An improved qPCR protocol for rapid detection and quantification ofClostridium difficilein cattle feces

Abstract

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 TaqMan 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.

Identification of risk factors influencing Clostridium difficile prevalence in middle-size dairy farms

“…Studies reporting prevalence of C. difficile in calves reported prevalence from 6 to 22% [ 10 , 22 – 24 , 43 , 46 , 47 ], and even 56% in calves less than 7 days old [ 36 ]. The use of qPCR as a screening method made C. difficile detection more sensitive [ 27 ], which most likely accounted for the higher C. difficile prevalence in this study compared to other studies. Several sampling stages over a prolonged period are also more likely to identify bacteria in the investigated population.…”

“…For total DNA isolation, SmartHelix™ First DNAid kit (IFB, Slovenia) was used as described previously [ 27 ]. Clostridium difficile 16S rDNA gene was detected using an improved quantitative PCR (qPCR) that has the lowest detection (7.72 CD cells/g feces) and quantification limit (77.2 CD cells/g feces) published to date [ 27 ]. Calf fecal samples were analyzed individually when pooled fecal samples tested positive on qPCR.…”

Quantification of Clostridioides (Clostridium) difficile in feces of calves of different age and determination of predominant Clostridioides difficile ribotype 033 relatedness and transmission between family dairy farms using multilocus variable-number tandem-repeat analysis

“…This is the first suggestion of a possible calf to calf and farm to farm transmission of C. difficile . However, due to lower sensitivity of the culture method compared to qPCR [ 36 ] and the history of calf trading between these two farms, we could also assume that transmission of this clonal C. difficile isolate could have happened before the start of our study.…”

“…Calves fecal samples were tested for C. difficile specific fragment of 16S gene using a quantitative real-time PCR (qPCR) reported by Bandelj et al [ 36 ] with a LOD and LOQ of 7.72 CD cells/g feces and 77.2 CD cells/g feces, respectively. Samples ( n = 243) were retested in duplicates and in 1:10 dilutions to evaluate for possible inhibitory effects of the matrix.…”

Reference genes in real-time PCR

“…Some breakthrough moments for qPCR and reference genes were the 1st International qPCR Symposium in Germany (March 2004), continued further on in different countries as lead by Prof. Stephen Bustin, and the publication of MIQE Guidelines which goes for Minimum Information for Publication of Quantitative Real-Time PCR Experiments (Bustin et al 2009 ; Bustin et al 2010 ; Derveaux et al 2010 ). At the same time, implementation of MIQE goes with varying effectiveness, for example the report by Bandelj et al ( 2013 ) was actually the first study concerning Clostridium difficile applying those guidelines and improving previous protocol from 2005. They achieved much greater precision and sensitivity to a large extent by testing various TaqMan universal PCR master mixes as suggested by MIQE.…”