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Method for detecting cardiac troponin I based on CRISPR Cas13d

A technique for cardiac troponin and analyte, applied in the field of fluorescence detection, can solve the problem of detecting non-nucleic acid molecules without Cas13d, and achieve the effect of improving accuracy

Pending Publication Date: 2022-08-02
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many reports on its in vitro detection application, and there is currently no literature report on the detection of non-nucleic acid molecules based on Cas13d

Method used

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  • Method for detecting cardiac troponin I based on CRISPR Cas13d
  • Method for detecting cardiac troponin I based on CRISPR Cas13d
  • Method for detecting cardiac troponin I based on CRISPR Cas13d

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] CRISPR RspCas13d protein expression and purification, the specific operation includes the following steps:

[0057] 1. 3 μl of pET28a-MH6-RspCas13d plasmid and E. coli DE3 competent cell BL21 (50 μl) were placed in an ice bath and incubated in a clean bench for 10 min;

[0058] 2. After the competent cells were heat-shocked in a 42°C water bath for 90s, they were immediately taken out and placed in an ice bath to cool for 5min;

[0059] 3. Add 450 μL of anti-antibody LB liquid medium to the competent cells introduced into the plasmid in step 2, invert and mix, and incubate at 37°C with shaking at 220 rpm for 1 hour;

[0060] 4. Centrifuge and aspirate 400 μL of the supernatant of the medium in step 3 and discard it, and resuspend the remaining bacterial liquid, spread it evenly on the AMP-resistant LB solid plate medium, and cultivate it upside down at 37 degrees for 12 hours;

[0061] 5. Pick a single clone into 5ml of AMP-resistant LB liquid medium and culture at 37°...

Embodiment 2

[0069] CRISPR RNA synthesis, the specific operation includes the following steps:

[0070] The CRISPR RNA required for the present invention was obtained by HyperScribe TM T7 High Yield RNA SynthesisKit (Apexbio) was synthesized by in vitro transcription.

[0071]

[0072] The reagents required for the synthesis process are shown in the table above, and the main steps are as follows:

[0073] 1. Prepare crRNA-F and crRNA-R DNA primers in DEPC water (see the primer sequence list) and determine the concentration to 10 μM in a micro UV spectrophotometer;

[0074] 2. The above ssDNA was annealed in annealing buffer (20 mM Tris-HCl, pH=8.0, 100 mM NaCl, 8 mM MgCl at a ratio of 1:1) 2 ) annealed and assembled into double-stranded DNA;

[0075] 3. The DNA double-stranded DNA in step 2 is mixed according to the added amount of each reagent in the table above, and then mixed with the pipette tip and incubated at 37 degrees for 16 hours;

[0076] 4. Add 3 μL of DNase I 10× react...

Embodiment 3

[0079] CRISPR RNA purification by spin column method, the specific operation includes the following steps:

[0080] The RNA purification kit used in this example is Monarch RNA Cleanup Kit T2040 (50 μg) (NewEngland Biolabs). RNA is bound to the silica adsorption column by a high-concentration salt ion binding buffer, and washed with a low-salt ion elution buffer. The impurities bound to the silica column are removed, so as to achieve the purpose of RNA purification.

[0081] Refer to the manufacturer's instructions for the specific operation process. The main process is as follows:

[0082] 1. Add 20 μL of RNase-free double-distilled water to the Mix obtained in step 5 in Example 2 to a volume of 50 μL;

[0083] 2. Add 100 μL RNACleanup binding buffer to Mix in step 1;

[0084] 3. Add 150μL of ethanol (purity > 95%), mix by pipetting with pipette tip;

[0085] 4. Transfer all the mixture in 3 to the spin column, centrifuge at 12000rpm for 30s, and discard the flow-through l...

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Abstract

The invention discloses a method for detecting an acute myocardial infarction marker cTnI (cardiac troponin I) based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas13d gene editing protein. A sample is incubated on an elisa plate to form an antibody-cTnI compound, two ssDNAs are annealed to form a double-chain cTnI DNA aptamer with a T7 promoter sequence, and the aptamer and the cTnI-antibody compound are incubated and combined to form an antibody-cTnI-aptamer ternary sandwich structure. The structure can be recognized by T7 RNA polymerase, transcripts and expresses activator RNA, and can be recognized by a CRISPR Cas13d-crRNA compound, and the side cutting activity generated by activation can effectively cut RNA fluorescent reporter molecules to generate certain fluorescent signals. The Cas13d gene editing protein is applied to accurate detection of the cardiac marker cTnI for the first time.

Description

technical field [0001] The invention belongs to biological detection technology, in particular to a fluorescence detection method for detecting cardiac troponin I in vitro by CRISPR Cas13d gene editing protein. Background technique [0002] Acute myocardial infarction (AMI) is a common clinical heart disease caused by acute coronary occlusion and blood flow interruption caused by myocardial ischemic necrosis. Because of its high lethality and harm, the accurate diagnosis of AMI has very important clinical value. Current specific myocardial markers for monitoring AMI are myoglobin (MYO), creatine kinase isoenzyme (CK-MB) and cardiac troponin I (cTnI). Among them, cTnI has the best myocardial specificity, and after the occurrence of AMI, cardiac troponin I (cTnI) in myocardial tissue is rapidly released into the blood and can maintain a time window of 5-10 days, so it can be used as a cardiac injury Pre-screening is also one of the most effective ways to diagnose AMI. It is...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58C12N15/115C12N15/113
CPCG01N33/6893G01N33/581G01N33/582C12N15/115C12N15/113G01N2800/324G01N2333/4712C12N2310/16C12N2310/20
Inventor 冯致远张晶晶
Owner NANJING UNIV
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