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

一种快速、灵敏的 PCR 检测方法，用于检测 HIV-1 特异性 DNA 和 开发了培养物上清液中的 RNA 和血清中的病毒 RNA。 感染 100ng p24 的 H9 和 U937 细胞的培养上清液 每天分析 5000 万个细胞的病毒 p24 抗原 抗原、DNA 和 RNA。为了进行 DNA 分析，将培养物上清液 在非阴离子洗涤剂存在下加热并使用等分试样 用于 PCR。来自病毒 gag、env 和 nef 区域的引物对 基因组用于共扩增。 RNA 提取是从 通过硫氰酸胍/苯酚/一步法制备样品 CHCl3 萃取并用异丙醇沉淀。 PCR 产物为 通过狭缝印迹或放射性标记液体杂交进行分析 寡核苷酸探针，然后进行 PAGE 和放射自显影。以此 方法1-10拷贝可以使用8E5细胞标准检测。病毒式 艾滋病患者 100 ul 血清中存在的 RNA 或同等量的 RNA 培养上清液中可检测到 0.2 pg p24 抗原 感染H9细胞。培养物中检测到病毒 DNA 和 RNA 感染后 1 天感染细胞的上清液，同时显着 直到服用 2-3 天后才检测到 p24 抗原水平 使用了病毒。在用 AZT 处理的细胞的培养上清液中， 感染后 3 天和 7 天未检测到病毒 RNA 或 DNA，并且 处理表明上清液 PCR 可能有助于监测 抗病毒活性。在血清对照样品中未观察到活性 或未感染的H9细胞培养液。我们的结果表明 PCR 上清液可能有助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。该方法已被应用 成功地从其他体液（例如尿液）中检测病毒， 汗水和唾液。我们目前正在将其应用扩展到更多 通过使用非同位素系统快速检测 化学发光或荧光引物/探针可实现 结合这些技术进行单拷贝检测。

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.