食品中四种致病菌的多重PCR检测方法的建立和应用

发布时间：2018-04-11 21:34

本文选题：多重PCR + 沙门氏菌　；参考：《福建农林大学》2013年硕士论文

【摘要】：当前社会,由食物源病原微生物引起的人类疾病和死亡病例越来越引起人们的关注。为了保障自身的健康,居民们对食物供给的安全性和食用性有了更多的需求。这些需求要求当前的食品检测技术必须不断发展进步,与时俱进。传统微生物检测方法虽然仍被使用,但是它耗时费力,并且检测灵敏度也不高,并且需要受过专业培训经验丰富的检测人员才能胜任。分子生物学的的发展极大地改变了食品检测技术。免疫学技术、分子生物学技术、自动化以及信息化技术的正向推动作用已经被被大家看在眼里。我们应该勇于探索将不同的检测技术联合使用的可能性。最新的检测技术正向着病原微生物定量检测和同时检测多个靶标微生物或毒素这个方向发展。相比于传统PCR,多重PCR含有更高的技术含量,把两对以上的引物加入到同一个反应体系中,使它们针对各自的目的基因经行同时扩增,最终可以同时得到不同病原微生物的检测结果,大大缩短了检测时间,同时也节约了检测成本。本研究意在建立一种可用于同时检测四种食源性肠道致病菌的多重PCR方法。此前,没有针对弗氏柠檬酸杆菌的分子生物学检测方法。首先依据沙门氏菌(Salmonella spp)的fimY基因,弗氏枸橼酸杆菌(Citrobacter freundii)的ldh基因,奇异变形杆菌(Proteus mirabilis)的idsC基因和迟缓爱德华氏菌(Edwardsiella tarda)的fimA基因的保守序列设计了四对特异性引物,其PCR扩增产物大小依次为161bp、336bp、396bp、526bp。然后建立了针对四种致病菌的单重PCR检测体系,并通过试验确定了特异性和灵敏度。通过测序确定特异性极高,体系中检测到沙门氏菌的灵敏度为39CFU；体系中检测到弗氏柠檬酸杆菌的灵敏度为72CFU；体系中检测到奇异变形杆菌的灵敏度为6CFU；体系中检测到迟缓爱德华氏菌的灵敏度为7CFU。这为之后建立多重PCR检测体系提供了很好的参考。在四种细菌的单重PCR基础上,根据对多重PCR反应的主要影响因素,如退火温度,引物浓度,dNTP浓度和Mg2+浓度等,将这些因素展开试验进行优化,建立了四种细菌的多重PCR检测体系。研究结果表明该多重PCR反应体系对4株病原菌能扩增出清晰特异的条带。并且体系中沙门氏菌的检测灵敏度为39CFU；弗氏柠檬酸杆菌的检测灵敏度为7CFU；奇异变形杆菌的检测灵敏度为57CFU；迟缓爱德华氏菌的检测灵敏度为6CFU。人工染菌样品检测结果显示,通过预增菌后,样品中目的菌浓度在10CFU/ml数量级时结果为阳性,说明本研究建立的多重PCR方法具有结果可靠、灵敏度高、方便快捷的优点。本检测体系十分适用于针对四种食源性肠道致病菌的检测,在食品加工过程以及食品进出口环节都有很广的应用空间。并且为研发快速检测以上四种食源性肠道致病菌的试剂盒提供了重要技术保障。同时,本研究还初步探索了利用基质辅助激光解析电离飞行时间质谱仪对四种细菌的检测方法,通过与己建立的单重PCR方法比较,可以得知该方法具有特异性高、操作便捷的优点。
[Abstract]:In current society, caused by a food source pathogenic microorganism in human diseases and deaths caused by more and more attention. In order to protect their own health, the residents of food safety and food have more demand. The demand of food inspection technology current must be continuous development progress, conventional methods of keeping pace with the times. Although it is still used, but it is time-consuming, and the detection sensitivity is not high, and the need for trained experienced inspectors to be competent. Molecular biology development has greatly changed the food inspection technology. Immunological techniques, molecular biology techniques, positive role in promoting automation and information technology have been for everyone to see in the eyes. We should have the courage to explore the possibility of the combined use of different detection techniques. The new detection technology is disease The quantitative detection of microbes and the simultaneous detection of multiple target microorganisms or toxins have developed in this direction.
Compared to the conventional PCR, multiplex PCR with higher technology content, to more than two pairs of primers were added to the same reaction system, making them for their target genes by simultaneous amplification can be obtained at the same time, the final detection results of different pathogenic microorganisms, shorten testing time, but also saves the cost of detection.
This study aimed to establish a kind of can be used for simultaneous detection of four foodborne pathogens of intestinal multiple PCR method. Previously, no molecular biological detection methods for c.freundii. Firstly on the basis of Salmonella (Salmonella spp) fimY gene, Citrobacter freundii (Citrobacter freundii) LDH gene, proteus coli (Proteus mirabilis) idsC gene and delayed Edward's bacteria (Edwardsiella tarda) conserved sequence of fimA gene was designed four pairs of specific primers, PCR amplification of 161bp, 336bp, 396bp, 526bp. and then set up for the four single PCR pathogen detection system, and determine the specific and through the test. To determine the sensitivity of high specificity by sequencing system, detect sensitivity of Salmonella 39CFU system; detected c.freundii sensitivity to 72CFU body; The sensitivity of the Proteus mirabilis detected in the system was 6CFU. The sensitivity of Edward's bacteria detected in the system was 7CFU., which provided a good reference for the establishment of multiple PCR detection system after that.
In a single PCR based on four kinds of bacteria, according to the main influencing factors of response to multiple PCR, such as annealing temperature, primer concentration, dNTP concentration and Mg2+ concentration, these factors test optimization, establish multiple PCR detection system of four kinds of bacteria. The results showed that the multiplex PCR reaction system of 4 strains of pathogenic bacteria can be amplified and specific. The detection sensitivity of Salmonella in the system is 39CFU; the detection sensitivity of c.freundii was 7CFU; Bacillus proteus detection sensitivity is 57CFU; Edwardsiella detection sensitivity is 6CFU.
Artificial infection sample test results showed that the pre enrichment, the purpose of bacteria concentration in the samples in the level of 10CFU/ml when the result is positive, that the multiplex PCR method established in this study is reliable, high sensitivity, convenient advantages. The detection system is very suitable for the detection of four food borne pathogens needle intestine. Have a very wide application space in the process of food processing and food import and export sectors. Provides important technical guarantee for the development of pathogens and kits for rapid detection of more than four species of food borne.
At the same time, this study also explored the detection method of four kinds of bacteria by matrix assisted laser desorption ionization time of flight mass spectrometry. Comparing with the established single PCR method, we can see that this method has the advantages of high specificity and convenient operation.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

【参考文献】