【摘要】：棘球蚴病是由寄生于中間宿主的肝臟、肺臟及其他部位的棘球絳蟲(chóng)(Echinococcus spp.)幼蟲(chóng)——棘球蚴引起的一種人獸共患寄生蟲(chóng)病，廣泛分布于世界各地，它不僅威脅人類(lèi)的健康，而且給畜牧業(yè)生產(chǎn)帶來(lái)嚴(yán)重經(jīng)濟(jì)損失，被認(rèn)為是新興和再現(xiàn)疾病。其傳染源為感染棘球絳蟲(chóng)的終末宿主——犬科動(dòng)物，因此對(duì)傳染源進(jìn)行準(zhǔn)確檢測(cè)和流行病學(xué)調(diào)查，為及時(shí)采取驅(qū)蟲(chóng)、撲殺等防治措施控制傳染源，評(píng)價(jià)現(xiàn)有防控效果和制定相關(guān)防治措施等提供重要依據(jù)。 目前，棘球絳蟲(chóng)終末宿主的檢測(cè)診斷及流行病學(xué)調(diào)查主要以剖檢法為主，免疫診斷和分子生物學(xué)檢測(cè)技術(shù)在不斷發(fā)展和日益完善中。本研究以棘球絳蟲(chóng)(細(xì)粒棘球絳蟲(chóng)G1基因型和多房棘球絳蟲(chóng))線粒體nad5基因?yàn)榘袠?biāo)分子，分別設(shè)計(jì)4條特異性引物，建立棘球絳蟲(chóng)LAMP方法，檢測(cè)棘球絳蟲(chóng)終末宿主感染。本實(shí)驗(yàn)建立的用于檢測(cè)終末宿主細(xì)粒棘球絳蟲(chóng)G1基因型和多房棘球絳蟲(chóng)感染的LAMP方法特異性強(qiáng)且重復(fù)性好，與犬科動(dòng)物其他常見(jiàn)絳蟲(chóng)(如石渠棘球絳蟲(chóng)、泡狀帶絳蟲(chóng)、帶狀帶絳蟲(chóng)、多頭帶絳蟲(chóng)、豆?fàn)顜Ы{蟲(chóng)、犬復(fù)孔絳蟲(chóng))之間均無(wú)交叉反應(yīng)。 將多房棘球蚴基因組DNA定量至10ng/μl，并進(jìn)行10倍倍比稀釋?zhuān)源藶槟０暹M(jìn)行敏感性檢測(cè)。與參考的PCR方法進(jìn)行比較，該LAMP方法與PCR具有相同的敏感性，均能檢測(cè)到10-3ng水平。以家犬為實(shí)驗(yàn)動(dòng)物，人工感染細(xì)粒棘球蚴和多房棘球蚴后，收集感染后不同天數(shù)的糞樣品(f-DNA)進(jìn)行LAMP和PCR檢測(cè)，結(jié)果表明LAMP方法在感染后第12天即可擴(kuò)增出目的條帶，而PCR方法在感染后第17天方可擴(kuò)增出目的條帶，表明該LAMP方法可用于多房棘球絳蟲(chóng)終末宿主的早期診斷；此外，將每克糞中蟲(chóng)卵定量進(jìn)行LAMP檢測(cè)，結(jié)果表明LAMP能夠檢測(cè)到每克糞便中含有4個(gè)蟲(chóng)卵。 將細(xì)粒棘球絳蟲(chóng)(G1基因型)基因組DNA定量到10ng/μl，，并進(jìn)行10倍倍比稀釋?zhuān)c參考的PCR方法進(jìn)行比較，結(jié)果表明該LAMP方法敏感性比PCR高100倍，在對(duì)f-DNA進(jìn)行相同處理檢測(cè)時(shí)也得到類(lèi)似的試驗(yàn)結(jié)果，即LAMP方法敏感性比PCR高100倍。對(duì)人工感染犬f-DNA進(jìn)行LAMP、PCR及糞抗原ELISA檢測(cè)，結(jié)果表明LAMP方法在感染后第22天即可擴(kuò)增出目的條帶，而PCR方法在感染后第26天方可擴(kuò)增出目的條帶，糞抗原ELISA在感染后第25天其OD值明顯升高，表明該LAMP方法可用于細(xì)粒棘球絳蟲(chóng)(G1基因型)終末宿主的早期診斷；此外，所建立的LAMP方法可檢測(cè)到每克糞便中含有5個(gè)蟲(chóng)卵。 應(yīng)用已建立的LAMP方法對(duì)我國(guó)甘肅省天�？h(n=30)和青海省治多縣(n=48)、久治縣(n=9)和達(dá)日縣(n=132)犬糞樣品進(jìn)行多房棘球絳蟲(chóng)和細(xì)粒棘球絳蟲(chóng)(G1基因型)感染的檢測(cè)，結(jié)果顯示天�？h30份樣品全為陰性，青海部分地區(qū)多房棘球絳蟲(chóng)和細(xì)粒棘球絳蟲(chóng)(G1基因型)終末宿主的感染率分別為16.40%和13.76%。 本實(shí)驗(yàn)建立了特異、敏感的多房棘球絳蟲(chóng)和細(xì)粒棘球絳蟲(chóng)G1基因型終末宿主糞LAMP檢測(cè)方法，可用于感染犬早期診斷。應(yīng)用LAMP檢測(cè)方法對(duì)我國(guó)甘肅省和青海省多房棘球絳蟲(chóng)和細(xì)粒棘球絳蟲(chóng)終末宿主進(jìn)行了流行病學(xué)調(diào)查，取得了預(yù)期結(jié)果，為制定相關(guān)防治措施提供了重要參考依據(jù)，為研制棘球絳蟲(chóng)終末宿主快速診斷試劑盒奠定了良好基礎(chǔ)。
[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
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2496053