食源性致病菌特异性靶点发掘系统及数据库的构建
发布时间：2018-09-05 07:39
【摘要】：及时准确地检测和鉴定食源性致病菌对于提高公共卫生水平和保障食品安全是非常重要的,因此任何检测和鉴定食源性致病菌的方法必须是灵敏的、特异的、快速的。传统微生物检测方法如平皿计数法、生化鉴定法耗时较长,一般需要5-6天完成全部鉴定过程。近年来,基于核酸序列的各种分子检测方法如PCR法、芯片法等由于其操作简单、结果稳定、省时等优点已广泛应用于快速检测和鉴定食源性致病菌。然而,由于检测靶点序列变异、靶点基因水平迁移等因素的存在导致了食源性致病菌检测的假阴性、假阳性结果。因此,食源性致病菌检测靶点的特异性和数量限制了基于核酸序列的分子检测方法应用能力。 本研究以构建食源性致病菌特异性靶点发掘系统及相关数据库、评价部分食源性病原菌特异性靶点为目的,开展了一系列研究,主要研究 内容和结果如下: 1.食源性致病菌特异性靶点发掘系统SMM-system构建 本研究开发的软件SMM-system在不改变BLAST核心算法的前提下,以整合调用BLAST程序的方式,完成目标序列与基因组数据库BLASTN比对与分析筛选工作。SMM-system软件设有四个功能模块,各功能模块线性组合可以发掘食源性致病菌同源性序列及特异性序列,也可单独使用执行各自功能。SMM-system是一个高通量特异性靶标序列发掘工具,可以发掘属特异性、种特异性、甚至血清型特异性核酸序列。SMM-system可用于筛选风险物种以及食品工业、诊断学和分类学研究。在网站http://foodsafety.sjtu.edu.cn/SMM-system.html可免费下载SMM-system。 2.副溶血弧菌irgB基因特异性评价与多重PCR体系构建 利用SMM-system软件发掘出23个副溶血弧菌特异性蛋白编码序列(protein-coding sequences, CDSs),选择其中编码铁调控毒力调控蛋白的irgB (vp2603)基因作为靶点序列建立PCR检测方法。PCR检测结果表明293株副溶血弧菌为模板的PCR反应全部为阳性、11株其它弧菌和35株非弧菌菌株模板为全部阴性扩增。该PCR方法扩增片段为369 bp,扩增灵敏度为0.17pg副溶血弧菌纯基因组DNA/PCR。此外,构建了扩增irgB、tdh和trh基因的三重PCR检测体系,用以检测和鉴定副溶血弧菌及有毒菌株。实验数据显示,irgB基因序列为新的副溶血弧菌特异性靶点,同时这也表明应用SMM-system系统进行基因组序列比对鉴定特异性靶点是有效的。 3.沙门氏菌特异性靶点发掘与PCR体系构建 利用SMM-system软件发掘出214个沙门氏菌特异性靶点CDS,其中59个为假设蛋白,64个为推测基因,91个赋予功能注释。对18个沙门氏菌特异性CDS分别设计了18对PCR引物,其中7对特异性引物(分别靶向SC1286、SC1431、SC1598、SC2172、SC2225、SC2471和SC4361基因)用于扩增101株沙门氏菌基因组模板和30株非沙门氏菌基因组模板,扩增结果分别为100%阳性结果、100%阴性结果;而11对非特异性引物用于扩增上述131株菌株基因组模板时,出现了假阴性或/和假阳性检测结果。基于这7对特异性引物的PCR方法对人工污染牛奶样品进行检测,乙型副伤寒沙门氏菌S. enterica Paratyphi B (CMCC 50094)和猪霍乱沙门氏菌S. enterica Choleraesuis (ATCC 13312)培养物富集培养10h后,所有人工污染牛奶的PCR检测结果都呈现阳性,PCR引物的检测限均小于1 cfu/ml。此外,本研究选择三对引物(分别靶向SC1286,SC1598和SC4361基因)用于构建一个多重PCR体系。 4.食源性致病菌特异性靶点数据库SMDB构建 从NCBI基因组资源库(ftp://ftp.ncbi.nih.gov/genomes/bacteria/)下载了1052个细菌基因组(all.fna.tar.gz文件),CDS (all.ffn.tar.gz文件)和CDS注释信息(all.ptt.tar.gz文件),下载了23种食源性致病菌共150个基因组数据,数据下载日期截止至2010年3月1日。利用SMM-system软件筛选了23种食源性致病菌特异性靶点,构建了特异性靶点数据库SMDB。SMDB数据库内容主要包含16种食源性疾病的相关信息、23种食源性致病菌相关信息、6588个特异性靶点序列信息及相关注释信息、23种食源性致病菌分子检测相关文献共852篇。SMDB数据库为公共数据库,有助于于开展各种食源性疾病的诊断、检测和治疗以及预防食源性疾病暴发。SMDB数据库网站访问链接为http://202.120.41.154/。
[Abstract]:Timely and accurate detection and identification of foodborne pathogens is very important to improve public health and food safety. Therefore, any method for detection and identification of foodborne pathogens must be sensitive, specific, and rapid. Traditional microbiological detection methods such as plate counting, biochemical identification method takes a long time, generally needs 5- In recent years, various molecular detection methods based on nucleic acid sequences, such as PCR and microarray, have been widely used for rapid detection and identification of foodborne pathogens due to their advantages of simple operation, stable results and time-saving. However, due to the detection of target sequence variation, target gene horizontal migration and other factors exist to guide the detection of target genes. Therefore, the specificity and quantity of detection targets of foodborne pathogens limit the application of DNA sequence-based molecular detection methods.
