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Detection method and application of EBV based on CRISPR-Cas12a and G quadruplex-heme

A detection method, the technology of RPA-1, is applied in biochemical equipment and methods, measurement/inspection of microorganisms, and introduction of foreign genetic material using carriers, etc. It can solve problems such as time-consuming, cumbersome test procedures, and difficulty in meeting on-site detection. Achieve the effect of simple equipment, simple detection method and high accuracy

Active Publication Date: 2019-06-25
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 EBV based on CRISPR-Cas12a and G quadruplex-heme
  • Detection method and application of EBV based on CRISPR-Cas12a and G quadruplex-heme
  • Detection method and application of EBV based on CRISPR-Cas12a and G quadruplex-heme

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Experimental program
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Effect test

Embodiment 1

[0080] The principle of the RPA-Cas12a-G4 / Hemin detection method: After Cas12a forms a ternary complex with EBV crRNA and the target DNA amplified by RPA, the activated Cas12a can not only cut the target EBV DNA, but also convert the PS2. Strand fragmentation prevents PS2.M from forming G-quadruplexes and G-quadruplex / Hemin complexes, inhibiting ABTS2-H 2 o 2 The redox reaction of the system, the absorbance response value of the system decreases sharply. The present invention is thus a "Turn-off" detection mode.

[0081] 1. Sensitivity detection of RPA-Cas12a-G4 / Hemin detection method

[0082] (1) Dilute the recombinant plasmid PUC57 / Bam recombinant plasmid so that its concentration is 0, 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 copies / ul (copies / μl).

[0083] (2) RPA amplification: the above-mentioned PUC57 / Bam recombinant plasmids with different concentrations were used as templates, and RPA amplification was performed with EBV RPA primers. The RPA reaction...

Embodiment 2

[0087] Example 2 RPA-Cas12a-G4 / Hemin detection method detection effect evaluation

[0088] Serum samples were collected: 6 serum samples from EBV-infected patients and 6 healthy individuals. All were confirmed by qPCR detection.

[0089] 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:

[0090] (1) Add 20 μl proteinase K (Proteinase K) into a clean 1.5ml centrifuge tube.

[0091] (2) Add 200 μl of human serum sample into the centrifuge tube.

[0092] (3) Add 200 μl of Carrier RNA working solution, and vortex for 15 sec to mix.

[0093] (4) Incubate at 56°C for 15 minutes.

[0094] (5) Add 250 μl of absolute ethanol, vortex for 15 sec, and mix thoroughly. Place at room temperature for 5min.

[0095] (6) Briefly centrifuge to collect the liquid attached to the tube wall and cap.

[0096] (7) Carefully transfer all the solu...

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Abstract

The invention provides a detection method and application of EBV based on CRISPR-Cas12a and G quadruplex-heme. The method comprises the following steps: taking DNA extracted from EBV patient serum asa template, adding an RPA reaction system containing an amplification primer pair, a re-swelling buffer solution and ddH2O into freeze-dried enzyme powder, then adding MgAc, uniformly mixing and incubating to obtain a PRA product; dissolving PS2. M DNA in a Tris-HCL solution, adding a Hemin solution into the solution to prepare a PS2.M / Hemin solution; adding FnCas12a and EBV crRNA to the Tris-HClbuffer solution, executing incubation to prepare a Cas12a / crRNA complex solution; incubating an RPA product, the PS2.M / Hemin solution, the Cas12a / crRNA complex solution and determining absorbance after treatment with ABTS and H202. The detection method is rapid, 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 EBV based on CRISPR-Cas12a and G quadruplex-heme. Background technique [0002] B virus (Epstein-Barrvirus; EBV) belongs to the herpes virus family, mainly invades B lymphocytes, causes infectious mononucleosis, and is closely related to the occurrence of various lymphomas. EBV is widely infected in the population, and more than 90% of the adult serum EBV antibodies are positive. [0003] Under physiological conditions, under the action of the body's immune system, EBV reactivation and antiviral immune control of EBV-specific CD8+ T cells are in a state of balance, so that EBV is in an inactive state and remains latent in human lymphoid tissue for a long time. Lifelong carrier of the virus without disease. Previous EBV detection methods mainly include detection of EBV-specific antibodies in serum by immunoenzyme sta...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6806C12N15/70
Inventor 邓敏刘万里邢珊
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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