Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR)

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Abstract

An internally controlled high-input PCR method, termed HIV-1 Mega-PCR was developed to lower the detection limit of HIV-1 DNA polymerase chain reaction (PCR) and to improve its value as a complementary diagnostic test. It is based on PCR amplification of two target sequences in the gag gene of HIV-1 following the selective capture of the targeted sequence and removal of unselected DNA from up to 500 μg of DNA. Efficient selection and amplification was monitored by inclusion of two mimic plasmids. The method was evaluated with buffy coat cells from healthy blood donors which were spiked with blood from 106 different HIV-1-infected individuals, and with 107 HIV-1 seronegative control buffy coats. All specimens from HIV-infected individuals were positive by a PCR protocol using 1 μg of patient DNA. Amplification of 1 μg DNA of the 106 spiked, diluted samples resulted in 68 double positive, 14 single positive, and 24 double negative reactions. In the Mega-PCR, the average input was 260 ± 84 μg DNA containing an estimated 1.1 ± 0.6% of spiked patient DNA. Of the 106 samples tested by Mega-PCR, 102 were positive and three negative. One failed to select the mimic plasmid. Among the 107 negative buffy coat controls, none was false-positive and four exhibited a failure of the internal reaction control. Application of HIV-1 Mega-PCR to clinical specimens from seroreverting newborns of HIV-infected mothers and seroindeterminate, PCR-negative specimens revealed no indication for HIV infection, whereas three samples from confirmed, HIV-1-infected but PCR negative individuals showed evidence of the presence of HIV-1 DNA. Mega-PCR lowers the detection limit of an individual analysis to approximately 0.01 HIV-1 DNA copies/μg of applied DNA and may help to confirm or exclude HIV-1-infection in difficult situations diagnostic. J. Med. Virol. 72:1–9, 2004. © 2004 Wiley-Liss, Inc.

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