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CN-122012806-A - CRISPR-Cas13a system for detecting rift valley fever virus and application

CN122012806ACN 122012806 ACN122012806 ACN 122012806ACN-122012806-A

Abstract

The invention discloses a CRISPR-Cas13a system for detecting a rift valley fever virus and application thereof, wherein the CRISPR-Cas13a system for detecting the rift valley fever virus comprises Cas13a protein and crRNA or a complex formed by the two, the rift valley fever virus target point sequence is SEQ ID NO.1, the crRNA sequence is shown as SEQ ID NO.2, the crRNA comprises an anchoring sequence for combining with the Cas13a protein and a guide sequence for targeting the rift valley fever virus target point sequence, and the rift valley fever virus target point sequence is positioned at 23-390 th position of a rift valley fever virus S gene. The crRNA can realize high-sensitivity and high-specificity detection of the valley fever virus nucleic acid by activating the Cas13a, the sensitivity reaches 1copy (1 copy/. Mu.L), and the crRNA has important application value.

Inventors

  • HAN YAO
  • WANG JING
  • QI JIANRONG
  • LI HAO
  • SUN YANSONG

Assignees

  • 中国人民解放军军事科学院军事医学研究院

Dates

Publication Date
20260512
Application Date
20251124

Claims (10)

  1. 1. The crRNA target for detecting the rift valley fever virus is characterized in that the rift valley fever virus target sequence is SEQ ID NO.1.
  2. 2. The CRISPR-Cas13a system for detecting the rift valley fever virus is characterized in that the CRISPR-Cas13a system comprises Cas13a protein and crRNA or a complex formed by the two, the crRNA comprises an anchor sequence for combining with the Cas13a protein and a guide sequence for targeting a rift valley fever virus target sequence, and the rift valley fever virus target sequence is shown as SEQ ID NO. 1.
  3. 3. The CRISPR-Cas13a system of claim 2, wherein the crRNA sequence is set forth in SEQ ID NO. 2.
  4. 4. The CRISPR-Cas13a system of claim 2 or 3, wherein said Cas13a protein is LwCas a protein.
  5. 5. A kit for detecting a rift valley fever virus comprising the CRISPR-Cas13a system for detecting a rift valley fever virus of any one of claims 2-4.
  6. 6. The kit according to claim 5, wherein the kit further comprises a RAA amplification primer for specifically amplifying the valley fever virus target sequence, and the RAA amplification primer consists of a single-stranded DNA molecule shown in SEQ ID NO.4 and a single-stranded DNA molecule shown in SEQ ID NO. 5.
  7. 7. Any one of the following substances: A1 A crRNA as set forth in any one of claims 2 to 4; A2 A complex of the Cas13a protein and the crRNA, or both as set forth in any one of claims 2-4; a3 A primer set as described in claim 6.
  8. 8. Any of the following applications: B1 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for the detection or assisted detection of a rift valley fever virus or a nucleic acid thereof; B2 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for the preparation of a product for detecting or aiding in the detection of a rift valley fever virus or a nucleic acid thereof; B3 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for detecting or aiding in the detection of whether a test sample contains a rift valley fever virus or a nucleic acid thereof; B4 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for the preparation of a product for detecting or aiding in the detection of the presence or absence of a rift valley fever virus or a nucleic acid thereof in a sample to be tested; b5 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for screening or aiding in the screening of a split valley fever virus controlling drug; B6 Use of the CRISPR-Cas13a system of any one of claims 2-4 or the kit of claim 5 or 6 or the substance of claim 7 for the preparation of a product for screening or co-screening of a split valley fever virus controlling drug; b7 Use of a substance according to claim 7 for the preparation of a kit according to claim 5 or 6.
  9. 9. A method for detecting or aiding in the detection of a rift valley fever virus comprising the steps of: C1 Using nucleic acid of a sample to be detected as a template, and carrying out RAA amplification by using a primer pair consisting of a single-stranded DNA molecule shown as SEQ ID NO.4 and a single-stranded DNA molecule shown as SEQ ID NO.5 to obtain a RAA product; C2 Preparing a CRISPR-Cas13a detection system comprising the RAA product, the Cas13a protein as described in any one of claims 2-4, the crRNA as described in any one of claims 2-4, the report RNA, NTP, T RNA polymerase, the rnase inhibitor, while replacing the PCR product with water as a negative control; C3 Reacting the CRISPR-Cas13a detection system, and detecting a reaction product, thereby judging whether a sample to be detected contains the rift valley fever virus.
  10. 10. The method of claim 9, wherein in step C1), the RAA amplification is performed at a temperature of 42℃for 30 minutes.

