroprevalence of anti-HAV IgG in adults of 4 cities in China.</p><p><b>METHODS</b>Serum samples were collected from 2390 local residents aged between 20 to 88 years from Beijing, Shanghai, Wuhan and Guangzhou. The anti-HAV IgG in sera was detected with a microparticle enzyme immunoassay (MEIA).</p><p><b>RESULTS</b>The anti-HAV IgG seroprevalence in female of 30 to 39 years in Beijing (64.58%, 62/96) was higher than that in male (45.57% 36/79)) (x(2) = 6.358, P = 0.012). It increased with age in adults of Beijing and Guangzhou. The rates were 54.22 % (90/166), 56.00% (98/175) and 67.18% (88/131) for the 20-, 30- and 40-49 age groups in Beijing (x(2) = 4.76, P = 0.03); and 52.83% (56/106), 52.50% (63/120), 82.46% (94/114), 89.80% (88/98) and 96.77% (60/62) for the 20-, 30-, 40-, 50- and 60-88 age groups in Guangzhou, respectively (x(2) = 72.58, P less than 0.01). This trend was not found in Shanghai and Wuhan (x2 = 0.96, 2.99; P = 0.33, 0.08 respectively). The seroprevalence rates of anti-HAV IgG in the 20 to 39 age group of Beijing, Shanghai, Guangzhou and Wuhan were 55.13% (188/341), 63.93% (429/671), 52.65% (119/226) and 78.37% (308/393), respectively.</p><p><b>CONCLUSION</b>The seroprevalence rates of anti-HAV IgG in young adults aged 20 to 39 years of the four cities are relatively low, and HAV vaccination should be suggested for the susceptible population of this age group in China.</p>

Comparison among four kits in detection of anti-SARS-CoV IgG in sera of SARS patients / 中华流行病学杂志

<p><b>OBJECTIVE</b>To compare the sensitivity and specificity of four kits for detection of anti-severe acute respiratory syndrome (SARS)-CoV IgG in sera of SARS patients.</p><p><b>METHODS</b>Anti-SARS-CoV IgG was detected in 99 serial sera from 18 SARS patients and in 123 negative reference sera, using two enzyme linked immunosorbent assays (EIA No. A and No. B) and two indirect immunofluorescence assays (Australian IFA and Euroimmun IFA).</p><p><b>RESULTS</b>Anti-SARS-CoV IgG was not detected in sera collected from SARS patients at the first week after onset by any of the four kits, however, it was detectable in sera obtained at the second week of illness by EIA No. B, and two IFA, but not by EIA No. A, with the positive rates of 57.1% (4/7), 57.1% (4/7) and 42.9% (3/7), respectively. The anti-SARS-CoV IgG was first determined in sera on the 9th day by Euroimmun IFA, 12th day by EIA No. B, 13th day by Australian IFA, and 16th day by EIA No. A. The positive rates of antibody on the 3rd week after onset were 84.2% (16/19), 94.7% (18/19), 78.9% (15/19) and 52.6% (10/19) respectively. They were identical since the 4th week after the disease onset. Through detection of 123 negative reference sera, the specificity of EIA No. A and two IFA was 100%, with exception of 94.9% for EIA No. B.</p><p><b>CONCLUSION</b>The sensitivity and specificity of the two IFAs were relatively higher than that of the two EIAs. The quality of the two homemade EIAs should be improved.</p>