| Cockroaches is one of the important medical vectors monitored at the frontier port area. Cockroaches in the biological classification is arthropoda, insecta, blattaria. Cockroaches live in the human living environment. It is not only harassment, bites, damaged items, but also media or storage host of pathogens. Cockroaches carry and disseminate bacteria, fungi, viruses and parasites. It can cause intestinal, respiratory, skin and other multi-system diseases. Cockroaches cause a serious hazard to human health.With the global economic integration and the development of international trade, the number of passengers and international traffic of transport increase obviously. Pathogens carried by vector spread between ports in different countries with transport, containers, cargo, mail and other. It provide convenient conditions for the international spread of cockroaches and pathogens. Insect-borne diseases have a clear upward trend.Pathogen detection technology currently has some limitations. Although, in recent years, variety detection methods have been developed based on microbiology, chemistry, immunology and molecular biology experimental techniques for the detection and identification of pathogenic microorganisms, it can not meet the need for simultaneous and rapid detection of multiple pathogens. Based on mastering the composition of the background of cockroaches at Changchun Longjia international airport, pathogens carried by cockroaches are detected by multiplex PCR combined with DNA microarray in this study. It is important to prevent and control disease caused by cockroaches in the port area.Objectives:1. Through establishing the method of multiplex PCR combined with DNA microarray to detect 11 kinds of pathogens, to provide effective detection for clinical diagnosis of disease, food poisoning testing, import and export food inspection, pathogens carried by vector.2. By mastering the composition of the background of cockroaches at Changchun Longjia international airport, to strengthen monitor and control of cockroaches at frontier port and immigration transport, and provide scientific guidance.3. Through using the method of multiplex PCR combined with DNA microarray to detect pathogens carried by cockroaches, to make scientific analysis and forecasts the risk of transmission of infectious diseases caused by cockroaches, and protect health and safety of frontier port, transportation and cargo.Methods:1. Oligonucleotide microarray was developed. Eleven pathogens including Shigella, Listeria monocytogenes, Escherichia coli O157, Staphylococcus aureus, Bacillus cereus, Campylobacter jejuni, Proteusbacillus vulgaris, C.perfringens, Vibiro parahaemolyticus, Salmonella typhi, Salmonella enteritidis was detected by oligonucleotide microarray. Primers and oligonucleotide probes were designed and synthesized. The multiplex PCR products were hybridizes with DNA microarray, which contained specific probes of eleven pathogens. Scanner was used to determinant the types of bacterium. The specificity and sensitivity of DNA microarray was detected.2. In order to investigate the kind, composition, density and the seasonal fluctuation of cockroaches, a baseline survey, from April.2008 to Mar.2009, was carried out at Changchun Longjia international airport. Cockroaches were captured by "box trapping method". Four different habitats were office, food production plant, dining area, cargo space for the survey area.3. Three types methods of extracting DNA of pathogens carried by cockroaches including boiling method, kit method. Pathogens carried by cockroaches are detected by DNA microarray combined with multiplex PCR.Results:1. A series of simulation sample from a mixture of different DNA samples were detected by multiplex PCR combined with DNA microarray. The result showed that simulation DNA sample could be hybridized specifically with probes. DNA microarray is 10 times the sensitivity of PCR.2. From April.2008 to Mar.2009,3600 catching boxes were placed at Changchun Longjia international airport and 2919 boxes was recoveried. Blattella germanica was dominant species. Density of cockroaches was different in different habitats. Dining area is highest density of cockroaches which was 4.08 per box. The annual average density of cockroaches was 1.36 per box. Density of cockroaches throughout the year reached its peak in January and July. 3. Purity and concentration of DNA which was extracted by three types methods including boiling method, kit method, SDS method were significant differences. Ultimately, SDS method as the method of extracting DNA was used in this study.4.11 kinds of target pathogens in this study were detected from cockroaches at Changchun Longjia international airport. Detection rate of pathogens were Staphylococcus aureus 30%, Shigella 100%, Escherichia coli 0157 50%, Salmonella enteritidis 10%, Salmonella typhi 45%, Bacillus cereus 15%, Campylobacter jejuni 100%, C.perfringens 80%, Listeria monocytogenes 55%, Vibiro parahaemolyticus 5%, C.perfringens 5%. The DNA of pathogens carried by cockroaches were sequenced. The sequencing results were compared with standard sequences of GenBank and the consistency are more than 85%.Conclusions:1. Multiplex PCR and gene chip technology enables the integration of two technologies complement each other. This detection system has high sensitivity and specificity through multiplex PCR amplification of genes and DNA microarray hybridization technology. Detection of a variety of pathogens and multiple samples at the same time can be achieved.2. Blattella germanica is dominant species. Density of cockroaches was different in different habitats. Dining area is highest density of cockroaches. Density of cockroaches was different in different seasons. Density of cockroaches throughout the year reached its peak in January and July.3. The method of extracting genome DNA of pathogens carried by cockroaches was optimized. SDS method as the method of extracting DNA was used. The method is simple, extraction efficiency, low cost, easy-to-site testing.4. In this study, the pathogens carried by cockroaches were detected by multiplex PCR combined with DNA microarray.11 kinds of target pathogens were detected from cockroaches at Changchun Longjia international airport.Innovation:1. The detection method of multiplex PCR combined with DNA microarray was established.11 kinds of pathogens can be detected simultaneously, rapidly and accurately.2. For the first time, a baseline survey of cockroaches was carried out at Changchun Longjia international airport. We mastered the kind, composition, density and the seasonal fluctuation of cockroaches. 3. For the first time, the method of multiplex PCR combined with DNA microarray to detect pathogens carried by cockroaches was used. The feasibility of detecting multiple pathogens directly from the vector was discussed. It will provide help for detection of pathogens carried by vector at frontier port.
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