Abstract
Clostridium difficile is known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Toxinogenic strains of the bacterium produce toxins A (TcdA) and B (TcdB), which are associated with the pathogenicity. The standard methods for diagnosis of C. difficile infection include the cell cytotoxicity assay and the culture of a toxinogenic strain. Due to the long turnaround time of these methods, more rapid methods are preferred. Enzyme immunoassays are fast, but lack sensitivity. Therefore, real-time PCR methods have been developed.
The real-time PCR described in this chapter detects tcdB, the gene coding for toxin B. Since toxin A-negative, toxin B-positive strains have been reported to cause disease as well, these strains can also be detected by this method which uses an automated STAR-MagnaPure method for the optimum isolation of DNA from feces. An internal control is included as well to control for inhibition of the PCR method.
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Acknowledgments
We would like to thank Ingrid Sanders for her help in optimizing the DNA isolation protocol and real-time PCR conditions. We also would like to thank the department of Clinical Microbiology Laboratory for providing the correct protocols for the use of the MagnaPure and CFX96 systems.
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van den Berg, R.J., Bakker, D., Kuijper, E.J. (2013). Diagnosis of Clostridium difficile Infection Using Real-Time PCR. In: Wilks, M. (eds) PCR Detection of Microbial Pathogens. Methods in Molecular Biology, vol 943. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-353-4_16
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DOI: https://doi.org/10.1007/978-1-60327-353-4_16
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