多重PCR技术同时高效检测食源性致病菌的研究

发布时间：2018-04-20 02:17

本文选题：食品药品安全 + 致病菌　；参考：《吉林大学》2017年硕士论文

【摘要】：随着经济与科技的快速不断进步,食品药品安全问题也变得越来越多,近些年在我国各地的食品药品微生物安全风险监测中就检出几十种致病菌,由于传统检测方法耗费时间较长,操作繁杂,我们以用多重PCR技术改进检测方法达到高效快速食源性致病菌的目的。我们设计了特异性良好的引物,这些致病菌的引物退火温度都在55℃左右,扩增条带是呈现梯度,并且优化了反应体系和条件。多重PCR是基于核酸的方法,检测目标病原体中的特异性基因,能避免出现假阳性的结果。我们根据目前常被监测到的食源性致病菌,并选择由吉林省食品药品监督管理局提供的菌株:金黄色葡萄球菌、乙型副伤寒沙门氏菌、铜绿假单胞菌、枯草芽孢杆菌、短小芽孢杆菌、大肠埃氏菌、黑曲霉菌为检测的目的菌株。首先是提取致病菌基因组,由于黑曲霉菌是真菌所以选择的基因组提取试剂盒与细菌的不同,之后设计特异性引物,是把所设计的引物进行两两配对,并且设置阴性对照组,然后进行PCR扩增,通过观察琼脂糖凝胶电泳图,可以判断该两对引物能否交叉扩增。然后是进行多重PCR反应。最后以制备的模拟致病菌污染的牛奶样品和模拟致病菌污染的牛奶对照组样品为模板进行多重PCR反应。我们从查到的七种致病菌的总共18个基因中,筛选并验证了七种致病菌的特异性基因分别是乙型副伤寒沙门氏菌为mgt C基因、大肠埃希氏菌为uid A基因、金黄色葡萄球菌nuc基因、短小芽孢杆菌gyr B基因、枯草芽孢杆菌rpo A基因、铜绿假单胞菌Opr L基因、黑曲霉fum8基因。以这些为目的基因设计了特异性引物用软件设计出了特异性较好的引物,长度大小为:黑曲霉菌的引物长度为665 bp、乙型副伤寒沙门氏菌的引物长度为500 bp、短小芽孢杆菌的引物长度为403 bp、枯草芽孢杆菌的因为长度为349 bp、金黄色葡萄球菌的引物长度为280 bp、大肠埃希氏菌的引物长度为210 bp、铜绿假单胞菌的引物长度105 bp。然后优化了较好的反应条件和反应体系,而且有较高的稳定性,其检测限可以达到1.2 CFU/m L,较目前开发的Ge XP-PCR技术检测六种致病菌的检测限分别为420,310,270,93,85和66 CFU/m L等提高了至少50-350倍,并且多重PCR检测方法较其他基于生物传感器和免疫学的检测方法具有成本低,稳定性好等优点,同时仍然可以不断增加同时检测的菌株数目。我们在用多重PCR检验牛奶样品的初步应用时,其中除大肠杆菌的其他6种致病菌的检测限可以达到102 CFU/m L,并且有5种种致病菌的检测限可达10 CFU/m L,但是也发现大肠杆菌只能在104 CFU/m L的活菌数浓度才能扩增出目的条带,可能的原因:1)在第二章中提到了进行多重PCR反应应该要对增加反应体系中Mix的量以及反应条件的循环数等,所以本实验改进之处就是在以牛奶为模板时可以根据检测的菌种数调整反应体系和条件,一般的,检测目的菌种数目越多,循环数越多,Mix用量也越多,并且以后再以其他类样品如肉类,豆制品等食品为模板时也应适当增加Mix的量或者循环数等;2)同样也可能是大肠杆菌的引物对的Tm值为58.7℃和62.9℃,较其他引物对高,而这也是由于反应条件没进行调整造成的,所以在检测不同食品的应用中都要调整反应条件。我们也检测了吉林大学药学院赠予的无菌眼药水,并且以无菌水为阴性对照组,结果为该眼药水是无致病的,并且检测的时间为4 h,因此我们在本实验中建立的方法为较传统方法数3-7d的检测时间快。
[Abstract]:With the rapid and continuous progress of economy and science and technology, the problem of food and drug safety has become more and more. In recent years, dozens of pathogenic bacteria have been detected in the monitoring of the safety risk of food and drug microorganisms in all parts of our country. Because of the long time consuming and complicated operation, we use multiple PCR technology to improve the detection method to achieve high efficiency. The aim of rapid foodborne pathogenic bacteria. We designed specific primers. The primers of these pathogens were annealed at about 55 degrees centigrade. The amplified bands were gradient, and the reaction system and conditions were optimized. Multiple PCR is a nucleic acid based method to detect specific genes in the target pathogen and avoid false positive. Results. We are based on the food borne pathogens that are currently monitored and selected by the strains provided by the food and Drug Administration of Jilin Province: Staphylococcus aureus, paratyphoid salmonella, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus subtilis, Escherichia coli and Aspergillus niger. The genome of the pathogenic bacteria was taken from the genomic Extraction Kit selected by Aspergillus niger, and then the specific primers were designed. 