2019

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Browsing 2019 by Author "Asma Akhter"

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    Comparative analaysis of pcr and lipa methods for hcv genotypes screening in pakistan
    (UMT,Lhr, 2019) Asma Akhter
    Hepacivirus C is a sole standard virus that fits into the "falviviridae" family. HCV has the size of 9500 kilo Dalton and its genome consists of 9000 nucleotides. The genome of HCV contains 3000 poly proteins that are structural and nonstructural e.g. E1, E2, P7, NS2, NS4B, NSSA. High prevalence of HCV is found in Asia and Africa, while low occurrence in Australia, North America, Western and North Europe. HCV is spread among individuals of all age groups and gender throughout the world, and it spread via unsafe medical treatment and transfusion of blood without screening, tattooing, drug abuse with contaminated syringes and mother to fetus also. HCV causes acute or chronic infection and become a main cause of hepatocellular carcinoma. HCV has 6 main genotypes (1 - 6) and also their subtypes (a, b, c). These genotypes are on the basis of polymorphism in the 5' UTR. Most prevalent genotype is 3a in Pakistan. There are different methods for HCV genotype detection e.g. Real time PCR, reverse hybridization and sequencing. We used two different techniques for genotype detection, one is nested PCR and others is Line Probe Assay (LIPA).In nested PCR there are 2 sets of primers used for HCV genotype detection and results were analyzed by gel electrophoresis while the LIPA method is a reverse hybridization line probe assay in which biotinylated DNA PCR amplicons were hybridized to immobilized oligonucleotide probes and results were analyzed via banding pattern on cellulose strips. Epidemiological studies displayed the dispersal frequency of HCV that varies among different regions of world. Preventive strategies and therapies can be improved by the HCV genotype evaluation in different regions. Among 416 studied patients ratio of various HCV genotypes was different I different districts of Punjab. Most common genotypes were 3a (14.66%) and 1b (12.01%), 1(2.62%). While 2b (0.25%), 2a (0.17%), 4 (0.11%), 5a (0.37%) and 6 (0.28%) remained less predominant. Outcomes of present study were same as the previous different studies, as the 3a genotype is most frequent type of HCV in different districts of Punjab. For detection of HCV genotypes there is a need to develop the validated assay that is more reliable and sensitive for un typeable samples. This study shows the comparison between Nested PCR and LIPA method results and also showing the more reliable results on Nested PCR technique, because only 5 samples were un typeable on Nested PCR as compared to LIPA on which 33 samples were showing un typable result out of total 416 patients. We concluded that Nested PCR is more reliable, sensitive and cost-effective method as compared to LIPA method and most prevailing HCV genotype in Pakistan is 3a, which is clearly analyzed by Nested PCR.