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CRISPR nucleic acid detection kit for detecting novel coronavirus (2019-nCoV)

A coronavirus and kit technology, applied in the field of CRISPR nucleic acid detection kits, can solve problems such as insufficient sensitivity, easy contamination, and high requirements for instruments

Active Publication Date: 2020-06-12
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain deficiencies in the existing detection technologies. The colloidal gold method is simple and portable, but the sensitivity is insufficient; Gene sequencing is stable, reliable, and has certain sensitivity, but the sample processing is complicated and requires high equipment

Method used

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  • CRISPR nucleic acid detection kit for detecting novel coronavirus (2019-nCoV)
  • CRISPR nucleic acid detection kit for detecting novel coronavirus (2019-nCoV)
  • CRISPR nucleic acid detection kit for detecting novel coronavirus (2019-nCoV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Novel coronavirus nucleic acid detection kit and detection method based on CRISPR-Cas13a system 1. Novel coronavirus nucleic acid detection kit based on CRISPR-Cas13a system

[0065] (1) Preparation of mRNA standard

[0066] The new coronavirus N gene is sequence 3, and the 992-1019th position of sequence 3 is the target sequence of COVID-19 crRNA.

[0067] mRNA is transcribed from the PCR amplification product of the above-mentioned plasmid to simulate viral nucleic acid. The specific method steps are as follows:

[0068] (1) PCR amplification

[0069] Using the synthetic DNA molecule shown in Sequence 3 as a template, PCR amplification was carried out with the system shown in Table 1 below to obtain a PCR product.

[0070] The PCR reaction system was prepared as shown in Table 1.

[0071] Table 1. PCR amplification system

[0072] name volume DNA molecule shown in sequence 3 2μL nCoVnp-F1 2μL nCoVnp-R1 2μL ExTaq Mix...

Embodiment 2

[0139] Example 2. Condition optimization and exploration of detection method based on CRISPR-Cas13a system

[0140] 1. Screening of optimal RT-RAA amplification primers

[0141] Taking the mRNA standard product prepared in one (one) in Example 1 as a template, RT-RAA amplification was carried out with the method in (1) of Example 1, and the primers were each primer combination shown in Table 3 ( Combinations such as figure 2 shown) to obtain RT-RAA amplification products.

[0142] The amplification product with water as template was used as a negative control.

[0143] Take 20 μL of the amplified product, add 20 μL of chloroform: Tris equilibrium phenol = 1:1 and mix an equal amount of the reaction product, shake and centrifuge for 10 minutes, discard the supernatant mixture, shake and mix, and centrifuge at 10,000 rpm for 10 minutes , Aspirate 10 μL of supernatant, add 3 μL of 6*Loading Buffer, mix well and run on agarose gel.

[0144] detect as figure 2As shown, the r...

Embodiment 3

[0155] Embodiment 3. Sensitivity detection of the detection method of novel coronavirus nucleic acid based on CRISPR-Cas13a system

[0156] The mRNA standard substance prepared in one (one) of embodiment 1 utilizes RNase-free water to carry out 10-fold serial gradient dilution, obtains the solution that contains different concentrations of novel coronavirus gene mRNA, detects according to the method for two in embodiment 1, The RT-RAA products in Table 7 were replaced with solutions containing different concentrations of novel coronavirus gene mRNA, so that the concentration of mRNA in the reaction system was 10 5 -10 -1 copies / test, and set the amplification product of water as template as negative control.

[0157] Test results such as Figure 4 shown, 10 5 -10 1 The "T" line disappears and the "C" line appears in the copies / test group; 10 0 copies / test group, 10 -1 Both the "T" and "C" lines in the copies / test group and the negative control group were visible. Judgm...

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Abstract

The invention discloses a CRISPR nucleic acid detection kit for detecting a novel coronavirus (2019-nCoV). The invention provides a kit for detecting the novel coronavirus. The kit comprises a CRISPR-Cas13a system for detecting the novel coronavirus and lateral flow test paper matched with the CRISPR-Cas13a system for use. The CRISPR-Cas13a system comprises a1) a crRNA protein and an LwCas13a protein which are independently packaged; a2) a reporter RNA consisting of 20 U; and a3) an RT-RAA amplification primer used for amplifying a target sequence of a sample to be detected, wherein a 5'tail end of one primer in the RT-RAA amplification primer is provided with an area recognized by T7 RNA polymerase. According to the CRISPR nucleic acid detection test paper, high-sensitivity, high-specificity and convenient detection on a novel coronavirus nucleic acid can be realized through the CRISPR-Cas13a system, and the sensitivity reaches 10copies / test.

Description

technical field [0001] The invention relates to a CRISPR nucleic acid detection kit for detecting a novel coronavirus (2019-nCoV), belonging to the technical field of molecular diagnosis. Background technique [0002] Corona Virus Disease 2019 (COVID-19) is a Class B infectious disease caused by a new type of coronavirus (2019-nCoV) that causes acute infection in humans. The characteristics of specific drugs and no vaccine prevention. New coronavirus detection technologies include viral gene sequencing, detection of IgM and IgG antibodies, fluorescence quantitative PCR, constant temperature amplification chip nucleic acid detection technology, etc. However, the existing detection technologies all have certain shortcomings. The colloidal gold method is simple and portable, but has insufficient sensitivity; constant temperature amplification methods including LAMP and RPA have high sensitivity, but are prone to contamination during operation; fluorescence quantitative PCR met...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6804C12Q1/6844C12R1/93
CPCC12Q1/6804C12Q1/6844C12Q1/701C12Q2525/161C12Q2521/327C12Q2565/625C12Q2563/131
Inventor 李浩寇志华孙岩松周育森董雪王彦贺何雷赵忠鹏孙世慧谷宏婧
Owner ACADEMY OF MILITARY MEDICAL SCI
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