摘要
肉制品和乳制品中的多种致病菌是影响食品安全和威胁人类身体健康的重要因素。目前国内外对肉制品和乳制品中致病菌的检测方法还多停留在检测效率低、目标单一的水平上。本研究采用DNA聚合酶链式反应(Polymerase Chain Reaction, PCR)结合变性高效液相色谱(Denaturing High Performance Liquid Chromatography, DHPLC)高通量检测方法,对肉制品和乳制品中多种致病菌的高通量检测、属种鉴定、血清分型、流行株检测等检测技术进行了系统研究,搭建相应技术平台。并且通过应用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS),对单核细胞增生性李斯特氏菌的鉴定分型和溯源进行了系统的研究。本研究取得的试验结果如下：
1.在国内外首次应用PCR结合DHPLC技术,建立了针对肉制品中常见的沙门氏菌、金黄色葡萄球菌、单核细胞增生性李斯特氏菌、空肠弯曲菌、小肠结肠炎耶尔森氏菌、D溶血性链球菌、产肠毒素大肠埃希氏菌、肠出血性大肠杆菌O157：H7、致病性大肠杆菌和肠侵袭性大肠杆菌等10种致病菌的单一致病菌的PCR-DHPLC检测方法,并且建立了可以同时检测沙门氏菌、金黄色葡萄球菌、单核细胞增生性李斯特氏菌、空肠弯曲菌、小肠结肠炎耶尔森氏菌、β溶血性链球菌等六种致病菌的多重PCR-DHPLC高通量同步检测方法和四种致泻性大肠杆菌的多重PCR-DHPLC高通量同步检测方法。
2.在国内外首次应用PCR结合DHPLC技术,建立了乳制品中常见的阪崎肠杆菌、粘质沙雷氏菌、绿脓杆菌、普通变形杆菌、奇异变形杆菌、肺炎克雷伯氏菌、气味沙雷氏菌、致病性嗜水气单胞菌等8种致病菌的各单一致病菌的快速高通量检测方法。并且建立了可同时检测阪崎肠杆菌、粘质沙雷氏菌、绿脓杆菌、普通变形杆菌等四种致病菌的多重PCR-DHPLC检测方法以及奇异变形杆菌、肺炎克雷伯氏菌、气味沙雷氏菌、致病性嗜水气单胞菌等四种致病菌的多重PCR-DHPLC检测方法。
3.在国内外首次应用PCR结合DHPLC技术,通过设计并筛选出特异性的引物,在菌属的水平上,建立了食品中李斯特氏菌属的PCR-DHPLC快速检测方法。
4.在国内外首次应用PCR结合DHPLC技术,建立了食品中李氏菌三种致病菌即单核细胞增生性李斯特氏菌、绵羊李斯特氏菌和英诺克李斯特氏菌的多重PCR-DHPLC高通量检测方法。
5.在国内外首次应用PCR结合DHPLC技术,建立了食品中单核细胞增生性李斯特氏菌血清型中临床感染率高的主要血清型(1/2a和4b)的分型多重PCR-DHPLC高通量检测方法。
6.以单核细胞增生性李斯特氏菌的流行株分类(ECⅠ、ECⅡ、ECⅢ)为检测对象,应用DHPLC的非变性条件下的鉴定技术,在流行株分类的水平上,在国内外首次建立了食品中单核细胞增生性李斯特氏菌流行株分类的高通量检测方法。
7.以李斯特氏菌属的6个分类菌种(单核细胞增生性李斯特氏菌、绵羊李斯特氏菌、英诺克李斯特氏菌、威尔斯李斯特氏菌、西尔李斯特氏菌、格氏李斯特氏菌)为检测对象,应用DHPLC的部分变性条件下的高通量分型鉴定技术,在属以下的菌种分类水平上,在国内外首次建立了食品中李斯特氏菌属6个分类菌种的高通量分种鉴定方法。
8.本研究收集了37株单核细胞增生性李斯特氏菌分离株,应用MALDI-TOF-MS采集获取独特的蛋白质指纹图谱,汇总成标准图谱,在国内外首次建立了单核细胞增生性李斯特氏菌鉴定数据库。并且在数据库信息的基础上,对37株单核细胞增生性李斯特氏菌分离株进行了聚类分型和溯源研究。
综上所述,本研究在国内外首次建立了肉制品和乳制品中多种致病菌的PCR-DHPLC快速、精准、高通量同步检测技术体系,并且建立了单核细胞增生性李斯特氏菌的MALDI-TOF-MS鉴定数据库,开展了系统的单核细胞增生性李斯特氏菌的聚类分型和溯源研究。上述研究成果对于食源性致病菌的快速鉴定和主动监测以及重大食源性疾病的预防和溯源具有重要的意义。
Many kinds of pathogenic bacteria in meat and dairy products are the fatal factors to the food safety and physical health of human beings. Currently, the domestic and foreign inspection methods of detecting the pathogenic bacteria are mostly on the level of low efficiency and single target. This research has established the detection system of the pathogenic bacteria in meat and dairy products, species identifying, serotyping and epidemic strain detection using Polymerase Chain Reaction (PCR) combining with denaturing high performance liquid chromatography (DHPLC). Meanwhile the research has studied the identifying, genotyping and tracing of Listeria using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). The results of the research are as follows:
1. The individual detection methods for 10 common pathogenic bacteria in the meat products, including Salmonella, Staphylococcus aureus, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Streptococcus hemolyticus, Enterotoxigenic E. coli, Enterohemorrhagic E.coli O157:H7, Enteropathogenic E.coli, Enteroinvasive E.coli were established using PCR-DHPLC. On the base of above, the simultaneous detection systems for six pathogenic bacteria, including Salmonella, Staphylococcus aureus, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Streptococcus hemolyticus were established and the simultaneous detection systems for four pathogenic bacteria, including Enterotoxigenic E. coli, Enterohemorrhagic E.coli O157:H7, Enteropathogenic E.coli, Enteroinvasive E.coli were also established. All the above systems were developed for the first time worldwide.
