Open AccessAn improved qPCR protocol for rapid detection and quantification of C lostridium difficile in cattle feces
Author(s) -
Bandelj Petra,
Logar Katarina,
Usenik Alenka M.,
Vengust Modest,
Ocepek Matjaz
Publication year - 2013
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12102
Subject(s) - clostridium difficile , feces , microbiology and biotechnology , clostridium , clostridiaceae , biology , bacteria , antibiotics , genetics , toxin
Clostridium difficile ( CD ) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real‐time PCR ( qPCR ) protocol for CD detection defined by Penders et al . ( FEMS Microbiol Lett, 243 , 2005, 141–147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD ‐spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used T aq M an universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD ‐spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD ‐spiked feces, R 2 between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans.