Abstract:

Objective To establish a method for rapid detection of three toxin genes of Bacillus cereus nhe， cytK and entFM based on multiplex PCR，and to evaluate its efficiency. Methods The DNA of Bacillus cereus was extracted by boiling method. The specific toxin genes of Bacillus cereus nhe， cytK and entFM were used as target genes， and the specific primers were designed by NCBI primer-BLAST 5.0 software to optimize the reaction system. The triple PCR detection system was established and the specificity， sensitivity and stability were detected. Results The Bacillus cereus DNA was extracted by boiling method with a concentration of 227 mg·L^－1 and a purity of 1.50－1.60. The pathogenic genes of three toxins of Bacillus cereus were detected simultaneously by multiplex PCR system. When the annealing temperature was 56 ℃， the amplification fragments of Bacillus cereus gene primer nhe499， gene primer cytK191 and gene primer entFM363 were 499， 191 and 363 bp， respectively. The bands of three toxin genes of Bacillus cereus amplified by PCR system were clear and bright， and there were no non-specific bands， and no amplification with Staphylococcus aureus， Escherichia coli and Pseudomonas aeruginosa， indicating that the primers of nhe499， cytK191 and entFM363 of Bacillus cereus genes had strong specificities， the three primers did not interfere with each other in the same reaction system， and the lowest detection limit of multiplex PCR system was 10^－1 mg·L^－1. The stability test was consistent with the self-made preparation of multiple PCR system every 6 months. Conclusion The established multiplex PCR detection system has high sensitivity and specificity for three toxin genes of Bacillus cereus nhe499， cytK191 and entFM363， the detection results are accurate and stable， and the detection system has good stability， which is suitable for rapid detection of three different pathogenic toxin genes of Bacillus cereus.

Key words: Bacillus cereus, Toxin gene, Multiplex polymerase chain reaction, Sensitivity, Boiling method