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弓形虫荧光定量PCR检测方法的建立与初步应用

Establishment and application of real-time PCR for the detection of Toxoplasma gondii

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【作者】 候照峰王尚尚贾红臧传佼朱鸿飞周永华陶建平

【Author】 HOU Zhao-feng;WANG Shang-shang;JIA Hong;ZANG Chuan-jiao;ZHU Hong-fei;ZHOU Yong-hua;TAO Jian-ping;Coll of Vet Med Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou Univ;Beijing Veterinary Research Institute,Chinese Academy of Agricultural Sciences;Jiangsu Institute of Parasitic Diseases;

【机构】 扬州大学兽医学院江苏省动物重要疫病与人兽共患病防控协同创新中心中国农业科学院北京畜牧兽医研究所江苏省寄生虫病防治研究所

【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%)。

【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.

【基金】 “十一五”国家科技支撑计划重点项目(2010BAD04B02);江苏省高校优势学科建设工程资助项目
  • 【文献出处】 中国兽医杂志 ,Chinese Journal of Veterinary Medicine , 编辑部邮箱 ,2015年01期
  • 【分类号】S852.7
  • 【被引频次】16
  • 【下载频次】478
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