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

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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.