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

doi:10.7668/hbnxb.2014.03.012

Abstract

Abstract: 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.

Key words: Pseudomonas aeruginosa, 16S rDNA, Real-time quantitative PCR

CLC Number:

S855.1

SONG Yue, CHENG Kun, LIANG Xiu-li, WANG Ya-bin, FU Tong, HAN Li-qiang, WEI Zhan-yong. Development and Preliminary Application of a SYBR Green I Real-time PCR Method for Detection of Pseudomonas aeruginosa[J]. journal1, 2014, 29(3): 59-63. doi: 10.7668/hbnxb.2014.03.012.

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http://www.hbnxb.net/EN/Y2014/V29/I3/59

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