关键词: 蜡样芽孢杆菌, cytK, nhe, 双重核酸试纸条

Abstract:

Objective To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereuscytK and nhe toxin genes based on polymerase chain reaction（PCR）and colloidal gold technique，and to evaluate its specificity， sensitivity， repeatability and stability. Methods Bacillus cereus DNA was extracted by boiling method. Specific primers were designed with Bacillus cereus cytK and nhe as the target genes. Clonal transformation was used to identify the PCR products. The optimal labeling amounts of colloidal gold-labeled streptavidin， quality control line （C line）， cytK detection line （T₁） and nhe detection line （T₂） were determined. The nucleic acid test strips were assembled and its specificity，sensitivity，reproducibility and stability were evaluated. Results The DNA concentration of Bacillus cereus was 248 mg·L^-1，and the purities were 1.8-2.0. After cloning and plasmid sequencing，the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100%. Under the condition of pH 7.0， the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL； the optimal marking amount was 2.00 g·L^-1 for the quality control line （C line）， 0.550 g·L^-1 for cytK gene detection line （T₁） and 0.2 g·L^-1 for nhe gene detection line （T₂）. In the specificity test， positive result on the test strips was seen only for Bacillus cereus，and no cross-reactivity was observed for Staphylococcus aureus， Escherichia coli， Pseudomonas aeruginosa and Bacillus subtilis， which were consistent with the electrophoresis results. Sensitivity assay showed that even when DNA concentration was reduced to 10^-2 mg·L^-1， three bands （C line， T₁ line and T₂ line） could be observed， and the detection limit of the test strip was one-tenth of agarose gel electrophoresis （10^-1 mg·L^-1）. The nucleic acid test strips were verified by different operators in different laboratories， and the results were consistent. The stability of the test strips was verified at the 6th，9th and 12th months， and the results showed good stability. Conclusion The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity，achieving short-term visual detection.

Key words: Bacillus cereus, cytK, nhe, Dual nucleic acid test strips