One-pot dual-CRISPR/Cas12b-mediated dUTP-LAMP for rapid and sensitive detection of foodborne pathogens without carryover contamination

被引:2
作者
Zhang, Chao [1 ]
Luo, Zisheng [1 ,2 ,3 ]
Lin, Xingyu [1 ,2 ,3 ]
机构
[1] Zhejiang Univ, Coll Biosyst Engn & Food Sci, State Key Lab Fluid Power & Mechatron Syst, Hangzhou 310030, Peoples R China
[2] Zhejiang Univ, Key Lab Agriprod Postharvest Handling, Minist Agr & Rural Affairs, Hangzhou 310058, Peoples R China
[3] Zhejiang Univ, Ningbo Res Inst, Ningbo 315100, Peoples R China
基金
中国国家自然科学基金;
关键词
Dual-sgRNA mediated-CRISPR/Cas12b; New poly-U PAM; Design mechanism; Without nucleic acid extraction; Contamination-free; DNA; ELIMINATION; SEQUENCES;
D O I
10.1016/j.fbio.2024.104069
中图分类号
TS2 [食品工业];
学科分类号
0832 ;
摘要
Rapid and accurate detection of foodborne pathogens is of great significance for ensuring food safety. Currently, the CRISPR-Dx based on Cas12b, a new and better performing Cas enzyme, is very rare, and the vast majority require a two-step method, facing aerosol contamination. In this work, we reported a one-pot dual-CRISPR/ Cas12b mediated dUTP-LAMP method for rapid and sensitive detection of foodborne pathogens without nucleic acid extraction and contamination-free. The design mechanism of sgRNAs in the one-pot CRISPR/Cas12bmediated LAMP method was investigated in detail for the first time. Moreover, a novel PAM sequence UUN of AapCas12b was discovered, and combined with dUTP-LAMP system for one-pot dual-sgRNA detection without carryover contamination. Our developed method can achieve high specificity, nucleic acid extraction-free detection of 2.33 x 104 CFU/mL Salmonella within 1 h. Furthermore, accurate detection of Salmonella was successfully performed in real samples of milk, fruit and egg, demonstrating the practical significance of the developed method.
引用
收藏
页数:7
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