CAJ | 학술논문

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 rD-NA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 Green Ⅰ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。
위료건립록농간균적SYBR Green I실시정량PCR검측방법，이편경가쾌첩、방편적검측록농간균，근거16S rDNA고도보수적특성，재록농간균16S rDNA보수구설계1대인물，이용보통PCR기술확증출록농간균16S rD-NA보수구277 bp적편단，병극륭도pMD-18 T재체상，순화적질립작위모판진행SYBR Green Ⅰ형광정량PCR확증병제작표준곡선，건립료록농간균적형광정량PCR검측방법。병대방법적령민성、특이성、중복성진행평개，우진일보재림상실천중진행검험。결과현시，소건립적방법대표준양품적최소검출농도위28고패/μL。병차특이성검험결과현시여상견적균군몰유교차반응，중복성량호。재림상중검측의사록농간균감염병료55빈，용소건립적형광정량방법검측출양성병료40빈，이보통적PCR방법검측출양성병료32빈。표명건립적실시형광정량PCR구유특이、민감、쾌속、정량、중복성호등우점，가이재림상중용우록농간균적검측。
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 aerug-inosa 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 re-al-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 bacte-ria . 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.