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General bacterium and virus detection system and method based on CRISPR-Cas14a and application

A technology for virus detection and bacteria, applied in the field of general bacteria and virus detection systems, can solve the problems of increasing detection difficulty and detection cost

Pending Publication Date: 2021-07-30
HAINAN MICROKRYPTON BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The above conditions limit the application of the nucleic acid detection system based on the CRISPR-Cas system, increasing the difficulty and cost of detection; at the same time, it also highlights the need for a CRISPR-Cas nucleic acid detection system that does not use PAM and universal sgRNA

Method used

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  • General bacterium and virus detection system and method based on CRISPR-Cas14a and application
  • General bacterium and virus detection system and method based on CRISPR-Cas14a and application
  • General bacterium and virus detection system and method based on CRISPR-Cas14a and application

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preparation example Construction

[0041] (2) Preparation of general sgRNA

[0042]A template for universal sgRNA was amplified by PCR from the pUC19 vector containing the T7 promoter, the nucleotide target sequence (SEQ ID NO.1) matching the sequence shown in Table 1, and sgRNA resistance. Use the amplified PCR product as a DNA template, transcribe the sgRNA in vitro at 37°C for 16 hours with the T7 Rapid High-yield RNA Synthesis Kit, and then use the RNA Purification Kit (NEB) to purify to obtain a universal sgRNA and store it at -70°C for future use .

[0043] Table 1 Sequence of universal sgRNA

[0044]

preparation example

[0046] (1) Culture of Escherichia coli and preparation of cDNA

[0047] Escherichia coli was purchased from China Industrial Culture Collection Center (CICC) and cultured according to the manufacturer's instructions. That is, use LB medium (10g / L trypsin, 5g / L yeast extract and 10g / L sodium chloride) to inoculate Escherichia coli, keep a constant temperature at 37°C, and cultivate overnight at 180rpm, until it grows to 10 8 After CFU / mL, the bacteria were centrifuged at 10000rpm for 10 minutes, and the bacterial RNA kit (E.Z.N.A. Bacterial RNA Kit, OMEGA) was used to extract bacterial RNA.

[0048] The bacterial RNA extracted above was subjected to a thermal cycle condition of 42°C for 60 minutes using the PrimeScript II first-strand cDNA synthesis kit (Takara), and then subjected to a thermal cycle condition of 70°C for 5 minutes, and finally, E. coli was obtained. cDNA.

[0049] (2) Culture of Salmonella typhi and preparation of cDNA

[0050] Salmonella typhi was purcha...

Embodiment 1

[0067] (1) Preparation of Escherichia coli TSPE

[0068] The target region (SEQ ID NO.2) of E. coli was selected by MegAlign software (DNASTAR). Primers were designed by Premier software according to the target region, wherein the forward primer (SEQ ID NO.3) was combined with a Tag tag, and the reverse primer (SEQ ID NO.4) was combined with a biotin tag. The sequences are shown in Table 2.

[0069] 200nM Escherichia coli-specific forward and reverse primers (wherein the forward primer has a Tag sequence and the reverse primer has a biotin sequence) and six kinds of bacteria such as Escherichia coli and the cDNA of SARS-CoV-2 are used as templates respectively, Add DreamTaq TM Hot-start DNA polymerase (Thermo) adopts traditional PCR technology to carry out PCR amplification to prepare TSPE products respectively.

[0070] The PCR system is: DreamTaq TM Hot-start DNA polymerase 1 μL, 10X buffer 5 μL, 10 mM dNTPs 1 μL, upstream and downstream primers (forward and reverse prim...

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Abstract

The invention relates to a general bacterium and virus detection system and method based on CRISPR-Cas14a, and application of the general bacterium and virus detection system and method based on CRISPR-Cas14a. The detection system comprises a CRISPR-Cas14a system, TSPE and magnetic nanoparticles; the CRISPR-Cas14a system comprises a general sgRNA (small guide ribonucleic acid) and Cas14a; the TSPE is a tag specific primer amplification system and comprises a tag forward primer containing a Tag sequence and a reverse primer containing a biotin labeling sequence; and the magnetic nanoparticles may be linked to a reverse primer of TSPE. The detection system has universal sgRNA, PAM is not needed in the detection process, the detection process is simplified, and the application range is wider. The detection method is simple to operate, and compared with a traditional detection method, the process of preparing sgRNA for multiple times is omitted, the purification process (TSPE) of a tag specific primer amplification system is simplified, and the operation difficulty and the production cost in the detection process are reduced. The application specifically refers to the application of the detection system provided by the invention in detection of DNA without PAM sequences.

Description

technical field [0001] The invention relates to the technical field of medical detection technology, in particular to a CRISPR-Cas14a-based general bacteria and virus detection system, method and application. Background technique [0002] Many life-threatening infectious diseases usually have similar signs and symptoms. In order to further determine how to use the drug, it is necessary to clarify the specific type of bacteria or virus that the patient is infected with. Nowadays, nucleic acid detection systems can be used to identify the species of bacteria or viruses, and have the advantages of high sensitivity and fast diagnosis. [0003] In the field of virus and bacteria detection, commonly used nucleic acid detection systems mainly rely on the CRISPR-Cas system. The CRISPR-Cas system (clustered into regularly interspaced short palindromic repeats and related proteins) is an adaptive immune system in bacteria that can integrate invading foreign DNA; when the pathogen inv...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/70
CPCC12Q1/6844C12Q1/689C12Q1/701C12Q2521/327C12Q2525/161
Inventor 宋凤阁崔倩杨治庆
Owner HAINAN MICROKRYPTON BIOTECHNOLOGY CO LTD
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