酶切法去除荧光定量聚合酶链反应体系中内源性核酸污染  

Elimination of the endogenous nucleic acid in the real-time quantitative polymerase chain reaction by restriction endonuclease digestion

作　　者：刘健刚 汪求精 刘明刚[2] 宋昭 陈乾[2] LIU Jiangang;WANG Qiujing;LIU Minggang;SONG Zhao;CHEN Qian(Department of Neurosurgery, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong 518000, P. R. China;Department of Neurosurgery, Shenzhen Children＇s Hospital, Shenzhen, Guangdong 518026, P. R. China)

机构地区：[1]南方医科大学深圳医院神经外科,广东深圳518000 [2]深圳市儿童医院神经外科,广东深圳518026

出　　处：《华西医学》2018年第6期744-747,共4页West China Medical Journal

基　　金：深圳市科技计划项目(JCYJ20150403100317065)

摘　　要：目的建立去除荧光定量聚合酶链反应(polymerase chain reaction,PCR)体系中内源性核酸污染的方法,用于降低或排除颅内术后易感染细菌的荧光定量PCR检测出假阳性的概率。方法首先去除荧光定量PCR反应体系中的内源性核酸污染;随后利用多重PCR技术,以混合菌DNA作为模板进行革兰菌特异性鉴定;并研究去除污染后的多重PCR技术检测混合细菌DNA的检测限和灵敏度。结果建立的方法能够快速有效地去除荧光定量PCR体系内的内源性核酸污染,并对PCR体系中Taq酶的活性以及扩增效率均无影响,最低检测限为102 CFU/m L(金黄色葡萄球菌和绿脓假单胞菌),与培养法相同。酶切法对PCR体系中酶的活性及扩增效率均无明显影响,且对PCR反应体系及引物特异性没有影响(循环数Ct=32,荧光强度ΔRn=200)。过滤法却使PCR扩增效率显著下降(循环数Ct=32,荧光强度ΔRn=150)。结论酶切法去除内源性核酸污染对荧光定量PCR检测限及灵敏度均无影响,操作简便快速,降低了PCR检测的假阳性概率,能及时准确地诊断颅内细菌感染。Objective To establish a method that can eliminate the pollution of endogenous nucleic acid in the real-time quantitative polymerase chain reaction（PCR） reaction system, which can be used to reduce or eliminate the false positive rate of real-time PCR assay in detection of postoperative intracranial bacteria infection. Methods At first,eliminated the pollution of endogenous nucleic acid in the real-time PCR reaction system. Then, with mixed bacteria DNA as a template, multiple PCR was used to specifically identify the gram-negative bacteria. Meanwhile, evaluated the text line and sensitivity of the multiple PCR after eliminating pollution in detecting the DNA of the mixed bacteria. Results The method established could quickly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system,and it didn＇t affect the Taq enzyme activity and the amplification efficiency in PCR system, with the minimum detection limit of 102 CFU/m L（Staphylococcus aureus and Pseudomonas aeruginosa）, which was the same to the culture method.The enzyme cutting method had no significant effect on the activity and amplification efficiency of the enzyme in PCR system, It had no effect on PCR reaction system and primer specificity（Ct=32, ΔRn=200）. However, the filtration method significantly reduced the PCR amplification efficiency（Ct=32, ΔRn=150）. Conclusions This method can easily and rapidly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and greatly reduce the false positive of PCR detection. It is able to timely and accurately diagnose the intracranial bacteria infection, which is significant for clinical testing.

关 键 词：颅内感染 荧光定量聚合酶链反应 内源核酸污染 

分 类 号：R440[医药卫生—诊断学]