本文選題：多重PCR + 基因芯片�。� 參考：《吉林大學(xué)》2011年博士論文

【摘要】：蜚蠊是我國在國境口岸地區(qū)監(jiān)測(cè)的重要醫(yī)學(xué)媒介生物之一,它在生物分類中屬節(jié)肢動(dòng)物門,昆蟲綱,蜚蠊目。蜚蠊生存于人類居住的環(huán)境中,它不僅騷擾、叮咬、損壞物品,而且是多種病原菌的傳播媒介或儲(chǔ)存宿主。蜚蠊可以攜帶并傳播細(xì)菌、真菌、病毒和寄生蟲,能夠引起腸道、呼吸道、皮膚等多系統(tǒng)疾病的發(fā)生,對(duì)人類健康構(gòu)成嚴(yán)重危害。 隨著全球經(jīng)濟(jì)一體化,國際貿(mào)易的不斷發(fā)展,國際間通行的交通工具和旅客的數(shù)量明顯上升,媒介生物及其攜帶的病原體借助交通工具、集裝箱、貨物、郵件等在不同國家口岸間傳播,這給蜚蠊及其攜帶的病原體在國際間的傳播帶來了便利條件,蟲媒傳染病有明顯上升的趨勢(shì)。 目前檢測(cè)病原菌的技術(shù)具有一定的局限性。近年來,已發(fā)展了多種基于微生物學(xué)、化學(xué)、免疫學(xué)和分子生物學(xué)的實(shí)驗(yàn)技術(shù)用于檢測(cè)和鑒別病原微生物,但還不能滿足同時(shí)快速檢測(cè)多種病原體的需要。本研究建立了多重PCR結(jié)合基因芯片技術(shù)檢測(cè)11種病原菌的方法,并在掌握長春龍嘉國際機(jī)場(chǎng)口岸內(nèi)蜚蠊的本底情況基礎(chǔ)上,對(duì)口岸內(nèi)蜚蠊攜帶的病原菌進(jìn)行了檢測(cè),對(duì)有效地預(yù)防和控制蜚蠊在口岸地區(qū)引起疾病的傳播具有重要意義。 目的： 1.建立多重PCR結(jié)合基因芯片技術(shù)的方法對(duì)11種常見病原菌進(jìn)行檢測(cè),為臨床疾病診斷、食物中毒檢測(cè)、進(jìn)出口食品檢驗(yàn)、媒介生物病原菌檢測(cè)等工作提供有效的檢測(cè)方法。 2.掌握長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊的本底情況,為加強(qiáng)國境口岸及出入境交通工具蜚蠊的監(jiān)測(cè)與控制提供科學(xué)指導(dǎo)。 3.運(yùn)用多重PCR結(jié)合基因芯片技術(shù)對(duì)長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊攜帶的病原菌進(jìn)行檢測(cè),對(duì)蜚蠊傳播傳染病的風(fēng)險(xiǎn)程度作出科學(xué)分析和預(yù)測(cè),保障國境口岸和交通工具及貨物的衛(wèi)生安全。 方法： 1.研制寡核苷酸基因芯片檢測(cè)常見的11種病原菌。以蠟樣芽孢桿菌、大腸桿菌0157、金黃色葡萄球菌、傷寒沙門氏菌、腸炎沙門氏菌、志賀氏菌、變形桿菌、產(chǎn)氣莢膜梭菌、空腸彎曲菌、副溶血性弧菌、單核細(xì)胞增生李斯特菌為檢測(cè)的靶細(xì)菌,設(shè)計(jì)特異性引物及探針,制備寡核苷酸芯片。靶細(xì)菌特異性基因經(jīng)多重PCR擴(kuò)增后,與帶有11種特異性探針的基因芯片雜交,用掃描儀掃描,判定病原菌種類。對(duì)基因芯片的特異性和靈敏度進(jìn)行檢測(cè)。 2.掌握長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊本底情況。選擇口岸內(nèi)4種不同生境分別為辦公區(qū)、食品生產(chǎn)廠區(qū)、餐飲區(qū)、倉儲(chǔ)場(chǎng)地為調(diào)查區(qū),采用“盒式誘捕法”捕獲蜚蠊,調(diào)查2008年4月至2009年3月間長春龍嘉國際機(jī)場(chǎng)口岸內(nèi)蜚蠊本底情況,掌握蜚蠊的種群構(gòu)成、密度和季節(jié)消長規(guī)律。 3.對(duì)煮沸法、試劑盒法、SDS法三種提取蜚蠊攜帶病原菌DNA的方法進(jìn)行比較。運(yùn)用已建立的多重PCR結(jié)合基因芯片技術(shù)對(duì)提取的病原菌DNA進(jìn)行檢測(cè),掌握長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊攜帶病原菌的種類。 結(jié)果： 1.將不同的病原菌DNA樣本混合,配制成一系列模擬樣本,對(duì)這些模擬樣本采用優(yōu)化的多重PCR體系和基因芯片雜交體系進(jìn)行檢測(cè)分析,結(jié)果顯示混合樣本DNA均能與相對(duì)應(yīng)的探針進(jìn)行特異性雜交,證明本研究研制的基因芯片具有良好的特異性�；蛐酒臋z測(cè)靈敏度比PCR方法檢測(cè)靈敏度高10倍。 