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BioMed Research International
Volume 2013 (2013), Article ID 875437, 6 pages
Research Article

Evaluation of Multiplex PCR with Enhanced Spore Germination for Detection of Clostridium difficile from Stool Samples of the Hospitalized Patients

1Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
2Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
3Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand
4Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
5Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
6Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Received 23 November 2012; Revised 31 January 2013; Accepted 16 February 2013

Academic Editor: Joy Scaria

Copyright © 2013 Surang Chankhamhaengdecha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.