5种分离纯化粪便中隐孢子虫卵囊方法的比较

Comparison of five methods in separating Cryptosporidium oocysts from feces

[目的]比较5种分离纯化粪便中隐孢子虫卵囊的方法。[方法]分别用饱和氯化钠漂浮法、Sheather's蔗糖漂浮法、甲醛-乙醚沉淀法、不连续蔗糖密度梯度离心法和Percoll密度梯度离心法对感染小鼠粪样中的隐孢子虫卵囊进行分离纯化,浓集回收的卵囊利用鼠抗隐孢子虫卵囊壁单克隆抗体间接免疫荧光法检测卵囊数,作定量比较,同时定性比较回收卵囊液的纯度。[结果]Percoll密度梯度回收的卵囊数量最多,而且杂质少;不连续蔗糖密度梯度离心法和Sheather's蔗糖漂浮法次之,但前者回收的卵囊液中杂质较少;饱和氯化钠漂浮法获得的卵囊液中杂质也较少,但卵囊数次之;甲醛-乙醚沉淀法获得的卵囊液中检出的数量最少,且杂质最多。[结论]不连续蔗糖密度梯度离心法是一种经济、有效的卵囊分离纯化方法。

[ Objective] To compare the results of separation of Cryptosporidium oocysts from feces using five solutions. [ Methods] Cryptosporidium oocysts were separated from feces of infected mice using isopycnic percoll gradients, discontinuous sucrose gradients, formaldehyde - aether sedimentation, Sheather＇s flotation, and saturated sodium chloride. Cryptosporidium cocysts were detected with monoclonal antibody against Cryptosporidium parvum oocyst. [ Results ] Recovered oocysts using isopycnic Percoll gradients, and discontinuous sucrose gradients, were essentially free of debris and bacteria. Isopycnic Pereoll gradients recovered the most ocysts. Oocysts recovered by discontinuous sucrose gradients and Sheather＇s flotation methods were the second, and formaldehyde - aether sedimentation recovered the least oocysts. [ Conclusion ] Discontinuous sucrose gradients is an economic and effective method to separate Cryptosporidium oocysts from feces.