Establishment and evaluation of RT-LAMP for HIV-1 P24 gene detection in comparison with RT-PCR, and COBAS TaqMan real time RT-PCR among Sudanese

Abstract

Abstract Background: A rapid, simple, cost efficient, nucleic acid based test for detecting HIV-1 in resource poor or field settings is highly desirable. Recent molecular techniques based on genomic sequence detection such as RT- PCR, real-time PCR are sensitive and rapid, but these methods use thermocycling equipment and so are impractical for field applications and for use in less developed laboratories. Loop-mediated isothermal amplification (LAMP) is a new developed technique which can amplify nucleic acids DNA and RNA (LA1\/IP and RT-LAMP, respectively) with high specificity, sensitivity and rapidity under isothermal conditions. Objective: The objective of this study is establishment and evaluation of RT- LAIVIP, for HIV-1 P24 gene in comparison with RT-PCR, and COBAS TaqMan real time RT-PCR. Materials and Methods: Ninety EDTA anticoagulated blood samples were collected, 70 from HIV -1 positive participants (ELISA, and western blot), and the remaining 20 samples were collected from HIV-1 negative participants (ELISA), all samples were tested for HIV-1 P24 gene with RT-LAIVIP and RT-PCR, nine HIV-1 positive sample viral load were detected using COBAS® TaqMan® HIV-1 Test, version 2.0 (v2.0) for use with the high pure system. Results: All samples were tested for HIV-1 P24 gene using RT-LAl\/[P and RT- PCR, 68/70 were positive with RT-LAl\/[P (97.1%), and 61/70 were positive with RT-PCR (87.1%). All 20 HIV-1 negative sample were negative with RT-LAMP 20/20 (100%) and 18 were negative with RT-PCR 18/20 (90 The detection limit of RT-LAl\/[P assay were detected by diluting RNA with 6.69 X105 HIV-1 copies/ml, and 1.3 X104 copies/ml, the reproducible 100% detection limit of the RT-LAMP assay was 130 and 162 copies/ml, compared to 522 and 325 copies/ml with RT-PCR. Conclusion: I RT-LAMP is an inexpensive, rapid assay and valuable tool for the detection of HIV-1 RNA in plasma. The RT-LA1\/IP was performed under isothermal conditions at 63 °C and no sophisticated equipments were needed, which make it more reliable for use in developing countries, like Sudan.