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Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system

A Mycobacterium tuberculosis detection kit technology, applied in the field of Mycobacterium tuberculosis complex detection kits, can solve the problems of expensive instruments and equipment, difficult to popularize in low- and middle-income areas, and lack of detection methods for amplification products, etc., to achieve Get rid of the dependence on precision instruments, improve the detection sensitivity, and broaden the effect of application prospects

Active Publication Date: 2019-12-06
MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Gene Xpert automatic detection system recommended by the WHO uses PCR technology to detect the Mycobacterium tuberculosis complex. The sensitivity and specificity are good, and there is no need to wait for a long time for the results. However, the equipment is expensive and difficult to use in low- and middle-income areas. universal
In recent years, various constant and isothermal amplification technologies such as LAMP and RPA have emerged, which can be used for on-site detection, but there are problems such as the lack of effective detection methods for amplification products.

Method used

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  • Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system
  • Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system
  • Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: CRISPR-Cas12a protein expression, purification, activity verification

[0063] 1) Purchase the expression LbCas12a protein plasmid from addgene, transform the BL21 strain, and culture the LB plate at 37°C overnight;

[0064] 2) Pick a single bacterium from the LB plate and insert it into 5mL LB medium (100ug / mL Amp), culture at 37°C for 3-5h;

[0065] 3) Put 5mL of bacterial liquid into 1L LB liquid medium (100ug / mL Amp), culture at 37°C until OD60≈0.6, add 0.3mmol / L IPTG, and induce at 16°C for 16h;

[0066] 4) Put the cultured bacterial liquid into a centrifuge, centrifuge at 5000r / min for 10-15min, discard the supernatant, collect the bacterial cells, and suspend the bacterial cell pellet after centrifugation in 25mL lysis buffer (50mM Tris-HCl, pH 7.5, 500mM NaCl, 5% (v / v) glycerol, 1mM TCEP, 0.5mM PMSF and 0.25mg / ml lysozyme);

[0067] 5) Place in ice bath, ultrasonic crushing, 26 times per cycle, ultrasonic 6s, interval 5s, a total of 2 to 4 cycles ac...

Embodiment 2

[0074] Example 2: CRISPR-Cas12a detection of Mycobacterium tuberculosis complex target sequence selection and gRNA, amplification primer screening

[0075] 1) Target selection: Compared with the insertion sequence IS6110, although the copy number of the insertion sequence IS1081 in the Mycobacterium tuberculosis complex is relatively low (5-7 repeated sequences), it exists in all the Mycobacterium tuberculosis complexes. In order to avoid missed detection as much as possible, the insertion sequence IS1081 was selected as the target for the primer design of the recombinase polymerase amplification reaction and the target for CRISPR-Cas12a targeted detection.

[0076] 2) gRNA screening: Obtain the insert sequence IS1081 sequence information from NCBI, use CRISPR-DT software to design and score gRNAs, select gRNAs with a score not lower than 0.5, and use IS1081 plasmid to screen suitable gRNAs. Specifically, the 20uL system contains 1×buffer (50 mM NaCl, 10mM Tris-HCl, 10mM MgCl ...

Embodiment 3

[0089] Embodiment 3: Reaction system sensitivity, specificity investigation

[0090] 1) Sensitivity investigation of the reaction system: the IS1081 plasmid was used as a template, and a series of serial dilutions (448.3 amol, 44.83 amol, 4.483 amol, 0.4483 amol, 0.08966 amol, 0.04483 amol) were performed, and the nucleic acid DNA of non-tuberculous mycobacteria was used as a negative template , with H 2 O was used as a blank control, and each was repeated 3 times to investigate the sensitivity of the system. The result is as image 3 shown. Its minimum detection limit is 0.04483 amol, and its linear regression equation is Y=136613+84616x, R 2 = 0.973.

[0091] 2) Specificity investigation of the reaction system: select Pseudomonas aeruginosa, Escherichia coli, Mycoplasma pneumoniae, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Mycobacterium intracellulare, avium Mycobacterium, Mycobacterium kansasii, Mycobacterium scrofu...

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Abstract

The invention belongs to the technical field of nucleic acid detection, and relates to a mycobacterium tuberculosis complex detection kit and method based on a CRISPR-Cas12a system. According to the kit, the detection sensitivity is improved by adopting a recombinase polymerase amplification technology, CRISPR-Cas12a is used for specifically targeting a mycobacterium tuberculosis complex target sequence and then activating the bypass cutting activity of Cas12a, and a mycobacterium tuberculosis complex can be sensitively and specifically detected from sputum. The kit and method have the advantages of being non-invasive, capable of detecting the mycobacterium tuberculosis complex frequently and repeatedly, high in detection speed and the like. Compared with an existing PCR detection method,the method based on the CRISPR-Cas12a system does not need a thermal cycler with heating and cooling functions, can detect the mycobacterium tuberculosis complex in sputum through fluorescence reading, has the advantages of being low in cost, easy and convenient to operate, high in sensitivity, good in specificity and the like and is suitable for clinical large-scale application.

Description

[0001] (1) Technical field [0002] The invention relates to a detection kit and detection method for Mycobacterium tuberculosis complex based on CRISPR-Cas12a system. [0003] (2) Background technology [0004] Tuberculosis (TB) is an infectious disease that threatens human health for a long time. Its pathogen is Mycobacterium tuberculosis, and about one-third of the world's population is infected with Mycobacterium tuberculosis. According to the statistics of the World Health Organization, there were nearly 10 million new cases of tuberculosis in the world in 2017, and about 1.6 million people died of tuberculosis. China is one of the 22 countries with a high burden of tuberculosis in the world, and the number of tuberculosis patients ranks second in the world. There are about 1 million new cases in my country every year, and about 130,000 people die of tuberculosis. Therefore, tuberculosis is still a global health problem that threatens human health. [0005] Studies have...

Claims

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Application Information

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IPC IPC(8): C12Q1/6848C12Q1/689C12Q1/04
CPCC12Q1/6848C12Q1/689C12Q2521/507C12Q2531/119C12Q2522/101C12Q2537/1376C12Q2521/301C12Q2563/107
Inventor 刘景丰刘小龙许海坡蔡志雄董秀清
Owner MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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