【作者】 杨芳；

【导师】 李秀娟； 徐保红； 魏永巨；

【作者基本信息】 河北师范大学 ， 分析化学， 2011， 硕士

【摘要】 在世界范围内,沙门菌是引起食物中毒最常见的致病菌,目前由沙门菌引起的食品安全、公共卫生等问题较为突出。因此建立一种准确、快速、实时、高通量的沙门菌鉴定方法对食品日常监测和沙门菌爆发疫情的调查等具有重要的意义,本研究以条码基因理论为基础并结合焦磷酸测序技术检测、鉴定沙门菌。论文内容主要包括四部分:1、沙门菌条码基因的初步确定通过查阅大量中外文献,选择鉴定沙门菌最常用的invA、iroB、hns、hisJ、hilA、fimY基因作为研究对象,在Genbank数据库中下载这六个基因相应的基因序列,通过多重序列比对确定每个基因的目的片段。采用Assay design软件设计特异性PCR扩增引物和测序引物并初步确定了沙门菌的条码基因。2、沙门菌条码基因焦磷酸测序检测方法的建立优化PCR扩增体系和反应条件以及焦磷酸测序体系以提高本实验的可操作性。PCR扩增体系为:2×GoTaq Master Mix 12.5μl,上游引物和下游引物各5 pmol,DNA模板2μl,加无菌去离子水至25μl。反应条件为:94℃预变性5 min,94℃变性20 s,56℃退火30 s,72℃延伸20 s,35个循环,72℃延伸5 min。焦磷酸测序体系采用20μl的PCR产物加入量和优化的碱基加入程序。10株沙门菌和10株非沙门菌的测序结果显示沙门菌均测序出相应的核酸序列,非沙门菌均未测序出DNA序列,表明该方法可用于沙门菌的鉴定。对测序序列在线Blast比对分析发现测序的DNA片段均存在多态性位点,这些碱基的多样化并不会对沙门菌鉴定的准确性产生影响。3、大量已知菌株对该方法的验证用已建立的方法对205株沙门菌和60株非沙门菌同时进行鉴定,结果显示所有沙门菌均测序出相应的DNA序列,非沙门菌中只有一株鲍氏志贺氏菌测序结果与沙门菌hilA基因的条码基因一致,于是在沙门菌条码基因中删除了该基因以避免出现假阳性结果。通过对大量已知菌株的鉴定进一步验证了所建立检测方法的准确性和可行性,同时体现了该方法操作简便、快速、准确、高通量的特点。本实验最终确定的鉴定沙门菌的条码基因为:invA基因条码基因:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG。iroB基因条码基因:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG。hns基因条码基因:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。hisJ基因条码基因:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。fimY基因条码基因:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。4、沙门菌条码基因焦磷酸测序检测方法的实际应用通过在食物中毒检测和食源性疾病监测的实际应用中表明了沙门菌条码基因焦磷酸测序方法与国标法(GB/T 4789.4-2008)对沙门菌的检测结果一致,同时显示了该方法节时省力、准确、实时的优点。更多还原

【Abstract】 Salmonella species are the major pathogenic bacteria of foodborne bacterial diseaseas in the worldwide. At present, the prominent problem is that Salmonella species is an important reason of food security and public health. Therefore, development of an accurate, rapid, real-time and high throughput identification method can significantly support thefood daily monitoring and Salmonella species outbreak investigation. This study is based on the DNA barcode theory and combines the pyrosequencing technique in order to identify Salmonella species.Four parts were included in this thesis:Firstly, Salmonella DNA barcode was preliminarily determined. invA, iroB, hns, hisJ, hilA, fimY gene which the most commonly used to identify Salmonella were selected from the sino-foreign literature. The six genes corresponding sequences were download in Genbank database and multiple sequence alignment determine the purpose fragments of each gene. The six specific PCR primer sets and sequencing primers were designed using Assay design software and preliminarily determined the DNA barcode.Secondly, Salmonella DNA barcode pyrosequencing method was established and Salmonella DNA barcode sequence was determined.Through optimizing the PCR amplification system and PCR reaction conditions and pyrosequencing conditions, the experiment maneuverability were enhanced. PCR mixture contained the following: 12.5μl 2×Go Taq Master Mix, 5pmol of each primer, 2μl of DNA template and added ddH2O until 25μl. The PCR reaction: after initial denaturation at 94°C for 5 min, 35 cycles of amplification included denaturation at 94°C for 20 s, annealing at 56°C for 30 s, and elongation at 72°C for 20 s. The reaction concluded with a 5 min extension phase at 72°C. 20μl of biotinylated PCR products and optimition of bases join procedure were used in the pyrosequencing system. 10 Salmonella strains and 10 non-Salmonella strains by prosequencing showed that only all of Salmonella strains obtained the correct DNA sequences. Some polymorphism sites were founded in the DNA sequence by online Blast tool analysis, but the base variation will not affect the accuracy of Salmonella detection.Thirdly, the new mthod were verified by a large number of known strains. 205 Salmonella strains and 60 non-Salmonella strains were identified by this newmethod, and the result showed that all Salmonella strains obtained DNA sequence which correspond with Salmonella DNA barcode. In non-Salmonella strains, only Shigella boydii DNA sequence was compatibile with the hilA DNA barcode of Salmonella. To avoid the false positive result, hilA gene was excluded in Salmonella identification barcode gene panel. Through a large number of strains further verified the accuracy and feasibility of this method. It indicated that pyrosequencing was a easy to operate, rapid, accurate, sensitive and high-throughput method. So the DNA barcode sequence to identify the Salmonella was determined in the study: (A)invA gene barcode:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG.(B)iroB gene barcode:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG.(C)hns gene barcode:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。(D)hisJ gene barcode:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。(E)fimY gene barcode:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。Finally, practical application of Salmonella DNA barcode pyrosequencing method.Through practical application which the food poisoning detection and food-borne disease monitoring, it showed that the result by Salmonella DNA barcode pyrosequencing method was same to the traditional cultural method (GB/T GB 4789.4-2008), and showed advantages of this method: efficiency, accuracy, real time.更多还原