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Detection method and application of HBV based on CRISPR-Cas12a and G-quadruplex hemin

A detection method, the technology of RPA-1, is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Accuracy, simple equipment, low cost effect

Active Publication Date: 2019-06-04
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, conventional PCR detection requires sophisticated instruments and cumbersome test procedures, and takes a long time, which is difficult to meet the requirements of on-site detection in non-laboratory environments

Method used

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  • Detection method and application of HBV based on CRISPR-Cas12a and G-quadruplex hemin
  • Detection method and application of HBV based on CRISPR-Cas12a and G-quadruplex hemin
  • Detection method and application of HBV based on CRISPR-Cas12a and G-quadruplex hemin

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Experimental program
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Effect test

Embodiment 1

[0091] The principle of the RPA-Cas12a-G4 / Hemin detection method: After Cas12a forms a ternary complex with HBV crRNA and RPA-amplified target DNA, the activated Cas12a can not only cut the target HBV DNA, but also convert PS2. Strand fragmentation prevents PS2.M from forming G-quadruplexes and G-quadruplex / Hemin complexes, inhibiting ABTS2-H 2 o 2The redox reaction of the system, the absorbance response value of the system decreases sharply. The present invention is thus a "Turn-off" detection mode.

[0092] Preparation of HBV positive control substance: the recombinant plasmid containing HBsAg gene fragment was used as positive control. Preparation method: Synthesize HBsAg gene fragment (its nucleotide sequence is shown in SEQ ID NO.3) by chemical synthesis method, and clone into PUC57 vector to obtain recombinant plasmid. The inserted sequence was confirmed to be correct by Sanger sequencing. Cultivate the recombinant plasmid bacteria overnight at 37 degrees, extract th...

Embodiment 2

[0099] Example 2 RPA-Cas12a-G4 / Hemin detection method detection effect evaluation

[0100] Serum samples were collected: 5 serum samples from HBV patients and 5 healthy people. All were confirmed by qPCR detection.

[0101] Viral nucleic acid extraction: The viral genome DNA / RNA extraction kit (DP315) of Beijing Tiangen Biochemical Technology Co., Ltd. was used for nucleic acid extraction. The main steps are as follows:

[0102] (1) Add 20 μl proteinase K (Proteinase K) into a clean 1.5ml centrifuge tube.

[0103] (2) Add 200 μl of human serum sample into the centrifuge tube.

[0104] (3) Add 200 μl of Carrier RNA working solution, and vortex for 15 sec to mix.

[0105] (4) Incubate at 56°C for 15 minutes.

[0106] (5) Add 250 μl of absolute ethanol, vortex for 15 sec, and mix thoroughly. Place at room temperature for 5min.

[0107] (6) Briefly centrifuge to collect the liquid attached to the tube wall and cap.

[0108] (7) Carefully transfer all the solution and flocc...

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Abstract

The present invention provides a detection method and an application of HBV based on CRISPR-Cas12a and G-quadruplex hemin. Processes are as follows: DNA extracted from serum of HBV patients is used asa template, a RPA reaction system comprising an amplification primer pair, rehydration buffer and ddH2O is added to lyophozyme powder, then MgAc is added, the materials are mixed evenly and the mixture is incubated to obtain a RPA product; PS2.M DNA is dissolved in a Tris-HCL solution and a Hemin solution is added to the solution to obtain a PS2.M / Hemin solution; FnCas12a and HBV crRNA are addedto a Tris-HCl buffer and incubated to prepare a Cas12a / crRNA complex solution; and the RPA product, PS2.M / Hemin solution and Cas12a / crRNA complex solution are incubated, and ABTS and H2O2 are added for treatment, and absorbance is measured. The detection method is fast, low in cost, high in accuracy, high in sensitivity and simple in equipment.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection method and application of HBV based on CRISPR-Cas12a and G quadruplex-heme. Background technique [0002] Hepatitis is a serious disease that is widely prevalent in the world and seriously endangers human health and even life. At present, it is mainly divided into hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, and hepatitis G. According to the route of transmission, it can be divided into two categories. Among them, the main routes of transmission of hepatitis B, C, D, and G are blood transmission, iatrogenic transmission, mother-to-child transmission, and sexual transmission. Among them, blood transmission and sexual transmission Transmission is the most common route. Hepatitis A and E are mainly transmitted by the fecal-oral route. [0003] In 1985, when the PCR method became a routine experimental method, it led to expon...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
Inventor 刘万里邓敏邢珊
Owner SUN YAT SEN UNIV CANCER CENT
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