摘要
目的探讨微滴数字聚合酶链反应(ddPCR)检测含有目的基因的PUC57质粒过程中出现的问题及解决方法。方法用ECORⅠ酶37℃孵育含有目的基因的PUC57质粒2h和4h,然后65℃孵育20min,灭活ECORⅠ酶。用伯乐QX200TM微滴式数字PCR分别检测PUC57质粒和经ECORⅠ酶酶切后的PUC57质粒,同时对酶切后的PUC57质粒分多天进行检测,对检测结果进行比较。结果含有目的基因的PUC57质粒经ECORⅠ酶酶切2h和4h后的检测值与质粒理论值之间差异无统计学意义(t=-0.192、-0.403,P>0.05),同时酶切与没有酶切的PUC57质粒检测值之间差异有统计学意义(Z=-4.194,P<0.05)。酶切后的检测值与PUC57质粒理论值相符合。酶切后的PUC57质粒随着放置时间的延长,检测值逐渐降低。结论用伯乐QX200^(TM)微滴式数字PCR检测含有目的基因的PUC57质粒时要先酶切再检测,酶切时间只需2h即可将质粒酶切完全,同时酶切的PUC57质粒要尽快检测。
Objective To explore the problems and solutions in the detection of PUC57 plasmid containing the target gene by droplet digital polymerase chain reaction(ddPCR).Methods PUC57 plasmid containing the target gene was incubated with ECORⅠenzyme at 37℃for two hours and four hours,then incubated with 20 min at 65℃to inactivate ECORⅠenzyme.The PUC57 plasmid and PUC57 plasmid after ECORⅠenzyme digestion were detected by using QX200 TMDroplet Digital PCR(Bio-Rad),respectively,and the PUC57 plasmids were detected for many days after the enzyme digestion,and the results were compared.Results The PUC57 plasmid containing target gene had no statistically significant difference between the detection value of two hours and four hours after enzyme digestion and the theoretical value of plasmid(t=-0.192,-0.403,P>0.05).Meanwhile,there was a statistical difference(Z=-4.194,P<0.05)between the detection of PUC57 plasmid after ECORⅠenzyme digestion and the detection without enzyme digestion.The value of enzyme digestion was consistent with the theoretical value of PUC57 plasmid.The PUC57 plasmid decreased gradually with the prolongation of the time after enzyme digestion.Conclusion When the PUC57 plasmid containing the target gene is detected by QX200TM Droplet Digital PCR(Bio-Rad),the enzyme digestion is first and then tested.The enzyme digestion time is only two hours,and the plasmid can be cut completely,and the PUC57 plasmid which is cut by enzyme should be detected as soon as possible.
作者
杨德平
刘维薇
YANG Deping;LIU Weiwei(Department of Clinical Laboratory,Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital,Shanghai 201318,China;Department of Clinical Laboratory,Shanghai Tenth People′s Hospital,Tongji University,Shanghai 200072,China;Department of Clinical Laboratory,Shanghai Skin Disease Hospital,Shanghai 200070,China)
出处
《国际检验医学杂志》
CAS
2019年第6期653-656,共4页
International Journal of Laboratory Medicine
基金
国家自然科学基金项目(81370067
81572061)
上海市新优青人才计划(XYQ2013095)
上海申康医院发展中心辅助科室能力建设项目(SHDC22014006)
关键词
微滴数字聚合酶链反应
PUC57质粒
酶切
droplet digital polymerase chain reaction
PUC57plasmid
enzyme digestion
