CHARACTERIZATION OF PREVALENT ECHINOCOCCUS GRANULOSUS GENOTYPES THROUGH MODIFIED PCR-RFLP TECHNIQUE
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Abstract
Background:
Cystic echinococcosis is a zoonotic, neglected tropical disease caused by various genotypes of cestode parasite Echinococcus granulosus. Although, echinococcosis causes enormous financial and health impacts, no gold standard diagnostic technique is available. Genetic characterization of these prevalent cestode at species level is essential for disease management and appropriate control measures.
Objective:
We aimed to investigate the genetic diversity of echinococcus granulosus using a modified Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based assay in infected population.
Methodology:
A total of 18 human hydatid cyst samples were collected from various hospitals of Southern Punjab and Islamabad Capital Territory region of Pakistan. Extracted DNA was used for PCR amplification of mitochondrial NADH dehydrogenase subunit 1 (Nad1) gene followed by sequencing and phylogenetic analysis using Molecular evolutionary Genetic Analysis (MEGA) Software. The entire sequences were fed into NEBcutter V2.0 to select a single restriction enzyme followed by invitro confirmation through PCR-RFLP.
Results:
Amplification on the Nad1 gene was observed in 100% of the samples processed. The Basic Local Alignment Tool (BLAST) and phylogenetic tree analysis revealed 83.3% E. granulosus (G1-G3 genotypes), 11.1% E. multilocularis and 5.6% E. Canadensis (G6 genotype). The use of the BfaI enzyme in PCR-RFLP analysis revealed that all of the 18 samples were assigned consecutive genotypes as observed in the sequencing.
Conclusion:
The current study concluded that the BfaI enzyme could be used for the genotypic analysis of echinococcosis in developing and frequently affected countries. It will be a cost-effective and easy technique compared to sequencing, which will aid in developing novel therapeutic and control strategies for the parasite.
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