微滴式数字PCR评价保存时间和冻融对细菌基因组DNA稳定性的影响  

Effects of Storage Time and Freeze-Thawing on the Stability of Bacterial Genomic DNA Evaluated by Droplet Digital PCR

作　　者：薛盼盼 张京云[1] XUE Pan-Pan;ZHANG Jing-Yun(National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《生物技术通讯》2020年第5期571-575,579,共6页Letters in Biotechnology

基　　金：“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项(2018ZX10712001-014)。

摘　　要：目的:定量分析5个常用的保存温度下,保存时间和反复冻融对沙门菌和志贺菌基因组DNA的影响。方法:将沙门菌和志贺菌基因组DNA分别于室温(20~25℃)、4℃、-20℃、-40℃和-80℃保存10 d,其中3个冻存温度的核酸每天冻融1次;以微滴式数字PCR检测靶标基因拷贝数的变化,以指数回归模型进行曲线拟合,分析靶标拷贝数随冻融次数(或天数)的变化趋势。结果:菌株核酸在4℃存放时,10 d以内靶标拷贝数保持稳定。冻存时,10次冻融后靶标拷贝数仅剩余25.3%~31.7%(沙门菌)和8.6%~18.4%(志贺菌);即使仅1次冻融,靶标拷贝数也会下降到初始值的71.5%~75.4%(沙门菌)和67.5%~76.1%(志贺菌)。室温(20~25℃)存放的核酸拷贝数变化趋势与-80℃保存冻融时类似。结论:本研究测定细菌靶标DNA拷贝数随冻融次数的变化趋势,可为研究者选择合适的细菌DNA保存方式和评估冻融对试验结果的影响提供数据依据。Objective:To quantitatively analyze the effects of storage time and repeated freeze-thawing on genomic DNA of Salmonella and Shigella.Methods:The purified DNA of Salmonella and Shigella were stored for 10 days at room temperature(20~25℃),4℃,-20℃,-40℃,and-80℃;and the DNA stored at the last three temperatures were frozen and thawed once a day.The copy numbers of the target genes were detected by droplet digital PCR(ddPCR).The curves were fitted by exponential regression model to analyze the trend of the changes of target copy number with the number of freeze-thaw cycles(or days).Results:When the DNA were stored at 4℃,the copy numbers of target genes remained stable within 10 days.During cryopreservation,the target copy number decreased to 25.3%~31.7%(Salmonella)and 8.6%~18.4%(Shigella)of the initial value after 10 freeze-thaw cycles.The target copy number decreased to 71.5%~75.4%(Salmonella)and 67.5%~76.1%(Shigella)of the initial values even after only one freeze-thaw cycle.The change trend of target copy number of DNA stored at room temperature was similar to that at-80℃.Conclusion:This study described the change trend of copy number of bacterial target genes with freeze-thaw cycle,which can provide reference for researchers to select appropriate bacterial DNA preservation methods and evaluate the impact of freeze-thaw on test results.

关 键 词：细菌基因组DNA 冻融 保存 微滴式数字PCR 沙门菌 志贺菌 

分 类 号：Q78[生物学—分子生物学]