DETECTION OF FALSE-NEGATIVE RESULTS IN NESTED PRIMER PCR OF PROVIRAL HIV-1 DNA

A control template for a competitive nested primer PCR of the HIV-1 ga g region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment aft er amplification (wild-type: 115 bp). To provide an internal control f or the individual PCR runs, six copies of this nested primer control p lasmid were introduced into a reaction tube containing the specific sa mple (under the PCR conditions used, this copy number reproducibly gav e a positive PCR signal). The results of our study show the feasibilit y of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild -type sequences, and examination of samples from a cohort of HIV-1-ser opositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, whic h without the use of the control template would have yielded a false-n egative result.