In this study, a series of studies were carried out to construct a food-borne pathogenic bacteria specific target detection system and related databases, and to evaluate some food-borne pathogenic bacteria specific targets.
The content and results are as follows:
1. establishment of SMM-system system for specific target detection of foodborne pathogens
The software SMM-system developed in this study can complete the alignment and analysis of target sequence and genome database BLASTN without changing the core algorithm of BLAST. The software SMM-system has four functional modules, and the linear combination of each functional module can discover the homologous sequence of foodborne pathogenic bacteria. SMM-system is a high-throughput specific target sequence discovery tool that can discover genus-specific, species-specific, and even serotype-specific nucleic acid sequences. SMM-system can be used for screening risk species as well as for food industry, diagnostics and taxonomic research. At http://foodsa Fety.sjtu.edu.cn/SMM-system.html can download SMM-system. for free.
2. specific evaluation of irgB gene of Vibrio parahaemolyticus and construction of multiple PCR system
Twenty-three protein-coding sequences (CDSs) of Vibrio parahaemolyticus were detected by SMM-system software. The irgB (vp2603) gene encoding the iron-regulated virulence protein was selected as the target sequence for PCR detection. PCR results showed that all 293 strains of Vibrio parahaemolyticus were positive and 11 strains of Vibrio parahaemolyticus were positive. The amplified fragment was 369 BP and the amplified sensitivity was 0.17 PG. In addition, a triple PCR system was established for the detection and identification of Vibrio parahaemolyticus and its toxic strains by amplifying irgB, TDH and TRH genes. The gB gene sequence is a new specific target for Vibrio parahaemolyticus, which also shows that the SMM-system is effective in identifying specific targets for genomic sequence alignment.
3. Salmonella specific target mining and construction of PCR system
214 specific target CDS of Salmonella were detected by SMM-system software, 59 of which were hypothetical proteins, 64 speculative genes and 91 functional annotations. 18 pairs of PCR primers were designed for 18 specific CDS of Salmonella. Seven pairs of specific primers (targeting SC1286, SC1431, SC1598, SC2172, SC2225, SC2471 and SCC4361 genes respectively) The amplification results of 101 Salmonella genomic templates and 30 non-Salmonella genomic templates were 100% positive and 100% negative respectively, while 11 pairs of non-specific primers were used to amplify the genomic templates of 131 strains above, and false negative or/or false positive results were found. Methods the detection of artificially contaminated milk samples was carried out. After the enrichment of 10h * S. enterica Paratyphi B (CMCC 50094) and Salmonella typhimurium S. enterica Choleraesuis (ATCC 13312) culture, the detection results of all artificially contaminated milk were positive, and the detection limits of the primers were less than 1. In this study, three pairs of primers (targeting SC1286, SC1598 and SC4361 genes respectively) were selected to construct a multiplex PCR system.
4. construction of specific target database for foodborne pathogens SMDB
A total of 1 052 bacterial genomes (all.fna.tar.gz file), CDS (all.ffn.tar.gz file) and CDS annotation information (all.ptt.tar.gz file) were downloaded from the NCBI Genome Database (ftp://ftp.ncbi.nih.gov/genomes/bacteria/), and a total of 150 genomic data of 23 foodborne pathogens were downloaded by the date of March 1, 2010. System software screened 23 specific targets of foodborne pathogens, and constructed a specific target database SMDB. SMDB database, which mainly contains 16 kinds of foodborne diseases related information, 23 kinds of foodborne pathogens related information, 6588 specific target sequence information and related annotation information, 23 kinds of foodborne pathogens molecular detection related information. A total of 852 papers were published. The SMDB database is a public database which is helpful for the diagnosis, detection and treatment of various foodborne diseases and the prevention of outbreaks of foodborne diseases. The link to the SMDB database website is http://202.120.41.154/.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R155.5
本文编号：2223624
[Abstract]:Timely and accurate detection and identification of foodborne pathogens is very important to improve public health and food safety. Therefore, any method for detection and identification of foodborne pathogens must be sensitive, specific, and rapid. Traditional microbiological detection methods such as plate counting, biochemical identification method takes a long time, generally needs 5- In recent years, various molecular detection methods based on nucleic acid sequences, such as PCR and microarray, have been widely used for rapid detection and identification of foodborne pathogens due to their advantages of simple operation, stable results and time-saving. However, due to the detection of target sequence variation, target gene horizontal migration and other factors exist to guide the detection of target genes. Therefore, the specificity and quantity of detection targets of foodborne pathogens limit the application of DNA sequence-based molecular detection methods.
