AEGiS-14IAC: A rapid procedure to detect a single copy of HIV-1 virion RNA.

14th International AIDS Conference

Barcelona, Spain - July 7-12, 2002

A rapid procedure to detect a single copy of HIV-1 virion RNA.

Int Conf AIDS 2002 Jul 7-12; 14:(abstract no.. B10226)

Saito Y, Tanaka R, Hanabusa H, Kato S
Keio University, Tokyo, Japan

BACKGROUND: It is often required in clinical settings to judge the presence of HIV-1 in materials to avoid the risk of infection completely. Thus, we established a rapid procedure by assessing minimal requirement in whole steps from RNA isolation to detection of PCR-amplified products that was able to detect a single copy of HIV-1 in sample solutions.

METHODS: One milliliter of sample solutions were centrifuged at 23,500 x g for 1 hour. RNA was purified from the precipitate with RNAeasy Mini Kit (QIAGEN) and subjected to RT-nested PCR using Ex Taq (Takara, Japan) and GeneAmp PCR System 9700. The PCR-amplified products were electrophoresed and photographed under UV light. The concentration of HIV-1 virions was evaluated by direct determination of HIV-1 RNA in virus stock solutions with competitive RT-nested PCR.

RESULTS: In the experiment starting solutions containing 250 virions, the yield of HIV-1 RNA was almost 100% without interference of the presence of culture medium, plasma or Percoll. When experiments using solutions containing one virion on average were repeated ten times, five positive results were obtained, which was in concord with the score of 6.3 expected from Poison distribution. It took 4.5 hours to complete the whole procedure.

CONCLUSIONS: Our protocol to detect HIV-1 virion RNA is simple, rapid, and highly sensitive. This is especially applicable for assessment of the infection risk in in-vitro fertilization using processed sperm from infected males.

Keywords: AEGIS, HIV-1, Virion, Single Person, Reverse Transcriptase Polymerase Chain Reaction, Polymerase Chain Reaction, HIV-1 Reverse Transcriptase, Japan, Human, Male, methods

020707
B10226