摘要
为建立一种快速、敏感、特异的猪弓形虫检测方法,根据弓形虫保守基因序列,设计一套特异性引物和FAM荧光素标记的MGB探针;通过对PCR反应体系和反应条件进行优化筛选,建立了猪弓形虫实时荧光定量PCR检测方法,并对此PCR检测方法进行了特异性、敏感性、重复性试验;利用所建立的方法对60份疑似弓形虫感染的临床样品进行了检测。结果显示:建立的猪弓形虫实时荧光定量PCR检测方法在101~107拷贝/μL模板范围内有很好的线性关系;对弓形虫重组阳性质粒出现阳性扩增信号,但对阴性对照的水和其他7种病原对照未扩增出特异性曲线;最低检测模板浓度为10拷贝/μL;自60份疑似猪弓形虫感染样品中检出32份阳性,并且和克隆测序结果一致。结果表明,本研究建立的猪弓形虫实时荧光定量PCR检测方法可用于猪弓形虫的快速检测,从而为猪弓形虫病的诊断提供了特异、敏感、高通量的方法。
In order to establish a rapid,sensitive and specific method for detection of Toxoplasmas gondii(T. gondi),a pair of specific primers and FAM(fluorescein)-labeled MGB probes were designed based on the conserved gene sequences of T. gondii. Followed by optimizing PCR reaction system and conditions,a real-time fluorescent PCR assay was established,and the specificity,sensitivity and reproducibility test were carried out;then 60 clinical samples suspected of T. gondii infection were tested by the established method. The results showed that the method expressed a good linear relationship when the template was within the range of 101–107 copies/μL. Specificity test showed that positive signal was observed only when amplifying the recombinant plasmids of T. gondii,rather than the water of negative contrast or other 7 pathogens;the minimum concentration of detection template was 10 copies/μL;32 out of 60 suspected samples were detected positive,and the result was consistent with that of cloning and sequencing. As a conclusion,the established method in this study could be used for rapid detection of T. gondii,which provided a specific,sensitive and high-throughput method for the identification of toxoplasmosis.
作者
袁淑萍
谢彩华
王东方
班付国
闫若潜
Yuan Shuping;Xie Caihua;Wang Dongfang;BanFuguo;Yan Ruoqian(Henan Provincial Center for Animal Disease Control and Prevention,Zhengzhou,Henan 450008,China)
出处
《中国动物检疫》
CAS
2020年第5期106-110,共5页
China Animal Health Inspection
基金
河南省科技创新人才计划项目(174200510003)。
作者简介
通信作者:闫若潜。
