检出限
重组酶聚合酶扩增
多重位移放大
核酸
化学
DNA
聚合酶链反应
清脆的
放大器
分子信标
分子生物学
生物化学
生物
环介导等温扩增
色谱法
基因
DNA提取
寡核苷酸
作者
Yayun Jiang,Chaochuan Zheng,Ming Jin,Ruolan Zhou,Qiaoli Wu,Fuyuan Huang,Yongliang Lou,Laibao Zheng
标识
DOI:10.1021/acs.jafc.2c08888
摘要
Accurate, rapid, and sensitive pathogenic detections play an important role in food safety. Herein, we developed a novel CRISPR/Cas12a mediated strand displacement/hybridization chain reaction (CSDHCR) nucleic acid assay for foodborne pathogenic colorimetric detection. A biotinylated DNA toehold is coupled on avidin magnetic beads and acts as an initiator strand to trigger the SDHCR. The SDHCR amplification allowed the formation of long hemin/G-quadruplex-based DNAzyme products to catalyze the TMB-H2O2 reaction. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a was activated to cleave the initiator DNA, resulting in the failure of SDHCR and no color change. Under optimal conditions, the CSDHCR has a satisfactory linear detection of DNA targets with a regression equation Y = 0.0531*X - 0.0091 (R2 = 0.9903) in the range of 10 fM to 1 nM, and the limit of detection was determined as 4.54 fM. In addition, Vibrio vulnificus, one foodborne pathogen, was used to verify the practical application of the method, and it showed satisfactory specificity and sensitivity with a limit of detection at 1.0 × 100 CFU/mL coupling with recombinase polymerase amplification. Our proposed CSDHCR biosensor could be a promising alternative method for ultrasensitive and visual detection of nucleic acids and the practical application of foodborne pathogens.
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