Simultaneous Amplification of Multiple HIV-1 DNA Sequences from Clinical Specimens by Using Nested-Primer Polymerase Chain Reaction

Abstract

A sensitive and specific polymerase chain reaction (PCR) protocol with nested primers was developed for simultaneous amplification of three independent human immunodeficiency virus type 1 (HIV-1) DNA sequences from clinical specimens. DNA samples were first amplified with gag, pol, and env outer primer pairs and then with the corresponding three inner primer pairs in the same two-step reaction. Detection of the different amplification products was readily accomplished by simple agarose gel electrophoresis of the reaction product, even when starting with a single copy of HIV-1 DNA. Equivalent amounts of the three PCR products were generated, provided that the relative concentrations of the inner primer pairs were optimized. In addition, a beta-globin control primer pair could be conveniently included in the internal amplification step to verify that the DNA sample was suitable for PCR analysis. One nested multiplex PCR test was sufficient to detect HIV-1 DNA in all of 80 HIV-1-seropositive individuals and none of 50 HIV-1-seronegative healthy blood donors. The nested multiplex PCR procedure provides an attractive means for simple, rapid, and cost-effective direct detection of HIV-1 DNA in patient samples.

Antiretroviral therapy with protease inhibitors in human immunodeficiency virus type 1- and human herpesvirus 8-coinfected patients

“…The supernatant containing the DNA was transferred to another tube and DNA was precipitated with isopropanol, collected by centrifugation, washed with 70% ethanol, resuspended in TE buffer (10-mM Tris-HCl, pH 8, 0.2-mM EDTA), and evaluated spectrophotometrically. All DNA samples were confirmed to be suitable for PCR analysis by successful amplification of HIV-1 DNA targets [Zazzi et al, 1993].…”

Detection of Antibodies to Human Immunodeficiency Virus (HIV) That Recognize Conformational Epitopes of Glycoproteins 160 and 41 Often Allows for Early Diagnosis of HIV Infection

“…The MZ 13/14 outer primer pairs for gag and 25 cycles of amplification were followed by the MZ 8/9 inner primer pairs and 30 cycles of amplification. The assay protocol was identical to the initial report of this assay [29]. We evaluated the amplified DNA products by agarose gel electrophoresis and ethidium bromide staining.…”

“…In general, earlier studies to determine whether PCR for HIV DNA in blood cells would shorten the diagnostic window found little improvement by use of single amplification regimens [20][21][22][23][24][25][26]. In addition, some studies showed that single PCR amplification regimens were unable to detect the few HIV DNA copies present in ∼10 6 peripheral blood lymphocytes (PBL) during the window period and that nested PCR assays were needed [27][28][29][30].…”

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression

“…Forty-two uninfected subjects were analyzed as controls. PBMC DNA from the HIV-1-seropositive subjects and the uninfected controls was prepared by sodium dodecyl sulfate-proteinase K lysis, phenol-chloroform extraction, and ethanol precipitation as previously described [Zazzi et al, 1993a] and quantitated by spectrophotometry. PCR quality was confirmed by a 24-cycle amplification of the GH20/PC04 human beta-globin region [Saiki et al, 1988].…”