2.2008年4月至2009年3月間,在長春龍嘉國際機(jī)場(chǎng)口岸共布放粘捕盒3600盒,有效回收2919盒,共捕獲蜚蠊3957只。德國小蠊是長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊的優(yōu)勢(shì)種屬。不同生境下蜚蠊密度不同,餐飲區(qū)蜚蠊密度最高,為4.08只/盒；口岸蜚蠊平均密度為1.36只/盒。全年12個(gè)月中,蜚蠊密度在1月份和7月份分別達(dá)到高峰。 3.煮沸法、試劑盒法、SDS法三種方法提取蜚蠊攜帶病原菌DNA,得到的DNA純度和濃度有顯著性差異,最終確定SDS方法作為本研究提取蜚蠊攜帶病原菌DNA的方法。 4.基因芯片檢測(cè)蜚蠊攜帶病原菌結(jié)果表明,本研究設(shè)定的11種靶細(xì)菌均在長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊中被檢出,各病原菌的檢出率分別為金黃色葡萄球菌30%、志賀氏菌100%、大腸桿菌015750%、腸炎沙門氏菌10%、傷寒沙門氏菌45%、蠟樣芽胞桿菌15%、空腸彎曲菌100%、變形桿菌80%、單核細(xì)胞增生李斯特菌55%、副溶血性弧菌5%、產(chǎn)氣莢膜梭菌5%。蜚蠊攜帶病原菌DNA進(jìn)行普通PCR擴(kuò)增得到的產(chǎn)物進(jìn)行純化測(cè)序,測(cè)序結(jié)果與GenBank上的標(biāo)準(zhǔn)序列的一致性均達(dá)到85%以上。 結(jié)論： 1.多重PCR與基因芯片技術(shù)的整合可以實(shí)現(xiàn)兩種技術(shù)的優(yōu)勢(shì)互補(bǔ),通過多重PCR的基因放大作用和基因芯片的探針雜交技術(shù)使此種檢測(cè)體系達(dá)到較高的靈敏度和特異度,可實(shí)現(xiàn)對(duì)多種病原菌、多個(gè)樣本的同時(shí)檢測(cè)。 2.長春龍嘉國際機(jī)場(chǎng)口岸內(nèi)蜚蠊的優(yōu)勢(shì)種屬為德國小蠊；不同生境的蜚蠊密度不同,餐飲區(qū)蜚蠊密度最高；不同季節(jié)蜚蠊密度不同,1月份和7月份為蜚蠊密度較高。 3.本研究對(duì)蜚蠊攜帶病原菌基因組DNA的提取方法進(jìn)行了優(yōu)化。確定了SDS法作為蜚蠊攜帶病原菌基因組DNA的提取方法,該方法具有操作簡單、提取效率高、成本低、易于現(xiàn)場(chǎng)檢測(cè)等特點(diǎn)。 4.本研究運(yùn)用多重PCR結(jié)合基因芯片技術(shù)對(duì)蜚蠊攜帶病原菌進(jìn)行檢測(cè)。結(jié)果顯示11種目標(biāo)病原菌在長春龍嘉國際機(jī)場(chǎng)口岸蜚蠊中均被檢出。 本研究的創(chuàng)新點(diǎn)： 1.建立了一種多重PCR結(jié)合基因芯片技術(shù)的檢測(cè)方法,能同時(shí)對(duì)11種常見病原菌進(jìn)行快速、準(zhǔn)確地檢測(cè)。 2.首次對(duì)長春龍嘉國際機(jī)場(chǎng)口岸的蜚蠊進(jìn)行本底調(diào)查,掌握了該地區(qū)蜚蠊的種群構(gòu)成、密度、季節(jié)消長規(guī)律等情況； 3.首次運(yùn)用多重PCR結(jié)合基因芯片技術(shù)對(duì)蜚蠊攜帶的病原菌進(jìn)行檢測(cè),探討直接從媒介生物樣品中同時(shí)檢測(cè)多種病原菌的可行性,為國境口岸媒介生物攜帶病原菌的檢測(cè)提供幫助。
[Abstract]:Cockroaches are one of the important medical medium organisms in the frontier port area of our country . It is an arthropod door , an insect class and a cockroach in the biological classification . The cockroach can carry and spread bacteria , fungi , viruses and parasites , which can cause the occurrence of multiple systemic diseases such as intestinal tract , respiratory tract and skin , which can cause serious harm to human health .


