Real-time PCR Method For The Detection Of Clostridium Difficile And Cryptosporidium Parvum In Biological Samples

Objective:To establish real-time PCR assays based on TaqMan probe chemistry for thedetermination of the Clostridium difficile with or without toxin genes and theCryptosporidium parvum oocysts in biological samples inclouding chicken and humanstools. To validate the real-time PCR assay by applying it to the detection of thetoxigenic Clostridium difficile and Cryptosporidium parvum oocysts in diarrheapediatric stool samples.Methods:1. The efficiency of DNA extraction was evaluated by testing the DNA contentsand OD260/OD280ratios extracted from standard Clostridium difficile with4differentDNA extraction methods. The specific primers and probes were designed according tothe housekeeping gene tpi and toxin tcdA/tcdB genes for real-time PCR system ondetecting pathogenic Clostridium difficile. The experiment was conducted with chickenand fece samples spiked with10⁰to10⁶CFU/ml of standard Clostridium difficile toanalyze its sensitivity. The specifity for detecting Clostridium difficile and interferencefrom other bacterial were also assayed.2. The DnaJ-like protein gene was selected as target gene and special primers andtaqman probes within the conserved regions of the gene were designed for real-timePCR system on detecting C.parvum. Blank fecal sample spiked with standard culturefluid containing2.6to2.6×10⁵oocysts of C.parvum was tested for the sensitivity of themethod. The specificity for detecting Cryptosporidium parvum oocysts and interferencefrom other enteric parasites and bacteria were assayed too.3. Forty three clinical diarrhea samples were collected from pediatric hospital. Allsamples were analyzed for the presence of Clostridium difficile and Cryptosporidiumparvum oocysts with above established real-time PCR methods, and results were compared with those obtained by conventional detection methods. Clostridium difficileselective medium was appied for identification of Clostridium difficile whilemicroscopic examination for determination C.parvum oocysts. The consistency of theresults obtained by different methods was analyzed with Chi-square test.Results:1. With described real-time PCR for screening assay all Clostridium difficiletrains tested were tpi-positive and all non-Clostridium difficile trains yielded noamplification products. The detection limits for Clostridium difficile in pure culture,artificially contaminated chicken sample and fecal sample are10cfu/ml,102cfu/ml and103cfu/ml respectively. Among3atandard Clostridium difficile trains only one trainwith toxin genes showed tcdA/B positive in real-time PCR for pathogenic Clostridiumdifficile assay. The detection limits for toxigenic C.difficile with tcdA/B genes are2.5×10^-3ng DNA. Both amplification reactions showed a good linear relationshipbetween Ct and DNA amounts which yielded the R values of0.9975and0.9984fortcdA and tcdB gene respectively.2. The real-time PCR assay for C.difficile could detect oocyst of C.difficile withno amplification on other enteric parasites and bacteria.The detection limits for oocystand the extracted DNA of C.difficile in pure culture are26oocysts and2.5×10^-1ng. Thedetection limit for C.difficile in artificially contaminated fecal sample is2.6×10³oocysts.3. In53collected clinical diarrhea samples, five samples were detectedClostridium difficile positive using traditional Clostridium difficile selective mediumtest while no C.parvum oocyst found present in samples by microscopicallyexamination. In comparision,12samples detected Clostridium difficile positive and noC.parvum oocyst found in all samples by using above real-time PCR methods.C.difficile in12positive samples were approved to be all toxigenic Clostridium difficilecontaining tcdA and tcdB genes with real-time PCR for pathogenic Clostridium difficileassay. The amplification products were then verified to be identical to tcdA and tcdBgenes using DNA sequence analysis.Conclusions: Our developed real-time PCR based on TaqMan probe chemistry revealed theability for identification of C.difficile and C.parvum oocysts with high specificity andsensitivity. They are more suitable for the detection of C.difficile and C.parvumoocysts present in the clinical diarrhea sample than traditional methods.