Integration of random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) and DNA sequencing in search for strain-specific pharmacological targets in Echinococcus granulosus

Abstract

Echinococcus granulosus a parasite characterized with intra-species variability and genetic studies show existence of 10 genotypes (G1-G10). Host specificity and different susceptibility to intermediate hosts has also been demonstrated. Better understanding of this parasitosis can assist in designing appropriate control and preventive measures and its management based on strain-molecular peculiarity. Thus, the necessity for identification and characterization of all strains. A total of 96 hydatid cysts from which either protoscolces or germinal membranes were extracted followed by DNA extraction and then, amplification by random amplification of polymorphic DNA (RAPD). The RAPD products with distinctive bands were cloned in pGEMT-Easy vector and recombinant DNA subjected to sequencing. Twelve oligonucleotides primers were designed from recombinant DNA sequences and strains-specific PCR were conducted. The PRC products amplified by primers P1F2R2 (Ta=66°C, 35 cycles) and P1F1R1TXPCRs (Ta = 66°C, 35 cycles) showed specie-specificity. Analysis of the DNA sequences showed homologies to some important molecules like laminin-binding protein and glutathione transferase. Notwithstanding that the present study indicates partial success on attaining distinctive strain-specific DNA sequences, the resultant polymerase chain reaction (PCR) products were not strain-discriminatory. It is speculated that incorporation of more restriction endonucleases and well-adjusted reaction conditions strain-distinctive PCR-restriction fragment lenght polymorphism (RFLP) can be designed.

Key words: Echinococcus granulosus, strain-specificity, random amplification of polymorphic DNA-polymerase chain reaction RAPD-PCR, DNA sequencing.