SIMULTANEOUS DETECTION OF HIV-1 AND HCV GENOMIC SEQUENCES BY PCR

通过 PCR 同时检测 HIV-1 和 HCV 基因组序列

• 批准号: 3804833
• 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/3804833
• 关键词: AIDS; AIDS /HIV diagnosis; communicable disease diagnosis; complementary DNA; diagnosis design /evaluation; diagnostic tests; enzyme linked immunosorbent assay; hepatitis C virus; human immunodeficiency virus 1; human tissue; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; rapid diagnosis; serology /serodiagnosis; southern blotting; virus RNA

A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1), specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti- HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01, SN02, SN03 and SN04) based on the HCV prototype sequence of clones 37b and 81 for HCV, and SK 38/39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization and Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the two primer pairs, excluding the primer sequences. The primer pairs were able to detect as few as 1-3 copies and 10-20 copies of genomic RNA for HCV and HIV-1, respectively, when compared with 8E5 cells as the single copy standard. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to coamplify specific sequences from two different viral genomes in the same reaction mixture could facilitate the simultaneous detection of multiple viral sequences, thus offering the possibility of simultaneous diagnosis of more than one viral agent in serum samples of symptomatic and asymptomatic individuals.

采用聚合酶链反应（PCR）技术， 设计用于丙型肝炎的共扩增和同时检测 病毒（HCV）和人类免疫缺陷病毒1型（HIV-1），特异性 同一血清样本中的序列 从13个样本中提取基因组RNA， 在ELISA中对抗-HCV和抗- HIV-1 提取的RNA逆转录为cDNA并进行扩增 使用基于HCV的巢式引物对（SN 01、SN 02、SN 03和SN 04）， HCV的克隆37 b和81以及HIV-1的SK 38/39的原型序列 同步 通过液体杂交分析PCR产物， 32 P末端标记寡核苷酸探针的Southern印迹杂交 从两个引物对之间的区域，不包括引物 序列的 引物对能够检测少至1-3个拷贝， HCV和HIV-1基因组RNA的拷贝数分别为10-20， 以8 E5细胞作为单拷贝标准品。 13例均检出HCV-RNA (100%）的样本进行了检测;在11个（85%）样本中检测到HIV-RNA。 的 能够从两种不同的病毒基因组中共同扩增特定序列， 相同的反应混合物可以促进同时检测 多个病毒序列，从而提供了同时 在有症状和有症状的患者的血清样本中诊断出一种以上的病毒因子， 无症状个体。