犬棘球绦虫粪LAMP检测方法的建立和应用
发布时间：2019-06-10 01:20
【摘要】：棘球蚴病是由寄生于中间宿主的肝脏、肺脏及其他部位的棘球绦虫(Echinococcus spp.)幼虫——棘球蚴引起的一种人兽共患寄生虫病，广泛分布于世界各地，它不仅威胁人类的健康，而且给畜牧业生产带来严重经济损失，被认为是新兴和再现疾病。其传染源为感染棘球绦虫的终末宿主——犬科动物，因此对传染源进行准确检测和流行病学调查，为及时采取驱虫、扑杀等防治措施控制传染源，评价现有防控效果和制定相关防治措施等提供重要依据。 目前，棘球绦虫终末宿主的检测诊断及流行病学调查主要以剖检法为主，免疫诊断和分子生物学检测技术在不断发展和日益完善中。本研究以棘球绦虫(细粒棘球绦虫G1基因型和多房棘球绦虫)线粒体nad5基因为靶标分子，分别设计4条特异性引物，建立棘球绦虫LAMP方法，检测棘球绦虫终末宿主感染。本实验建立的用于检测终末宿主细粒棘球绦虫G1基因型和多房棘球绦虫感染的LAMP方法特异性强且重复性好，与犬科动物其他常见绦虫(如石渠棘球绦虫、泡状带绦虫、带状带绦虫、多头带绦虫、豆状带绦虫、犬复孔绦虫)之间均无交叉反应。 将多房棘球蚴基因组DNA定量至10ng/μl，并进行10倍倍比稀释，以此为模板进行敏感性检测。与参考的PCR方法进行比较，该LAMP方法与PCR具有相同的敏感性，均能检测到10-3ng水平。以家犬为实验动物，人工感染细粒棘球蚴和多房棘球蚴后，收集感染后不同天数的粪样品(f-DNA)进行LAMP和PCR检测，结果表明LAMP方法在感染后第12天即可扩增出目的条带，而PCR方法在感染后第17天方可扩增出目的条带，表明该LAMP方法可用于多房棘球绦虫终末宿主的早期诊断；此外，将每克粪中虫卵定量进行LAMP检测，结果表明LAMP能够检测到每克粪便中含有4个虫卵。 将细粒棘球绦虫(G1基因型)基因组DNA定量到10ng/μl，，并进行10倍倍比稀释，与参考的PCR方法进行比较，结果表明该LAMP方法敏感性比PCR高100倍，在对f-DNA进行相同处理检测时也得到类似的试验结果，即LAMP方法敏感性比PCR高100倍。对人工感染犬f-DNA进行LAMP、PCR及粪抗原ELISA检测，结果表明LAMP方法在感染后第22天即可扩增出目的条带，而PCR方法在感染后第26天方可扩增出目的条带，粪抗原ELISA在感染后第25天其OD值明显升高，表明该LAMP方法可用于细粒棘球绦虫(G1基因型)终末宿主的早期诊断；此外，所建立的LAMP方法可检测到每克粪便中含有5个虫卵。 应用已建立的LAMP方法对我国甘肃省天祝县(n=30)和青海省治多县(n=48)、久治县(n=9)和达日县(n=132)犬粪样品进行多房棘球绦虫和细粒棘球绦虫(G1基因型)感染的检测，结果显示天祝县30份样品全为阴性，青海部分地区多房棘球绦虫和细粒棘球绦虫(G1基因型)终末宿主的感染率分别为16.40%和13.76%。 本实验建立了特异、敏感的多房棘球绦虫和细粒棘球绦虫G1基因型终末宿主粪LAMP检测方法，可用于感染犬早期诊断。应用LAMP检测方法对我国甘肃省和青海省多房棘球绦虫和细粒棘球绦虫终末宿主进行了流行病学调查，取得了预期结果，为制定相关防治措施提供了重要参考依据，为研制棘球绦虫终末宿主快速诊断试剂盒奠定了良好基础。
[Abstract]:The echinococcosis is an echinococcus spp that is caused by the liver, lung and other parts of the intermediate host. ) The parasitic disease of a human and animal caused by the larvae _ echinosphere is widely distributed around the world, which not only threatens the health of human beings but also brings serious economic losses to the production of animal husbandry, and is considered to be a new and reproducible disease. The source of infection is the terminal host _ canine, which is infected with echinococcus, so that the source of infection can be accurately detected and epidemiological, so as to provide an important basis for the control of the infection source, the evaluation of the existing prevention and control effect and the development of related prevention and control measures. At present, the detection and diagnosis of the terminal host of the echinocandin and the epidemiological investigation are mainly based on the cross-cutting method, and the immunodiagnosis and the molecular biology detection technology are continuously developed and perfected. In that present study,4 specific primers were designed to detect the terminal host sense of echinococcus and the terminal host of echinocula, by using the nad5 gene as the target molecule. The LAMP method for detecting the G1 genotype of the terminal-host fine-grained echinococcus and the infection of the multi-chamber echinococcus is strong and the repeatability is good, and the LAMP method which is used for detecting the infection of the terminal host fine-grained echinococcus and the multi-chamber echinococcus is strong and the repeatability is good, and the LAMP method is similar to that of the other common insect pests of the canine (such as the echinococcus, the vesicular-like insect, the bubble-like insect, the belt-like worm and the multi-head belt). There was no cross-inversion between the worm, the bean-like, the worm, and the canyworm. The template should be sensitive to the quantification of the polyacanthracantha genomic DNA to 10 ng/. mu.l and 10-fold-to-fold dilution. Compared with the reference PCR method, the LAMP method has the same sensitivity as that of the PCR, and can be detected to be 10-3n. G. The results showed that the