Tobramycin detection system and method based on CRISPR-Cas12a

A technology of tobramycin and detection system, applied in measurement devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of high false positive rate, long operation time, lack of ultraviolet chromophore, etc. The effect of visualization and quantitative detection

Active Publication Date: 2021-11-12
南京百利特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Detection of tobramycin remains challenging due to aminoglycoside antibiotics lacking a UV chromophore or fluorophore
In addition, traditional detection methods often re...

Method used

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  • Tobramycin detection system and method based on CRISPR-Cas12a
  • Tobramycin detection system and method based on CRISPR-Cas12a
  • Tobramycin detection system and method based on CRISPR-Cas12a

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Embodiment 1

[0035]A CRISPR-Cas12a-based tobramycin detection system, including: aptamer probe AP, CrRNA, AsCas12a, KF polymerase, and reporter probes modified with fluorescent groups and quenching groups at both ends;

[0036] The sequence of the aptamer probe AP is: 5'-ATC ATT TGG AGG AAC TGG AGT CAC AAG CTGAGG ATG TGA CTC CAG GCA CTT AGT CAC A-3' (SEQ ID NO: 1);

[0037] The sequence of the CrRNA is: 5'-UAA UUU CUA CUC UUG UAG AUG CUU GUG ACU CCA GUUCCU C-3' (SEQ ID NO: 2);

[0038] The sequence of the reporter probe is: 5'-FAM-TTATT-BHQ1-3'.

[0039] The detection principle diagram of the tobramycin detection system of the present invention is shown in figure 1 . The designed aptamer probe AP of the present invention comprises two functional areas: one is the aptamer sequence of tobramycin, and the other is a signal transduction sequence used for KF polymerase binding and extension for signal amplification, These two regions are designed to partially hybridize to form a stem-loop st...

Embodiment 2

[0049] The present invention uses the CRISPR-Cas12a system as the signal output element, and the concentration of crRNA directly leads to the sensitivity of the CRISPR-Cas12a system to recognize the trigger chain. In order to explore the optimal crRNA concentration of the CRISPR-Cas12a system, different concentrations of crRNA (0nM, 5nM, 10nM, 15nM, 20nM, 25nM, 30nM, 50nM, 80nM, 100nM, 200nM), constant double-strand trigger strand (ds- Activator) at a concentration of 1 nM. The result is as figure 2 As shown, the fluorescence intensity reaches the highest when the crRNA concentration is 20nM, and the fluorescence intensity will decrease when it is greater than 20nM, so the crRNA concentration of 20nM is used for analysis in the present invention to ensure higher detection efficiency.

Embodiment 3

[0051] The response of the CRISPR-Cas12a system to different concentrations of the trigger chain: In order to verify the characteristics of the CRISPR-Cas12a system, different concentrations of the trigger chain (ds-Activator) were designed to explore the concentration response range and the minimum concentration of the CRISPR-Cas12a system to the trigger chain. The concentration of the response. Such as image 3 The fluorescence intensity curves generated by the CRISPR-Cas12a system under different concentrations of the trigger chain. It can be seen that when the excitation wavelength is 480nm, there is an emission peak at 520nm. According to the fluorescence intensity at 520nm, the correlation between the fluorescence intensity measured within the trigger chain concentration range and the trigger chain concentration can be drawn. The result is as follows Figure 4 shown. Figure 4 The inset of the graph shows a linear relationship between the fluorescence intensity and the...

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Abstract

The invention discloses a tobramycin detection system based on CRISPR-Cas12a. The tobramycin detection system comprises an aptamer probe AP, CrRNA (Ribonucleic Acid), AsCas12a protein, KF polymerase and a reporter probe, wherein two ends of the reporter probe are respectively modified with a fluorophore and a quenching group; the sequence of the aptamer probe AP is as shown in SEQ ID NO:1; and the sequence of the CrRNA is as shown in SEQ ID NO:2. Compared with the prior art, the aptamer probe in the detection system can be specifically combined with tobramycin and change the configuration, trigger DNA capable of being recognized by the CRISPR-Cas12a system is generated under the action of KF polymerase, finally, the fluorescent probe is cut in the CRISPR-Cas12a system to generate a fluorescence signal, and through blue light irradiation and fluorescence intensity analysis, visual and quantitative detection of tobramycin can be realized, and the detection sensitivity is high.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a CRISPR-Cas12a-based tobramycin detection system and detection method. Background technique [0002] The CRISPR-Cas system is a type of adaptive immune system present in bacteria and archaea. In recent years, the CRISPR-Cas system has been widely studied and has become a molecular biology tool that can efficiently cut target genes. In addition to genome editing, CRISPR-Cas systems have also been applied to bioassays. For example, SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter) biosensors have realized the use of CRISPR-Cas system to detect nucleic acid targets, and then applied to Zika virus, new coronavirus, etc. Infectious disease quarantine work. Unlike the Cas9 protein, Cas12a can bind to single-stranded guide CRISPR RNA (crRNA) to form a complex (CRISPR-Cas12a), which...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/64G01N21/6428G01N21/643G01N2021/6417Y02A50/30
Inventor 李大为凌燊吕蓓
Owner 南京百利特生物科技有限公司
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