Jun Luo1, Junping Yu2, Hang Yang2, Hongping Wei3. 1. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100039, China. 2. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China. 3. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China. Electronic address: hpwei@wh.iov.cn.
Abstract
OBJECTIVE: The methods combining culture and quantitative PCR(qPCR) offer new solutions for rapid antibiotic susceptibility testing(AST). However, the multiple steps of DNA extraction and cold storage of PCR reagents needed make them unsuitable for rapid high throughput AST. In this study, a parallel culture-qPCR method was developed to overcome above problems. METHOD: In this method, bacteria culture and DNA extraction automatically and simultaneously completed through using a common PCR instrument as a controllable heating device. A lyophilized 16S rDNA targeted qPCR reagent was also developed, which was stable and could be kept at 4°C for long time and at 37°C for about two months. RESULT: Testing of 36 P. aeruginosa isolates and 28 S. aureus isolates showed that the method had good agreements with the standard broth microdilution method, with an overall agreement of 97.22% (95% CI, 85.83-99.51) for P. aeruginosa and 96.43% (95% CI, 79.76-99.81) for S. aureus. This method could test 12 samples against a panel of up to 7 antibiotics simultaneously in two 96-well PCR plates within 4h, which greatly improves the testing efficiency of the culture-qPCR method. CONCLUSION: With rapidness to obtain results and the capabilities for automation and multiple-sample testing, the parallel culture-qPCR method would have great potentials in clinical labs.
OBJECTIVE: The methods combining culture and quantitative PCR(qPCR) offer new solutions for rapid antibiotic susceptibility testing(AST). However, the multiple steps of DNA extraction and cold storage of PCR reagents needed make them unsuitable for rapid high throughput AST. In this study, a parallel culture-qPCR method was developed to overcome above problems. METHOD: In this method, bacteria culture and DNA extraction automatically and simultaneously completed through using a common PCR instrument as a controllable heating device. A lyophilized 16S rDNA targeted qPCR reagent was also developed, which was stable and could be kept at 4°C for long time and at 37°C for about two months. RESULT: Testing of 36 P. aeruginosa isolates and 28 S. aureus isolates showed that the method had good agreements with the standard broth microdilution method, with an overall agreement of 97.22% (95% CI, 85.83-99.51) for P. aeruginosa and 96.43% (95% CI, 79.76-99.81) for S. aureus. This method could test 12 samples against a panel of up to 7 antibiotics simultaneously in two 96-well PCR plates within 4h, which greatly improves the testing efficiency of the culture-qPCR method. CONCLUSION: With rapidness to obtain results and the capabilities for automation and multiple-sample testing, the parallel culture-qPCR method would have great potentials in clinical labs.
Authors: Tucker Maxson; Candace D Blancett; Amanda S Graham; Christopher P Stefan; Timothy D Minogue Journal: PLoS One Date: 2018-12-13 Impact factor: 3.240
Authors: Ana I Cubas-Atienzar; Christopher T Williams; Abhilasha Karkey; Sabina Dongol; Manandhar Sulochana; Shrestha Rajendra; Glyn Hobbs; Katie Evans; Patrick Musicha; Nicholas Feasey; Luis E Cuevas; Emily R Adams; Thomas Edwards Journal: J Glob Antimicrob Resist Date: 2021-09-03 Impact factor: 4.035