Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit

A detection kit and detection method technology are applied in the field of human papillomavirus rapid detection kits, which can solve the problems of difficulty in popularization, difficulty in interpretation of results, and inability to interpret results in visualization, and achieve convenient operation and eliminate non-specific color development. , the effect of easy operation

Pending Publication Date: 2020-08-21
亚能生物技术(深圳)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although these CRISPR / Cas-based technologies can achieve rapid and sensitive detection of nucleic acid molecules, they still need the help of a fluorescence detector, and the results cannot be interpreted visually, which brings difficulties to the popularization of this technology in grassroots inspection institutions
[0010] At present, there are examples of using nucleic acid test strips to interpret the results, but the test strips used will produce serious non-specific color development, which brings great difficulties to the result interpretation

Method used

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  • Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit
  • Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit
  • Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit

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Embodiment 1

[0074] A detection method based on CRISPR / Cas12 and nucleic acid test paper, the following takes the detection of human β-actin (ACTB) as an example to describe the operation steps of the method:

[0075] 1) Search for the 5'-TTTN-3' sequence in the human β-actin region to design and synthesize crRNA; design primer sequences upstream and downstream of crRNA, and the crRNA and primers used in the present invention were synthesized by Shanghai Sangong. Synthesize and use thio-modified reporter molecules, and make nucleic acid test strips, the structure and combination of reporter molecules and test strips used in the present invention are as follows figure 1 and figure 2 shown.

[0076] The primers, crRNA, and reporter sequences used in this example are shown in Table 1 below:

[0077] Table 1

[0078]

[0079] 2) Samples from healthy volunteers were taken with throat swabs, added to the rapid lysis solution formulated in Table 2 below, and lysed at 80° C. for 5 minutes. ...

Embodiment 2

[0093] A human papillomavirus detection kit, including crRNA, Cas12, RPA upstream and downstream primers, buffer system and single-stranded DNA reporter molecule; specific design and use are as follows:

[0094] 1) Search for the 5'-TTTN-3' sequence in the conserved L1 gene region of human papillomavirus, design and synthesize crRNA; design primer sequences upstream and downstream of crRNA, the crRNA and primers used in the present invention are synthesized by Shanghai Sangong . Synthesize and modify the reporter molecules, and make nucleic acid test strips, the structures and combinations of the reporter molecules and test strips used in the present invention are as follows: Figure 5 and Image 6 shown.

[0095] In this example, primers, crRNA, and reporter molecule sequences are selected as shown in Table 5 below.

[0096] table 5

[0097]

[0098] 2) Take RPA reaction particles, and add appropriate proportions of RPA hydration buffer, RPA upstream and downstream pri...

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Abstract

The invention relates to the technical field of gene detection, in particular to a nucleic acid detection method based on CRISPR / Cas and nucleic acid test paper and a human papilloma virus rapid detection kit. The human papilloma virus detection kit comprises crRNA, Cas12, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule, the sequence of the reportermolecule is 5 '-NNN......-3', the length of the reporter molecule is 3-30 basic groups, FAM, DIG and Biotin labeling is carried out, and meanwhile modification for preventing DNA enzyme or cas12 degradation after activation is carried out on nucleic acid molecules. The CRISPR / Cas and nucleic acid test paper-based nucleic acid detection method and the human papilloma virus rapid detection kit havethe advantages that innovative reporter molecule design and modification are adopted, and corresponding test paper strips are combined, so that non-specific color development is completely eradicated, and the kit has remarkable significance in clinical practical application.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a nucleic acid detection method based on CRISPR / Cas and nucleic acid test paper and a rapid detection kit for human papillomavirus. Background technique [0002] The diagnostic industry is an important part of the biopharmaceutical industry. China's diagnostic industry is currently in the initial stage of investment in product development and production, and the growth period has not yet come. China's current diagnostics market is less than 1 / 10 of the global market. Generally speaking, currently, in projects with wide clinical application and broad market prospects (such as hepatitis, sexually transmitted diseases and pregnancy test series in immune reagents, enzymes, lipids, liver function, blood sugar, urine test series in clinical biochemistry, etc.) ), the technical level of major domestic manufacturers has basically reached the international advanced level; the PCR ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/708C12Q1/6844C12Q2521/507C12Q2521/327C12Q2527/101C12Q2565/625
Inventor 汤光辉龚姣姣薛良
Owner 亚能生物技术(深圳)有限公司
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