AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained...AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.展开更多
AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutat...AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutation ofGBV-C/HGV.METHODS Quantitative RT-PCR for detectionpf HCV RNA concentration in serum samples,RT-nested PCR with two sets of primers fordetection of GBV-C RNA,RT-PCR ELISA with twosets of primers for detection of HGV RNA,nucleotide sequence and putative amino acidsequence analysis.RESULTS The positive rates of GBV-C RNA atthe 5’-NCR and NS3 region in 211 serums amplesfrom the patients with HCV infection were 31.8%and 22.8% respectively.The positive rates ofHGV RNA at the 5’-NCR and NS5 region in thesame samples were 47.9% and 31.8%respectively.The total positive rate of GBV-C/HGV RNA was as high as 55.5%.HCV copynumbers in the patients without GBV-C/ HGVcoinfection were statistically higher than that inthe patients with GBV-C/ HGV coinfection(P【0.01).Frequent mutation of nucleotideresidue was present in the amplificationproducts.Frameshift mutation was found in twosamples with GBV-C NS3 region nucleotidesequences.All nucleotide sequences fromamplification products showed higher homologyto HGV genome than to GBV-C genome even though part of the sequences were amplifiedwith GBV-C primers.CONCLUSION A high frequency of GBV-C/ HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/ HGV RNA. The primers derived from the 5 -NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/ HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/ HGV genomes were random mutation of nucleotide residue.展开更多
目的探究低载量HBV DNA S区基因扩增的可行性并对实验条件进行优化,为隐匿性HBV感染(OBI)患者HBV DNA S区基因突变检测提供依据。方法采用传统巢式PCR和自建两轮PCR方法扩增6例低HBV DNA载量(100~200 IU/mL)和22例更低HBV DNA载量(20~99...目的探究低载量HBV DNA S区基因扩增的可行性并对实验条件进行优化,为隐匿性HBV感染(OBI)患者HBV DNA S区基因突变检测提供依据。方法采用传统巢式PCR和自建两轮PCR方法扩增6例低HBV DNA载量(100~200 IU/mL)和22例更低HBV DNA载量(20~99 IU/mL)的血清样本中HBV DNA S区基因,并对引物序列、引物量、PCR产物模板稀释倍数、退火温度、PCR反应循环数、PCR总反应体系等条件进行优化。PCR扩增产物经琼脂糖凝胶电泳后,切割目的条带凝胶进行克隆测序,然后对克隆测序结果进行核酸序列BLAST比对确认。结果设计3对巢式PCR引物(P1~P3),扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化后,6例低HBV DNA载量的血清样本中仅2例经巢式PCR扩增出HBV DNA S区基因特异性靶序列,22例更低HBV DNA载量样本无一例扩增成功。自建两轮PCR法设计了P4~P15共12对引物,扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化并筛选出P13为最佳引物后,6例低HBV DNA载量的血清样本全部扩增出HBV DNA S区基因特异性靶序列;15例(15/22,68.18%)更低HBV DNA载量的样本扩增出HBV DNA S区基因特异性靶序列,经PCR产物克隆测序均证实为HBV DNA S区基因,该15例样本中HBV DNA载量最低为20.1 IU/mL。结论基于引物P13自建的两轮PCR法更适用于低载量HBV DNA S区基因的扩增,扩增效率和特异性均优于传统巢式PCR;扩增产物可进一步应用于OBI者HBV DNA S区基因序列突变分析。展开更多
ALM To determine the frequencies of HGV and TTVinfections in blood donors in Hangzhou.METHODS RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleo...ALM To determine the frequencies of HGV and TTVinfections in blood donors in Hangzhou.METHODS RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed.``RESULTS Thirty-two ( 15.8%) and 30 (14.8%) of the 203serum samples were positive for HGV RNA and TTV DNA,respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA.Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3serum samples compared with the reported HGV and TTV sequences was 89.36%. 87.94%, 88.65% and 63.51%.65.77% and 67.12%. respectively.``CONCLUSION The infection rates of HGV and/or TTV inblood donors are relatively high. and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.展开更多
文摘AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.