Along with the global economic integration , the development of international trade , the number of vehicles and passengers passing internationally , the media and the pathogens carried by the media spread among different ports through vehicles , containers , goods , mails , etc . , which brings convenience for the spread of the cockroach and its carried pathogens in the international community , and the insect vector infectious disease has obvious ascending trend .


In recent years , a variety of experimental techniques based on microbiology , chemistry , immunology and molecular biology have been developed for the detection and identification of pathogenic microorganisms .


Purpose :


1 . The methods of establishing multiplex PCR and gene chip technology were used to detect 11 common pathogenic bacteria , which provided an effective detection method for the diagnosis of clinical disease , food poisoning detection , import and export food inspection , and the detection of vector biological pathogens .


2 . To master the situation of the cockroach at the port of Changchun Longjia International Airport and provide scientific guidance to strengthen the monitoring and control of the Cockroaches at frontier ports and exit vehicles .


3 . Using multiplex PCR combined with the gene chip technology to detect the pathogenic bacteria carried by the cockroach at the port of Changchun Longjia International Airport , scientific analysis and prediction of the risk degree of the spread infectious disease of the cockroach are made , so as to guarantee the hygienic safety of the frontier port and the vehicle and the goods .


Method :


1 . The oligonucleotide microarray was developed to detect the common 11 kinds of pathogenic bacteria . The specific primers and probes were designed for the detection of Salmonella , Escherichia coli 0157 , Staphylococcus aureus , Salmonella typhimurium , Salmonella enteritidis , Shigella spp . , Bacillus proteus , Clostridium perfringens , aerogenes , vibrio parahemolyticus and listerias . The specific primers and probes were designed to hybridize with the gene chips with 11 specific probes . The specific primers and probes were used to scan the target bacteria . The specificity and sensitivity of the gene chip were tested .