In this study, a series of studies were carried out to construct a food-borne pathogenic bacteria specific target detection system and related databases, and to evaluate some food-borne pathogenic bacteria specific targets.
The content and results are as follows:
1. establishment of SMM-system system for specific target detection of foodborne pathogens
The software SMM-system developed in this study can complete the alignment and analysis of target sequence and genome database BLASTN without changing the core algorithm of BLAST. The software SMM-system has four functional modules, and the linear combination of each functional module can discover the homologous sequence of foodborne pathogenic bacteria. SMM-system is a high-throughput specific target sequence discovery tool that can discover genus-specific, species-specific, and even serotype-specific nucleic acid sequences. SMM-system can be used for screening risk species as well as for food industry, diagnostics and taxonomic research. At http://foodsa Fety.sjtu.edu.cn/SMM-system.html can download SMM-system. for free.
2. specific evaluation of irgB gene of Vibrio parahaemolyticus and construction of multiple PCR system
Twenty-three protein-coding sequences (CDSs) of Vibrio parahaemolyticus were detected by SMM-system software. The irgB (vp2603) gene encoding the iron-regulated virulence protein was selected as the target sequence for PCR detection. PCR results showed that all 293 strains of Vibrio parahaemolyticus were positive and 11 strains of Vibrio parahaemolyticus were positive. The amplified fragment was 369 BP and the amplified sensitivity was 0.17 PG. In addition, a triple PCR system was established for the detection and identification of Vibrio parahaemolyticus and its toxic strains by amplifying irgB, TDH and TRH genes. The gB gene sequence is a new specific target for Vibrio parahaemolyticus, which also shows that the SMM-system is effective in identifying specific targets for genomic sequence alignment.
3. Salmonella specific target mining and construction of PCR system
214 specific target CDS of Salmonella were detected by SMM-system software, 59 of which were hypothetical proteins, 64 speculative genes and 91 functional annotations. 18 pairs of PCR primers were designed for 18 specific CDS of Salmonella. Seven pairs of specific primers (targeting SC1286, SC1431, SC1598, SC2172, SC2225, SC2471 and SCC4361 genes respectively) The amplification results of 101 Salmonella genomic templates and 30 non-Salmonella genomic templates were 100% positive and 100% negative respectively, while 11 pairs of non-specific primers were used to amplify the genomic templates of 131 strains above, and false negative or/or false positive results were found. Methods the detection of artificially contaminated milk samples was carried out. After the enrichment of 10h * S. enterica Paratyphi B (CMCC 50094) and Salmonella typhimurium S. enterica Choleraesuis (ATCC 13312) culture, the detection results of all artificially contaminated milk were positive, and the detection limits of the primers were less than 1. In this study, three pairs of primers (targeting SC1286, SC1598 and SC4361 genes respectively) were selected to construct a multiplex PCR system.
4. construction of specific target database for foodborne pathogens SMDB
A total of 1 052 bacterial genomes (all.fna.tar.gz file), CDS (all.ffn.tar.gz file) and CDS annotation information (all.ptt.tar.gz file) were downloaded from the NCBI Genome Database (ftp://ftp.ncbi.nih.gov/genomes/bacteria/), and a total of 150 genomic data of 23 foodborne pathogens were downloaded by the date of March 1, 2010. System software screened 23 specific targets of foodborne pathogens, and constructed a specific target database SMDB. SMDB database, which mainly contains 16 kinds of foodborne diseases related information, 23 kinds of foodborne pathogens related information, 6588 specific target sequence information and related annotation information, 23 kinds of foodborne pathogens molecular detection related information. A total of 852 papers were published. The SMDB database is a public database which is helpful for the diagnosis, detection and treatment of various foodborne diseases and the prevention of outbreaks of foodborne diseases. The link to the SMDB database website is http://202.120.41.154/.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R155.5
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