To establish a novel,sensitive,and visualized polymerase cross-linking spiral reaction(PCLSR)method for rapid detection of emetic Bacillus cereus producing emetic toxin,specific primers were designed using the structural synthase gene cesB of the bacterium as the target sequence.The reaction system and amplification conditions were optimized.The results showed that PCLSR method obtained the best amplification effect at 64℃for 50min,0.3mol/L dNTP and 8U/tube Bst polymerase.To evaluate the specificity of the PCLSR assay established in this study,5 strains of Bacillus cereus and 12 strains of clinically common bacteria were used as templates.It showed that the method was effective in detecting 5 strains of Bacillus cereus,while negative amplification was performed on the other 12 strains of clinically common bacteria with good specificity.After diluting 10 times the ratio of producing vomiting toxin Bacillus cereus(1×107cfu/mL-1×100cfu/mL),the PCLSR method,LAMP method,and real-time PCR method were used for detection.The results showed that the detection limits of PCLSR method and real-time PCR were both up to 1×102cfu/mL,while the detection limit of LAMP method was 1×103 cfu/mL,indicating that the PCLSR method established in this study was highly sensitive.50 artificially contaminated emetic B.cereus feed and sterilized milk samples were simultaneously detected using"National Standard"bacterial culture,PCLSR,qPCR,and LAMP methods.The results showed that the positive rate of PCLSR method and qPCR method for detecting emetic B.cereus was 30%(15/50),and the negative rate was 70%(35/50),and the coincidence rate of both methods reached 100%.The positive rate of emetic B.cereus detected by bacterial culture method and LAMP method was 24%(12/50),and the negative rate was 76%(38/50),with a coincidence rate of 80%for both methods.In summary,the PCLSR assay established in this study has strong specificity,high sensitivity,and does not require complex equipment or expensive reagents and consumables to complete the detection of emetic B.cereus in a short period of time.The establishment of this method provides a new technical means for grassroots detection departments to detect emetic B.cereus and conduct epidemiological investigations of this bacterium.
维普
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