Development of a novel quantitative PCR analysis method for HIV-1

Detta är en Master-uppsats från Lunds universitet/Tillämpad biokemi; Lunds universitet/Teoretisk kemi

Sammanfattning: Biological pharmaceuticals are the answer to many severe diseases of today’s society. Some of these are protein-based drugs and can be produced from human blood plasma. In order to guarantee the safety of patients, absence of any pathogens needs to be ensured. Such a pathogen is HIV-1, which holds high mutation rate and a large variation in its genome. The aim of this project was to develop novel primers and probes for detection of a wide range of subtypes of HIV-1, using the TaqMan qPCR detection system. Three targets within conserved parts of the genome were selected in the regions of LTR5´, Pol and LTR3´. The primers and probes were optimized regarding concentrations, salt content and compatibility. The sensitivity of detection showed promising results with a value as low as 8.18 IU. The selectivity did not result as preferred, with the best combination of primers and probe possible to detect 7 out of 8 subtypes of the most common genotype, M. If combining the primers and probes of all targets suggested here, detection of all tested subtypes was possible. Summarizing the results, the primers and probe targeting the Pol region shows promising data. Optimization regarding the sequences of the Pol primers and probe and additional evaluation of compatibility between all targets need to be studied, in order to see, if the method can meet the standards and further be implemented into the experimental routine for HIV-1 detection.

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