11th International AIDS Conference Vancouver, British Columbia — July 7-12, 1996

Int Conf AIDS 1996 Jul 7-12; 11:7 (abstract no. Mo.A.160)
Gleeson T, Montpetit M; HIV Genetics, Ottawa, Ontario, Canada. Fax: 613-957-7258. E-mail: mmontpetit@hpb.hwc.ca.

OBJECTIVE: To determine the effects of primer-template mismatches on Roche Amplicor Diagnostic polymerase chain reaction (PCR) results for HIV-1 strains not belonging to genetic subtype B.

METHODS: Computer analysis (OLIGSAN program) of homology between the Roche Amplicor PCR primer pair SK431/SK462 and 100 HIV-1 strains from the 8 Group M and Group O subtypes of HIV-1 revealed considerable mismatching. Site-directed mutagenesis of cloned SK431 and SK462 amplification primers was performed to generate the mismatches found in the computer analysis of primer homology. Amplification and detection of the original and mutagenized targets was performed initially according to the manufacturer's instructions. Targets incorrectly diagnosed were subjected to further amplification attempts using different reaction conditions to identify suitable PCR reaction parameters for proper diagnosis.

RESULTS: Concerns about the limited detection of HIV-1 strains and subtypes by the Roche Amplicor HIV-1 PCR system have been confirmed. While limited divergence at the 5' ends of the primers had little effect on diagnostic accuracy the widespread mismatching found in a number of subtypes severely limits the diversity of HIV-1 detected by the Roche Amplicor system. The use of the same primers in the Roche Amplicor Monitor System designed for viral load measurement may cause falsely low readings for patients with viral strains not members of subtype B.

960707
MoA160

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