Description

CRISPR-Cas13a system for detecting rift valley fever virus and application Technical Field The invention belongs to the technical field of molecular diagnosis, and particularly relates to a CRISPR-Cas13a system for detecting a rift valley fever virus and application thereof. Background Rift Valley Fever Virus (RVFV) is an arthropod-transmitted Virus belonging to the genus sand fly Virus of the family butterfly Virus of the order bunyaviridae. The RVFV genome consists of three enveloped, segmented, single-stranded negative-sense RNA fragments, named L (LARGE SEGMENT), M (Medium Segment) and S (SMALL SEGMENT) fragments, respectively, the L fragment encodes only one protein, RNA-dependent RNA polymerase, the M fragment encodes four proteins, two glycoproteins Gn and Gc, which form the viral envelope, and two non-structural proteins NSm and Gn/NSm fusion proteins, and the S fragment encodes two proteins, the nucleoprotein gene NC and the non-structural protein gene NSs, respectively. Mosquitoes are the most important transmissible animals of RVFV, mainly by biting host animals or humans, and also by directly contacting the blood of infected animals, breathing air around slaughtered infected animals, drinking the raw milk of infected animals, etc. The infection of the crowd can cause mild symptoms such as fever, headache, back pain, dizziness, anorexia, photophobia and the like, and serious symptoms such as vision loss, serious headache, confusion and the like, and the disease can also cause the death rate of adults to be 0.5-2 percent, and the death rate of infants to be up to 28 percent. Therefore, developing an accurate, rapid, sensitive and simple detection technology for the rift valley fever virus is important for the prevention and control of the rift valley fever. Clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats, CRISPR) and related proteins (Cas) are one type of acquired immune system, found at the earliest in archaebacteria. With the intensive research, researchers find that after the Cas13a protein is combined with target RNA, the Cas13a protein is activated, non-target RNA can be cut, and target RNA can be indirectly detected by detecting target groups through connecting target groups which can be detected on two ends of the non-target RNA, namely a CRISPR-Cas13a detection method. At present, three methods for clinical examination and diagnosis of heat and heat of the rift valley are respectively serological immunodetection, nucleic acid detection and virus separation culture. Virus isolation and culture are the most direct diagnostic basis, but the operation is extremely complicated. Compared with the nucleic acid detection method, the immunological detection method has the advantages of complex operation, lower sensitivity and longer time consumption, and is unfavorable for on-site rapid detection. The PCR nucleic acid detection relies on a fluorescent quantitative PCR instrument for experiments, which is not beneficial to screening in areas with weak medical conditions. In summary, the existing detection method cannot meet the requirements of rapidness, simplicity and convenience, and meanwhile, high sensitivity is maintained, and development of a rapid, simple, high-sensitivity and high-specificity detection method for detecting the rift valley fever virus is urgently needed. Disclosure of Invention Based on the technical problems in the prior art, the CRISPR-Cas13a system for detecting the rift valley fever virus and the application thereof are provided, and the detection method based on the combination of the RT-RAA detection technology and the lateral flow chromatography test paper has the technical advantages of simple operation, stable system and wide application in the clinical molecular diagnosis field, and realizes the high-sensitivity detection of corresponding pathogens. In order to achieve the aim, 5 crrnas are designed according to the rift valley fever virus gene sequence based on the principle of the CRISPR-Cas13a system and the selection principle of the target sequence, and the crrnas with the best activating effect on the CRISPR-Cas13a system are preferably used for rift valley fever virus nucleic acid detection. The first object of the invention is to provide a crRNA target for detecting the rift valley fever virus, and the rift valley fever virus target sequence is SEQ ID NO.1. A second object of the present invention is to provide a CRISPR-Cas13a system for detecting a rift valley fever virus, the CRISPR-Cas13a system comprising a Cas13a protein and a crRNA, or a complex formed by both; the crRNA comprises an anchor sequence for combining with Cas13a protein and a guide sequence for targeting a rift valley fever virus target sequence, wherein the rift valley fever virus target sequence is shown as SEQ ID NO. 1. In certain embodiments, the crRNA sequence is set forth in SEQ ID No. 2. In certain embodiments, the