隐孢子虫卵囊基因组的提取及以保守基因为靶标的PCR检测  

GENOMIC DNA EXTRACTION FROM CRYPTOSPORIDIUM PARVUM OOCYSTS AND PCR DETECTION BASED ON TARGET GENE 18S rRNA

作　　者：王尚[1] 王华然[1] 王福玉[1] 李迎凯[1] 尹静[1] 刘超[1] 

机构地区：[1]军事医学科学院卫生学环境医学研究所,天津300050

出　　处：《解放军预防医学杂志》2009年第1期18-21,共4页Journal of Preventive Medicine of Chinese People's Liberation Army

基　　金：国家科技支撑计划项目课题(No.2006BAD01B05-03)

摘　　要：目的探讨提高PCR检测微小隐孢子虫卵囊方法的特异性和敏感性,为防止隐孢子虫病流行提供检测依据。方法应用BALB/c小鼠感染扩增卵囊,不连续蔗糖密度梯度离心法从粪便中纯化卵囊,通过冻融和超声法破碎卵囊,然后以酚/氯仿/异戊醇法提取基因组DNA作为PCR扩增模板,根据隐孢子虫保守基因18S rRNA,设计一对特异性的引物,扩增约450bp的目的片段。结果超声处理法对隐孢子虫卵囊的破囊率为92%,明显高于冻融法的86%。2个组抽提的DNA作为模板均扩增出目的片段,但超声处理组敏感性高于冻融处理组,分别能检测到卵囊2个/mL和20个/mL。结论超声法对微小隐孢子虫卵囊的破碎效果优于冻融法,经改进的PCR检测微小隐孢子虫卵囊方法具有特异性好和敏感性强的特点。Objective To enhance the specificity and sensitivity of PCR assay of Cryptosperidium oocysts, so as to provide the basis for detection of Cryptosporidiosis epidemic. Methods A number of Cryptosporidium parvum oocysts from infected BALB/c mice were obtained by amplification method. The fecal materials of mice were collected from which oocysts were isolated and purified with discontinuous sucrose gradients technique. The oocysts were cracked by freezing and thawing method, as well as sonic cracking method. Extracted DNA was amplified by PCR targeting a 450 bp fragment of the conservative gene 18S rRNA. The PCR products were checked by the Agarose Gel Electrophoresis. Results The cracking rate of oocysts by freezing and thawing method（86% ） was lower than that by sonic cracking method（92% ）. The extracted DNA template by both methods could amplify the target fragment, but the detection sensitivity by sonic method was higher than that by freezing and thawing method （detection limit： 2 oocysts/mL vs 20 oocysts/mL）. Conclusion The cracking effect of sonic cracking method on oocysts is better than that of freezing and thawing. The modified PCR assay of Cryptosporidium parvum was characterized by its better sensitivity and specificity.

关 键 词：隐孢子虫卵囊 基因组提取 18S RRNA基因 PCR 

分 类 号：R531.5[医药卫生—内科学] R392-33[医药卫生—临床医学]