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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent for detecting 2019-nCov and application of CRISPR reagent

A 2019-ncov, reagent technology, applied in the field of medical diagnosis, which can solve the problems of sample contamination and complicated operation

Pending Publication Date: 2020-10-30
宜明(苏州)细胞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technologies still have many shortcomings: 1. PCR technology requires precise temperature system control, which cannot be realized in some underdeveloped areas; 2. Detection sensitivity and specificity still need to be further improved; 3. PCR technology can only be used for For the detection of DNA fragments, RNA fragments need to be added to a reverse transcription process in advance, the operation is complicated, and the operation of opening the tube is easy to cause sample contamination
However, this technology has not been applied to the detection of 2019-nCov

Method used

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  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent for detecting 2019-nCov and application of CRISPR reagent
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent for detecting 2019-nCov and application of CRISPR reagent
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent for detecting 2019-nCov and application of CRISPR reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Screening of CRISPR reagents for detection of 2019-nCov

[0055] Screen a set of CRISPR reagents for the detection of 2019-nCov, including amplification primer pairs and crRNA; and optimize the reagent volume of the entire detection system to obtain the best detection results in the shortest time.

[0056] experimental method:

[0057] 1. Through RPA amplification, agarose gel electrophoresis screening for amplification primer pairs with high efficiency.

[0058] 1. Two pairs of primers were designed for the nucleoprotein gene N of COVID-2019:

[0059] (1) N1 primer pair:

[0060] RPA-N1F: gaaatTAATACGACTCACTATAgggAAATTTCAAAGATCAAGTCATTTTGCTGAATA (SEQ ID No: 1), wherein, the lowercase letters and the sequence between them are the T7 sequence, and the rest of the sequence is the forward primer sequence of the targeting sequence.

[0061] RPA-N1R: TTTCATCAGCCTTCTTCTTTTTGTCCTTTTTA (SEQ ID No: 2).

[0062] Its targeting sequence is:

[0063] AAATTTCAAAGATCAA...

Embodiment 2

[0170] Embodiment 2: preparation contains nCov-N, the pseudovirus plent-N of nCov-S, plent-S

[0171] 1. Construction of lentiviral vector

[0172] 1. Primer design

[0173] Table 10 Primer Sequence

[0174]

[0175]

[0176] 2. Experimental process

[0177] 1), PCR

[0178] Centrifuge briefly after receiving the primers, and add (nmol number*10) μL H 2 O, diluted to 100 μM mother solution, then transferred to a 1.5mL centrifuge tube, diluted 10 times to become PCR working solution.

[0179] PCR system: (unit: μL)

[0180] Table 11: PCR system

[0181]

[0182] Primers and templates correspond as follows:

[0183] Table 12: Primers, Templates

[0184]

[0185] Reaction procedure:

[0186] Table 13: Reaction Program

[0187]

[0188] After the PCR reaction, 2 μL of the PCR product was separated and detected by 1% agarose gel electrophoresis. If the size of the target band was correct, the PCR product was purified using a DNA purification kit.

[0189] ...

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Abstract

The invention provides a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent for detecting 2019-nCov and application of the CRISPR reagent in preparation of a kit for detectingthe 2019-nCov. The CRISPR reagent comprises an RPA (Recombinase Polymerase Amplification) primer pair and corresponding crRNA. Based on the CRISPR molecular diagnosis technology, through coupling of RPA-CRISPR, two-stage amplification of 'sequence amplification' (RPA completion) and 'enzymatic cascade' (Cas enzyme completion) can be realized, so that 2019-nCov is detected with high sensitivity andhigh specificity under the condition of a simple instrument (metal bath).

Description

technical field [0001] The invention relates to the field of medical diagnosis, in particular to a CRISPR reagent for detecting 2019-nCov and its use in preparing a kit for detecting 2019-nCov. Background technique [0002] Coronaviruses are a family of large RNA viruses known to cause colds as well as more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). 2019 Novel Coronavirus (2019-nCov), named SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11, 2020, can cause 2019 Novel Coronavirus Disease (COVID-2019), the virus from 2019 Novel Coronavirus Disease It is a new strain of coronavirus that has never been found in humans before, and it is referred to as a new coronavirus (nCov) for short. Common signs of people infected with 2019-nCoV include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q1/6804C12Q2521/507C12Q2521/101C12Q2521/327C12Q2565/625
Inventor 孙秀莲郭学双崔若茗
Owner 宜明(苏州)细胞生物科技有限公司
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