两种锥虫双重实时荧光PCR检测方法的建立

Establishment of a Duplex Fluorescence RT-PCR Assay for Detecting two Try pa no somatid

通过比较美洲锥虫和非洲锥虫基因组的保守性与特异性,设计2套锥虫特异性引物和探针用于核酸检测,美洲锥虫和非洲锥虫分别使用HEX和FAM荧光基团标记探针,建立美洲锥虫和非洲锥虫的双重实时荧光PCR检测方法,并对建立的方法进行灵敏度、特异性和重复性分析。结果显示,新建立的双重实时荧光PCR检测方法只对2种锥虫阳性对照模板有特异性扩增,与健康人血液、健康人尿液、钩端螺旋体、丝虫阳性对照样本均无交叉反应。2套引物和探针的检测灵敏度均可达到最低10^(-4)拷贝/mL。美洲锥虫和非洲锥虫阳性对照模板的2种稀释度重复检测10次均有较好的重复性。结果表明,建立的2种锥虫双重实时荧光PCR检测方法灵敏度高,特异性强,重复性好,可用于对我国口岸输入性锥虫病例的早期检测和分型鉴别。

Conservation and specificity of the genomes of Trypanosoma cruzi and Trypanosoma brucei were compared.Specific primers and probes were designed for nucleic acid detection of these two species.The probes for Trypanosoma cruzi and Trypanosoma brucei were labeled by HEX and FAM fluorophore respectively.To establish a duplex fluorescence RT-PCR assay for detecting Trypanosoma cruzi and Trypanosoma brucei.Meanwhile,the sensitivity,specificity and reproducibility of duplex fluorescence RT-PCR assay were also analyzed.The result was that the specificity of the method was high on detection the nucleic acid of Trypanosoma cruzi and Trypanosoma brucei genomes.There were no cross reactions with healthy human blood,healthy human urine,positive control samples of leptospirosis and filariasis.Detection sensitivities of two sets of primers and probes can reach a minimum of 10^(-4) copies/mL.Duplicate detections for ten times had better repeatability at two dilutions of positive control DNA templates of American trypanosomiasis and African trypanosomiasis.The conclusion was that the duplex fluorescence RT-PCR assay for detecting Trypanosoma cruzi and Trypanosoma brucei genomes was highly sensitive,specific and repeatable.It can be used for early detection and typing identification of imported trypanosomiasis cases.