生菜污染隐孢子虫卵囊洗脱与巢式PCR检测方法的研究

Study on the method of oocyst elution and nested PCR detection of Cryptosporidium contaminated with lettuce

目的比较生菜表面污染隐孢子虫卵囊的洗脱与检测方法。方法对生菜表面人工"种植"的隐孢子虫卵囊的洗脱次数、振荡方式、振荡频率、振荡时间、洗脱液种类等进行比较,以及3种商业化DNA提取试剂盒提取水溶液中隐孢子虫卵囊DNA的效果进行比较。结果采取往返振荡,振荡频率为100次/min,振荡时间为1 h,洗脱液采用1%Alconox■R时,其总体回收率最高,达71.40%。不同DNA试剂盒比较显示,土壤DNA提取试剂盒与粪便DNA提取试剂盒较组织DNA提取试剂盒提取水溶液中隐孢子虫卵囊DNA的质与量均高。结论本研究建立了巢式PCR检测生菜表面微小隐孢子虫卵囊的检测方法,为蔬菜的食品安全提供新的技术方法。

Cryptosporidium is an important zoonotic intestinal protozoa,which has leading caused many food-borne and water-borne cryptosporidiosis outbreaks worldwide.In order toompare the elution and detection methods for Cryptosporidium oocysts contaminated on lettuce,the elution times,oscillation patterns,oscillation frequency,oscillation time,and elution solution of Cryptosporidium oocysts by artificial‘seeding’on vegetable surface were compared,and three commercialized DNA extraction kits were compare used to extract oocyst DNAs.The results showed that the overall recovery rate was 71.40% when the oscillation frequency was 100 times/min,the oscillation time was 1 h,and the elution solution was 1% Alconox■,which had the highest overall oocysts recovery rate.The higher DNAs in quality and quantity of the Cryptosporidium oocyst were obtained between soil DNA extraction kit and stool DNA extraction kit compared with tissue DNA extraction kit.And,the soil DNA kit is better if considering the time cost,and stool DNA kit is better if considering the economic cost.In conclusion,in this study we develop a nested PCR detection method for Cryptosporidium oocysts on lettuce surface,which provided a new detection method for food safety of vegetables.