核酸试纸条快速检测蜡样芽胞杆菌entFM毒素基因方法的建立

目的 基于核酸试纸条技术，建立一种快速检测蜡样芽胞杆菌entFM毒素基因的方法。 方法 煮沸法提取蜡样芽胞杆菌DNA，以蜡样芽胞杆菌entFM毒素基因作为靶基因，采用NCBI primer-BLAST 5.0软件设计特异性引物并通过克隆转化鉴定PCR产物，建立并组装核酸试纸条，评价核酸试纸条的特异性、灵敏度和稳定性。 结果 蜡样芽胞杆菌DNA浓度为300 mg?L^－1，纯度约为1.60。PCR产物阳性条带经切胶回收、克隆转化和测序比对，与GenBank数据库中已登记的entFM相似性为100%。在pH值7.0条件下，每100 μL 胶体金溶液加入3.3 μg链霉亲和素浓度标记，质控线浓度为1.8 g?L^－1，检测线浓度为1 g?L^－1时，硝酸纤维素膜上质控线与检测线均可与PCR产物反应出现清晰的红色条带。按照最适条件组装成核酸试纸条，PCR产物6 μL，样品展开液100 μL，检测10 min后可观察结果。核酸试纸条特异性与电泳结果一致，仅蜡样芽胞杆菌为阳性结果，与金黄色葡萄球菌、铜绿假单胞杆菌、大肠埃希菌和沙门氏菌皆无交叉反应，为阴性结果；灵敏度检测，核酸试纸条DNA质量浓度同时降至10^－3 mg?L^－1仍可准确检测，普通PCR电泳结果显示DNA质量浓度同时降至10^－1 mg?L^－1出现目的条带，核酸试纸条比普通PCR灵敏度提高100倍；稳定性检测，核酸试纸条在第6、9和12个月进行检测稳定性一致。 结论 建立的核酸试纸条检测蜡样芽胞杆菌entFM毒素基因灵敏度高、特异性强且具有良好稳定性，适用于快速鉴别蜡样芽胞杆菌entFM毒素基因。

Establishment of method for rapid detection of Bacillus cereus entFMtoxin gene using nucleic acid test strips

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.