Optimal conditions for detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction with nested primers

An assessment of optimal conditions for nested primer amplification of low copy number target DNA sequences was made using a human immunodeficiency virus type 1 (HIV-1) model. In this polymerase chain reaction (PCR) strategy, an outer primer pair is first used to amplify the target sequence and a fraction of the amplification product is further amplified with a pair of inner (nested) primers. Several methodological parameters were evaluated, including number of cycles in the first and second step of the reaction, proportion of preamplified material to be used as the template for the second amplification, concentrations of primers, deoxynucleotides, and Taq DNA polymerase in the outer and inner PCR. The two-step PCR required minimal amounts of reaction components and was shown to be highly flexible, resulting in exquisite and specificity over a wide range of technical conditions. Potential drawbacks of this practical and effective amplification procedure are also discussed.