RAPID AND QUANTITATIVE DETECTION BY PCR OF HIV-1 SPECIFIC DNA AND RNA

通过 PCR 快速定量检测 HIV-1 特异性 DNA 和 RNA

• 批准号: 3792569
• 负责人: I K HEWLETT
• 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/3792569
• 关键词: AIDS; AIDS /HIV diagnosis; SDS polyacrylamide gel electrophoresis; autoradiography; human immunodeficiency virus 1; human tissue; nucleic acid hybridization; polymerase chain reaction; rapid diagnosis; virus DNA; virus RNA

A rapid and sensitive PCR assay for detection of HIV-1 specific DNA and RNA in culture supernatants and viral RNA in serum was developed. Culture supernatant from H9 and U937 cells infected with 100ng of p24 antigen/ 50 million cells was analysed on a daily basis for viral p24 antigen, DNA and RNA. For analysis of DNA, culture supernatant was heated in the presence of nonanionic detergent and an aliquot was used for PCR. Primer pairs from the gag, env and nef regions of the viral genome were used for co-amplification. RNA extraction was achieved from samples by a single step procedure of guanidine thiocyanate/ phenol/ CHCl3 extraction and precipitation with isopropanol. PCR products were analyzed by slot-blot or liquid hybridization with radiolabelled oligonucleotide probes followed by PAGE and autoradiography. By this method 1-10 copies could be detected using the 8E5 cell standard. Viral RNA present in 100 ul of serum from an AIDS patient or an equivalent of 0.2 pg of p24 antigen could be detected in culture supernatant from infected H9 cells. Viral DNA and RNA were detected in culture supernatant of infected cells at 1 day post-infection while significant levels of p24 antigen were not detected until 2-3 days at the dose of virus used. In the culture supernatants from the cells treated with AZT, no viral RNA or DNA was detected at 3 and 7 days post infection and treatment suggesting that PCR on supernatants may be useful to monitor antiviral activity. No activity was observed in control samples of serum or uninfected H9 cell culture fluid. Our result suggests that PCR on supernatants may be useful in monitoring co-cultures from infected individuals or patients undergoing therapy, as well as to monitor infection in in vitro studies. The methodology has been applied successfully for detecting virus from other body fluids such as urine, sweat and saliva. We are currently extending its application for more rapid detection by using non-isotopic systems either by using chemiluminescent or fluorescent primers/ probes that would achieve single copy detection in conjunction with these techniques.

一种快速灵敏的检测HIV-1特异性DNA和 建立了培养上清液中的RNA和血清中的病毒RNA。 100 ng p24感染H9和U937细胞的培养上清 每天对抗原/5000万个细胞进行病毒p24的分析 抗原、DNA和RNA。用于DNA分析的培养上清液为 在非阴离子洗涤剂存在下加热，并使用等分 用于聚合酶链式反应。来自病毒Gag、env和nef区的引物对 用基因组进行共扩增。RNA的提取是从 硫氰酸胍/苯酚/一步法制备样品 三氯甲烷的提取和异丙醇沉淀。扩增产物为 缝隙印迹或放射性标记液体杂交法分析 寡核苷酸探针，然后是PAGE和放射自显影。借此 方法使用8E5细胞标准可检测到1-10个拷贝。病毒式传播 在100毫升艾滋病患者或相当于艾滋病患者的血清中存在RNA 培养上清液中可检测到0.2pg的p24抗原 感染H9细胞。在培养物中检测到病毒DNA和RNA 感染后1天的感染细胞上清液，但显著 直到2-3天才能检测到p24抗原水平。 使用了病毒。在用AZT处理的细胞的培养上清中， 在感染后3天和7天未检测到病毒RNA或DNA， 建议对上清液进行聚合酶链式反应的处理可能有助于监测 抗病毒活性。在对照血清样本中未观察到活性。 或未感染的H9细胞培养液。我们的结果表明， 培养上清液可用于监测受感染的共培养物 接受治疗的个人或患者，以及监测 体外研究中的感染。该方法论已被应用于 成功地从尿液等其他体液中检测病毒， 汗水和唾液。我们目前正在扩展其应用程序，以获得更多 使用非同位素系统的快速检测通过使用 化学发光或荧光底物/探针将实现 单拷贝检测与这些技术相结合。