绿脓杆菌SYBR Green Ⅰ实时定量PCR检测方法的建立及初步应用  

Development and Preliminary Application of a SYBR Green Ⅰ Real-time PCR Method for Detection of Pseudomonas aeruginosa

作　　者：宋月[1,2] 程琨[1] 梁秀丽[3] 王亚宾[2] 付彤[2] 韩立强[2] 魏战勇[1] 

机构地区：[1]河南省动物性食品安全重点实验室,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002 [3]安阳工学院,河南安阳455000

出　　处：《华北农学报》2014年第3期59-63,共5页Acta Agriculturae Boreali-Sinica

基　　金：河南省重大科技专项(111100110300);国家质检总局科技计划项目(2013QK233)

摘　　要：为了建立绿脓杆菌的SYBR Green I实时定量PCR检测方法,以便更加快捷、方便的检测绿脓杆菌,根据16S rDNA高度保守的特性,在绿脓杆菌16S rDNA保守区设计1对引物,利用普通PCR技术扩增出绿脓杆菌16S rDNA保守区277 bp的片段,并克隆到pMD-18 T载体上,纯化的质粒作为模板进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立了绿脓杆菌的荧光定量PCR检测方法。并对方法的灵敏性、特异性、重复性进行评价,又进一步在临床实践中进行检验。结果显示,所建立的方法对标准样品的最小检出浓度为28拷贝/μL。并且特异性检验结果显示与常见的菌群没有交叉反应,重复性良好。在临床中检测疑似绿脓杆菌感染病料55份,用所建立的荧光定量方法检测出阳性病料40份,而普通的PCR方法检测出阳性病料32份。表明建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可以在临床中用于绿脓杆菌的检测。To development the SYBR Green I real-time quantitative PCR assay for detection of Pseudomonas aeruginosa quicker and more convenient.According to the characteristics of highly conserved of Pseudomonas aeruginosa 16S rDNA,we designed a pair of primers to establish the quantitative PCR methods.We got a 277 bp region of the Pseudomonas aeruginosa 16S rDNA was amplified using normal PCR.Then sub-cloned to pMD-18 T vector and acquired the recombinant plasmid,which served as template to conduct the standards curve of the SYBR Green I real-time PCR.And the sensitivity,specificity and reproducibility of our method were evaluated,and further testing was done in clinical practice.The sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 28 coppies /μL.Specificity testing results showed that it has no cross-react with common environmental bacteria.Then the established method was used to detect the clinical samples.The results showed that 40 positive samples out of 55 suspicious positive samples could be observed by real-time PCR and 32 positive samples could be detected by normal PCR.These results indicated that the SYBR Green I real-time PCR we established in present study showed the characteristics of sensitivity and specificity,and could be used in clinical diagnosis and epidemiological investigation for Pseudomonas aeruginosa.

关 键 词：绿脓杆菌 16S RDNA 荧光定量PCR 

分 类 号：S852.61[农业科学—基础兽医学]