LAMP method can be used for the detection of different days of post-infection by LAMP and PCR. The results show that the LAMP method can be amplified on the 12th day after infection. The PCR method can be used for the early diagnosis of the terminal host of the multi-chamber echinocandin, and the LAMP detection is carried out on each gram of the eggs, the result shows that the LAMP can detect that the LAMP is contained in each gram of the faecal matter The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results of the test, that is, the LAMP method sensitivity ratio PCR The results showed that the LAMP method can amplify the target band on the 22nd day after the infection, and the PCR method can amplify the target band on the 26th day after infection, and the faecal antigen ELISA is the OD of the 25-day post-infection. The value is obviously increased, indicating that the LAMP method can be used for the early diagnosis of the terminal host of the fine-grained echinococcus (G1 genotype); in addition, the established LAMP method can detect that the LAMP method comprises the following steps: There were 5 eggs. The established LAMP method was used to carry out the multi-chamber echinococcus and the fine-grained echinococcus (G1 gene) in Tianzhu County (n = 30) and Zhixian (n = 48), Jiuzhi County (n = 9) and Dazhan (n = 132) in Gansu Province. The results showed that the infection rate of 30 samples in Tianzhu county was negative, and the infection rate of the terminal host in the multi-chamber echinococcus and the fine-grained echinococcus (G1 genotype) in the Qinghai-Tibet region was 16.40%, respectively. and 13.76%. The experiment has established a specific and sensitive method for detecting the terminal host manure of a multi-chamber echinococcus and a fine-grained echinococcus, which can In that early diagnosis of the infected dog, an epidemiological survey was conducted on the terminal host of the echinodiscus and the fine-grained echinococcus in Gansu and Qinghai province by the LAMP detection method, and the expected results were obtained, and the relevant prevention and control measures were set up. provides an important reference basis for the development of a fast diagnostic test for the terminal host of the echinococcus
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2496053
[Abstract]:The echinococcosis is an echinococcus spp that is caused by the liver, lung and other parts of the intermediate host. ) The parasitic disease of a human and animal caused by the larvae _ echinosphere is widely distributed around the world, which not only threatens the health of human beings but also brings serious economic losses to the production of animal husbandry, and is considered to be a new and reproducible disease. The source of infection is the terminal host _ canine, which is infected with echinococcus, so that the source of infection can be accurately detected and epidemiological, so as to provide an important basis for the control of the infection source, the evaluation of the existing prevention and control effect and the development of related prevention and control measures. At present, the detection and diagnosis of the terminal host of the echinocandin and the epidemiological investigation are mainly based on the cross-cutting method, and the immunodiagnosis and the molecular biology detection technology are continuously developed and perfected. In that present study,4 specific primers were designed to detect the terminal host sense of echinococcus and the terminal host of echinocula, by using the nad5 gene as the target molecule. The LAMP method for detecting the G1 genotype of