Development and validation of a novel quantitative PCR analysis method for HIV-1
Sammanfattning: The major problem when developing primers and probe sets for detection of HIV-1 is its high genetic variability and strong ability to mutate. HIV-1 is divided into four groups, M, N, O, P based on genetic similarities, and there are more than 70 different circulating recombinant forms. The aim of this master thesis was to develop new primers and probe for a novel quantitative PCR analysis method for detection of a broad range of genotypes of HIV-1. Four target sequences with conserved parts were chosen: the pol gene at around nucleotide position 2000 in the reference sequence HXB2, the pol gene at around nucleotide position 4000, the nef gene at around nucleotide position 9000 and the gag gene at around nucleotide position 800. The primers and probe set targeting the pol gene, pol4000, showed best potential. The primer and probe concentrations were optimized, and the best concentration on both primers and probe was 900 nM. To test the ability to detect a broad range of genotypes of HIV a genotype test was conducted. The result showed that pol4000 could amplify all tested subgroups in group M, but failed to amplify group O and N. Detected replicates in percent from three independent assessor were compared against the average nucleotide mismatches for each genotype and a regression analysis showed that 27.5% of the detected result could be explained by nucleotide mismatches (p< 0.01). Moreover, the results showed that there are potential in the primers and probe set, pol4000, but there is a need for further investigation to be able to determine if pol4000 will function in the new routine analysis by Octapharma AB.
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