水果及蔬菜表面环孢子虫卵囊检测方法的建立

Development of method for detecting Cyclospora oocysts on the surface of fresh fruits and vegetables

为了建立检测果蔬表面人源卡耶坦环孢子虫(Cyclospora cayetanensis)卵囊的方法,分别用50,500个环孢子虫卵囊对环孢子虫卵囊阴性的新鲜果蔬进行人工污染,并设置阴性对照;使用含有0.1%Alconox,0.1%雕牌,0.1%立白、0.1%白猫的4种洗涤液和去离子水,在摇床中洗脱样品;然后,反复离心3次收集卵囊并提取卵囊DNA;最后,基于环孢子虫18S rRNA基因位点对卵囊DNA样品进行巢式PCR扩增.结果表明,卵囊污染吸附最佳时间为24 h,样品洗脱最佳时间为30 min.其中,只用去离子水时,生菜、西葫芦、黄瓜、西瓜、甜瓜和草莓的检测敏感性可达到50个卵囊,而西红柿可达到500个卵囊;当用不同洗涤剂时,各样品的检测敏感性均可达到50个卵囊.不同洗涤剂之间检测敏感性无显著差异.该研究初步建立检测水果、蔬菜污染环孢子虫卵囊的技术,采用此方法可特异、敏感、快速的检测果蔬表面环孢子虫卵囊,为食源性环孢子虫病暴发溯源提供技术支持.

In order to develop a method for detection of human Cyclospora cayetanensis on the surface of vegetables and fruits, 50 and 500 Cyclospora oocysts were artificially contaminated to the surface of fruits and vegetables, and a negative control was used. The samples were washed using different kinds of de- tergent （0.1% Alconox^（R） , 0. 1% Liby, 0. 1% Diaopai, and 0.1% Baimao） by the gas bath thermostats oscillator, then oocysts were collected after three times centrifuge and DNA was extracted. Finally, the DNA samples were detected by nested PCR amplification of the 18S rRNA gene. The results show that the optimal time for oocysts absorption to substance is 24 hours, and the optimal washing time for sam- ples is 30 minutes. When using 100 mL deionized water, the minimum detectable level of lettuce, zuc- chini, cucumbers, watermelons, melons and strawberries was 50 oocysts, respectively, but for tomatoes it was 500 oocysts. When using the 4 different kinds of detergent, the minimum detectable level of each sample could be 50 oocysts. There are no significant differences between different kinds of detergent.The method for the detection of Cyclospora oocysts adsorped to fruit or vegetables was developed primari- ly. It is quick, sensitive, and specific for the detection of the food-borne pathogen.