Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens

Zazzi, Maurizio; Romanò, Laura; Brasini, A.; Valensin, Pier Egisto

doi:10.1002/jmv.1890380304

Cited by 16 publications

(6 citation statements)

References 19 publications

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“…Various PCR procedures for the amplification of proviral DNA and the HIV RNA genome have been described (17, [25][26][27]. In the case of proviral DNA amplification, both actively replicating and quiescent viruses harbored in blood cells can be detected, whereas with RNA amplification only actively replicating viruses are detected and, therefore, diagnostic sensitivity is low (10,18).…”

Section: Discussionmentioning

confidence: 99%

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations

et al. 1995

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We used a colorimetric polymerase chain reaction (PCR)-based assay in kit form to detect directly human immunodeficiency virus type 1 (HIV-1) proviral gag sequences in peripheral blood cells from 68 healthy blood donors, 51 subjects at risk for HIV infection, 122 patients with HIV-1 infection, 11 patients with indeterminate Western blot (immunoblot) results, 4 blood donors HIV-1 positive by enzyme immunoassay, and 13 children born to HIV-1-seropositive mothers. The results obtained in the blood donors and HIV-1-infected patients demonstrated the high degree of diagnostic specificity and sensitivity of the PCR method. HIV-1 infection was excluded in 10 of the 11 patients with indeterminate Western blot results and in all four enzyme immunoassaypositive blood donors. A diagnosis of HIV infection was ruled out by negative PCR results in 5 of 13 children from seropositive mothers, which excluded vertical transmission of the infection in these cases; these children were younger than 3 months and had positive serological results. Two at-risk patients with negative serological results had positive PCR results. All results were confirmed by conventional PCR. In conclusion, colorimetric PCR, which is commercially available in kit form, is an easy and reliable technique that can be used to detect proviral HIV-1 genomes in blood cells, and despite the limitations owing to HIV genome variability, it is useful in the clinical setting for the diagnosis of HIV infection in selected categories of patients.

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Section: Discussionmentioning

confidence: 99%

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations

et al. 1995

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“…The HIV-1 gag region was ampli® ed using a MZ13 (0.2 mM, 5'-tac atc agg cca tat cac ct-3', 1224± 1243 of HIV-1 ARV-2 )/MZ14 (0.2 mM, 5'-gaa ccg gtc tac ata gtc tc-3', 1696-1677 of HIV-1 ARV-2 ) primer pair for initial ampli® cation of HIV-1 proviral DNA. A MZ8 (0.2 mM, 5'-gca tta tca gaa gga gcc ac-3', 1316-1335 of HIV-1 ARV-2 )/ MZ9 (0.2 mM, 5'-agg gta cta gta gtt cct gc-3', 1524-1505 of HIV-1 ARV-2 ) primer pair was subsequently used in the buffer with 0.025 U/ml Ex taq DNApolymerase, 0.2 mM dNTP, and 1.25 mM MgCl 2 (Takara, Tokyo, Japan) 12 . Ampli® ed DNA was visualized by agarose-electrophoresis coupled with ethidium bromide staining.…”

Section: Detection Of Hiv-1 and Hiv-2 Proviral Dnamentioning

confidence: 99%

Seronegative HIV-2 carriers in India

Kageyama¹,

Maniar²,

Iwasaki³

et al. 2000

Int J STD AIDS

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The discordant cases of seronegative, but culture and proviral HIV-2 DNA positive were found in Mumbai, India. This was corroborated by the successful isolation of HIV-2-RNA in culture medium, HIV-2 cDNA sequence determination and the detection of the antigen. The sequence of the isolated HIV-2 genomic RNA does not seem to be altered to the extent that the change will alter antibody binding. Furthermore, antibody from the same individual (even at 8 months from initial sampling) from whom HIV-2 was isolated did not react with the antigen of this strain. Those evidences imply that extremely low or non-production of the antibody may be due to suboptimal immune stimulation due to extremely slow HIV-2 replication. This low virus-load may be responsible for the negative antibody results in the HIV-2 carriers.

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“…Prepared DNA (0.15 µg) was subjected to PCR with competitor DNA, serially diluted and adjusted at appropriate concentrations as reported elsewhere (Menzo et al, 1992;Piatak et al, 1993). The primers MZ8 (5′-GCAT-TATCAGAAGGAGCCAC-3′,1316-1335 of ARV2) and MZ9(AGGGTACTAGTAGTTCCTGC-3′, 1524-1505) had been reported previously (Zazzi et al, 1992), and were designed to amplify an internal fragment of either 209 bp (from wild-type HIV-1 target sequences) or 189 bp (from the pMZ∆L competitive template). A plasmid (pMZ∆L) containing a 20 bp internal deletion was prepared.…”

Section: Competitive Dna Pcrmentioning

confidence: 99%

Extract of Prunella Vulgaris Spikes Inhibits HIV Replication at Reverse Transcription in vitro and can Be Absorbed from Intestine in vivo

Kageyama

Kurokawa

Shiraki

2000

Antivir Chem Chemother

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It has been reported that extracts of the spike of Prunella vulgaris (PS) exhibit anti-HIV activity at the adsorption and reverse transcription stages. In this study, the actual activity of PS in cells, kinetic analysis of the inhibitory activity of PS against HIV reverse transcriptase and the feasibility of oral administration were examined. First, to clarify whether this extract shows anti-HIV activity in cells in vitro, the number of copies of proviral DNA in HIV-exposed cells was calculated. The number of copies was significantly decreased in cells cultured in the presence of PS extract, but not in the presence of dextran sulphate. The activity of PS extract in the cells was also assessed by the drug addition test, during and after HIV adsorption. PS extract and dextran sulphate suppressed HIV production to similar levels when added after HIV adsorption. However, only PS extract suppressed HIV production at the same concentration when the drugs were added during HIV adsorption. Presumably, the penetration of the PS extract into the cells was required for this activity. Secondly, fractionated PS inhibited HIV reverse transcription in a non-competitive manner. This fractionated PS kept anti-HIV activity, but inhibited HIV replication and adsorption to a lesser extent compared to dextran sulphate. Lastly, an active component(s) was detected in plasma in vivo, after injection into the intestine, which demonstrates the feasibility of oral administration dosing.

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Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens

Cited by 16 publications

References 19 publications

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations

Seronegative HIV-2 carriers in India

Extract of Prunella Vulgaris Spikes Inhibits HIV Replication at Reverse Transcription in vitro and can Be Absorbed from Intestine in vivo

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