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Visual rapid nucleic acid detection method of bovine nodular skin disease virus

A detection method and nodularity technology, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of high cost, insensitive detection method, long time, etc., and achieve specificity High efficiency, accurate and reliable detection results, and low detection cost

Active Publication Date: 2021-08-27
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to establish a rapid nucleic acid detection method for bovine nodular skin disease virus. The method uses RPA amplification technology to amplify the nucleic acid by designing RPA specific primers, and the product is digested by Cas12a, and the fluorescent signal Virus detection can not only quickly detect and get results, but also solve the shortcomings of current detection methods such as insensitivity, long time, and high cost.

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  • Visual rapid nucleic acid detection method of bovine nodular skin disease virus
  • Visual rapid nucleic acid detection method of bovine nodular skin disease virus
  • Visual rapid nucleic acid detection method of bovine nodular skin disease virus

Examples

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Embodiment 1

[0071] Example 1: Screening of sgRNA for bovine nodular skin disease gene visualization detection

[0072] 1.1 Determination of target genes

[0073] Using LSDV / Russia / Saratov / 2017 (GenBank: MH646674.1) as the standard, compared all the viruses in the genus of capipoxvirus in NCBI, and found the most conserved gene sequence. The gene name in LSDV is LSDV orf068 , within the scope of the gene, search for the PAM sequence, and find that there are 6 sgRNAs within the range of 401bp, the sequences are shown in SEQ ID NO:1-6, and their positions in the genome are as follows figure 1 shown.

[0074] The fragment was amplified with the sequence shown in PCR-F / R (SEQ ID NO:7, SEQ ID NO:8), the amplification program was: 98°C, 5min; 35 cycles (98°C, 10s; 58°C, 15s; 72°C, 30s) 72°C, 5min. The amplification system is: PremixSTAR Max Premix (TAKARA product number: R045A): 25 μL; PCR-F / R: 2.5 μL each; DNA template: 1 μL; double distilled water to make up 50 μL. The result is as figur...

Embodiment 2

[0086] Embodiment 2: Screening of RPA primers

[0087] The 401bp fragment in Example 1.1 was constructed into the pUC57 plasmid and named pUC57-orf068. According to the formula: copy number = (concentration × 6.022 × 10 23 ) / (full length of plasmid×1×10 9 ×650) to calculate the copy number, pUC57-orf068 was diluted to different copy numbers as the template for RPA reaction. According to the RPA primer design principle, 4 pairs of primers (SEQ ID NO.18-25) were designed for the orf068 gene, and the amplified product contained sgRNA1. RPA amplification was performed for each copy number of plasmids using different primer pairs. The RPA reaction configuration is as follows: Primer free Buffer: 29.5 μL; DEPCwater: 12.2 μL; RPA-F / R. Mix well and add to a TwistAmp Basic reaction to fully dissolve, then add 2.5μL of 280mM magnesium acetate to each system. Finally, 1 μL of plasmid DNA of each copy number was added, and reacted at 37° C. for 40 minutes. Take 3 μL of the RPA produ...

Embodiment 3

[0095] Embodiment 3: This detection method compares with qPCR detection sensitivity

[0096] 1. Detection sensitivity to plasmids

[0097] Each Cas12a digestion reaction system of the second pair of primers in Example 2 was transferred to a black 96-well microtiter plate, then 80 μL of DEPC water was added, and after mixing, the fluorescence value was measured with a multifunctional microplate reader. Each reaction was repeated in triplicate. The result is as Figure 8 . It shows that the detection sensitivity can also reach 5 copies / μL, which is consistent with the result of naked eye observation in Example 2. The plasmid diluted in Example 2 was used as a template to verify the sensitivity of qPCR. The detection target of this qPCR method is also the LSDVorf068 gene, and the RPA amplification region completely includes the qPCR amplification region. The detection results of qPCR primers (SEQ ID NO:26-27) are shown in Table 1. It is found that the sensitivity of qPCR is a...

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Abstract

The invention discloses a visual rapid nucleic acid detection method of bovine nodular skin disease virus (LSDV), the method is characterized in that an RPA primer is designed for orf068 gene of bovine nodular skin disease virus, amplification is carried out, then Cas12a protein is added into an amplification product, enzyme digestion reaction is carried out, the reaction system comprises the Cas12a protein, sgRNA and a nucleic acid probe, and the signal emitted by the nucleic acid probe can be detected. The sensitivity for detecting orf068 plasmids is 5 copies / [mu] L, the sensitivity for detecting LSDV and goat pox virus (GTPV) is 0.1 TCID50 / [mu] L, the diagnostic sensitivity for detecting clinical samples is 96.3% (95% CI: 81.0%, 99.9%), the specificity is high, the changes in color are observed by eyes for result judgement, and the method has the advantages of simplicity, convenience, rapidness, suitability for field detection and the like.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a nucleic acid visualization detection technology mediated by a CRISPR-Cas12a system, a method and a kit for rapidly detecting bovine nodular skin disease (LSD). Background technique [0002] Lumpy skin disease (LSD) is a skin disease caused by Lumpy skin disease virus (LSDV), characterized by fever, skin edema and local hard nodules. Will seriously hinder the development of the cattle industry. LSD can cause huge financial losses. For example, the milk production of dairy cows is reduced, the growth performance of beef cattle is reduced, the bulls are temporarily or permanently sterile, and the utilization value of hides is destroyed. The incidence rate of the disease is between 2% and 45%, and the fatality rate is as high as 20%. Dairy cows are more susceptible and have been listed as one of the diseases that must be notified by the World Organization for Animal Hea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107C12Q2521/327
Inventor 郭爱珍姜传文谢胜松陈颖钰耿元晨陶大刚徐兵荣胡长敏陈建国陈曦
Owner HUAZHONG AGRI UNIV
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