Method for detecting SARS-CoV-2 69: 70del site

A sars-cov-269 and sars-cov-2 technology, applied in the field of molecular diagnosis, can solve the problems of time-consuming, high instrument precision requirements, and massive data analysis

Active Publication Date: 2022-01-11
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this technique is important for identifying new variants and enabling real-time tracking of VOCs, it is time-consuming and requires extensive data analysis that cannot be performed in all laboratories
Other detection methods are mainly based on RT-qPCR. Although it is faster than gene sequencing methods, the sample processing is complicated and requires high instrument precision.

Method used

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  • Method for detecting SARS-CoV-2 69: 70del site
  • Method for detecting SARS-CoV-2 69: 70del site
  • Method for detecting SARS-CoV-2 69: 70del site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1, the design and preparation of crRNA and PCR primers for the present invention

[0068] 1. CRRNA design and preparation for the present invention

[0069] (1) Synthesis of primer sequence

[0070] The invention is designed with wild sites in SARS-COV-2 69: 70DEL site. CrRNA's 5 'end with a repeating sequence of 39 nT, which can be combined with LWCas13a protein, 5'-gggauuuacuaccccaaaaaaaaaaaaagggguaaaaac-3', design as a single-stranded DNA sequence as a template to repeat sequence + target sequence, T7 sequence (5'- TaatacgActCACTATAGGG-3 ') + part repeat sequence (5'-GatttagActaccccaa-3') is an upstream primer, and the reverse complementary sequence of about 20 bp downstream of the target sequence is taken as a downstream primer (Table 1). The sequences in Table 1 are synthesized.

[0071]

[0072] (2) PCR amplification

[0073] The sequence shown in Table 1 is synthesized from Beijing Tianyi Huiyuan Company. According to the research method in the literature, ...

Embodiment 2

[0105] Example 2, PCR-CRISPR / Cas13a 69 detector 2 SARS-CoV-: establishing site method 70del

[0106] In this study, PCR amplification of the target nucleic acid, the SARS-CoV-2 69: 70del detected target sequence to be transcribed to give the corresponding ssRNA, with the above crRNA: Mut6970-crRNA1, Mut6970-crRNA2, Mut6970-crRNA3, Mut6970 -crRNA4, Wt6970-crRNA1, Wt6970-crRNA2, Wt6970-crRNA3, Wt6970-crRNA4 detected, comparing different signal strength crRNA choose a stronger fluorescence signal and a high sensitivity crRNA crRNA subsequent detection. Specific works as follows: The first step in using the target specific primer sequence (by denaturation, annealing, extension process), having a period of T7 transcripts of the primer sequences in the 5 'end, such that the double stranded DNA was amplified by PCR (dsDNA) can be transcribed and T7RNA polymerase recognition. The second step, the amplification product take-out portion of T7 RNA polymerase was added, LwCas13a protein can...

Embodiment 3

[0119] Example 3, SARS-CoV-2 69: 70del sensitivity and specificity of detecting wild crRNA

[0120] 1, SARS-CoV-2 69: 70del optimal screening crRNA

[0121] Was screened to a higher detection sensitivity, the detection time is shorter the crRNA, using the previously described construct wild-type, 69: 70del mutant ssRNA and design primers 6970-Primer 1-F, and 6970-Primer 1-R, PCR was performed amplification. And treated with 4 kinds Mut6970-crRNA ssRNA sequence detector 2, respectively.

[0122] Synthesis (1) crRNA primer sequence

[0123] Table 1 Synthesis of pieces of sequence, in Example 1, Step 1 of the synthesis method of Embodiment press and prepared crRNA.

[0124] (2) The procedure of Example 1 in the embodiment 3 mutant and wild-type RNA template RNA obtained were serially diluted to give 69 different concentrations: solution 70del RNA gene fragments with gene fragments of different concentrations of wild 69:70, followed by concentration 10 5 copies / μL, 10 4 copies / μL,...

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Abstract

The invention discloses a CRISPR-Cas13a system for detecting an SARS-CoV-2 69: 70del site and crRNA used by the CRISPR-Cas13a system. The system comprises a Cas13a protein and the crRNA; a target spot sequence of the SARS-CoV-2 69: 70del site is located at the 21753 site to the 21786 site of an SARS-CoV-2 genome, and 6 tacatg basic groups are deleted; and a crRNA sequence of the SARS-CoV-2 69: 70del site is as shown in SEQ ID NO: 3. Experiments prove that the crRNA provided by the invention can be used for realizing high-sensitivity and high-specificity detection on nucleic acid at the SARS-CoV-2 69: 70del site by activating the Cas13a, and the sensitivity reaches a single copy (1copy/test). The method has an important application value.

Description

Technical field [0001] The present invention belongs to the technical field of molecular diagnostics, specifically relates to 2 SARS-CoV-69 for detecting: 70del site CRISPR-Cas13a crRNA system and its use. Background technique [0002] COVID-19 is a disease coronavirus 2 (severe acute respiratorysyndrome coronavirus 2, SARS-CoV-2) caused by the severe acute respiratory syndrome. Over time, SARS-CoV-2 there have been many new concern mutants (VOCs). B.1.1.7 lineage (VOC202012 / 01, 501Y.V1) first appeared in southern England, characterized in that the spectrum of 17 mutations, including substitutions and 14 amino acids and three in ORF 1 a / b, ORF8, Spike (S ) and N gene deletion within the framework region 2. S gene region located in the 69: 70del has been shown to have potential biological significance. 69: 70del is SARS-CoV-2 spike protein (Spike protein, S protein) a histidine at position 69 n-terminal domain (NTD) in (H69) and valine at position 70 (V70) deletion mutations, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/113C12Q1/70C12Q1/686
CPCC12N9/22C12N15/113C12Q1/701C12Q1/686C12N2310/10C12Q2521/327
Inventor 孙岩松李浩韩尧牛梦伟董雪
Owner ACADEMY OF MILITARY MEDICAL SCI
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