Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis

摘要

Cryptosporidiumspecies subtypes are generally identifiedviaDNA sequencing of thegp60gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products,gp60is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay inCryptosporidiumsubtyping nomenclature.This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementionedgp60nomenclature system, in order to differentiate betweenCryptosporidium parvum gp60subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region ofgp60.? Nestedgp60primers were designed to amplify a target region of <200 base pairs for effective HRM analysis? This method presents a rapid, sensitive, cost effective alternative to conventional sequencing.? This method is highly flexible and may be applied to other loci in order to facilitate multi-locus analysis and improve the discriminative abilities of the method.