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Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens
Author(s) -
Zazzi Maurizio,
Romano Laura,
Brasini Agostina,
Valensin Pier Egisto
Publication year - 1992
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.1890380304
Subject(s) - polymerase chain reaction , ethidium bromide , primer (cosmetics) , primer dimer , nested polymerase chain reaction , dna , microbiology and biotechnology , biology , virology , multiple displacement amplification , hot start pcr , polymerase chain reaction optimization , chemistry , dna extraction , genetics , gene , multiplex polymerase chain reaction , organic chemistry
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. © 1992 Wiley‐Liss, Inc.