Open AccessDetection of False-Negative Results in Nested Primer PCR of Proviral HIV-1 DNA
Author(s) -
Klaus F. Zimmermann,
Barbara Plaimauer,
Daniela Schögl,
Josef W. Mannhalter
Publication year - 1997
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/97235st04
Subject(s) - primer (cosmetics) , biology , nested polymerase chain reaction , human immunodeficiency virus (hiv) , polymerase chain reaction , primer dimer , dna , virology , provirus , genetics , microbiology and biotechnology , gene , genome , chemistry , multiplex polymerase chain reaction , organic chemistry
A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wildtype template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wildtype sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.