title: Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens.
abstract: A highly sensitive two-step polymerase chain reaction (PCR) method was evaluated for detection of human immunodeficiency virus type 1 (HIV-1) DNA in clinical specimens. The product resulting from the first amplification reaction is used as the template for the second PCR with an internal (nested) primer pair. Even when starting from a single copy of HIV-1 DNA, the double PCR product was readily detected by direct visualization in ethidium bromide-stained agarose gels. Amplification of minute amounts of HIV-1 DNA was successful in a considerable excess of HIV-1 negative DNA than reported previously. All of 85 HIV-1-infected individuals were PCR-positive with at least two of the three sets of primers used, 252 of 255 amplifications allowing unambiguous visualization of a unique DNA fragment of the expected size. The two-step amplification protocol is simple and rapid and fulfills the requirements of sensitivity and specificity for use in a clinical laboratory.
authors.affiliation: Department of Molecular Biology, University of Siena, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Zazzi
authors.forename: L
authors.initials: L
authors.lastname: Romano
authors.forename: A
authors.initials: A
authors.lastname: Brasini
authors.forename: P E
authors.initials: PE
authors.lastname: Valensin
mesh_terms: D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000097:blood* / Q000737:chemistry; D005069:Evaluation Studies as Topic; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral