title: Nested PCR assays with novel primers yield greater sensitivity to Tanzanian HIV-1 samples than a commercial PCR detection kit.
abstract: To investigate the efficacy of the SK431/SK145 primer pair and two nested primer assays in amplifying African HIV-1 samples, a total of 35 Tanzanian PBMC samples were examined. These were assayed by two HIV-1 specific nested in-house PCR assays and a commercial HIV-1 PCR kit (GeneAmp) using SK431/SK145 as the primer pair. One of the nested PCR assays has been evaluated previously (old assay), whereas the modified assay was constructed from the HIV-1 sequence alignment released in August 1993. The modified nested primer assay showed increased sensitivity in the gag and env regions compared to the old nested primer assay. However, both the old and the modified nested primer assays displayed higher sensitivity for the detection of Tanzanian HIV-1 proviruses than the GeneAmp assay. When two regions were used (gag and env) as targets for the amplification, the modified nested primer assay detected 97.1% (34/35) of the proteinase K lysed samples, compared to 68.6% (24/35) using the SK431/SK145 primer pair (P < 0.01**). The results indicate that the SK431/SK145 primer pair may be less suitable when HIV-1 samples from Africa are analysed. The results also show that continuous modification of primer sequences can improve and maintain high sensitivity for the detection of highly divergent HIV-1 strains.
authors.affiliation: Department of Virology, University of Umed, Sweden.
authors.forename: O
authors.initials: O
authors.lastname: Grankvist
authors.forename: L
authors.initials: L
authors.lastname: Walther
authors.forename: U
authors.initials: U
authors.lastname: Bredberg-Rådén
authors.forename: E
authors.initials: E
authors.lastname: Lyamuya
authors.forename: F
authors.initials: F
authors.lastname: Mhalu
authors.forename: A
authors.initials: A
authors.lastname: Gustafsson
authors.forename: G
authors.initials: G
authors.lastname: Biberfeld
authors.forename: G
authors.initials: G
authors.lastname: Wadell
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000302:isolation & purification*; D019286:Endopeptidase K / Q000378:metabolism; D005260:Female; D015686:Gene Products, env / Q000235:genetics; D015684:Gene Products, pol / Q000235:genetics; D016346:Gene Products, vif / Q000235:genetics; D016655:HIV Core Protein p24 / Q000235:genetics; D015658:HIV Infections / Q000453:epidemiology / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013636:Tanzania / Q000453:epidemiology; D054320:vif Gene Products, Human Immunodeficiency Virus
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D015686:Gene Products, env; D015684:Gene Products, pol; D016346:Gene Products, vif; D016655:HIV Core Protein p24; D054320:vif Gene Products, Human Immunodeficiency Virus; D019286:Endopeptidase K