the terminal-host fine-grained echinococcus and the infection of the multi-chamber echinococcus is strong and the repeatability is good, and the LAMP method which is used for detecting the infection of the terminal host fine-grained echinococcus and the multi-chamber echinococcus is strong and the repeatability is good, and the LAMP method is similar to that of the other common insect pests of the canine (such as the echinococcus, the vesicular-like insect, the bubble-like insect, the belt-like worm and the multi-head belt). There was no cross-inversion between the worm, the bean-like, the worm, and the canyworm. The template should be sensitive to the quantification of the polyacanthracantha genomic DNA to 10 ng/. mu.l and 10-fold-to-fold dilution. Compared with the reference PCR method, the LAMP method has the same sensitivity as that of the PCR, and can be detected to be 10-3n. G. The results showed that the LAMP method can be used for the detection of different days of post-infection by LAMP and PCR. The results show that the LAMP method can be amplified on the 12th day after infection. The PCR method can be used for the early diagnosis of the terminal host of the multi-chamber echinocandin, and the LAMP detection is carried out on each gram of the eggs, the result shows that the LAMP can detect that the LAMP is contained in each gram of the faecal matter The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results showed that the sensitivity of the LAMP method was 100 times higher than that of the PCR. The results of the test, that is, the LAMP method sensitivity ratio PCR The results showed that the LAMP method can amplify the target band on the 22nd day after the infection, and the PCR method can amplify the target band on the 26th day after infection, and the faecal antigen ELISA is the OD of the 25-day post-infection. The value is obviously increased, indicating that the LAMP method can be used for the early diagnosis of the terminal host of the fine-grained echinococcus (G1 genotype); in addition, the established LAMP method can detect that the LAMP method comprises the following steps: There were 5 eggs. The established LAMP method was used to carry out the multi-chamber echinococcus and the fine-grained echinococcus (G1 gene) in Tianzhu County (n = 30) and Zhixian (n = 48), Jiuzhi County (n = 9) and Dazhan (n = 132) in Gansu Province. The results showed that the infection rate of 30 samples in Tianzhu county was negative, and the infection rate of the terminal host in the multi-chamber echinococcus and the fine-grained echinococcus (G1 genotype) in the Qinghai-Tibet region was 16.40%, respectively. and 13.76%. The experiment has established a specific and sensitive method for detecting the terminal host manure of a multi-chamber echinococcus and a fine-grained echinococcus, which can In that early diagnosis of the infected dog, an epidemiological survey was conducted on the terminal host of the echinodiscus and the fine-grained echinococcus in Gansu and Qinghai province by the LAMP detection method, and the expected results were obtained, and the relevant prevention and control measures were set up. provides an important reference basis for the development of a fast diagnostic test for the terminal host of the echinococcus
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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相关期刊论文 前3条
1 张亚楼;刘开泰;刘继文;杨晓燕;温浩;苗玉清;马旭东;齐新伟;刘涛;;巢式PCR在鉴别多房棘球绦虫及细粒棘球绦虫基因亚型中的应用[J];地方病通报;2008年03期
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3 苏效东;;果洛州人群棘球蚴病患病现况调查[J];科技信息;2011年19期
本文编号：2496053
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