从粪便中分离隐孢子虫用于全基因组测序的研究  

A study involving whole genome sequencing of Cryptosporidium DNA extracted from fecal specimens

作　　者：杨凤坤[1] 赵宏丽 栾天蔚 舒晶[1] YANG Feng-kun ZHAO Hong-li LUAN Tian wei SHU Jing(Department of Parasitology, Harbin Medical University, Harbin 150081, China Daoli District＇s People） Hospital)

机构地区：[1]哈尔滨医科大学寄生虫学教研室,黑龙江哈尔滨150081 [2]道里区人民医院

出　　处：《中国病原生物学杂志》2017年第9期830-832,共3页Journal of Pathogen Biology

基　　金：黑龙江省教育厅科学技术研究项目(No.12541280)

摘　　要：目的使用新的方法直接从少量粪便样品中分离和纯化隐孢子虫卵囊,并进行全基因组扩增(whole genome amplification,WGA),从而获得符合全基因组测序要求的隐孢子虫DNA。方法 10份新鲜牛源隐孢子虫粪便样品,用蔗糖和氯化铯离心法分离卵囊,然后使用免疫磁珠试剂盒纯化;用漂白水进行卵囊表面消毒,以除去细菌及真菌等病原微生物。采用QIAamp DAN Mini kit试剂盒提取基因组DNA,然后采用REPLI-g MIDI kit试剂盒进行WGA,最后,采用定量PCR对基因组DNA进行量和纯度检测。结果分离纯化后的卵囊计数为2.81×104个/g^2.90×107个/g。提取基因组DNA后进行WGA,所有扩增产物均为大分子片段,表明WGA成功。WGA产物的DNA浓度为55.3ng/μl^357.5ng/μl。隐孢子虫SSU rRNA基因的定量PCR扩增显示,10份样品纯化WGA产物的CT值范围为9.75~17.76,其中有7个样品CT<15,满足全基因组测序要求。结论直接从粪便中分离和纯化隐孢子虫卵囊再进行WGA,可使隐孢子虫的基因组DNA浓度和纯度能达到测序要求,该方法使隐孢子虫全基因测序常规化是可行的。Objectives To develop and use a new method for preparation of Cryptosporidium oocysts and whole genome amplification（WGA）to facilitate whole genome sequencing directly from stool specimens. Methods Sucrose and cesium chloride density gradient separation was used to isolate and purify Cryptosporidium oocysts from 10 fresh stool specimens from cattle.Oocysts were further purified via immunomagnetic separation（IMS）using the Dynabeads AntiCryptosporidiumkit.Finally,oocysts were treated with bleach to remove any attached bacteria or fungi.Genomic DNA was extracted using the QIAamp DNA Mini kit and then REPLI-g Midi Kit was used to generate DNA of sufficient quantity and purity for analysis of the complete Cryptosporidiumgenome.The quality of WGA products was analyzed using quantitative PCR（qPCR）based on the SSU rRNA gene. Results After oocyst purification,the oocyst count ranged between 2.81×104/g and 2.90×107/g.After WGA,electrophoresis of the products resulted in large molecules on the gel,indicating that amplification was successful.The concentration of purified WGA DNA varied between 55.3ng/μl and357.5ng/μl.The ten WGA products had relatively low CT values ranging from 9.75 to 17.76.Seven CT values were less than 15,indicating that Cryptosporidium genomic DNA was present in these specimens in high concentrations.Conclusion The current results indicated that the strategy described here can be used to effectively isolate and purify Cryptosporidium DNA directly from fecal specimens for whole genome sequencing.The method developed in this study should make routine whole genome sequencing of Cryptosporidium sppfeasible.

关 键 词：隐孢子虫 纯化卵囊 免疫磁珠分离 全基因组扩增 定量PCR 

分 类 号：R382.3[医药卫生—医学寄生虫学]