肠球菌SYBR Green I实时定量PCR检测方法的建立及应用

Establishment and preliminary application of a SYBR Green I real-time PCR method for detection of Enterococcus

为了建立肠球菌的实时定量PCR检测方法,以便更加快捷、准确检测肠球菌.根据肠球菌16S rDNA高度保守的特性,在保守区设计1对引物,对其扩增条件进行探索,建立荧光定量PCR检测肠球菌的方法,并对方法的灵敏性、特异性、重复性进行评价,并在临床实践中进行检验.结果表明,所设计的引物能够在阳性样品中扩增出144 bp的目的片段,并且与常见的菌群如绿脓杆菌、大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、变形杆菌等均不发生特异性扩增,检测的灵敏度为17拷贝·μL-1,在临床应用中,荧光定量PCR检测阳性率比普通PCR高18.87%.

To establish the SYBR Green I real-time quantitative PCR assay methods for quicker and more convenient detection of Enterococcus. A pair of primers were designed according to the highly conserved characteristics of Enterococcus 16S rDNA, and the sensitivity, specificity and reproducibility of our method were evaluated, and further testing was done in clinical practice. A 144 bp region of the Enterococcus 16S rDNA was amplified using our primers, and had no cross-react with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Proteus and other common envi- ronmental bacteria. The sensitivity analysis showed that the developed SYBR Green I real-time PCR method could detect 17 coppies μL^-1. In clinical practices, the established method was used to de- tect 61 suspicious positive samples, and the results showed that the positive rate using the SYBR Green I real-time quantitative PCR is 18.87% higher than that of normal PCR.