The development and clinical application of papillomavirus genotyping by DNA chip / 中华流行病学杂志

<p><b>OBJECTIVE</b>To develop a new platform for genotyping human papillomavirus (HPV) and to investigate its effect in clinical application.</p><p><b>METHODS</b>A pair of common primers of 18 HPV subtypes for PCR, was designed in HPV conservative L1 region. Genotyping probes for detecting 15 high-risk HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, together with 3 low-risk HPV 6, 11 and 42 were selected respectively from Genbank and fixed on membrane to make DNA chip. PCR amplification and DNA chip technology were optimized. 100 clinical samples were used to investigate the effect of HPV genotyping DNA chip. Veracity of the genotyping results was verified by sequencing.</p><p><b>RESULTS</b>From the 100 clinical samples, 30 were found to be HPV positive, including high-risk HPV subtypes 16, 18, 33, 45, 51, 58, and 66, and low-risk HPV 6, 11 and 42. The sensitivity tested by standard samples was up to 10 copies of HPV DNA.</p><p><b>CONCLUSION</b>The HPV genotyping system developed here with DNA chip showed high sensitivity and specificity, suitable to be applied in clinical practice for HPV diagnosis and investigation on the prevalence of HPV sub-types.</p>

Development and application of a new hepatitis C virus genotyping method with polymerase chain reaction-reverse blot dot technique / 中华流行病学杂志

<p><b>OBJECTIVE</b>Using polymerase chain reaction-reverse blot dot (PCR-RDB) technique to establish a new method for hepatitis C virus (HCV) genotyping and to study the distribution of HCV genotypes in Foshan area.</p><p><b>METHODS</b>HCV primers and probes were designed in 5'-untranslated region (nt-1-nt-299) of HCV. HCV RNA in serum was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin-labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype 1a to 3b) on Biodyne C membrane to genotype HCV by color development while adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluorescent quantitative (FQ)-PCR. 60 FQ-PCR-positive HCV sera from Foshan area were genotyped using this assay.</p><p><b>RESULTS</b>All 60 sera could be successfully genotyped by PCR-RBD. 50 (83.3%) cases were found to be genotype 1b, 2 (3.3%) as genotype 1a and 2 (3.3%) as genotype 2a while 5 (8.0%) to be mixture of genotype 1a and 1b, and 1 (1.7%) to be mixture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. The results of PCR-RDB genotyping methods coincided with sequence analysis.</p><p><b>CONCLUSION</b>Newly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies. The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area.</p>

Establishment of a new HBV genotyping method with PCR-RBD and its application / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Using PCR-RDB to establish a new method for HBV genotyping, and to survey the distribution of HBV genotypes in the Foshan area.</p><p><b>METHODS</b>Biotin-labeled primers for amplification of HBV region X (nt1550-1789) were used to amplify extracted HBV DNA. HBV was genotyped by hybridization of the PCR products with immobilized specific probes (genotype A to F) on C membrane. Color development was achieved by adding POD and TMB. A judgment was made according to color reactions. The reliability of this new method was verified by gene sequencing. 300 samples of HBV DNA-positive sera from the Foshan area were genotyped using this assay.</p><p><b>RESULTS</b>Of the 300 sera genotyped by PCR-RBD, 147 (49.0%) cases were genotype B, 136 (45.3%) were genotype C, 1 (0.3%) genotype D, and 12 (4.0%) were mixtures of genotype B and C, and 4 (1.3%) were mixtures of genotype C and D. No genotype A, E or F were found. The results of PCR-RDB genotyping were consistent with the results obtained with sequence analysis.</p><p><b>CONCLUSION</b>This newly established HBV genotyping system proved to be sensitive, specific, precise and economic, and should be suitable for clinical practice and epidemic study. The results of HBV genotyping show that genotype B and C are the predominant genotypes in the Foshan area.</p>