从粪便中分离隐孢子虫用于全基因组测序的研究

发布时间：2018-03-03 10:04

本文选题：隐孢子虫　切入点：纯化卵囊　出处：《中国病原生物学杂志》2017年09期 　论文类型：期刊论文

【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CT15,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。
[Abstract]:Objective to isolate and purify Cryptosporidium oocysts directly from a small amount of fecal samples by a new method. The whole genome amplification was carried out to obtain Cryptosporidium sinensis DNA.Methods Ten samples of fresh bovine Cryptosporidium faeces were isolated by sucrose and cesium chloride centrifugation. Then purified by immunomagnetic bead kit, and disinfected with bleached solution to remove bacteria and fungi, genomic DNA was extracted by QIAamp DAN Mini kit kit, and then WGA was obtained by REPLI-g MIDI kit kit. Quantitative PCR was used to detect the quantity and purity of genomic DNA. Results the egg sac count after isolation and purification was 2.81 脳 10 4 / g, 2.90 脳 10 7 / g. The genomic DNA was extracted for WGA, and all the amplified products were macromolecular fragments. The results showed that the concentration of DNA of WGA was 55.3 ng / 渭 ll 357.5 ng / 渭 l.The quantitative PCR amplification of Cryptosporidium rRNA gene showed that the CT value range of purified WGA product from 10 samples of Cryptosporidium was 9.75 ~ 17.76, and CT15 of 7 samples met the requirement of genome sequencing. Conclusion\\\; The concentration and purity of genomic DNA of Cryptosporidium can meet the requirements of sequencing if WGA is used to isolate and purify Cryptosporidium oocysts from feces. This method is feasible to sequence the whole gene of Cryptosporidium.
【作者单位】：哈尔滨医科大学寄生虫学教研室;道里区人民医院;
【基金】：黑龙江省教育厅科学技术研究项目(No.12541280)
【分类号】：R382.3

【相似文献】