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CrRNA and CRISPR-Cas12a system for carbapenemase detection and application

A carbapenemase and protein technology, applied in the field of CRISPR-Cas12a system and CrRNA, can solve the problems of complicated operation, long time required, difficult to carry out in ordinary laboratories, etc., and achieves low cost and shortened detection time. Effect

Pending Publication Date: 2021-09-28
安徽医科大学第四附属医院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, clinical drug susceptibility testing methods for such strains include disc diffusion method, E-test, broth microdilution method, and microbial automated drug susceptibility testing method; phenotypic testing methods include CarbaNP test, mCIM test (modified carbon blue Mycelene inactivation test) and eCIM test (EDTA carbapenem inactivation test) test, enzyme inhibitor enhancement test, etc.; genotype detection methods include PCR, multiplex PCR, nested PCR, RT-PCR and microfluidic technology Combined to detect genotype or identify by sequencing; due to the need for expensive laboratory instruments, and these methods require a long time and complicated operations, it is difficult for ordinary laboratories to carry out

Method used

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  • CrRNA and CRISPR-Cas12a system for carbapenemase detection and application
  • CrRNA and CRISPR-Cas12a system for carbapenemase detection and application
  • CrRNA and CRISPR-Cas12a system for carbapenemase detection and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] A CrRNA used for carbapenemase detection in this embodiment.

[0040] The core of the CRISPR-Cas12a detection method lies in CrRNA, so the selection of CrRNA is directly related to the effectiveness of the final detection method.

[0041] The base sequence of CrRNA in this example is shown in SEQ ID NO.1, and the design idea is shown in figure 1(The design of crRNA is obtained according to the 20 bases behind the PAM TTTN site recognized by Cas12a). The CrRNA is used to guide the Cas12a protein to recognize and bind to the sequence amplified by LAMP to cut the target sequence. At the same time, the Cas12a protein trans-cuts any single-stranded DNA in the reaction system.

[0042] Specifically, any single-stranded DNA in the reaction system is mainly the LAMP amplification primer in the CRISPR-Cas12a system, including primer F3, primer B3, primer BIP, primer FIP, primer LB, and primer LF, and their sequences are as follows: Shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO....

Embodiment 2

[0044] A CRISPR-Cas12a system for detecting carbapenemase in this embodiment includes Cas12a protein, crRNA, fluorescein isothiocyanate-quencher double-labeled probe, and LAMP amplification product. Wherein, the amplification primers of the LAMP amplification product include: primer F3, primer B3, primer BIP, primer FIP, primer LB, primer LF, the sequences are respectively as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, Shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7.

[0045] At the same time, the CRISPR-Cas12a system also includes the reagents required for the LAMP amplification system and the Cas12a cleavage reaction system.

[0046] Specifically, the LAMP amplification system is as follows: primer F3 (0.2 μM), primer B3 (0.2 μM), primer BIP (1.6 μM), primer FIP (1.6 μM), primer LB (0.4 μM), primer LF (0.4 μM) , 1.4mM dNTP, 6mM MgSO 4 , 1× isothermal amplification buffer, 320U / mL Bst 2.0 warm-start DNA polymerase, fluorescein isothiocyanate, 2 μL target DNA template, and add ddH...

Embodiment 3

[0049] A method for the rapid detection of carbapenemase based on CRISPR-Cas12a of this embodiment, using the CRISPR-Cas12a system of Example 2 to detect the sample to be tested, the method is as follows:

[0050] 1. Extraction of DNA from samples to be tested:

[0051] The sample DNA was crudely extracted by boiling method and used as the DNA template for the subsequent LAMP reaction.

[0052] 2. Primer design and LAMP amplification:

[0053] (1) Use primer explorerV5 to design the nucleotide sequence LAMP primer and CrRNA of the target gene fragment NDM and send it to the company for synthesis. Wherein, the target gene fragment NDM is the nucleotide sequence starting from the 563 position of the carbapenemase NDM gene corresponding to the 5' end, and the outer primers F3 and B3 sequences (5'-3') of the nucleotide sequence As shown in SEQ ID NO.2 and SEQ ID NO.3, the sequences (5'-3') of the internal primers FIP and BIP are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0054] (2...

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Abstract

The invention relates to the technical field of medical treatment, and provides CrRNA for carbapenemase detection with the base sequence as shown in SEQ ID NO. 1. The CrRNA is used for guiding Cas12a protein identification and combined to a LAMP amplified sequence to cut a target sequence, and meanwhile, the Cas12a protein is used for trans-cleavage of any single-stranded DNA in a reaction system. The invention further provides a CRISPR-Cas12a system and a method for rapidly detecting the carbapenemase based on the CRISPR-Cas12a, as well as application of the CRISPR-Cas12a system in the preparation of a carbapenemase detection kit. The method can be used as a clinical large-scale crowd detection method. Compared with a traditional diagnosis detection mode, the detection mode has the advantages that the detection time is greatly shortened, operators are not needed to be repeatedly trained, and cost is low.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a CrRNA and CRISPR-Cas12a system and application for carbapenemase detection. Background technique [0002] Klebsiella pneumoniae is the most important type of bacteria in the Enterobacteriaceae Klebsiella genus (commonly known as Klebsiella pneumoniae), and the diseases caused by it account for more than 95% of Klebsiella infections. After Klebsiella pneumoniae infects the human body, symptoms such as cough, sputum, fever, chills, and dyspnea will appear. The "carbapenemase resistance gene" carried by Klebsiella pneumoniae to antibiotic resistance is currently a hot spot of global concern question. This type of drug-resistant bacteria has a strong ability to spread in the hospital, and the clinical treatment of the infection caused by it is limited and the effect is not good. Therefore, how to quickly, effectively and conveniently carry out molecular detection is very importa...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/04C12N15/11C12N15/113C12N9/22
CPCC12Q1/6844C12Q1/689C12N15/113C12N9/22C12N2310/20C12Q2531/119C12Q2521/327C12Q2563/107C12Q2565/625
Inventor 沈继录徐华铭唐浩夏兆新杨文苏朱毅
Owner 安徽医科大学第四附属医院
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