摘要
为了建立一种能够快速实现对鸡柔嫩艾美耳球虫(Eimeria tenella)和巨型艾美耳球虫(E.maxima)同时进行检测的双重PCR方法,基于2种艾美耳球虫各自的ITS-1基因筛选2对特异性引物,对制备的阳性DNA样品进行PCR扩增,凝胶电泳检测结果显示,分别在约450bp和150bp处出现目的条带。对PCR反应中的退火温度、MgCl2浓度、DNA模板量等进行优化。结果表明,当退火温度为59℃、MgCl2浓度为2.5mmol/L、DNA模板量为2μL时,双重PCR扩增结果最佳。特异性检测结果显示,所建立的双重PCR对环形泰勒虫、羊巴贝斯虫、隐孢子虫和蓝氏贾第虫的扩增结果均呈阴性,表明建立的方法具有较高的特异性。成功建立了柔嫩艾美耳球虫和巨型艾美耳球虫双重PCR检测方法,为临床中鸡球虫病的快速诊断提供了一种可靠的检测方法。
In order to establish a fast duplex PCR method to simultaneously detect Eimeria tenella and E.maxima of chickens,two pairs of specific primers of the ITS-1 gene from two Eimeria species were respectively screened.Two target bands (450 bp and 150 bp) were observed in agarose gel electrophoresis after PCR amplification of positive DNA samples.The duplex PCR system was then optimized with annealing temperature,MgCl 2 concentration and the amount of DNA template.The optimal amplification was achieved with conditions of annealing temperature at 59 ℃,MgCl 2 concentration at 2.5 mmol/L,and the amount of DNA template at 2 μL.Specificity test showed no amplification of duplex PCR to Theileria annulata,Babesia ovis,Cryptosporidium and Giardia lamblia ,indicating high specificity of duplex PCR system.These results indicated that a specific and efficient duplex PCR method was successfully established for detecting E.tenella and E.maxima ,providing a reliable method for quickly diagnose clinical chicken coccidiosis.
作者
范现成
马峋
张会军
唐欢
宋军科
赵光辉
FAN Xian-cheng;MA Xun;ZHANG Hui-jun;TANG Huan;SONG Jun-ke;ZHAO Guang-hui(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2019年第2期17-21,共5页
Progress In Veterinary Medicine
基金
国家重点研发计划项目(2017YFD0501200)
