多重PCR结合基因芯片技术检测国境口岸蜚蠊携带病原菌的应用研究

发布时间：2018-07-10 19:43

本文选题：多重PCR + 基因芯片　；参考：《吉林大学》2011年博士论文

【摘要】：蜚蠊是我国在国境口岸地区监测的重要医学媒介生物之一,它在生物分类中属节肢动物门,昆虫纲,蜚蠊目。蜚蠊生存于人类居住的环境中,它不仅骚扰、叮咬、损坏物品,而且是多种病原菌的传播媒介或储存宿主。蜚蠊可以携带并传播细菌、真菌、病毒和寄生虫,能够引起肠道、呼吸道、皮肤等多系统疾病的发生,对人类健康构成严重危害。随着全球经济一体化,国际贸易的不断发展,国际间通行的交通工具和旅客的数量明显上升,媒介生物及其携带的病原体借助交通工具、集装箱、货物、邮件等在不同国家口岸间传播,这给蜚蠊及其携带的病原体在国际间的传播带来了便利条件,虫媒传染病有明显上升的趋势。目前检测病原菌的技术具有一定的局限性。近年来,已发展了多种基于微生物学、化学、免疫学和分子生物学的实验技术用于检测和鉴别病原微生物,但还不能满足同时快速检测多种病原体的需要。本研究建立了多重PCR结合基因芯片技术检测11种病原菌的方法,并在掌握长春龙嘉国际机场口岸内蜚蠊的本底情况基础上,对口岸内蜚蠊携带的病原菌进行了检测,对有效地预防和控制蜚蠊在口岸地区引起疾病的传播具有重要意义。目的： 1.建立多重PCR结合基因芯片技术的方法对11种常见病原菌进行检测,为临床疾病诊断、食物中毒检测、进出口食品检验、媒介生物病原菌检测等工作提供有效的检测方法。 2.掌握长春龙嘉国际机场口岸蜚蠊的本底情况,为加强国境口岸及出入境交通工具蜚蠊的监测与控制提供科学指导。 3.运用多重PCR结合基因芯片技术对长春龙嘉国际机场口岸蜚蠊携带的病原菌进行检测,对蜚蠊传播传染病的风险程度作出科学分析和预测,保障国境口岸和交通工具及货物的卫生安全。方法： 1.研制寡核苷酸基因芯片检测常见的11种病原菌。以蜡样芽孢杆菌、大肠杆菌0157、金黄色葡萄球菌、伤寒沙门氏菌、肠炎沙门氏菌、志贺氏菌、变形杆菌、产气荚膜梭菌、空肠弯曲菌、副溶血性弧菌、单核细胞增生李斯特菌为检测的靶细菌,设计特异性引物及探针,制备寡核苷酸芯片。靶细菌特异性基因经多重PCR扩增后,与带有11种特异性探针的基因芯片杂交,用扫描仪扫描,判定病原菌种类。对基因芯片的特异性和灵敏度进行检测。 2.掌握长春龙嘉国际机场口岸蜚蠊本底情况。选择口岸内4种不同生境分别为办公区、食品生产厂区、餐饮区、仓储场地为调查区,采用“盒式诱捕法”捕获蜚蠊,调查2008年4月至2009年3月间长春龙嘉国际机场口岸内蜚蠊本底情况,掌握蜚蠊的种群构成、密度和季节消长规律。 3.对煮沸法、试剂盒法、SDS法三种提取蜚蠊携带病原菌DNA的方法进行比较。运用已建立的多重PCR结合基因芯片技术对提取的病原菌DNA进行检测,掌握长春龙嘉国际机场口岸蜚蠊携带病原菌的种类。结果： 1.将不同的病原菌DNA样本混合,配制成一系列模拟样本,对这些模拟样本采用优化的多重PCR体系和基因芯片杂交体系进行检测分析,结果显示混合样本DNA均能与相对应的探针进行特异性杂交,证明本研究研制的基因芯片具有良好的特异性。基因芯片的检测灵敏度比PCR方法检测灵敏度高10倍。 2.2008年4月至2009年3月间,在长春龙嘉国际机场口岸共布放粘捕盒3600盒,有效回收2919盒,共捕获蜚蠊3957只。德国小蠊是长春龙嘉国际机场口岸蜚蠊的优势种属。不同生境下蜚蠊密度不同,餐饮区蜚蠊密度最高,为4.08只/盒；口岸蜚蠊平均密度为1.36只/盒。全年12个月中,蜚蠊密度在1月份和7月份分别达到高峰。 3.煮沸法、试剂盒法、SDS法三种方法提取蜚蠊携带病原菌DNA,得到的DNA纯度和浓度有显著性差异,最终确定SDS方法作为本研究提取蜚蠊携带病原菌DNA的方法。 4.基因芯片检测蜚蠊携带病原菌结果表明,本研究设定的11种靶细菌均在长春龙嘉国际机场口岸蜚蠊中被检出,各病原菌的检出率分别为金黄色葡萄球菌30%、志贺氏菌100%、大肠杆菌015750%、肠炎沙门氏菌10%、伤寒沙门氏菌45%、蜡样芽胞杆菌15%、空肠弯曲菌100%、变形杆菌80%、单核细胞增生李斯特菌55%、副溶血性弧菌5%、产气荚膜梭菌5%。蜚蠊携带病原菌DNA进行普通PCR扩增得到的产物进行纯化测序,测序结果与GenBank上的标准序列的一致性均达到85%以上。结论： 1.多重PCR与基因芯片技术的整合可以实现两种技术的优势互补,通过多重PCR的基因放大作用和基因芯片的探针杂交技术使此种检测体系达到较高的灵敏度和特异度,可实现对多种病原菌、多个样本的同时检测。 2.长春龙嘉国际机场口岸内蜚蠊的优势种属为德国小蠊；不同生境的蜚蠊密度不同,餐饮区蜚蠊密度最高；不同季节蜚蠊密度不同,1月份和7月份为蜚蠊密度较高。 3.本研究对蜚蠊携带病原菌基因组DNA的提取方法进行了优化。确定了SDS法作为蜚蠊携带病原菌基因组DNA的提取方法,该方法具有操作简单、提取效率高、成本低、易于现场检测等特点。 4.本研究运用多重PCR结合基因芯片技术对蜚蠊携带病原菌进行检测。结果显示11种目标病原菌在长春龙嘉国际机场口岸蜚蠊中均被检出。本研究的创新点： 1.建立了一种多重PCR结合基因芯片技术的检测方法,能同时对11种常见病原菌进行快速、准确地检测。 2.首次对长春龙嘉国际机场口岸的蜚蠊进行本底调查,掌握了该地区蜚蠊的种群构成、密度、季节消长规律等情况； 3.首次运用多重PCR结合基因芯片技术对蜚蠊携带的病原菌进行检测,探讨直接从媒介生物样品中同时检测多种病原菌的可行性,为国境口岸媒介生物携带病原菌的检测提供帮助。
[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 .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R184

【引证文献】