›› 2003, Vol. 23 ›› Issue (1): 15-17.

• 论文 • 上一篇    下一篇

弓形虫PCR检测方法的建立及初步应用

  

  1. 上海市实验动物质量监督检验站 200032
  • 收稿日期:2002-06-06 出版日期:2003-01-25 发布日期:2013-03-19
  • 基金资助:

    上海市科技发展基金资助项目(004919071)

Establishment and Preliminary Application of Polymerase Chain Reaction for Toxoplasma gondii

  1. Shanghai Quality Monitoring Center Far Laboratory Animals,Shanghai 200032,China
  • Received:2002-06-06 Online:2003-01-25 Published:2013-03-19

摘要: 根据弓形虫P30基因序列设计二对引物分别进行了PCR一次扩增和套式扩增,结果这两对引物在一次扩增和套式扩增中分别出现预期的扩增片段914bp、522bp和522bp;套式扩增出现的条带比一次扩增出现的条带亮度明显增强。提示 套式扩增比一次扩增具有更高的敏感性。用外引物进行扩增后,最低可以检测到1pg的弓形虫DNA。说明 所建立的反应体系具有高度的敏感性。用内引物对人工感染弓形虫的ICR小鼠血液、组织进行弓形虫DNA的检测,结果均扩增出522bp的扩增带。根据B1基因序列设计的引物进行PCR一次扩增和半套式扩增。结果 均出现预期的扩增片段619bp、362bp和362bp;用半套式扩增检测感染弓形虫的ICR小鼠血液、组织时可扩增出522bp的扩增带,半套式扩增比一次扩增更敏感。

关键词: 弓形虫, 一次PCR, 套式PCR, 半套式PCR, ICR小鼠

Abstract: Two kinds of specific and sensitive methods for detecting Toxoplasma gondii was estab-lished by means of NT-PCR and One-Tube Hemi-Nested PCR. The DNAs were extracted from Toxooplasma gondii、Giardia lamblia and also blood、brain、liver、spleen、lung、kidney tissue of ICR mice infected with Toxoplasma gondii by routine methods. Two DNA fragments of the expected size (522 bp and 362 bp) was ampliflied from Toxoplasma gondii、blood、brain、liver、spleen、lung and kidney tissue by using NT-PCR and Hemi-Nested PCR rspectivelly, but no band was observed in Giardia lamblia. The sensitivity of the reaction was determined with different concentrations of genomic Toxoplasma gondii DNA,the DNA even less then lpg could be detected by us-ing single-step PCR.The NT-PCR or One-Tube Hemi-Nested PCR was more sensitive than sin-gle-step PCR.

Key words: Toxoplasma gondii, Single-step PCR, NT-PCR, One-Tube Hemi-Nested PCR