Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay

生物测定 沙门氏菌 检出限 G-四倍体 脱氧核酶 致病菌 反式激活crRNA 放大器 吸光度 血红素 化学 清脆的 适体 荧光染料 DNA 微生物学 生物 细菌 分子生物学 实时聚合酶链反应 色谱法 聚合酶链反应 基因 Cas9 生物化学 遗传学 血红素
作者
Lijuan Yin,Ninghui Duan,Si Chen,Yuan Yao,Jifeng Liu,Long Ma
出处
期刊:Sensors and Actuators B-chemical [Elsevier BV]
卷期号:347: 130586-130586 被引量:73
标识
DOI:10.1016/j.snb.2021.130586
摘要

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.
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