食源性致病菌特異性靶點發(fā)掘系統(tǒng)及數(shù)據(jù)庫的構建
發(fā)布時間：2018-09-05 07:39
【摘要】：及時準確地檢測和鑒定食源性致病菌對于提高公共衛(wèi)生水平和保障食品安全是非常重要的,因此任何檢測和鑒定食源性致病菌的方法必須是靈敏的、特異的、快速的。傳統(tǒng)微生物檢測方法如平皿計數(shù)法、生化鑒定法耗時較長,一般需要5-6天完成全部鑒定過程。近年來,基于核酸序列的各種分子檢測方法如PCR法、芯片法等由于其操作簡單、結果穩(wěn)定、省時等優(yōu)點已廣泛應用于快速檢測和鑒定食源性致病菌。然而,由于檢測靶點序列變異、靶點基因水平遷移等因素的存在導致了食源性致病菌檢測的假陰性、假陽性結果。因此,食源性致病菌檢測靶點的特異性和數(shù)量限制了基于核酸序列的分子檢測方法應用能力。 本研究以構建食源性致病菌特異性靶點發(fā)掘系統(tǒng)及相關數(shù)據(jù)庫、評價部分食源性病原菌特異性靶點為目的,開展了一系列研究,主要研究 內容和結果如下: 1.食源性致病菌特異性靶點發(fā)掘系統(tǒng)SMM-system構建 本研究開發(fā)的軟件SMM-system在不改變BLAST核心算法的前提下,以整合調用BLAST程序的方式,完成目標序列與基因組數(shù)據(jù)庫BLASTN比對與分析篩選工作。SMM-system軟件設有四個功能模塊,各功能模塊線性組合可以發(fā)掘食源性致病菌同源性序列及特異性序列,也可單獨使用執(zhí)行各自功能。SMM-system是一個高通量特異性靶標序列發(fā)掘工具,可以發(fā)掘屬特異性、種特異性、甚至血清型特異性核酸序列。SMM-system可用于篩選風險物種以及食品工業(yè)、診斷學和分類學研究。在網(wǎng)站http://foodsafety.sjtu.edu.cn/SMM-system.html可免費下載SMM-system。 2.副溶血弧菌irgB基因特異性評價與多重PCR體系構建 利用SMM-system軟件發(fā)掘出23個副溶血弧菌特異性蛋白編碼序列(protein-coding sequences, CDSs),選擇其中編碼鐵調控毒力調控蛋白的irgB (vp2603)基因作為靶點序列建立PCR檢測方法。PCR檢測結果表明293株副溶血弧菌為模板的PCR反應全部為陽性、11株其它弧菌和35株非弧菌菌株模板為全部陰性擴增。該PCR方法擴增片段為369 bp,擴增靈敏度為0.17pg副溶血弧菌純基因組DNA/PCR。此外,構建了擴增irgB、tdh和trh基因的三重PCR檢測體系,用以檢測和鑒定副溶血弧菌及有毒菌株。實驗數(shù)據(jù)顯示,irgB基因序列為新的副溶血弧菌特異性靶點,同時這也表明應用SMM-system系統(tǒng)進行基因組序列比對鑒定特異性靶點是有效的。 3.沙門氏菌特異性靶點發(fā)掘與PCR體系構建 利用SMM-system軟件發(fā)掘出214個沙門氏菌特異性靶點CDS,其中59個為假設蛋白,64個為推測基因,91個賦予功能注釋。對18個沙門氏菌特異性CDS分別設計了18對PCR引物,其中7對特異性引物(分別靶向SC1286、SC1431、SC1598、SC2172、SC2225、SC2471和SC4361基因)用于擴增101株沙門氏菌基因組模板和30株非沙門氏菌基因組模板,擴增結果分別為100%陽性結果、100%陰性結果;而11對非特異性引物用于擴增上述131株菌株基因組模板時,出現(xiàn)了假陰性或/和假陽性檢測結果�；谶@7對特異性引物的PCR方法對人工污染牛奶樣品進行檢測,乙型副傷寒沙門氏菌S. enterica Paratyphi B (CMCC 50094)和豬霍亂沙門氏菌S. enterica Choleraesuis (ATCC 13312)培養(yǎng)物富集培養(yǎng)10h后,所有人工污染牛奶的PCR檢測結果都呈現(xiàn)陽性,PCR引物的檢測限均小于1 cfu/ml。此外,本研究選擇三對引物(分別靶向SC1286,SC1598和SC4361基因)用于構建一個多重PCR體系。 4.食源性致病菌特異性靶點數(shù)據(jù)庫SMDB構建 從NCBI基因組資源庫(ftp://ftp.ncbi.nih.gov/genomes/bacteria/)下載了1052個細菌基因組(all.fna.tar.gz文件),CDS (all.ffn.tar.gz文件)和CDS注釋信息(all.ptt.tar.gz文件),下載了23種食源性致病菌共150個基因組數(shù)據(jù),數(shù)據(jù)下載日期截止至2010年3月1日。利用SMM-system軟件篩選了23種食源性致病菌特異性靶點,構建了特異性靶點數(shù)據(jù)庫SMDB。SMDB數(shù)據(jù)庫內容主要包含16種食源性疾病的相關信息、23種食源性致病菌相關信息、6588個特異性靶點序列信息及相關注釋信息、23種食源性致病菌分子檢測相關文獻共852篇。SMDB數(shù)據(jù)庫為公共數(shù)據(jù)庫,有助于于開展各種食源性疾病的診斷、檢測和治療以及預防食源性疾病暴發(fā)。SMDB數(shù)據(jù)庫網(wǎng)站訪問鏈接為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/.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R155.5
本文編號：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/.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R155.5
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