2. The individual detection methods for 8 common pathogenic bacteria in the dairy products, including Enterobacter.sakazakii, serratia macescens, Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Klebsiella peneumoniae, serratia odorifera, Aeromonas hydrophila were established using PCR-DHPLC. On the base of above, the simultaneous detection systems for four pathogenic bacteria, including Enterobacter.sakazakii, serratia macescens, Pseudomonas aeruginosa, Proteus vulgaris were established and the simultaneous detection systems for four pathogenic bacteria, including Proteus mirabilis, Klebsiella peneumoniae, serratia odorifera, Aeromonas hydrophila were also established. All the above systems were developed for the first time worldwide.
3. The rapid detection method for the family of Listeria was established for the first time worldwide.using PCR-DHPLC by designing and screening out specific primers.
4. The high throughput detection methods for 3 pathogenic bacteria of Listeria family, including Listeria monocytogenes, Listeria ivanovii, Listeria innocua were established using multiple PCR-DHPLC for the first time worldwide.
5. The high throughput detection methods for 2 main serotype (1/2a and 4b) with high clinical infection rate of Listeria monocytogenes were established using multiple PCR-DHPLC for the first time worldwide.
6. The high throughput detection methods for 3 main epidemic strains (ECⅠ、ECⅡ、ECⅢ) of Listeria monocytogenes were established using multiple PCR-DHPLC on the base of the identification function of DHPLC at non-denaturing condition for the first time worldwide.
7. The high throughput detection methods for 6 species of Listeria, including Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria seeligeri, Listeria grayi were established using multiple PCR-DHPLC on the base of the identification function of DHPLC at non-denaturing condition for the first time worldwide.
8. The identifying database for Listeria monocytogenes was established using MALDI-TOF-MS on the base of the standard MALDI-TOF-MS result charts of 37 isolated strains of Listeria monocytogenes. On the base of above, cluster subtyping and tracing research was done at the 37 isolated strains of Listeria monocytogenes.
Above all, this research has firstly developed the rapid, accurate and high throughput detection systems for common kinds of pathogenic bacterium of meat and dairy products. Also, the MALDI-TOF-MS identifying database for Listeria monocytogenes was established and cluster subtyping and tracing research was done systematically. The results of the above research have a signicant meaning to the rapid detection and initiative monitoring of food borne pathogenic bacteria as well as the prevention and tracing back of the serious food origin disease.
引文
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