Line eliminating method immunochromatography test paper and application thereof in CRISPR nucleic acid test

Abstract

The invention discloses line eliminating method immunochromatography test paper and application thereof in a CRISPR nucleic acid test. A kit provided by the invention comprises: 1) immunochromatography test paper; and 2) a CRISPR reaction system, wherein in a sample flow direction of the immunochromatography test paper sequentially comprises a sample pad containing a colloidal gold labelled antibody, an NC membrane contain a T line and a C line and water-absorbent filter paper; the T line is formed by streptavidin; the C line is formed by a secondary antibody of the colloidal gold labelled antibody; the CRISPR reaction system comprises report RNA, crRNA and Cas protein; and the Cas protein is in a second-class type V or type VI CRISPR system. Experiments of the invention prove that the test paper in the kit can realize high-sensitivity, high-specificity and convenient detection of multiple specific nucleic acids (pathogens, gene mutation and drug-resistant mutation) by binding a CRISPRnucleic acid detection system.

Description

technical field [0001] The invention relates to a "line elimination method" immunochromatographic test paper and its application in CRISPR nucleic acid detection, belonging to the technical field of molecular diagnosis. Background technique [0002] In recent years, CRISPR genome editing technology has triggered tremendous changes in the biological world. In 2017, researchers discovered the Leptotrichia wadei Cas13a protein (LwCas13a) that can be used for nucleic acid detection. By combining it with recombinant polymerase isothermal amplification technology (Reconbinase polymerase amplification, RPA), the system sensitivity was increased by 1 million times, and A highly sensitive and specific nucleic acid detection method SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) with a sensitivity of single copy and a specificity of single base was established. The system has been used for the detection of Zika and dengue virus in biological samples (blood or urine)...

AI Technical Summary

Problems solved by technology

The specific detection principle is: the two ends of the reporter RNA in the CRISPR reaction system are respectively modified with biotin and FAM. For positive samples, the double-labeled reporter RNA cut off by the Cas protein flows from the side of the sample pad to the binding pad due to capillary force. The FAM end of the reporter RNA will bind to colloidal gold particles, and the other end will be captured by streptavidin (C line) during chromatography, and the sheared reporter RNA-FAM-colloidal gold particle complex will bind to the T line The secondary antibody specifically binds to the T-line and C-line at the same time; however, in actual use, it is found that no matter the negative or positive sample, in the detection of LFCA test paper based on this detection principle, especially the sample concentration is low Although the color shades of the T line and C line in the negative and positive samples are different, bands will appear in both, which also brings certain difficulties to the interpretation of results that mainly rely on subjective naked eye observation.

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