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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。
Objective To isolate and purify Cryptosporidium oocysts directly from a small amount of stool sample using a new method and perform whole genome amplification (WGA) to obtain Cryptosporidium DNA that meets the requirements of genome-wide sequencing. Methods Ten samples of fresh Cryptosporidium parvum fecal samples were isolated from the oocysts by sucrose and cesium chloride centrifugation and then purified by immunomagnetic bead kit. The surface of the oocysts was disinfected with bleach to remove pathogenic microorganisms such as bacteria and fungi. Genomic DNA was extracted using the QIAamp DAN Mini kit and then the WGA was performed using the REPLI-g MIDI kit kit. Finally, the amount and purity of the genomic DNA were examined using quantitative PCR. Results The count of isolated and purified oocysts was 2.81 × 104 / g ~ 2.90 × 107 / g. WGA was extracted after genomic DNA was extracted and all amplified products were macromolecular fragments, indicating that WGA was successful. The DNA concentration of the WGA product ranged from 55.3 ng / μl to 357.5 ng / μl. Quantitative PCR amplification of Cryptosporidium SSU rRNA gene showed that the CT values of purified WGA products from 10 samples ranged from 9.75 to 17.76, of which 7 samples had CT <15, which met the requirement of genome-wide sequencing. Conclusion The isolation and purification of Cryptosporidium oocysts directly from feces and WGA can make the genomic DNA concentration and purity of Cryptosporidium reach the requirement of sequencing. This method is feasible to normalize the whole genome of Cryptosporidium.