Novel coronavirus detection kit based on Crisper

A technology for detection kits and coronaviruses, which can be used in the determination/inspection of microorganisms, resistance to vector-borne diseases, and measurement devices, etc. It can solve problems such as insufficient specificity, high dependence on special equipment, and insufficient sensitivity

Inactive Publication Date: 2020-08-14
宿迁市第一人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, most of the detection kits using the RT-PCR method on the market require a long time, are highly de...

Method used

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  • Novel coronavirus detection kit based on Crisper
  • Novel coronavirus detection kit based on Crisper
  • Novel coronavirus detection kit based on Crisper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Design and acquisition of crRNA targeting virus-specific sites

[0027] 1. Discovery of novel coronavirus detection sites based on CRISPR-LwaCas13a system

[0028] According to the latest "RT-PCR Detection Guide" issued by CDC, the inventor obtained the specific region of the new coronavirus. Design crRNA for these different regions and construct CRISPR / LwaCas13a system for research. The results show that the detection site based on the CRISPR / LwaCas13a system has a good detection effect with the region sequence shown in SEQ ID NO.1-2.

[0029] Table 1

[0030]

[0031] 2. Design of crRNA targeting specific regions

[0032] Since the CRISPR-Cas13a system is a targeted DNA gene editing system, in which Cas13a combines with crRNA to form a monitoring complex, the crRNA design requires: crRNA includes a protein anchor sequence and a guide sequence, and the sequence format is: 5`-and Cas13a The protein-binding anchor sequence-crRNA guide sequence-3`, the gu...

Embodiment 2

[0038] Embodiment 2 Detection kit and detection method of novel coronavirus

[0039] 1. The composition of the new coronavirus detection kit

[0040] This kit includes:

[0041] (1) crRNA targeting the C-terminal partial sequence: its sequence is shown in any one of SEQ ID NO.3-4.

[0042] (2) crRNA targeting N-terminal partial sequence: its sequence is shown in any one of SEQ ID NO.5-6.

[0043] (3) A specific fluorescent probe (using a general molecular beacon purchased from Thermo Fisher, such as 5'- / 56-FAM / mArUrGrGrGrCmAmArArUrGrGrCmA / 3Bio / -3');

[0044] (4) LwaCas13a protein;

[0045] (5) RNase inhibitor, T7 polymerase, ribonucleic acid solution and magnesium chloride solution.

[0046] Preferably, in order to make highly sensitive and highly specific detection of trace amounts of 2019-nCov virus, it is necessary to pre-amplify the nucleic acid of the virus to increase the abundance. Therefore, the RPA amplification method is preferably used. The new coronavirus detec...

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Abstract

The invention provides a novel coronavirus detection kit based on Crisper. The crRNA comprises a crRNA targeting a C-terminal part sequence and a crRNA targeting an N-terminal partial sequence, wherein the sequence of the crRNA targeting the C-terminal part sequence is shown as any one of SEQ ID NO.3 and SEQ ID NO.4, and the crRNA targeting the N-terminal partial sequence is shown as any one of SEQ ID NO.5 and 6. The invention alsohe invention relates to the crRNA related to the C-terminal partial sequence and the N-terminal partial sequence of a novel coronavirus. Through application of a method for mutation detection by using the crRNA in combination with a CRISPR-LwaCas13a system, the method has the advantages of low cost, repeated detection for multiple times, simplicity, no dependenceon a special fluorescent quantitative PCR instrument, high detection speed, high sensitivity (the lowest detection limit reaches 10<-2> copies/[mu]L) and strong specificity, and can be used to detectwhether the target nucleic acid is contained or not in a short time through the change of a fluorescence signal.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a Crisper-based novel coronavirus detection kit. Background technique [0002] At present, most of the detection kits using the RT-PCR method on the market require a long time, are highly dependent on special equipment, are not specific enough, and have insufficient sensitivity to cause false negatives. Therefore, there is an urgent need to develop a fast, highly specific and highly sensitive virology detection kit. Contents of the invention [0003] The purpose of the present invention is to overcome the defects of the prior art and provide a detection kit for novel coronavirus, which has strong specificity, high sensitivity, and the lowest detection limit reaches 10 -2 Copy / uL or less. [0004] The present invention is achieved like this: [0005] One of the objects of the present invention is to provide a crRNA for detecting novel coronavirus, said crRNA comp...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/6844C12Q1/70
CPCC12N15/113C12Q1/6844C12Q1/701G01N2333/165C12Q2521/507C12Q2522/101Y02A50/30
Inventor 裴兵耿晨
Owner 宿迁市第一人民医院
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