本文關(guān)鍵詞：天津地區(qū)莫氏巴貝斯蟲PCR檢測法建立及綿羊線蟲病流行病學(xué)調(diào)查　出處：《天津農(nóng)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 綿羊 線蟲 莫氏巴貝斯蟲 PCR 序列 血液

【摘要】：羊寄生蟲病嚴(yán)重制約著養(yǎng)羊業(yè)的發(fā)展,被寄生蟲感染的羊只,生長發(fā)育遲緩。飼料回報率低,減少了產(chǎn)奶以及產(chǎn)肉量。嚴(yán)重時還會導(dǎo)致懷孕母羊早產(chǎn)、流產(chǎn),部分病羊會發(fā)生死亡,直接使經(jīng)濟(jì)收益下降。本試驗采集天津地區(qū)5個羊場的糞樣577份,采用多種方法檢測糞便中的寄生蟲卵,進(jìn)行天津地區(qū)綿羊感染線蟲狀況的調(diào)查。在此期間,發(fā)現(xiàn)部分羊的血液內(nèi)存在疑似巴貝斯蟲樣的原蟲,因此建立了其PCR檢測方法,且應(yīng)用該新建方法對50份綿羊全血樣品進(jìn)行檢測,與傳統(tǒng)的Giemsa染色法進(jìn)行比對,經(jīng)過序列比對,其同源性為98%。1.天津地區(qū)綿羊腸道線蟲的流行病學(xué)調(diào)查本試驗從2016年3月到2017年1月,對天津市5個羊場的577份綿羊糞便進(jìn)行寄生蟲檢測,其中有3個大型羊場,2個小型羊場。檢測方法包括,飽和氯化鈉漂浮蟲卵法、水洗沉淀法、碘染色法以及飽和蔗糖漂浮蟲卵法。結(jié)果:檢測到圓線蟲蟲卵,天津本地羊的感染率為6.97%。其中羔羊的感染率為12.86%,生長羊為6.74%,育肥羊為0.81%。羔羊的感染率明顯高于生長羊,而生長羊的感染率又大于育肥羊。從內(nèi)蒙古引進(jìn)的綿羊的感染率(78.26%)遠(yuǎn)遠(yuǎn)大于天津本地羊的感染率(6.97%);小型羊場的感染率(19.20%)大于大型羊場的感染率(3.20%)。結(jié)論,天津地區(qū)的綿羊線蟲感染情況雖不嚴(yán)重,但也不容忽視,要積極預(yù)防,重視引進(jìn)羊的寄生蟲感染情況的監(jiān)測。2.綿羊莫氏巴貝斯蟲PCR方法的建立及優(yōu)化本試驗通過染色鏡檢結(jié)果,選取核酸檢測樣本建立綿羊梨形蟲的分子檢測法,并設(shè)置退火溫度和循環(huán)次數(shù)的梯度來進(jìn)行優(yōu)化,選擴(kuò)增產(chǎn)物特異性高、產(chǎn)率穩(wěn)定的溫度和循環(huán)次數(shù)作為PCR檢測法的優(yōu)化結(jié)果。結(jié)果表明:PCR反應(yīng)的最佳體系為94℃預(yù)變性5 min;94°C變性1 min,56°C退火90 s,72°C延伸1 min,共循環(huán)39次;72°C延伸10 min,4°C保存。在本試驗中建立的PCR法具有良好的特異性。將特異性引物應(yīng)用于新建PCR方法。初步斷定本次檢測出的梨形蟲為莫氏巴貝斯蟲。3.新建綿羊巴貝斯蟲PCR方法的應(yīng)用應(yīng)用新建莫氏巴貝斯蟲PCR檢測方法對50份綿羊全血進(jìn)行檢測,與傳統(tǒng)的Giemsa染色法進(jìn)行比對并且選取兩條序列進(jìn)行測序。結(jié)果表明:在50份羊血樣品中,PCR法檢出的陽性樣品有13份,陽性率為26%(13/50),Giemsa染色法檢出的陽性樣品有8份,陽性率為16%(8/50)。PCR方法檢測出的陽性樣品,與Giemsa染色發(fā)檢出的陽性樣品符合率達(dá)100%。利用NCBI上BLAST序列比對,,序列與GenBank中莫氏巴貝斯蟲(登錄號:CP011906.1)參考序列的同源性為98%。本次PCR檢測出的綿羊血液原蟲為莫氏巴貝斯蟲。
[Abstract]:Sheep parasitosis seriously restricts the development of sheep industry. Sheep infected by parasites grow slowly. Feed returns are low, reducing milk production and meat production. In severe cases, it can also lead to premature birth of pregnant ewes and miscarriage. 577 fecal samples from 5 sheep farms in Tianjin were collected and the parasite eggs in feces were detected by various methods. During the investigation of nematodes infection in sheep in Tianjin area, it was found that some sheep had protozoa suspected to be Babes's in blood during this period, so the PCR detection method was established. The new method was used to detect 50 sheep whole blood samples and compared with the traditional Giemsa staining method. The epidemiological investigation of intestinal nematode of sheep in Tianjin was conducted from March 2016 to January 2017. Parasites were detected in 577 sheep feces from 5 sheep farms in Tianjin, including 3 large sheep farms and 2 small sheep farms. Results: the infection rate of nematode eggs in Tianjin local sheep was 6.97. The infection rate of lamb was 12.86 and that of growing sheep was 6.74%. The infection rate of lamb was significantly higher than that of growing sheep. The infection rate of growing sheep is higher than that of fattening sheep. The infection rate of sheep introduced from Inner Mongolia is much higher than that of Tianjin native sheep. The infection rate of small sheep farm (19.20) is higher than that of large sheep farm (3.20%). Conclusion although the infection rate of sheep nematode in Tianjin area is not serious, it should not be ignored and should be actively prevented. Attention should be paid to the monitoring of parasitic infection in introduced sheep. 2. Establishment and optimization of PCR method for Babes Mori in sheep. The nucleic acid detection samples were selected to establish the molecular detection method of piriform worm in sheep and the gradient of annealing temperature and cycle number were set up to optimize the selection of amplification products with high specificity. The stable yield temperature and cycle times were the optimum results of PCR detection. The results showed that the optimal reaction system was 94 鈩，

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