关键词: 蜡样芽胞杆菌, entFM毒素, 核酸试纸条, 煮沸法, 聚合酶链式反应

Abstract:

Objective To establish a method for rapid detection of Bacillus cereus entFM toxin gene based on nucleic acid test strip technology. Methods The Bacillus cereus DNA was extracted by boiling method， the Bacillus cereus entFM toxin gene was used as the target gene，the specific primers were designed using NCBI primer-BLAST 5.0， and the PCR products were identified by cloning and transformation；the nucleic acid test strips were established， and the specificity， sensitivity and stability of nucleic acid test strips were evaluated. Results The concentration of Bacillus cereus DNA was 300 mg?L^－1， and the purity was about 1.60. The positive bands of PCR products were 100% similar to entFM registered in GenBank database after gel cutting recovery， clone transformation and sequencing comparison. At the condition of pH value was 7.0， 3.3 μg streptavidin was added to each 100 μL colloidal gold solution for labeling， the quality control line concentration was 1.8 g?L^－1， the detection line concentration was 1 g?L^－1， and both the quality control line and the detection line on the nitro cellulose membrane could react with the PCR products to produce the clear red bands. The nucleic acid test strips were assembled according to the optimum conditions， with 6 μL of PCR product and 100 μL of sample developing solution， and the results could be observed after 10 min of detection. The specificity of the nucleic acid test strips was consistent with the electrophoresis results， only Bacillus cereus was a positive result， and there was no cross-reaction with Staphylococcus aureus， Pseudomonas aeruginosa， Escherichia coli and Salmonella， and they were negative results.The sensitivity test results showed that nucleic acid test strips could accurately detect when the DNA mass concentration droped to 10^－3 mg?L^－1 at the same time. The results of ordinary PCR electrophoresis showed that when the DNA mass concentration droped to 10^－1 mg?L^－1 at the same time， the target band appeared.The sensitivity of nucleic acid test strips was 100 times higher than that of ordinary PCR； for stability testing， nucleic acid test strips had the same stability in the 6th， 9th and 12th months. Conclusion The established nucleic acid test strips have high sensitivity， strong specificity and good stability for the detection of Bacillus cereus entFM toxin gene， which is suitable for rapid identification of Bacillus cereus entFM toxin gene.

Key words: Bacillus cereus, entFM toxin, Nucleic acid test strips, Boiling method, Polymerase chain reaction