SIMULTANEOUS DETECTION OF HBV AND HCV GENOMIC SEQUENCES BY PCR

通过 PCR 同时检测 HBV 和 HCV 基因组序列

• 批准号: 3804834
• 负责人: S NEDJAR
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH TRANSFUSION
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804834
• 关键词: alanine transaminase; antiviral antibody; communicable disease diagnosis; complementary DNA; diagnosis design /evaluation; diagnostic tests; gel electrophoresis; hepatitis B; hepatitis B virus group; hepatitis C virus; human tissue; molecular size; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; serology /serodiagnosis; serum; southern blotting; virus DNA; virus RNA

A sensitive and specific two stage polymerase chain reaction (PCR) method was devised for the simultaneous amplification and detection of genomic sequences of hepatitis B virus (HBV) and hepatitis C virus (HCV) in serum. RNA and DNA were extracted from sera. HCV-RNA was first reverse transcribed to cDNA. DNA sequences from both HCV (286 bp) and HBV (128 bp) were co-amplified by using primer pairs derived from conserved regions. PCR products were separated by agarose gel electrophoresis and their molecular size determined. Specificity of the PCR products for HBV and HCV sequences was confirmed by Southern blot analysis with 32P-end labeled oligonucleotide probes. Independent sets of sera, positive and negative by PCR for either HBV-DNA or HCV-RNA, were used as controls. Nine donor sera, antibody positive for both HBV (anti-HBc/HBs) and HCV (anti-c100-3) were tested. Sera from 5 patients with elevated alanine amino transferase (ALT) but negative for antibodies to HBV and HCV were also tested. Our assay detected HBV- and HCV-specific genomic sequences in 9/9 sera reactive for both HBV and HCV antibodies. Sera from patients with elevated ALT and antibody negative were all PCR positive for HCV-RNA, but negative for HBV-DNA. These results demonstrate that sera containing HBV and HCV sequences can be co-amplified by PCR and the products detected by agarose gel electrophoresis. Since this method is non-isotopic, it may be particularly useful in the clinical laboratory setting.

一种敏感、特异的两步法聚合酶链反应（PCR）方法 设计用于同时扩增和检测基因组 血清中B型肝炎病毒（HBV）和丙型肝炎病毒（HCV）的序列。 从血清中提取RNA和DNA。 HCV-RNA首次逆转 转录成cDNA。 HCV（286 bp）和HBV（128 bp）的DNA序列 通过使用来自保守区域的引物对共扩增。 PCR产物经琼脂糖凝胶电泳分离， 分子大小确定。 PCR产物对HBV和HCV的特异性 用32 P-末端标记的Southern印迹分析证实了序列的正确性。 寡核苷酸探针。 独立的血清组，阳性和阴性， HBV-DNA或HCV-RNA的PCR用作对照。 九份捐献者血清， HBV（抗-HBc/HBs）和HCV（抗-c100 -3）抗体均阳性， 测试. 5例丙氨酸氨基转移酶（ALT）升高患者的血清 但对HBV和HCV的抗体也检测为阴性。 我们的检测方法在9/9份血清中检测到HBV和HCV特异性基因组序列 对HBV和HCV抗体均具有反应性。 来自高血压患者的血清 ALT和抗体阴性者HCV-RNA均为PCR阳性， HBV-DNA 这些结果表明，含有HBV和HCV序列的血清可以被检测。 PCR共扩增，琼脂糖凝胶电泳检测 电泳 由于这种方法是非同位素的，因此它可能特别适合于 在临床实验室环境中有用。