Optimization of a Polymerase Chain Reaction System for Whole Genome Amplification of Human Immunodeficiency Virus Type 2 (HIV-2)

Abstract

BackgroundIn this study, attempts were made to amplify the whole genome of HIV-2 using polymerase chain reaction (PCR) from viral RNA and proviral DNA extracted from archived human plasma and whole blood, respectively. The aim this study is to develop a PCR system that can be used to amplify the entire genome of all known subtypes and recombinant forms of HIV-2MethodsProviral DNA and viral RNA were extracted from archived human whole blood and plasma using Zymo Research Viral DNA kit and Zymo Research Viral nucleic acids kit, respectively. Primers that target the conserved sites of the 3’ and 5’ long terminal repeat were selected based on in silico analysis using Snapgene® tool version 2.1.0. Eight overlapping Primers for PCR whole genome amplification of HIV-2 were also selected and used for overlap PCR amplification of whole genome of HIV-2. Long range and overlapping PCR of whole genome of HIV-2 were carried out using long range Kit (New England Biolabs Inc., Ipswich, MA, USA) and cDNA synthesized using NEB ProtoScript II for proviral DNA and viral RNA templates. Amplified whole genome of HIV-2 was gel purified and PCR confirmed using two sets of primers ENVF/ENVG and EB2/EB5 and gel electrophoresis.ResultsProviral DNA and viral RNA were successfully extracted from archived whole blood and plasma. Six primers were selected out of the 68 primer sequences retrieved using in silico analysis for long range single PCR amplification of HIV-2 whole genome. Primers P1/P8 and HIV2upA/HIV2lowA were successfully used in the whole genome amplification of HIV-2. Overlapping primers P1/P4, P6/P8, PolF/EnvG and Pol4F/EnvG covering the entire genome of HIV-2 were also successfully used in the whole genome amplification of HIV-2. The amplified whole genome fragment was confirmed to be HIV-2 by PCR using primers EB2/EB5 and EnvF/EnvG.ConclusionThe techniques of long-range and overlapping amplification of HIV-2 whole genome may be useful in HIV-2 genotyping using viral RNA and proviral DNA. This study has led to the selection and shown novel primer combinations of already existing primers which can be used to amplify the entire genome of HIV-2.Disclosures All authors: No reported disclosures.