Detection of toxin producing clostridium difficile using multiplex PCR

Clostridium difficile frequently causes infectious diarrhea in hospitalized and elderly people. Infection results from reduction of natural intestinal flora, allowing the spores to grow and spread via fomites. The severity of C. difficile infection depends on production of toxins A and B located on pathogenicity locus along with three other regulator genes involved in toxin regulation and secretion of toxins: tcdD, tcdE and tcdC. Toxin production is negatively regulated by tcdC, but the presence of a mutation at positions 117 and 184 of tcdC gene enhance the toxin production. Currently with the increasing demand for diagnosis of toxin producing C. difficile, we have developed a rapid multiplex PCR test based on the detection of gluD, tcdA, tcdB and tcdC gene along with corresponding mutations. The microarray based multiplex PCR assay, was initiated by designing the PCR primers and allele specific primer extension (ASPE) probes to target the desired region of the C. difficile. The Autogenomics INFINITI system was used to interpret the results for specificity of primer sets and PCR run conditions. Results showed custom designed primer sequences were specific and capable of amplifying tcdA, tcdB and tcdC gene, while amplification of the gluD and tcdC mutations need to be further optimized.