Development of Droplet Digital Polymerase Chain Reaction for Quantifying Escherichia Coli O157:H7
10.11895/j.issn.0253-3820.140569
- VernacularTitle:大肠杆菌O157:H7微滴数字PCR定量方法的建立
- Author:
Lianhua DONG
;
Ling ZHANG
;
Jun JING
;
Jiangnan WANG
;
Jing WANG
;
Weijun CHEN
- Publication Type:Journal Article
- Keywords:
Droplet digital polymerase chain reaction;
Escherichia coli O157:H7;
Copy number
- From:
Chinese Journal of Analytical Chemistry
2015;(3):319-324
- CountryChina
- Language:Chinese
-
Abstract:
A droplet digital polymerase chain reaction ( ddPCR) method for quantifying E. coli O157:H7 by targeting rfbE gene was developed. The probe concentration in ddPCR was optimized and the linearity range, precision, limit of detection ( LOD) and limit of quantification ( LOQ) were also evaluated. The optimized probe concentration was 300 nmol/L. The ddPCR response was linear over the E. coli O157:H7 genome DNA concentration range from 4 to 1. 25×105 copies in 20 μL ddPCR system and the linear correlation coefficient (R2) was 0. 999. The ddPCR precision (RSD) was less than 5% over the DNA concentration range from 760 to 88400 copies/20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL, respectively. Specificity test showed that the ddPCR was specific for detecting E. coli O157:H7. Both ddPCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat, pork and beef, which indicated that ddPCR method was suitable for detection of E. coli O157:H7 in food.
