从少量人源粪便中分离隐孢子虫用于全基因组测序的研究

Methods of whole genome sequencing using small volume stool samples of Cryptosporidium from human

目的直接从少量人源粪便样本中分离、纯化隐孢子虫卵囊,提取并扩增DNA,使其浓度和纯度符合全基因组测序(WGS)要求。方法2013年12月-2014年10月收集来自美国佛罗里达州、爱达荷州和缅因州11份少量人源隐孢子虫粪便样本,每份200 mg,使用免疫磁珠分离(IMS)进行纯化卵囊;2014年8-12月收集来自美国农业部52份牛源隐孢子虫粪便样本作为对照,每份2000 mg,采用饱和蔗糖、氯化铯密度梯度离心法和IMS等方法纯化卵囊。均使用QIAamp DNA Mini试剂盒提取基因组DNA,然后采用REPLI-g MIDI试剂盒进行全基因组扩增(WGA);最后通过定量PCR(qPCR)对WGA产物进行评估。结果11份人源隐孢子虫WGA产物的DNA浓度为68.00~257.80 ng/μL。qPCR对隐孢子虫小亚基rRNA基因(SSU rRNA)扩增显示,11个分离株的循环数阈值(Ct)范围为8.92~19.23,Ct值越小表明隐孢子虫基因组DNA浓度越高;其中有6个分离株Ct<15,54.54%的分离株满足WGS要求。52份牛源隐孢子虫WGA产物的DNA浓度为27.20~1333.30 ng/μL,WGA产物qPCR的Ct值范围为6.48~32.09,其中29个分离株Ct值<15,55.76%的分离株满足WGS要求。直接对少量人源粪便样本使用IMS纯化和对大量牛源样本多步骤纯化后通过WGA,符合WGS要求的样本量之间比较差异无统计学意义(P>0.05)。结论直接对临床上少量人源隐孢子虫粪便样本进行IMS纯化卵囊,可使隐孢子虫基因组DNA浓度和纯度达到测序要求,该方法可用于临床少量样本的全基因组测序。

Objective To obtain enough quality and quantity of DNA from small amounts of stool samples of human-derived Cryptosporidium for whole genome sequencing(WGS).Methods From December 2013 to October 2014,11 human-derived Cryptosporidium stool samples(200 mg each)were collected in Florida,Idaho and Maine,USA,and oocysts were purified using immunomagnetic separation(IMS);from August to December 2014,52 cattle-derived Cryptosporidium stool samples(2000 mg each)from United States Department of Agriculture served as controls,and the oocysts were purified with a sucrose gradient,cesium chloride density-gradient separation,and IMS.Genomic DNA was extracted from oocysts of human-derived and cattle-derived using QIAamp DNA Mini kit and the DNA was used to perform whole-genome amplification(WGA)using REPLI-g MIDI Kit.The quality of WGA products was assessed by quantitative PCR(qPCR).Results The concentrations of purified WGA DNA varied from 68.00 to 257.80 ng/μL from 11 human stool samples.Cycle threshold(Ct)value ranged from 8.92 to 19.23.The smaller of Ct value,the higher was the genomic DNA concentration of Cryptosporidium.The Ct values of 6 isolates were all less than 15,and 54.54%isolates was suitable for WGS.Meanwhile,the concentrations of purified WGA DNA varied from 27.20 to 1333.30 ng/μL from 52 cattle stool samples.Ct value ranged from 6.48 to 32.09.The Ct values of 29 isolates were all less than 15,and 55.76%isolates satisfied the requirement of WGS.There was no significant difference for the number of samples to suite for requirement of WGS between IMS method for human small volume stool samples and using multi-step purification methods for a large volume cattle stool samples(P>0.05).Conclusions By directly using IMS purification for a small number of clinical human-derived Cryptosporidium stool samples,the concentration and purity of Cryptosporidium genome DNA could meet WGS requirements.It showed that this method was feasible.