Method for detecting SARS-CoV-2 69-70del site based on RAA-CRISPR

A site-assisted detection technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as difficult to achieve rapid on-site detection

Active Publication Date: 2022-04-15
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these two technologies play a vital role in identifying new mutations, they both rely on professional equipment, laboratory facilities and testing personnel. and areas where it is difficult to achieve rapid on-site detection

Method used

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  • Method for detecting SARS-CoV-2 69-70del site based on RAA-CRISPR
  • Method for detecting SARS-CoV-2 69-70del site based on RAA-CRISPR
  • Method for detecting SARS-CoV-2 69-70del site based on RAA-CRISPR

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Experimental program
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Embodiment 1

[0062] Embodiment 1, be used for the design and preparation of crRNA of the present invention and PCR primer

[0063] 1. Design and preparation of crRNA used in the present invention

[0064] (1) Synthesis of primer sequences

[0065] The present invention designs crRNA at the SARS-CoV-2 69-70del site and the wild site respectively. The 5' end of the crRNA has a 39nt repeat sequence, which can bind to LwCas13a protein, 5'-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3', the single-stranded DNA sequence designed as a template is repeat sequence + target sequence, T7 sequence (5'- TAATACGACTCACTATAGGG-3') + partial repeat sequence (5'-GATTTAGACTACCCCAA-3') was used as the upstream primer, and the reverse complementary sequence about 20 bp downstream of the target sequence was used as the downstream primer (Table 1). Each sequence in Table 1 was synthesized.

[0066] Table 1. Template sequence and PCR amplification primer sequence for preparing crRNA used for mutation detection

...

Embodiment 2

[0105] Example 2, Establishment of RT-RAA-CRISPR detection method for SARS-CoV-2 69-70del site

[0106] In this study, RT-RAA technology was used to amplify the target nucleic acid, and the target sequence detected by SARS-CoV-2 69-70del was transcribed to obtain the corresponding ssRNA, using the above crRNA: Mut6970-crRNA1, Mut6970-crRNA2, Mut6970 -crRNA3, Mut6970-crRNA4, Wt6970-crRNA1, Wt6970-crRNA2, Wt6970-crRNA3, Wt6970-crRNA4 were detected, the signal strength of different crRNAs was compared, and the crRNA with strong fluorescent signal and high sensitivity was selected as the subsequent crRNA detection. The specific steps and principles are as follows: the first step uses specific primers to amplify the target sequence (through denaturation, annealing, and extension processes), and there is a section of T7 transcription sequence at the 5' end of the primer, so that the PCR amplification obtained double Stranded DNA (dsDNA) is recognized and transcribed by T7 RNA polyme...

Embodiment 3

[0122] Example 3, Specificity and sensitivity detection of SARS-CoV-2 69-70del and wild crRNA

[0123] 1. SARS-CoV-2 69-70del optimal crRNA screening

[0124] In order to screen crRNA with higher detection sensitivity and shorter detection time, RT-RAA was performed using the wild-type and 69-70del mutant ssRNA constructed above and the designed primers 6970-RAA-F1 and 6970-RAA-R1 Amplify. And use 4 kinds of Mut6970-crRNA to detect the ssRNA of 2 sequences respectively.

[0125] (1) Synthesis of crRNA primer sequence

[0126] Each sequence in Table 1 of Example 1 was synthesized, and crRNA was synthesized and prepared according to the method of step 1 in Example 1.

[0127] (2) The mutant RNA template obtained in step 3 in Example 1 and the wild-type RNA template were respectively gradiently diluted to obtain RNA solutions containing different concentrations of 69-70del gene fragments and different concentrations of 69-70 wild gene fragments: 10 5 copies / μL, 10 4 copies / μ...

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas13a (clustered regularly interspaced short palindromic repeats) system for detecting an SARS-CoV-2 69-70del site The system comprises an RT-RAA amplification primer, a Cas13a protein and a crRNA (Complementary Ribonucleic Acid), the RT-RAA primer pair is composed of a single-chain DNA molecule as shown in SEQ ID NO: 7 and a single-chain DNA molecule as shown in SEQ ID NO: 8; the target spot sequence of the 69-70del site of the SARS-CoV-2 is located at the 21753 site to the 21786 site of the SARS-CoV-2 genome, and the 69-70del site of the SARS-CoV-2 genome is lack of 6 tacatg basic groups; and the crRNA sequence of the 69-70del site of the SARS-CoV-2 is as shown in SEQ ID NO: 3. Experiments prove that high-sensitivity and high-specificity detection of SARS-CoV-2 69-70del site nucleic acid can be realized, and the sensitivity reaches a single copy. The method has an important application value.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a method for detecting the SARS-CoV-269-70del site based on RAA-CRISPR. Background technique [0002] COVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). Over time, multiple new variant strains of concern (VOCs) of SARS-CoV-2 have emerged. Lineage B.1.1.7 (VOC202012 / 01, 501Y.V1) first appeared in southern England, this lineage was characterized by 17 mutations, including 14 amino acid substitutions and 3 in ORF 1a / b, ORF8, Spike(S) and in-frame deletions in region 2 of the N gene. The 69-70del located in the S gene region has been proved to have potential biological significance. 69-70del is the deletion of the 69th histidine (h69) and the 70th valine (v70) in the n-terminal domain (ntd) of the SARS-CoV-2 spike protein (Spike protein, S protein) The mutation, 69-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCY02A50/30
Inventor 孙岩松李浩韩尧牛梦伟董雪杨兰聂优
Owner ACADEMY OF MILITARY MEDICAL SCI
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