AEGiS-11IAC: Sensitive detection of HIV-1 DNA by one-tube seminested PCR combined with solution hybridization-EIA.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Sensitive detection of HIV-1 DNA by one-tube seminested PCR combined with solution hybridization-EIA.

Int Conf AIDS 1996 Jul 7-12; 11:218 (abstract no. Tu.A.150)
Awatcharagarn P, Kunakorn M, Raksakait K, Petchclai B; Dept. of Pathology, Ramathibodi Hosp., Bangkok, Thailand. Fax: 662-246-4281. E-mail: ramkn@mahidol.ac.th.

OBJECTIVE: Single round polymerase chain reaction (PCR) followed by membrane hybridization, or nested PCR without hybridization are the usual strategies used for detection of HIV-1 DNA in peripheral blood cells. However, membrane hybridization is laborious and time-consuming while contamination remains the major problem of nested PCR. In this presentation, we showed the development of one-tube seminested PCR (OSPCR) combined with solution hybridization-enzyme immunoassay (SHEIA) which can achieve high sensitivity and avoid the problems previously mentioned. Method: A low annealing temperature nested primer were used altogether with the high annealing temperature outer primers in OSPCR which was successively run for 60 cycles. The PCR products was hybridized with digoxigenin probe in liquid phase, then captured by streptavidin microtitration plate, via the biotin group labeled onto one of the primer, and detected by calorimetric method. Result: Compared at same number of cycles, OSPCR has a higher sensitivity than the ordinary PCR In addition, SHELA showed quantitative optical density values in detection of OSPCR products from the template ranges of 5-1000 HIV-1 DNA copies.

CONCLUSION: The OSPCR combined with SHEIA provides a sensitive and rapid method for detection of HIV-1 DNA.

Keywords: AEGIS, Polymerase Chain Reaction, DNA Primers, Digoxigenin, Immunoenzyme Techniques, RNA Probes, Sensitivity and Specificity, Streptavidin, Biotin, ICA11

960707
TuA150