Premium
Novel quantitative T aq M an real‐time PCR assays for detection of C ryptosporidium at the genus level and genotyping of major human and cattle‐infecting species
Author(s) -
Burnet J.B.,
Ogorzaly L.,
Tissier A.,
Penny C.,
Cauchie H.M.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12103
Subject(s) - taqman , cryptosporidium , biology , genotyping , real time polymerase chain reaction , microbiology and biotechnology , cryptosporidium parvum , polymerase chain reaction , genotype , virology , feces , gene , genetics
Aims Development of T aq M an MGB real‐time PCR assays for quantitative typing of major cattle and human‐pathogenic C ryptosporidium species. Methods and Results Three specific T aq M an MGB real‐time PCR s, based on the SSU r RNA gene, were directed towards livestock‐restricted C ryptosporidium andersoni and C ryptosporidium bovis as well as both human‐pathogenic C ryptosporidium parvum and C ryptosporidium hominis . A generic T aq M an assay further identified all known C ryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific T aq M an protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non‐target DNA in environmental samples. Conclusions All T aq M an MGB real‐time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. Significance and Impact of the Study No molecular‐based method was yet available for the quantitative detection of C . andersoni and the cluster formed by C . bovis, C ryptosporidium ryanae and C ryptosporidium xiaoi . This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.