Journal
ANALYTICA CHIMICA ACTA
Volume 1231, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.aca.2022.340417
Keywords
Clustered regularly interspaced short; palindromic repeats; Multiple crRNAs; Amplification -free; Cytochrome b; Food adulteration
Categories
Funding
- Key R&D Program of Zhejiang Province
- Expert Workstation of Yunnan Prov-ince
- [2021C02059]
- [202105AF150060]
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This study demonstrates that combining multiple crRNAs in the CRISPR/Cas12a system can enhance detection sensitivity and establishes a multiple crRNAs enhanced CRISPR (meCRISPR) method for meat adulteration identification.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detec-tion procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/mu L extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adultera-tion, and genetically modified food detection.
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