22 pairs of primers were designed and the negative control group was set up and then amplified by PCR. By observing the agar gel electrophoresis, the two pairs of primers could be judged if the primers could be primed. Multiple PCR reactions were then carried out. In the end, milk samples contaminated by the simulated pathogenic bacteria and the milk control group samples contaminated with simulated pathogenic bacteria were used for multiple PCR responses. We screened and verified that the specific genes of the seven pathogenic bacteria were respectively B from the seven species of pathogenic bacteria found. Salmonella paratyphoid is Mgt C, Escherichia coli is UID A, Staphylococcus aureus NUC, Bacillus subtilis Gyr B, Bacillus subtilis RPO A, Opr L of Pseudomonas aeruginosa, and Aspergillus niger fum8 genes. The specific primers designed by these specific primers are better. Primer length is: the length of primers of Aspergillus niger is 665 BP, primer length of Salmonella paratyphi is 500 BP, the length of primers of Bacillus subtilis is 403 BP, the length of Bacillus subtilis is 349 BP, the length of primer of Staphylococcus aureus is 280 BP, the length of primer of Escherichia coli is 210 BP, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa. The primer length of monomonas aeruginosa was 105 bp., and the better reaction condition and reaction system were optimized, and the detection limit could reach 1.2 CFU/m L. The detection limit of six pathogenic bacteria detected by Ge XP-PCR technology is at least 50-350 times higher than that of 420310270,93,85 and 66 CFU/m L, respectively. Compared with other methods based on biosensor and immunology, the method has the advantages of low cost, good stability and so on. At the same time, we can continue to increase the number of strains detected at the same time. In the preliminary application of multiple PCR test milk samples, the detection limit of 6 kinds of pathogenic bacteria except Escherichia coli can reach 102 CFU/m L, And the detection limit of 5 pathogenic bacteria can be up to 10 CFU/m L, but it is also found that Escherichia coli can only amplify the target bands at 104 CFU/m L. Possible reasons: 1) in the second chapter, multiple PCR reactions should be added to the amount of Mix in the reaction system and the cycle number of the reaction conditions. The improvement of the test is that when milk is used as a template, the reaction system and conditions can be adjusted according to the number of tested bacteria. In general, the more the number of bacteria species, the more circulation, the more Mix is used, and in the future, the amount of Mix or the number of cycles should be appropriately increased with other samples such as meat, bean products and so on; 2 It is also possible that the Tm value of the primers of Escherichia coli is 58.7 and 62.9, higher than the other primers, and this is because the reaction conditions are not adjusted, so the reaction conditions should be adjusted in the application of different foods. We also tested the aseptic eyedrops given by the Jilin University College of pharmacy and used aseptic water as a result. In the negative control group, the result was that the eye drops were not pathogenic, and the time of detection was 4 h, so the method we established in this experiment was faster than the traditional method of 3-7d.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155;R440

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