天津地区莫氏巴贝斯虫PCR检测法建立及绵羊线虫病流行病学调查

发布时间：2018-01-04 04:13

  本文关键词：天津地区莫氏巴贝斯虫PCR检测法建立及绵羊线虫病流行病学调查　出处：《天津农学院》2017年硕士论文　论文类型：学位论文

  更多相关文章： 绵羊 线虫 莫氏巴贝斯虫 PCR 序列 血液

【摘要】：羊寄生虫病严重制约着养羊业的发展,被寄生虫感染的羊只,生长发育迟缓。饲料回报率低,减少了产奶以及产肉量。严重时还会导致怀孕母羊早产、流产,部分病羊会发生死亡,直接使经济收益下降。本试验采集天津地区5个羊场的粪样577份,采用多种方法检测粪便中的寄生虫卵,进行天津地区绵羊感染线虫状况的调查。在此期间,发现部分羊的血液内存在疑似巴贝斯虫样的原虫,因此建立了其PCR检测方法,且应用该新建方法对50份绵羊全血样品进行检测,与传统的Giemsa染色法进行比对,经过序列比对,其同源性为98%。1.天津地区绵羊肠道线虫的流行病学调查本试验从2016年3月到2017年1月,对天津市5个羊场的577份绵羊粪便进行寄生虫检测,其中有3个大型羊场,2个小型羊场。检测方法包括,饱和氯化钠漂浮虫卵法、水洗沉淀法、碘染色法以及饱和蔗糖漂浮虫卵法。结果:检测到圆线虫虫卵,天津本地羊的感染率为6.97%。其中羔羊的感染率为12.86%,生长羊为6.74%,育肥羊为0.81%。羔羊的感染率明显高于生长羊,而生长羊的感染率又大于育肥羊。从内蒙古引进的绵羊的感染率(78.26%)远远大于天津本地羊的感染率(6.97%);小型羊场的感染率(19.20%)大于大型羊场的感染率(3.20%)。结论,天津地区的绵羊线虫感染情况虽不严重,但也不容忽视,要积极预防,重视引进羊的寄生虫感染情况的监测。2.绵羊莫氏巴贝斯虫PCR方法的建立及优化本试验通过染色镜检结果,选取核酸检测样本建立绵羊梨形虫的分子检测法,并设置退火温度和循环次数的梯度来进行优化,选扩增产物特异性高、产率稳定的温度和循环次数作为PCR检测法的优化结果。结果表明:PCR反应的最佳体系为94℃预变性5 min;94°C变性1 min,56°C退火90 s,72°C延伸1 min,共循环39次;72°C延伸10 min,4°C保存。在本试验中建立的PCR法具有良好的特异性。将特异性引物应用于新建PCR方法。初步断定本次检测出的梨形虫为莫氏巴贝斯虫。3.新建绵羊巴贝斯虫PCR方法的应用应用新建莫氏巴贝斯虫PCR检测方法对50份绵羊全血进行检测,与传统的Giemsa染色法进行比对并且选取两条序列进行测序。结果表明:在50份羊血样品中,PCR法检出的阳性样品有13份,阳性率为26%(13/50),Giemsa染色法检出的阳性样品有8份,阳性率为16%(8/50)。PCR方法检测出的阳性样品,与Giemsa染色发检出的阳性样品符合率达100%。利用NCBI上BLAST序列比对,,序列与GenBank中莫氏巴贝斯虫(登录号:CP011906.1)参考序列的同源性为98%。本次PCR检测出的绵羊血液原虫为莫氏巴贝斯虫。
[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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