Korean J Clin Microbiol.  2009 Mar;12(1):11-16. 10.5145/KJCM.2009.12.1.11.

Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture

• Jang YH
• Chung J
• Baek S
• Park S
• Sung H
• Kim MN

• KMID: 1469964
• DOI: http://doi.org/10.5145/KJCM.2009.12.1.11

Abstract

BACKGROUND
We evaluated multiplex PCR for species identification and toxin typing to improve the sensitivity and turnaround time of toxigenic Clostridium difficile culture (TCDC).
METHODS
We performed multiplex PCR using primers targeting the species-specific gene, tpi, and the toxin genes, tcdA and tcdB. From January to March 2008, 528 stool specimens were tested with direct toxin assay (DT) using C. difficile Tox A/B II (Techlab, Blacksburg, USA) and TCDC. For 288 specimens from early study period, toxin production by C. difficile isolates of TCDC was measured by enzyme immunoassay with culture supernatants using VIDAS C. difficile Toxin A&B (CDAB;bioMerieux, Marcy-l'Etoile, France) and multiplex PCR with isolated colonies. For 240 specimens from late period, only multiplex PCR was used to test toxin production by the isolates.
RESULTS
During the early period, 29 C. difficile were isolated and their toxin-positive rates were 65.5% by PCR and 44.8% by CDAB (P<0.05). Among 528 stool specimens, the results of DT+/TCDC+, DT+/ TCDC-, and DT-/TCDC+ were 32 (6.1%), 33 (6.3%), and 10 (1.9%), respectively, when tested with PCR. 13.3% of total 75 positive specimens was detected only by TCDC. Of the 42 toxigenic C. difficile isolates, all were positive for tpi, 30 (71.4%) were tcdA+/tcdB+, and 12 (28.6%) were tcdA-/tcdB+.
CONCLUSION
TCDC using multiplex PCR for species identification and toxin typing is sensitive and rapid to be used as a routine diagnostic test.

Keyword

Clostridium difficile; Toxin; Polymerase chain reaction

MeSH Terms

Boron Compounds
Clostridium
Clostridium difficile
Diagnostic Tests, Routine
Immunoenzyme Techniques
Multiplex Polymerase Chain Reaction
Polymerase Chain Reaction
Boron Compounds

Figure

• 

  Fig. 1. Electrophoresis of the PCR products of nine isolates of C. difficile using DNA templates extracted with GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, USA) (A) and boiling method (B) in parallel. The bands on all lanes of A are consistently denser than those of B except lane 3. Lane M; 100 bp DNA ladder, Lane 1; negative control, Lane 2 and 3; positive for species specific gene (tpi) but negative for toxin genes (tcdA and tcdB), indicating nontoxigenic strains, Lane 4-7; positive for tpi, tcdA, and tcdB genes, indicating A+B+ toxigenic strains, Land 8-10; positive for tpi, and tcdB, but negative (deleted) for tcdA gene, indicating A-B+ toxigenic strains.

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