Comparison of in-house and commercial sample preparation and PCR amplification systems for detection of human immunodeficiency virus type 1 DNA in blood samples from Tanzanian adults

Abstract

This study compared the performance of several in-house nested PCR systems and the Amplicor human immunodeficiency virus type 1 (HIV-1) PCR kit in the detection of HIV-1 DNA in Tanzanian samples prepared by two different methods. All six of the in-house primer sets evaluated had a higher sensitivity for HIV DNA detection in samples prepared by the Amplicor PCR sample preparation method than in those prepared by the Ficoll-Isopaque (FIP) density gradient centrifugation method. A sensitivity of 100% was achieved by combining two in-house primer sets. The sensitivity of the standard Amplicor HIV-1 PCR kit was only 59%, whereas a modified Amplicor HIV-1 PCR test had a sensitivity of 98%. Our data show that Tanzanian samples prepared by the Amplicor preparation method are more suitable for HIV-1 PCR testing than samples prepared by the FIP method. The modified, but not the standard, Amplicor HIV-1 PCR kit provides an alternative to the nested in-house PCR technique for the diagnosis of HIV infection.

Strategies for diagnosis of HTLV‐I and ‐II

“…This conclusion is also supported by the fact that several of the samples were also negative in the commercial HTLV‐I and/or HTLV‐II PCR test. Finally, it should be mentioned that the cell lysis protocol used in the in‐house PCR has been found to be less robust than commercial protocols, 44 but this does not explain the difference in sensitivity between HTLV‐I and HTLV‐II.…”

V3 sequence analysis and biological characterization of HTV‐1 isolates from asymptomatic and early symptomatic Tanzanian individuals

“…These primers were also used in attempts to detect proviral DNA in PBMC lysates from HIV-1 group 0 isolates MVP-5 18019 1, MVP-2902/94 and MVP-7851/94 (kindly supplied by Dr. J. Eberle, Max von Pettenkofer Institute of Hygiene and Medical Microbiology, University of Munich, Germany). V3 (34) and pol (35) primer sequences and PCR conditions (36) have been previously described. The final PCR products were electrophoretically analyzed by applying 5 p1 of the second amplification product to 1.5% agarose gels containing 0.3 pg/ml ethidium bromide.…”

The Trophoblastic Epithelial Barrier Is Not Infected in Full-Term Placentae of Human Immunodeficiency Virus-Seropositive Mothers Undergoing Antiretroviral Therapy

“…Therefore, we performed PCR for detection of HIV DNA in the different placental cell preparations as well as the mothers' PBMC and the cord blood samples (CBMC). For PCR, cells were lysed (4) and nested PCR was performed in duplicate on 10 l of cell lysate corresponding to 10 5 cells, using primers JA79-JA82 and JA80-JA81 (1,25), known to amplify diverse genetic subtypes within group M of HIV-1 (25). For HIV-2-infected patient 3, the primer sets JA47-JA52 and JA48-JA49 (pol) were used (33).…”

“…Briefly, total RNA was extracted from purified trophoblastic cells using the Nuclisens method according to the manufactur- er's recommendation (Organon Teknika, Boxtel, The Netherlands) and reverse transcribed into cDNA (first-strand cDNA synthesis kit; Amersham Pharmacia Biotec, Uppsala, Sweden). Detection of HIV RNA was performed by using the previously described pol primers JA79-JA82 (1,25) and was negative for all placentae. The efficiency of this method was tested on blood donor PBMC spiked with a known amount of viral RNA.…”