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 91 4bp、5 2 2 bp和 5 2 2 bp;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 1 pg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 5 2 2 bp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 61 9bp、362 bp和 362 bp;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5 2 2 bp的扩增带 ,半套式扩增比一次扩增更敏感。
Two kinds of specific and sensitive methods for detecting Toxoplasma gondii was established by means of NT-PCR and One-Tube Hemi-Nested PCR. The DNAs were extracted from Toxoplasma 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 1pg could be detected by using single-step PCR. The NT-PCR or One-Tube Hemi-Nested PCR was more sensitive than single-step PCR.
出处
《上海实验动物科学》
2003年第1期15-17,20,34,共5页
Shanghai Laboratory Animal Science
基金
上海市科技发展基金资助项目 ( 0 0 49190 71)
