Use of whole blood and dried blood spot for detection of HIV-1 nucleic acids using reverse transcription loop-mediated isothermal amplification

Elsevier, Journal of Virological Methods, Volume 312, February 2023
Khan M.J.R., Bhuiyan M.A., Tabassum S., Munshi S.U.

For monitoring viral load (VL) or Early Infant Diagnosis (EID) of HIV-1, real-time Polymerase Chain Reaction (qPCR) is used to perform on plasma or Dried Blood Spot (DBS) sample. The qPCR method is expensive and requires sophisticated equipment. Therefore, there is a requirement for newer and cheaper technology for VL measurement or EID. In this analytical study, a Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) assay was optimized and applied for amplification of HIV nucleic acids (NA) extracted from plasma, heat-treated plasma, heat-treated whole blood and lysis buffer-treated dried blood spot (DBS). The amplified product of RT-LAMP assay was detected by color change of Hydroxy naphthol blue (HNB) dye, step ladder pattern band on agarose gel after electrophoresis and sigmoid-shaped curve in the real-time thermal cycler. Comparing the results from RT-LAMP testing of all conditions with the results obtained by RT-qPCR results, viewed as the gold standard; a relative analytical sensitivity and specificity of RT-LAMP was calculated as 100 % and 90 % respectively. The corresponding positive predictive value (PPV) and negative predictive value (NPV) were 93.75 % and 100 %, respectively. The percentage of agreement between the RT-LAMP and RT-qPCR was 88.46% and Cohen's kappa value was 0.75 shows a substantial agreement between the two tests. This study suggests that whole blood or DBS may be useful specimens for analysis by HIV-1 specific RT-LAMP, to provide a cost effective alternative to RT-qPCR for the detection of HIV-1 nucleic acid at the point of care, or in early infant diagnoses.