期刊
ANALYTICA CHIMICA ACTA
卷 1127, 期 -, 页码 225-233出版社
ELSEVIER
DOI: 10.1016/j.aca.2020.06.041
关键词
CRISPR-Cas13a biosensing; Staphylococcus aureus; Collateral RNA cleavage; Bacterial detection; Sensitivity and specificity; Food safety
资金
- National Natural Science Foundation of China [81503086, 21672161]
- Tianjin Municipal Science and Technology Committee [18PTSYJC00140, 19JCYBJC27800]
- Australian Research Council (ARC) [FT160100039]
- University of New South Wales (UNSW)
- Australian Research Council [FT160100039] Funding Source: Australian Research Council
The severity of foodborne diseases caused by foods contaminated by pathogens or their toxins creates an urgent need for the development of specific and sensitive method for detection of bacteria. In this study, taking advantages of CRISPR-Cas13a system, namely, the crRNA programmability and Cas13a collateral effect of promiscuous RNase activity upon target RNA recognition, we developed a bacterial sensing strategy with the name of CCB-Detection (CRISPR-Cas13a based Bacterial Detection). Staphylococcus aureus (S. aureus) was chosen as a model bacteria for validating the performance of CCB-Detection. Specifically, four steps were carried out: 1) simple extraction of genome DNA; 2) specific gene amplification by PCR; 3) in vitro transcription; and 4) the collateral effect cleavage of reporter RNA to report the analyte signal. It was observed that CCB-Detection was capable to successfully detect the target genomic DNA (gDNA) as low as 10(0) aM. The limit of detection (LOD) was 1 CFU/mL with a dynamic detection range of S. aureus from 10(0) to 10(7) CFU/mL. The entire sample-to-answer time for this biosensor was less than 4 h. CCB-Detection demonstrated satisfactory selectivity for S. aureus without interference from other bacteria. Furthermore, CCB-Detection was successfully applied for sensing S. aureus in real food samples with both known and unknown amounts bacteria (spiked ones and non-spiked ones) and its performance is comparable to the conventional culture-based counting method but with short assay time and high sensitivity. With desirable reliability, sensitivity, specificity and simplicity, herein proposed CCB-Detection could be extended and generalised for other bacterial detection, and has great potential to be used in a wide range of applications such as food safety inspection, disease diagnosis, environment monitoring, etc. (C) 2020 Elsevier B.V. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据