科研动态

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。

Development and Application of a Real-time PCR Method for Detection of Toxoplasma gondii 

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.

全文下载链接：https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202005020&v=MTYwNzhyTVB5clBlYkc0SE5ITXFvOUhaSVI4ZVgxTHV4WVM3RGgxVDNxVHJXTTFGckNVUjdxZlllWnNGeXpoVXI=