基质辅助激光解吸飞行时间质谱技术快速检测水产品中5种致病菌

Rapid determination of 5 kinds of pathogenic bacteria in aquatic products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

中文关键词:  致病菌  飞行时间质谱  单碱基延伸

英文关键词:pathogenic bacteria  matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  single nucleotide extension

中文摘要:

      目的 建立水产品中5种常见致病菌的自动化快速高分辨检测手段, 提高水产品致病微生物检测的效率和准确性。方法 设计引物对霍乱弧菌、副溶血性弧菌、沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏菌的特异基因owpW、tlh、invA、femA、prfA进行多重聚合酶链式反应(polymerase chain reaction, PCR)扩增; 设计特异探针以PCR产物为模板进行单碱基延伸, 延伸产物在基质辅助激光解吸飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS)上进行检测。基因owpW、tlh、invA、femA、prfA的探针分子量分别为4848、5435、5890、6560、7096 Da, 单基因延伸后探针的分子量分别为5119 Da(加A)、5697 Da(加T)、6137 Da(加C)、6822 Da(加T)、7383 Da(加G)。最终确定的体系方法通过重现性试验、特异性试验、灵敏度试验、人工污染水产样本检测试验进行验证。结果 以混合5种菌DNA的样本为模板做核酸质谱检测, 对应的5个探针可以同时延伸, 延伸效率大于80%, 在以干扰菌为模板的样本中不会检出上述5种菌, 检出沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特菌、副溶血性弧菌、霍乱弧菌的灵敏度分别可达到150、350、160、130、180 CFU/mL。结论 此方法展现出良好的重现性、特异性和灵敏度, 可以满足一次反应同时检测水产品中上述5种微生物的需求。

英文摘要:

      Objective To establish an automatic, fast and high-resolution detection method for 5 kinds of common pathogenic bacteria in aquatic products, and improve the efficiency and accuracy of detecting pathogenic microorganisms in aquatic products. Methods Genes of owpW, tlh, invA, femA, and prfA from Vibrio cholera, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, and Listeria monocytogenes were amplified by multiple polymerase chain reaction (PCR). PCR products were used as templates for single nucleotide extention and molecular weight of the extended probes was detected on the mass spectrometer. The molecular weight of probes for genes owpW、tlh、invA、femA and prfA were 4848, 5435, 5890, 6560 and 7096 Da. The molecular weights of the extended probes were 5119 Da (plus A), 5697 Da (plus T), 6137 Da (plus C), 6822 Da (plus T) and 7383 Da (plus G), respectively. This finally determined system was verified by reproducibility test, specificity test, sensitivity test and detection test of artificially contaminated aquatic samples. Results Using a sample of mixed DNA from 5 kinds of different bacteria as a template for nucleic acid mass spectrometry detection, the corresponding 5 kinds of probes could be extended simultaneously with an extension efficiency greater than 80%. The above 5 kinds of bacteria would not be detected in samples using interfering bacteria as templates. The sensitivity for detecting Salmonella, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, and Vibrio cholerae could reach 150, 350, 160, 130, and 180 CFU/mL, respectively. Conclusion This method demonstrates good reproducibility, specificity, and sensitivity, and has a high degree of automation, which can meet the detection needs of the above 5 kinds of microorganisms in aquatic products simultaneously.