Construction and evaluation of reference standards for detection and quantification of Klebsiella pneumoniae using real-time PCR
- Author:
Fei-Long SUN
1
Author Information
1. School of Life Science and Technology
- Publication Type:Journal Article
- Keywords:
Cloning vector;
Klebsiella pneumoniae;
Real-time PCR;
Standard
- From:Academic Journal of Xi'an Jiaotong University
;22(3):183-187
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct reference standards for detection and quantification of Klebsiella pneumoniae (K. pneumoniae) with SYBR Green I-based real-time PCR assay. Methods: Primers were designed based on the published sequence of the phoE gene of K. pneumoniae. The standard was prepared by cell culture, PCR and T-A clone methods, and was identified by colony PCR and DMA sequencing. Results: The standard curve showed a very good linear negative regression between threshold cycle (Ct) and Log starting quantity of copy number. The detection range was from 5.2 to 5.2 × 106 copies per reaction, and the detection limit was 6 copies per reaction. The coefficients of variance (CVs) of three parallel experiments were in the range of 0.05 %-0.91 %. Conclusion: The reference standards have high stability and reproducibility. They can be used in the quantitative detection of K. pneumoniae.
