【摘要】 根据Gen Bank中龚地弓形虫529 bp重复序列设计引物,以弓形虫RH株基因组DNA为模板,常规PCR扩增出长度212 bp的特异性保守序列,将其克隆到p GEM-T Easy载体中构建重组质粒标准品;以10倍倍比稀释的质粒标准品为模板,进行SYBR GreenⅠ实时荧光定量PCR扩增并制作标准曲线,建立弓形虫荧光定量PCR检测方法。将该方法用于临床病死猪的弓形虫检测。结果表明:用重组质粒制作的标准曲线循环阈值与模板浓度有良好的线性关系,溶解曲线特异相关系数0.997,平均试验间变异系数2.30%,能检出约0.05个速殖子;检测弓形虫YZ-1株与YZ-2株均为阳性,而检测水牛梭形肉孢子虫、犬等孢球虫、柔嫩艾美耳球虫基因组DNA均为阴性;检测24头病死猪,弓形虫阳性率为70.8%,略高于常规PCR方法的检出率(66.7%),但检测的69个组织样品中,荧光定量PCR方法的检出率(50.7%)明显高于常规PCR方法的检出率(34.8%)。更多还原

【Abstract】 The specific primers were designed targeting the 529 bp fragment of Toxoplasma gondii RH strain. A 212 bp specific andconsensus fragment was amplified and cloned into p GEM-T Easy vector. Standard curve was generated by using serial dilutions of re-combinant plasmid,showing that the correlation coefficient was 0.997. The mean interassay coefficient of variation was 2.30%. The detectionof T.gondii YZ-1 strain and YZ-2 strain were positive. The detection of Sarcocystis fusiformis,Isospora canis and Eimeria tenella werenegative. The twenty-four dead pigs from animal hospital were deteced with this method and conventional PCR. The positive rate was 70.8%as exmined by real-time fluorescent quantitative PCR,and 66.7% by conventional PCR. However,as to the detection results of 69 tissuesamples from 24 dead pigs,the positive rate(50.7%)by real-time fluorescent quantitative PCR was apparently higher than that(34.8%)by conventional PCR.更多还原