食源性致病菌特異性靶點(diǎn)發(fā)掘系統(tǒng)及數(shù)據(jù)庫(kù)的構(gòu)建
發(fā)布時(shí)間：2018-09-05 07:39
【摘要】：及時(shí)準(zhǔn)確地檢測(cè)和鑒定食源性致病菌對(duì)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(cè)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(cè)方法如平皿計(jì)數(shù)法、生化鑒定法耗時(shí)較長(zhǎng),一般需要5-6天完成全部鑒定過(guò)程。近年來(lái),基于核酸序列的各種分子檢測(cè)方法如PCR法、芯片法等由于其操作簡(jiǎn)單、結(jié)果穩(wěn)定、省時(shí)等優(yōu)點(diǎn)已廣泛應(yīng)用于快速檢測(cè)和鑒定食源性致病菌。然而,由于檢測(cè)靶點(diǎn)序列變異、靶點(diǎn)基因水平遷移等因素的存在導(dǎo)致了食源性致病菌檢測(cè)的假陰性、假陽(yáng)性結(jié)果。因此,食源性致病菌檢測(cè)靶點(diǎn)的特異性和數(shù)量限制了基于核酸序列的分子檢測(cè)方法應(yīng)用能力。 本研究以構(gòu)建食源性致病菌特異性靶點(diǎn)發(fā)掘系統(tǒng)及相關(guān)數(shù)據(jù)庫(kù)、評(píng)價(jià)部分食源性病原菌特異性靶點(diǎn)為目的,開(kāi)展了一系列研究,主要研究 內(nèi)容和結(jié)果如下: 1.食源性致病菌特異性靶點(diǎn)發(fā)掘系統(tǒng)SMM-system構(gòu)建 本研究開(kāi)發(fā)的軟件SMM-system在不改變BLAST核心算法的前提下,以整合調(diào)用BLAST程序的方式,完成目標(biāo)序列與基因組數(shù)據(jù)庫(kù)BLASTN比對(duì)與分析篩選工作。SMM-system軟件設(shè)有四個(gè)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(dú)使用執(zhí)行各自功能。SMM-system是一個(gè)高通量特異性靶標(biāo)序列發(fā)掘工具,可以發(fā)掘?qū)偬禺愋�、種特異性、甚至血清型特異性核酸序列。SMM-system可用于篩選風(fēng)險(xiǎn)物種以及食品工業(yè)、診斷學(xué)和分類(lèi)學(xué)研究。在網(wǎng)站http://foodsafety.sjtu.edu.cn/SMM-system.html可免費(fèi)下載SMM-system。 2.副溶血弧菌irgB基因特異性評(píng)價(jià)與多重PCR體系構(gòu)建 利用SMM-system軟件發(fā)掘出23個(gè)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(diào)控毒力調(diào)控蛋白的irgB (vp2603)基因作為靶點(diǎn)序列建立PCR檢測(cè)方法。PCR檢測(cè)結(jié)果表明293株副溶血弧菌為模板的PCR反應(yīng)全部為陽(yáng)性、11株其它弧菌和35株非弧菌菌株模板為全部陰性擴(kuò)增。該P(yáng)CR方法擴(kuò)增片段為369 bp,擴(kuò)增靈敏度為0.17pg副溶血弧菌純基因組DNA/PCR。此外,構(gòu)建了擴(kuò)增irgB、tdh和trh基因的三重PCR檢測(cè)體系,用以檢測(cè)和鑒定副溶血弧菌及有毒菌株。實(shí)驗(yàn)數(shù)據(jù)顯示,irgB基因序列為新的副溶血弧菌特異性靶點(diǎn),同時(shí)這也表明應(yīng)用SMM-system系統(tǒng)進(jìn)行基因組序列比對(duì)鑒定特異性靶點(diǎn)是有效的。 3.沙門(mén)氏菌特異性靶點(diǎn)發(fā)掘與PCR體系構(gòu)建 利用SMM-system軟件發(fā)掘出214個(gè)沙門(mén)氏菌特異性靶點(diǎn)CDS,其中59個(gè)為假設(shè)蛋白,64個(gè)為推測(cè)基因,91個(gè)賦予功能注釋。對(duì)18個(gè)沙門(mén)氏菌特異性CDS分別設(shè)計(jì)了18對(duì)PCR引物,其中7對(duì)特異性引物(分別靶向SC1286、SC1431、SC1598、SC2172、SC2225、SC2471和SC4361基因)用于擴(kuò)增101株沙門(mén)氏菌基因組模板和30株非沙門(mén)氏菌基因組模板,擴(kuò)增結(jié)果分別為100%陽(yáng)性結(jié)果、100%陰性結(jié)果;而11對(duì)非特異性引物用于擴(kuò)增上述131株菌株基因組模板時(shí),出現(xiàn)了假陰性或/和假陽(yáng)性檢測(cè)結(jié)果�；谶@7對(duì)特異性引物的PCR方法對(duì)人工污染牛奶樣品進(jìn)行檢測(cè),乙型副傷寒沙門(mén)氏菌S. enterica Paratyphi B (CMCC 50094)和豬霍亂沙門(mén)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(cè)結(jié)果都呈現(xiàn)陽(yáng)性,PCR引物的檢測(cè)限均小于1 cfu/ml。此外,本研究選擇三對(duì)引物(分別靶向SC1286,SC1598和SC4361基因)用于構(gòu)建一個(gè)多重PCR體系。 4.食源性致病菌特異性靶點(diǎn)數(shù)據(jù)庫(kù)SMDB構(gòu)建 從NCBI基因組資源庫(kù)(ftp://ftp.ncbi.nih.gov/genomes/bacteria/)下載了1052個(gè)細(xì)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(gè)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(diǎn),構(gòu)建了特異性靶點(diǎn)數(shù)據(jù)庫(kù)SMDB。SMDB數(shù)據(jù)庫(kù)內(nèi)容主要包含16種食源性疾病的相關(guān)信息、23種食源性致病菌相關(guān)信息、6588個(gè)特異性靶點(diǎn)序列信息及相關(guān)注釋信息、23種食源性致病菌分子檢測(cè)相關(guān)文獻(xiàn)共852篇。SMDB數(shù)據(jù)庫(kù)為公共數(shù)據(jù)庫(kù),有助于于開(kāi)展各種食源性疾病的診斷、檢測(cè)和治療以及預(yù)防食源性疾病暴發(fā)。SMDB數(shù)據(jù)庫(kù)網(wǎng)站訪問(wèn)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R155.5
本文編號(hào)：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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R155.5
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