基质辅助激光解吸电离飞行时间质谱技术分离鉴定生鲜猪肉中沙门氏菌/大肠杆菌类似菌

Isolation and identification of Salmonella/Escherichia coli analogues in raw pork by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

中文关键词:  生鲜肉  基质辅助激光解吸电离  飞行时间质谱法  条件致病菌  肠道病原菌

英文关键词:raw pork  matrix-assisted laser desorption ionization  time of flight mass spectrometry  opportunistic pathogen  intestinal pathogenic bacteria

中文摘要:

      目的 利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF-MS)技术鉴别生鲜猪肉中大肠杆菌和沙门氏菌的常规检验中出现的类似菌。方法 在郑州市不同地区分批次采集164份生鲜猪肉样品, 按照国标方法进行增菌培养, 选择性培养基HE和EMB对细菌进行分离纯化。通过MALDI-TOF-MS技术对疑似菌落进行鉴定; 提取被检菌株基因组DNA, 根据MALDI-TOF-MS鉴定出的细菌种类, 参考GenBank中相关菌株的已知基因序列分别设计引物, 进行PCR扩增、测序和序列比对, 以验证MALDI-TOF-MS鉴定结果。结果 经MALDI-TOF-MS方法鉴定, 从68个疑似菌株中分别检出柠檬酸杆菌(Citrobacter)6株、奇异变形杆菌(Proteobacteria)5株、肺炎克雷伯氏菌(Klebsiella pneumoniae)9株、绿脓杆菌(Pseudomonas aeruginosa)1株、阴沟肠杆菌(Enterobacter cloacae) 3株、不动杆菌(Acinetobacter)1株。PCR结果与MALDI-TOF-MS鉴定结果完全一致。结论 MALDI-TOF-MS可用于生鲜猪肉检验过程中大肠杆菌和沙门氏菌的分离鉴定。本次共检出6种在HE和EMB培养基上与沙门氏菌/大肠杆菌菌落特征类似的肠杆菌科其他细菌, 说明在生鲜猪肉中存在着一定程度的条件致病性肠道细菌污染, 因此具有一定的食品安全风险。

英文摘要:

      Objective To identify some similar bacteria appeared in routine examination for Escherichia coli and Salmonella in raw pork meat by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Methods Totally 164 raw pork samples were collected from different parts of Zhengzhou city, and they were routinely examined according to the national standard methods. The selective medium HE and EMB were used to separate and purify the bacteria and the suspected colonies were identified by MALDI-TOF-MS. Genomic DNA was extracted from the pure culture, PCR primers were designed based on the gene sequences published on the GenBank website, and PCR amplification were performed. Sequencing results were then analyzed and compared with those of the MALDI-TOF-MS indicators. Results Among the 68 test strains, 6 strains of Citrobacter, 5 strains of Proteobacteria, 9 strains of Klebsiella pneumoniae, 1 strain of Pseudomonas aeruginosa, 3 strains of Enterobacter cloacae, 1 strain of Acinetobacter were identified by MALDI-TOF-MS. The PCR results showed complete consistence with those of MALDI-TOF-MS. Conclusion MALDI-TOF-MS can be used for isolation and identification of Salmonella/E. coli analogues in raw pork. The 6 Enterobacteriaceae analogues of Salmonella/E. coli are present in raw pork products, which indicate that there is contamination of opportunistic intestinal bacteria in raw pork, and it might add to the food safety risks.