实验动物弓形虫荧光定量PCR检测方法的建立及应用

Establishment and Application of Fluorescent Quantitative PCR Assay for Laboratory Animal Toxoplasma gondii

目的建立实验动物弓形虫TaqMan探针实时荧光定量PCR检测方法,并对其进行初步应用。方法通过比对美国国家生物技术信息中心(NCBI)发表的弓形虫各毒株序列,选取其529 bp重复序列保守区域设计引物探针,建立弓形虫的荧光定量PCR方法。随后对方法的特异性和敏感性进行检验;取浓度为10^(6)~10^(2)copies/μL 10倍系列稀释的质粒标准品,每个浓度做3个平行,重复检测3次,计算组内和组间变异系数,评估方法的重复性和稳定性;用建立的荧光定量PCR方法及国标推荐的PCR方法检测14份犬组织样品和66份猪全血样品,以评估此方法在实际应用中的检测能力。结果建立的弓形虫荧光定量PCR方法在10^(8)~10^(1)copies/μL范围Ct值与质粒浓度呈线性关系,标准曲线Slope为-3.285,R^(2)值为1,扩增效率为10^(1).663%。可检测到的弓形虫最低浓度为10^(1)copies/μL;与其他14种实验猪常见病原不发生交叉反应;重复检测10^(6)~10^(2)copies/μL质粒标准品,组内变异系数小于1.21%,组间变异系数小于0.62%。用建立的弓形虫荧光定量PCR方法和国标PCR方法对14份犬组织样品和66份猪全血样品进行检测,结果显示,弓形虫荧光定量PCR方法和国标PCR方法检测的阳性率分别为10%(8/80)和7.5%(6/80),阳性符合率达到100%。结论建立的弓形虫荧光定量PCR方法可以有效地检测弓形虫,为实验动物弓形虫日常监测提供良好的方法。

Objective To establish and apply TaqMan probe real-time fluorescent quantitative PCR method for laboratory animal Toxoplasma gondii.Method By comparing the sequences of various strains of Toxoplasma gondii published by NCBI,the conservative region of 529 bp repetitive sequence was selected to design primers and probe,and a fluorescence quantitative PCR method was established.Subsequently,the specificity and sensitivity of the method was confirmed.Plasmid standards were obtained from 10^(6)to 10^(2)copies/μL in 10-fold series dilution,and 3 parallels were detected for each concentration and repeated 3 times.The coefficient of variation intra-assay and inter-assay was calculated to evaluate the reproducibility and stability of the method.14 canine tissue samples and 66 porcine whole blood samples were detected by the established quantitative PCR method and the PCR method recommended by national standard to evaluate the capability of this method in practical application.Result The established quantitative PCR method showed a linear relationship between Ct value and plasmid concentration in the range of 10^(8)~10^(1)copies/μL,the standard curve Slope was-3.285,R^(2)value was 1,and the amplification efficiency was 10^(1).663%.The lowest detectable concentration of Toxoplasma gondii was 10^(1)copies/μL.There was no cross reaction with other 14 common swine pathogens.Replicates were detected at 10^(6)to 10^(2)copies/μL plasmid standards,and the coefficient of variation intra-assay and inter-assay was less than 1.21%and 0.62%,respectively.The established quantitative PCR method and the national standard PCR method were used to detect the positive rate of 14 canine tissue samples and 66 porcine whole blood samples.The result showed that the positive rate of the established quantitative PCR method and the national standard PCR method were 10%(8/80)and 7.5%(6/80),respectively,and the positive coincidence rate reached 100%.Conclusion The established Toxoplasma gondii FQ-PCR method can effectively detect Toxoplasma gondii.It provides a good method for daily monitoring of toxoplasma gondii in laboratory animals.