2 . To master the situation of cockroach at the port of Changchun Longjia International Airport . Four different habitats in the port were selected as office area , food production plant area , catering area , storage yard as the investigation area , and the cockroach was captured by the " box - type trapping method " to investigate the background of cockroach in the port of Changchun Longjia International Airport between April 2008 and March 2009 .


3 . Compare the methods of boiling method , kit method and SDS method to extract pathogenic bacteria DNA from cockroach . Using the established multiplex PCR combined with the gene chip technology , the DNA of the extracted pathogenic bacteria was detected , and the species of the pathogenic bacteria borne by the cockroach at the port of Changchun Longjia International Airport were mastered .


Results :


1 . The DNA samples of different pathogenic bacteria were mixed and prepared into a series of simulated samples , and the optimized multiplex PCR system and gene chip hybridization system were used to detect the samples . The results showed that the mixed sample DNA could specifically hybridize with the corresponding probe . The results showed that the gene chip developed by this study has a good specificity . The detection sensitivity of the gene chip is 10 times higher than that of the PCR method .


2 . From April 2008 to March 2009 , a total of 3600 cases were distributed at the port of Changchun Longjia International Airport . 2919 cases were effectively recovered . 3957 species of cockroach were captured . The cockroach was the dominant species of cockroach at the port of Changchun Longjia International Airport . The density of cockroach in different habitats was different , the density of cockroach in catering area was the highest , which was 4.08 / box ;
The average density of cockroach at the port is 1.36 / box . In the 12 months of the year , the density of cockroach reached its peak in January and July respectively .


3 . Using three methods of boiling method , reagent kit and SDS method to extract DNA from cockroach , the purity and concentration of DNA were significantly different . Finally , it was determined that SDS method was used as a method to extract DNA from cockroach .


4 . The results showed that 11 kinds of target bacteria were detected in the cockroach in Changchun Longjia International Airport . The detection rates of each pathogenic bacterium were staphylococcus aureus 30 % , Shigella spp . 100 % , Escherichia coli 015750 % , Salmonella enteritidis 10 % , Salmonella typhimurium 45 % , Escherichia coli 015750 % , Vibrio parahemolyticus 5 % , Clostridium perfringens 5 % .


Conclusion :


1 . The integration of multiplex PCR and gene chip technology can realize the complementary advantages of the two technologies . Through the gene amplification of multiplex PCR and the probe hybridization technology of the gene chip , the detection system can achieve higher sensitivity and specificity , and the simultaneous detection of various pathogenic bacteria and multiple samples can be realized .


2 . The dominant species of Blattella germanica in Changchun Longjia International Airport Port is German cockroach ;
The density of cockroach in different habitats was different , and the density of cockroach in food and beverage area was the highest ;
The density of cockroach in different seasons was different , and the density of cockroach was higher in January and July .


3 . The method of extracting genomic DNA of pathogenic bacteria from cockroach was optimized . SDS method was used to extract genomic DNA of pathogenic bacteria . The method has the characteristics of simple operation , high extraction efficiency , low cost and easy on - site detection .


The results showed that 11 target pathogens were detected at the port of Changchun Longjia International Airport .


Innovative points for this study :


1 . The detection method of multiplex PCR combined with gene chip technology is established , which can quickly and accurately detect 11 common pathogenic bacteria at the same time .


2 . For the first time , the investigation of the cockroach in Changchun Longjia International Airport is carried out , and the population composition , density and seasonal fluctuation rule of the cockroach in the area are mastered .



3 . First , using multiplex PCR combined with gene chip technology to detect the pathogenic bacteria carried by the cockroach , the feasibility of simultaneously detecting various pathogenic bacteria from the media biological sample is discussed , and the help is provided for the detection of the pathogen in the media organism at the frontier port .
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R184

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳明愛;進(jìn)口加拿大油菜籽真菌與轉(zhuǎn)基因品系檢測(cè)技術(shù)研究[D];昆明理工大學(xué);2012年

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本文編號(hào)：2114470