Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides

Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be...

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Bibliographic Details
Published in:Experimental parasitology 2022-11, Vol.242, p.108366, Article 108366
Main Authors: Elwin, Kristin, Robinson, Guy, Pérez-Cordón, Gregorio, Chalmers, Rachel M.
Format: Article
Language:English
Subjects:
Animals
Cryptosporidiosis - diagnosis
Cryptosporidiosis - epidemiology
Cryptosporidium
Drinking water
Genotype
Genotyping
Oocysts - genetics
Real-time PCR
Real-Time Polymerase Chain Reaction - methods
SSU rRNA gene
Water - parasitology
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Summary:Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be undertaken post hoc and remains a specialist test, only undertaken in some laboratories. The benchmark method is nested PCR-sequencing of part of the SSU rRNA gene, but not all slides are typable and the workflow is cumbersome. We report the development, in-house validation and application of a real-time PCR-sequencing assay based on that gene, using a hydrolysis probe, for the detection and genotyping of all Cryptosporidium spp. The assay was investigated in two formats; a high volume DNA template for analysing all the DNA extracted from Cryptosporidium-positive water monitoring slides with
ISSN:0014-4894
1090-2449
1090-2449
DOI:10.1016/j.exppara.2022.108366