Meng Cheng,Caiwei Tan,Bo Xiang,Weihong Lin,Bolin Cheng,Xuechun Peng,Yihao Yang,Yongping Lin
Visualized gene detection based on the CRISPR‐Cas12/CRISPR‐Cas13 technology and lateral flow assay device (CRISPR‐LFA) has shown great potential in point‐of‐care testing sector. Current CRISPR‐LFA methodology mainly utilizes conventional immuno-based LFA test strips, which could visualize whether the reporter probe is trans -cleaved by Cas protein, indicating the target positive detection. However, conventional CRISPR‐LFA usually produces false-positive results in target negative assay. Herein, a nucleic acid C hain H ybridization‐based L ateral F low A ssay platform, named CHLFA, has been developed to achieve the CRISPR‐CHLFA concept. Different from the conventional CRISPR‐LFA, the proposed CRISPR‐CHLFA system was established based on the nucleic acid hybridization between the GNP‐probe embedded in test strips and ssDNA (or ssRNA) reporter from CRISPR (LbaCas12a or LbuCas13a) reaction, which eliminated the requirement of immunoreaction in conventional immuno-based LFA. The assay realized the detection of 1–10 copy of target gene per reaction within 50 min. The CRISPR-CHLFA system achieved highly accurate visual detection of target negative samples, thus overcoming the false-positive problem that often produced in assays using conventional CRISPR‐LFA. The CRISPR‐CHLFA platform was further adopted for the visual detection of marker gene from SASR‐CoV-2 Omicron variant and Mycobacterium tuberculosis (MTB), respectively, and 100% accuracy for the analysis of clinical specimens (45 SASR‐CoV-2 specimens and 20 MTB specimens) was obtained. The proposed CRISPR-CHLFA system could provide an alternative platform for the development of POCT biosensors and can be widely adopted in accurate and visualized gene detection.
Jiahui Li,Yuanyuan Zhang,Zhengshun Wen,Jiangbing Shuai,Xiaofeng Zhang,Jinru Zhou,Aiqian Ye,Linglin Fu
The delayed reporting of milk allergen residue detection results is a significant factor contributing to substantial losses for food production enterprises. Cumbersome operational procedures and high testing costs often lead to the abandonment of tests, resulting in frequent allergic reactions among milk-sensitive populations. Therefore, there is an urgent need for a novel, rapid, and low-cost detection method to mitigate enterprise losses and protect allergic individuals. This study investigates the formation mechanism of convection PCR, driven by Fast Taq polymerase in standard PCR tubes, and develops a fluorescence detection device for convection PCR. Through molecular docking and experimental validation of the trimmed β-LG aptamers, we elucidate their potential multi-site binding mechanism. Utilizing this mechanism, we designed a dual-strand hybridization magnetic bead detection system (dsA-SA-B), employing easily synthesized and amplified aptamer complementary strands as an indicator signal; Signal amplification was achieved within 15 min, while the entire detection process was completed in less than 50 min. This detection method allows for the rational selection of aptamer sequences based on actual testing needs, enabling the detection of hydrolyzed milk powder. The method demonstrated a linear detection range for β-LG from 5 to 100 μg/mL, with a detection limit as low as 0.25 μg/mL, effectively identifying hydrolyzed milk powder. This approach transforms protein detection into nucleic acid detection, and lays a solid foundation for multiplex PCR detection of eight major food allergens, facilitating the widespread detection of milk allergens and ultimately contributing to improved food safety and public health.
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