文摘AIM To detect infection rate of GBV-C/HGV inhepatitis C patients,to determine the methodsof higher sensitivity and the primers of higherefficiency for GBV-C/HGV RNA detection and tostudy the dominant subtype and mutation ofGBV-C/HGV.METHODS Quantitative RT-PCR for detectionpf HCV RNA concentration in serum samples,RT-nested PCR with two sets of primers fordetection of GBV-C RNA,RT-PCR ELISA with twosets of primers for detection of HGV RNA,nucleotide sequence and putative amino acidsequence analysis.RESULTS The positive rates of GBV-C RNA atthe 5’-NCR and NS3 region in 211 serums amplesfrom the patients with HCV infection were 31.8%and 22.8% respectively.The positive rates ofHGV RNA at the 5’-NCR and NS5 region in thesame samples were 47.9% and 31.8%respectively.The total positive rate of GBV-C/HGV RNA was as high as 55.5%.HCV copynumbers in the patients without GBV-C/ HGVcoinfection were statistically higher than that inthe patients with GBV-C/ HGV coinfection(P【0.01).Frequent mutation of nucleotideresidue was present in the amplificationproducts.Frameshift mutation was found in twosamples with GBV-C NS3 region nucleotidesequences.All nucleotide sequences fromamplification products showed higher homologyto HGV genome than to GBV-C genome even though part of the sequences were amplifiedwith GBV-C primers.CONCLUSION A high frequency of GBV-C/ HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/ HGV RNA. The primers derived from the 5 -NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/ HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/ HGV genomes were random mutation of nucleotide residue.
文摘目的探究低载量HBV DNA S区基因扩增的可行性并对实验条件进行优化,为隐匿性HBV感染(OBI)患者HBV DNA S区基因突变检测提供依据。方法采用传统巢式PCR和自建两轮PCR方法扩增6例低HBV DNA载量(100~200 IU/mL)和22例更低HBV DNA载量(20~99 IU/mL)的血清样本中HBV DNA S区基因,并对引物序列、引物量、PCR产物模板稀释倍数、退火温度、PCR反应循环数、PCR总反应体系等条件进行优化。PCR扩增产物经琼脂糖凝胶电泳后,切割目的条带凝胶进行克隆测序,然后对克隆测序结果进行核酸序列BLAST比对确认。结果设计3对巢式PCR引物(P1~P3),扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化后,6例低HBV DNA载量的血清样本中仅2例经巢式PCR扩增出HBV DNA S区基因特异性靶序列,22例更低HBV DNA载量样本无一例扩增成功。自建两轮PCR法设计了P4~P15共12对引物,扩增产物理论上包含整个HBV DNA S区基因。经过PCR扩增条件优化并筛选出P13为最佳引物后,6例低HBV DNA载量的血清样本全部扩增出HBV DNA S区基因特异性靶序列;15例(15/22,68.18%)更低HBV DNA载量的样本扩增出HBV DNA S区基因特异性靶序列,经PCR产物克隆测序均证实为HBV DNA S区基因,该15例样本中HBV DNA载量最低为20.1 IU/mL。结论基于引物P13自建的两轮PCR法更适用于低载量HBV DNA S区基因的扩增,扩增效率和特异性均优于传统巢式PCR;扩增产物可进一步应用于OBI者HBV DNA S区基因序列突变分析。
文摘ALM To determine the frequencies of HGV and TTVinfections in blood donors in Hangzhou.METHODS RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed.``RESULTS Thirty-two ( 15.8%) and 30 (14.8%) of the 203serum samples were positive for HGV RNA and TTV DNA,respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA.Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3serum samples compared with the reported HGV and TTV sequences was 89.36%. 87.94%, 88.65% and 63.51%.65.77% and 67.12%. respectively.``CONCLUSION The infection rates of HGV and/or TTV inblood donors are relatively high. and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.
