Ann Lab Med.  2015 May;35(3):306-313. 10.3343/alm.2015.35.3.306.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

• Kilic A
• Alam MJ
• Tisdel NL
• Shah DN
• Yapar M
• Lasco TM
• Garey KW

• KMID: 2363202
• DOI: http://doi.org/10.3343/alm.2015.35.3.306

Abstract

BACKGROUND
The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile.
METHODS
The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR.
RESULTS
A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%.
CONCLUSIONS
The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Keyword

Multiplex real-time PCR; Clostridium difficile; Toxin variant strains; Binary toxin

MeSH Terms

ADP Ribose Transferases/genetics
Bacterial Proteins/*genetics
Bacterial Toxins/*genetics
Clostridium difficile/isolation & purification/*metabolism
DNA, Bacterial/genetics/metabolism
Enterotoxins/genetics
Feces/*microbiology
Humans
Multiplex Polymerase Chain Reaction
Prospective Studies
Real-Time Polymerase Chain Reaction
Triose-Phosphate Isomerase/genetics
ADP Ribose Transferases
Bacterial Proteins
Bacterial Toxins
DNA, Bacterial
Enterotoxins
Triose-Phosphate Isomerase

Figure

• 

  Fig. 1 Amplification curves and dilution end-point standard curves of log10 genome equivalents versus threshold cycle number. The analytical sensitivity of this assay for tpi (A-B), tcdA (C-D), tcdB (E-F), and cdtA (G-H) was approximately 103 CFU/g.Abbreviations: CFU, colony forming unit; ΔRn, normalized reporter.

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