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CRISPR-Cas13 nucleic acid detection kit based on lightened RNA aptamer

A detection kit and the technology of the kit are applied in the field of nucleic acid detection, which can solve the problems of complicated operation process and high detection cost, and achieve the effects of simplified operation process, high sensitivity and cost saving.

Active Publication Date: 2021-04-06
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of CN107557455A has the following limitations: the preparation of its signal molecule needs to label fluorescent groups and quenching groups at both ends of the RNA, and at the same time, it is necessary to extract the RNA and first reverse transcribe it into DNA and then amplify it in the detection of the actual sample, resulting in detection The cost is extremely expensive and the operation process is also very complicated
[0008] At present, there is no technology for detecting nucleic acids that combines light-up RNA aptamers and CRISPR-Cas systems

Method used

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  • CRISPR-Cas13 nucleic acid detection kit based on lightened RNA aptamer
  • CRISPR-Cas13 nucleic acid detection kit based on lightened RNA aptamer
  • CRISPR-Cas13 nucleic acid detection kit based on lightened RNA aptamer

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1 Preparation of light-up RNA aptamer and signal recognition probe crRNA

[0069] In this example, the Broccoli aptamer was used as an example to prepare a bright-type RNA aptamer, and to prepare CRISPR-RNA (crRNA). The light-up RNA aptamer Broccoli aptamer can specifically bind to the nucleic acid dye DFHBI-1T as a beacon for Cas13 digestion. The signal recognition probe crRNA can specifically bind to the target RNA and play the role of molecular recognition.

[0070] Use the method of in vitro transcription to obtain Broccoli aptamer and crRNA, the steps are as follows:

[0071] Into a 100 μL centrifuge tube, add 10 μL 10×phi29 DNA polymerase buffer, 10 μL L-Broccoli aptamer, 10 μL promoter, denature at 90°C for 3-10 minutes, and react at room temperature for 30-90 minutes. Add 3 μL phi29 DNA polymerase, 2 μL dNTP mixture, and extend it to complete complementarity at 30°C for 30-90 minutes. Finally, add 20 μL of 5× transcription buffer, 2 μL of T7 RNA polym...

Embodiment 2

[0076] Embodiment 2 draws standard curve

[0077] Proceed as follows:

[0078]① Cultivate Bacillus cereus (B. cereus) at 37°C until the late logarithmic growth phase, and prepare live bacterial solutions with different concentrations using the gradient coefficient of normal saline to extract RNA.

[0079] ② Take 10 μL of the Broccoli aptamer prepared in Example 1, 3 μL of crRNA, and 4 μL of DFHBI-1T, add the RNA solution extracted from different concentrations of Bacillus cereus obtained in ①, and add 0.3 μL of Cas13 solution (1 pmol / μL) , add 19.7μL of water, and react at 37°C for 30min.

[0080] ③ Fluorescence was detected at an excitation wavelength of 468nm and an emission wavelength range of 498-560nm, with a detection step of 1nm, and the fluorescence value at the concentration of viable bacteria was recorded.

[0081] ④ Use the concentration of Bacillus cereus as the abscissa and the fluorescence value at this concentration as the ordinate to draw a standard curve. T...

Embodiment 3

[0083] Bacillus cereus quantity detection in embodiment 3 milk and rice

[0084] Add Bacillus cereus with different concentrations of viable bacteria to milk and rice, detect the number of viable bacteria in the sample after 48 hours of culture, and analyze the significant difference between each group and the sample with 100% viable bacteria. The steps are as follows:

[0085] ① Take 40g of cooked rice and 40ml of commercially available pure milk and put them into 4 Erlenmeyer flasks, and sterilize at 121°C for 20min.

[0086] ② Dilute the Bacillus cereus in the late logarithmic growth period to 10% with 0.85% normal saline. 5 CFU / mL.

[0087] ③ Centrifuge the bacterial solution prepared in ②, resuspend the bacterial solution with 70% isopropanol and normal saline respectively, treat at room temperature for 1 hour, centrifuge, and resuspend with normal saline to obtain dead bacteria and live bacteria.

[0088] ④ Take the viable and dead bacteria liquids prepared in ③, and m...

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Abstract

The invention discloses a CRISPR-Cas13 nucleic acid detection kit based on a lightening type RNA aptamer. The kit comprises the lightening type RNA aptamer, Cas13 protein, crRNA and nucleic acid dye; the lightening type RNA aptamer can be combined with nucleic acid dye to emit fluorescence, and if the lightening type RNA aptamer is sheared by Cas13 protein, fluorescence is quenched; the crRNA is a section of RNA, the crRNA and Cas13 protein can form a crRNA-Cas13 compound, and after the Cas 13-crRNA is combined with a target RNA sequence, the enzyme digestion activity of the Cas13 protein is activated, and an RNA aptamer is excised. The kit can detect living pathogens and RNA markers by detecting RNA, and compared with an existing nucleic acid detection method based on CRISPR-Cas13, the method does not depend on reverse transcription and nucleic acid amplification, target RNA sequence detection is completed in one step, the detection cost is low, the operation steps are simple, high specificity and sensitivity can be guaranteed, and industrial application is facilitated.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection. Background technique [0002] Infectious diseases caused by pathogenic bacteria or viruses are the most important factor threatening human health. In addition, peripheral blood nucleic acid is gradually recognized as a molecular marker including cancer, which can be used for early warning of diseases. In recent years, molecular detection of organisms such as pathogenic bacteria, viruses, and disease markers has been widely used, mainly including antibody detection and nucleic acid detection. [0003] Due to the importance of antibodies in serum immunity, antibody detection is very important in the field of molecular detection. For example, the patent application CN 111562370 A of Guangdong Kangjian Biotechnology Pharmaceutical Company discloses a virus antibody detection method based on colloidal gold, which can obtain fast and accurate virus detection results. However, compared with prot...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6813C12Q1/06C12R1/085C12R1/19C12R1/42
CPCC12Q1/689C12Q1/6813C12Q2521/327C12Q2525/161C12Q2563/107C12Q2545/113C12Q2525/205Y02A50/30
Inventor 邓锐杰张婷
Owner SICHUAN UNIV
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