Abstract:
ABSTRACT
Gene sequencing is a proven method of choice in detection of point mutations associated
with Human Immunodeficiency Virus (HIV) drug resistance. The method is however very
expensive and prohibitive in resource poor settings. This standard nucleotide sequencing
method costs $200-$500 and involves the use of expensive equipment. There is need
therefore to develop a cheaper method that is fast in detection of point mutations that are
responsible for HIV -1 drug resistance.
The objective of the study was to evaluate the use of mutagenically separated PCR in
detection of drug resistance mutations in HIV patients. The procedure involved the use of
sets of primers (molecular markers) that are able to differentiate between wild type and drug
resistance strain of virus. A total of 74 samples were collected from Mbagathi and Maragua
District hospitals. Samples were collected from HIV patients who have been on anti-
retroviral therapy for at least six months.
The assay involved single nucleotide polymorphism detection Polymerase Chain Reaction
that consisted of one-step Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
reaction and two subsequent rounds of competitive PCR. From the gel electrophoresis
experiments, bands of different base pairs were obtained. However, none of the bands
obtained were of the expected 190 bp for the mutant strains and 210 bp for the wild type
strains. For the 103 region with the 103 primers, amplicons of different band sizes were
amplified. Some amplicons had single bands and others had more than one band all of
different base pairs ranging from 100bp to 300bp. Samples amplified with 181 primers for
the 181 region had double bands of 90bp and 120bp. Hence further work should be done on
the samples to determine whether the mutations were as a result of mutations or not.
