犬弓首蛔虫卵卵壳裂解方法研究

Study on the Cracking Method of Toxocara canis Eggshell

[目的]为犬弓首蛔虫卵壳的鉴定奠定基础。[方法]采用3种联合作用方法对犬弓首蛔虫卵壳进行裂解,提取虫卵基因组DNA,然后用犬弓首蛔虫的ITS2特异性引物进行PCR扩增。[结果]DNA提取试剂对虫卵卵壳的作用不明显,PCR扩增结果未见预期片段;冻融后抽提DNA对虫卵卵壳有一定作用,但大多数虫卵还保留基本形态,PCR扩增结果也未见预期片段;冻融15次+蛋白酶K消化2次过夜,然后抽提DNA,虫卵大多数被裂解,利用PCR扩增到目的片段。[结论]破坏犬弓首蛔虫卵的卵壳采用冻融+蛋白酶K消化的联合方式效果明显,关键因素是作用时间。

[Objective] The research aimed to lay the foundation for the identification of Toxocara canis eggshell. [Method] The eggshell of T. canis was cracked by using three combined methods to extract genomic DNA from T. canis eggs. And ITS2 specific primers of T. canis were used for PCR amplification. [Result]The cracking effects of T. canis eggshell by using DNA extracting agent was not obvious,and target fragment wasn＇t seen after PCR. The method of extracting DNA after freeze-thawing had some cracking effects on T. canis eggshell,but most of eggshells still remained the original forms and target fragment wasn＇t seen after PCR. DNA was extracted from T. canis eggshell after they were frozen-thawed for 15 times and digested with protease K,then overnight. By using this method,most eggshells were cracked and target fragment was seen after PCR. [Conclusion] The method of freeze-thawing and digesting with protease K had obvious cracking effects on T. canis eggshell,and its key factor was action time.