摘要
目的用5种方法提取弓形虫速殖子基因组DNA,比较其效果。方法分别以试剂盒法、直接裂解法、煮沸法、酚-氯仿法和盐析法提取10倍系列稀释的弓形虫速殖子DNA,通过分光光度计、琼脂糖凝胶电泳和PCR比较其效果。结果酚-氯仿法提取的DNA纯度最高,煮沸法、盐析法、直接裂解法和试剂盒法依次降低;琼脂糖凝胶电泳可检测出由直接裂解法、酚-氯仿法、盐析法提取1×106个速殖子的基因组DNA;试剂盒法、直接裂解法、煮沸法、酚-氯仿法、盐析法提取的DNA经PCR扩增可检测最少速殖子的数量依次为1×104,无目的条带,1×102,1×101,1×103个。结论酚-氯仿提取的DNA纯度高,用于PCR扩增具有较高的敏感性,但操作相对繁琐;煮沸法操作简单,与除酚-氯仿法外其他3种方法相比具有较高的敏感性。其它3种方法提取的DNA纯度不高,用于PCR扩增的效果不如前面2种。
The objective of this work was to compare five DNA extraction methods including DNA extraction kit, schizolysis, boiling method, phenol-chloroform extraction and salt fractionation for Toxoplasma gondii tachyzoite and to evaluate their effects on PCR results. Tachyzoite DNA in 10-fold serial dilutions was extracted by these five methods. Then the DNA quantity and purity were detected by Eppendorf Biophotometer and agarose gel electrophoresis. It indicated that DNA purity extracted by phenol-chloroform extraction was the highest, following by boiling method, salt fractionation, schizolysis method and DNA extraction kit successively. Genome DNA from 1×106 tachyzoites extracted by schizolysis, phenol-chloroform extraction and salt fractionation could be detected by agarose gel electrophoresis. PCR detection results for amount of DNA extracted by extraction kit, schizolysis, boiling method, phenol-chloroform extraction and salt fractionation were 1×104, 0, 1×102, 1×101 and 1×103, respectively. It is concluded, Phenol-chloroform extraction is regarded as the best method to extract tachyzoite DNA, following by boiling method. DNA extracted by the other three methods is low in purity and the effect of PCR amplification is not so good as phenol-chloroform extraction and boiling method.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第4期333-336,共4页
Chinese Journal of Zoonoses
关键词
弓形虫速殖子
DNA提取
PCR
电泳
Toxoplasma gondii
tachyzoite
DNA extraction
PCR
agarose gel electrophoresis
