弓形虫生物检测靶标miR-191的鉴定

Identification of the Target miR-191 for the Biological Detection of Toxoplasma Gondii

目的建立弓形虫miRNA探针定量检测法。方法采用高通量测序技术筛选出不同弓形虫虫株间表达量高且差异不明显潜在靶标,并对其不同时间点(0 d、1 d、3 d、5 d、7 d)外周血的表达及诊断效能进行研究。结果高通量测序表明, miR-252、miR-3641、miR-509-5p、miR-1285、miR-140、miR128-1、miR-191、miR-3426具有作为血液中弓形虫生物检测靶标的潜在性。通过对感染弓形虫RH株和M49株小鼠的miR-191诊断效能分析后表明,敏感性分别为85%和95%,特异性均为100%。根据miR-191制备纳米金探针,对不同时间点感染弓形虫RH株和ME49株大鼠的血液靶标miR-191进行定量分析表明,感染后7 d内感染弓形虫miR-191表达情况相对稳定。制作弓形虫RH株、弓形虫ME49株、伯氏疟原虫、约氏疟原虫、隐孢子虫,鼠肝炎病毒和金黄色葡萄球菌感染的小鼠模型,与对照组比较分析表明,miR-191在感染弓形虫RH株和ME49株的小鼠模型中出现高表达,而在感染细菌、病毒和其它寄生虫的小鼠模型中均未出现表达。结论弓形虫miR-191具有作为检测靶标的应用价值。

Objective To establish the quantitative detection method of microRNAs（miRNAs） probe of Toxoplasma gondii. Methods High-throughput sequencing technology was used to screen out the high-level and non-significant potential targets between different strains of Toxoplasma gondii, and the expression and diagnostic efficiency of peripheral blood at different time points（0 d, 1 d, 3 d, 5 d, 7 d）were studied. Results High-throughput sequencing revealed that miR-252, miR-3641, miR-509-5 p,mi R-1285, miR-140, miR128-1, miR-191 and miR-3426 had potential as biological detection targets for Toxoplasma gondii. The miR-191 diagnostic efficiency of mice infected with Toxoplasma gondii RH strain and M49 strain showed that the specificity were 100%, and the sensitivity were 85% and 95%,respectively. Nanoscale gold probes were prepared by miR-191. The quantitative analysis of target miR-191 in blood of rats infected with Toxoplasma gondii RH strain and ME49 strain at different time points showed that miR-191 expression was relatively stable within 7 days after infection.The infection models of mice with Toxoplasma RH strain, Toxoplasma ME49 strain, Plasmodium bergi, Plasmodium yoelii,Cryptosporidium, mouse hepatitis virus and Staphylococcus aureus were made respectively. Compared with those of in control group, the miR-191 was highly expressed in mice infected with RH and ME49 strains of Toxoplasma gondii and no expression was found in mouse models infected with bacteria, virus and other parasites. Conclusions The miR-191 of Toxoplasma gondii has the application value as a detection target.