Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction

Abstract

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure. Key words: Clostridium difficile, polymerase chain reaction, bacterial toxins.

Molecular Analysis of Clostridium difficile PCR Ribotype 027 Isolates from Eastern and Western Canada

“…All strains were PCR probed for the virulence markers tcdA, tcdB, cdtA, and cdtB and the macrolide-lincosamidestreptogramin B resistance marker ermB (6,8,15). Thermocycler and reaction conditions for each marker were conducted in accordance with the corresponding previously published methods.…”

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city, Brazil

“…Pellet was resuspended in 300 µl of Milli-Q water and boiled for 17 min. After centrifugation (14,000 g, 10 min), supernatant was saved and used as template (McMillin et al 1992). Positive (C. difficile VPI 19463) and negative control strains of toxigenicity were included in PCR assays.…”

“…Multiplex-PCR reaction -The primers used were: Tox-A1 (5'-GGA AAT TTA GCT GCA GCA TCT GAC-3'); Tox-A2 (5'-TCT AGC AAA TTC GCT TGT GTT GAA-3'); Tox-B1 (5'-GGT GAT ATG GAG GCA TCA CCA CTA G-3') and Tox-B2 (5'-TCC AGG ATA AGT CTC CTC TAC GTT G-3') (McMillin et al 1992) (Gibco BRL Technologies). These primers amplified a characteristic 1,217-bp toxin A (gene tcdA) and 1,050-bp toxin B (gene tcdB) bands.…”

“…Recently, it has been developed a multiplex-polymerase chain reaction (PCR), which amplifies simultaneously toxins A and B genes and which can be used to distinguish both toxigenic and non-toxigenic C. difficile (McMillin et al 1992). …”

Detection and Transcription of Toxin DNA in a Nontoxigenic Strain of Clostridium difficile

“…Nontoxigenic strains, in contrast, lack these genetic determinants [2,11]. To this end, several groups have developed PCR-based techniques to detect the presence of pathogenicity determinants in C. diffıcile [11,14,15,[22][23][24]34], and thus rapidly diagnose C. diffıcile-induced diarrhea, colitis, or PMC.…”