Detection method of specific nucleic acid fragment based on CRISPR-Cas13a

A nucleic acid fragment and detection method technology, applied in the field of biological material detection, can solve problems such as easy contamination of samples, and achieve the effect of improving sensitivity and simplifying operation procedures

Inactive Publication Date: 2018-01-09
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a specific fragment detection method with

Method used

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  • Detection method of specific nucleic acid fragment based on CRISPR-Cas13a
  • Detection method of specific nucleic acid fragment based on CRISPR-Cas13a

Examples

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Embodiment 1

[0054] In this example, the KRAS gene is used to illustrate the detection of the target gene by the detection method (three-in-one) of the present invention, and to test the sensitivity of the method.

[0055] First, by annealing after primer synthesis, a fragment of the KRAS gene was synthesized as the target double-stranded DNA for detection.

[0056] Secondly, RPA primers were synthesized, the upstream primer was 5'-taatacgactcactataggctgctgaaaatgactgaatataaactt-3' (the 19 nucleotides at the 5' end were T7 promoter), and the downstream primer was 5'-ggatcatattcgtccacaaaatgattct-3'.

[0057] Again, by in vitro transcription, a crRNA targeting the KRAS gene fragment was synthesized with the sequence 5'-GACCACCCCAAAAATGAAGGGGACTAAAACccaccagctccaactaccaagtttat-3', where the uppercase letters are the anchor sequence and the lowercase letters are the guide sequence, which recognizes the 5'- ataaacttgtggtagttggagctggtgg-3'.

[0058] Finally, construct a 50 μL reaction system:

...

Embodiment 2

[0077] In this example, the KRAS gene and the KRAS-G12D mutant gene are used as the target genes to illustrate the detection method (three-in-one) of the present invention for the detection of target genes, and to test the specificity of the method.

[0078]首先,通过引物合成后退火的方法,分别合成了KRAS基因片段以及KRAS-G12D突变片段作为检测的目的双链DNA,序列分别为5'-ctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagctaattcagaatcattttgtggacgaatatgatcc-3'和5'-ctgctgaaaatgactgaatataaacttgtggtagttggagctgAtggcgtaggcaagagtgccttgacgatacagctaattcagaatcattttgtggacgaatatgatcc-3',其中 Capital A is the mutant base.

[0079] Secondly, RPA primers were synthesized, the upstream primer was 5'-taatacgactcactataggctgctgaaaatgactgaatataaactt-3' (the 19 nucleotides at the 5' end were T7 promoter), and the downstream primer was 5'-ggatcatattcgtccacaaaatgattct-3'.

[0080] Thirdly, since the Cas13a protein can generally tolerate one base mismatch, we synthesized the crRNA-KRAS targeting the KRAS wild-type gene fragment by...

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Abstract

The invention relates to the detection of biological materials, and particularly discloses a rapid detection method based on CRISPR-Cas13a which is suitable for a specific nucleic acid fragment in a biological sample. A to-be-detected nucleic acid fragment is amplified to obtain a transcription product; a guide RNA (Ribonucleic Acid) is designed by aiming at a target RNA sequence of a target gene,and an RNA molecule is reported by a signal added in a detection system; the amplification and read of the signal are realized by using the RNA enzymatic activity of the Cas13a protein, which is activated after recognizing the target RNA sequence and is not limited by the sequence, further the detection of the target gene is realized. The detection sensitivity of the method provided by the invention can reach 10 to 18mol/L, namely ammol level, and the specificity can be achieved for detecting the nucleic acid fragments with single-base difference.

Description

technical field [0001] The invention relates to the detection of biological materials, in particular to a CRISPR-Cas13a-based rapid detection method suitable for specific nucleic acid fragments in biological samples. Background technique [0002] With the development of human medicine, people have gradually realized that the research and diagnosis of diseases at the genetic level is the key to the realization of the medical concept of individualized precision medicine. For example, the rapid detection of circulating tumor DNA in the blood of tumor patients and the determination of the mutated genes in it can not only realize the early diagnosis of tumors and the mapping of mutated gene profiles, but also facilitate the subsequent targeted drug therapy. Provide evidence to avoid drug waste and drug resistance; after isolating and extracting pathogens present in biological samples of patients with viral or bacterial infections, the rapid identification of their genes by using ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
Inventor 聂广军赵潇郎佳妍
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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