Real-time quantitative PCR assay with TaqMan probe for rapid detection of colistin resistance mcr-1 gene
10.3760/cma.j.issn.0254-5101.2018.09.011
- VernacularTitle:黏菌素耐药细菌mcr-1基因TaqMan PCR快速检测方法的建立
- Author:
Wei SHANG
1
;
Wen DAI
;
Beifang CHEN
;
Mei WANG
;
Dayang ZOU
;
Baocheng CANG
Author Information
1. 中国人民解放军第一五三中心医院
- Keywords:
Colistin resistance;
mcr-1 gene;
PCR;
TaqMan probe
- From:
Chinese Journal of Microbiology and Immunology
2018;38(9):710-715
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a sensitive real-time quantitative PCR assay with TaqMan probe for rapid detection of mcr-1 gene in clinical isolated strains. Methods According to the mcr-1 gene sequence, a pair of specific primers and a TaqMan probe were designed. Moreover, a recombinant plasmid with mcr-1 gene was constructed as the positive standard. TaqMan probe-based fluorescence quantitative PCR assay was used to detect the colistin resistance gene mcr-1. The sensitivity, repeatability and specificity of the assay were evaluated. Results There was a good linear relationship between the initial template amount and Ct value (R2>0. 999). The lower limit of detection was 10 copies/μL, which was 100 times more sensitive than the conventional PCR. Results of test for specificity showed that only the strains carrying the mcr-1 gene were positive, while the remaining strains were negative. Coefficients of variation of intra-and inter-group repeatability tests were less than 1％. Two out of 150 clinical isolated strains carried mcr-1 re-sistance gene and both of them were identified as Escherichia coli. Conclusion TaqMan probe-based fluo-rescence quantitative PCR for the detection of colistin resistance gene mcr-1 was established with strong spe-cificity, high sensitivity and good repeatability. It could be used for the specific detection of clinical drug-re-sistant strains positive for mcr-1 gene and provide reference for pharmacotherapy.
