Development of Novel Methodology For the Molecular Differentiation of Cryptosporidium Parvum Gp60 Subtypes Via High Resolution Melting Analysis

Authors

Jennifer K. O'Leary, Department of Biological Sciences, Cork Institute of Technology
Liam Blake, Department of Clinical Microbiology, Cork University Hospital, Wilton, Cork, Ireland
Gerard D. Corcoran, Department of Clinical Microbiology, Cork University Hospital, Wilton, Cork, Ireland
Roy D. Sleator, Department of Biological Sciences, Cork Institute of TechnologyFollow
Brigid Lucey, Department of Biological Sciences, Cork Institute of TechnologyFollow

Document Type

Article

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Disciplines

Biology | Life Sciences | Microbiology

Publication Details

MethodsX

Abstract

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is 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 in Cryptosporidium subtyping 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 abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum gp60 subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region of gp60.

• Nested gp60 primers 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.

Recommended Citation

Jennifer K. O’ Leary, Liam Blake, Gerard D. Corcoran, Roy D. Sleator, Brigid Lucey, Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis, MethodsX, Volume 7, 2020, 101157, ISSN 2215-0161, https://doi.org/10.1016/j.mex.2020.101157.