体重差异双胎新生儿 粪便样本DNA提取的优化及分析

摘要：目的 优化新生儿粪便样本DNA提取方法，提取及分析体重差异双胎新生儿粪便样本DNA。方法 从7种DNA提取试剂盒方法中选择对成人粪便样本DNA提取效果最佳的QIAamp DNA Stool Mini Kit法为基准方法，通过钢珠打断前处理、DNA吸附柱收集全部裂解液上清和洗脱液重复洗脱的优化，建立了用于新生儿粪便样本DNA的提取方法。结果 该优化方法用于婴儿（出生1个月）粪便样本，结果显示DNA提取浓度平均提高了2.4倍。用于48对双胞胎新生儿出生第1天和第3天粪便样本DNA的提取，经酶标仪及PCR扩增检测，结果显示出生第1天粪便样本DNA提取率为32%，出生3天提取率达83%。RT-PCR显示新生儿第1天到第3天肠道微生物量呈现增长趋势。结论 优化的QIAamp DNA Stool Mini Kit法适用于新生儿粪便样本DNA的快速提取，为后续扩增子高通量测序和研究体重差异双胎新生儿肠道菌群构成规律奠定基础。

Optimization of extraction method and analysis of DNA from fecal samples of twin newborns with different body weights

Abstract: Objective To optimize the method for DNA extraction and analysis of DNA from the stool samples of twin newborns with different body weights. Methods From seven DNA extraction kits, the best method QIAamp DNA Stool Mini Kit for DNA extraction from adult stool samples was selected as the benchmark method. The method for DNA extraction from neonatal stool samples was established by optimizing the pretreatment of breakage with steel beads, collection of all supernatants of lysate with DNA adsorption column and repeated elution with eluent. Results For infant stool samples (one month after birth), the average concentration of DNA extraction increased by 2.4 times. When used to extract DNA from the feces of 48 pairs of twin newborns, the positive rate of DNA extraction was 32% on the first day of birth, and 83% on the third day of birth. RT-PCR showed that the intestinal microbial biomass of neonates increased from the first day to the third day. Conclusion The optimized QIAamp DNA Stool Mini Kit method is suitable for rapid extraction of DNA from fetal stool samples, which lays a foundation for subsequent high throughput sequencing of amplifiers and study on intestinal flora composition of twin newborns with weight differences.