Novel multiplex-PCR method for simultaneous detection of Clostridium difficile toxin A and toxin B and the binary toxin (cdtA/cdtB) genes applied on a Danish cohort

Abstract number: p1645

Olsen  K.E.P., Persson  S.


A new multiplex PCR method was developed for the detection of the Clostridium difficile toxin genes: tcdA, tcdB, cdtA and cdtB. This method was applied on 210 Clostridium difficile strains isolated from 175 Danish hospitalised patients with diarrhoea in the period from April to October 2005, in order to investigate the present toxin profiles and their correlation to sex and age.


A 5-gene multiplex PCR method was developed for the simultaneous amplification of the four Clostridium difficile toxin genes tcdA, tcdB, cdtA, cdtB and 16S rDNA as an internal positive control. Template DNA was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis.


Three different toxin profiles were detected in the Danish cohort: 43 tcdA+, tcdB+, cdtA+/cdtB+; 107 tcdA+, tcdB+, cdtA-/cdtB- and 24 non-toxigenic tcdA-, tcdB-, cdtA-/cdtB-. The prevalence of the binary toxin genes in this study was 25% of the clinical isolates.

More than half of the strains (62%) were isolated from the elderly part of the population (>59 years), and 74% of these strains displayed the tcdA+, tcdB+, cdtA+/cdtB+ profile. Of the non-toxigenic strains, 83% of the patients were females. One fourth of the strains isolated from children under 2 years of age were non-toxigenic. In four patients, two different toxin profiles were obtained from independent faecal samples.


This method offers a one-step, rapid and specific identification of Clostridium difficile toxin genes. This specific toxin profiling allows an evaluation of the pathogenic potential of the isolated Clostridium difficile and surveillance of emerging toxin profiles. Further studies of the isolated toxigenic Clostridium difficile strains will include gene deletion analyses of the tcdA and the tcdC (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Location: Oxford, UK
Presentation type:
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