Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR Green I.
In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential.
Wehrle
Esther
E
Hygiene and Technology of Milk, Department of Veterinary Sciences, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Oberschleissheim, Germany.
Didier
Andrea
A
Moravek
Maximilian
M
Dietrich
Richard
R
Märtlbauer
Erwin
E
D001409:Bacillus cereus / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics*; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D003967:Diarrhea / Q000382:microbiology; D005516:Food Microbiology; D006460:Hemolysin Proteins / Q000235:genetics*; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013045:Species Specificity; D044366:Transition Temperature
D001426:Bacterial Proteins; D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D006460:Hemolysin Proteins; D009930:Organic Chemicals; D011804:Quinolines; C083961:hemolysin BL protein, Bacillus; C098022:SYBR Green I