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Modified crrna fragment and African swine fever virus kit

A kit and fragment technology, applied in the field of modified CrRNA fragments and African swine fever virus kits, to achieve the effects of shortened detection time, strong specificity, and high sensitivity

Active Publication Date: 2022-04-01
厦门腾基医疗科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a modified CrRNA fragment and African swine fever virus kit, to solve one or more technical problems in the prior art, provide at least one beneficial selection or create conditions

Method used

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  • Modified crrna fragment and African swine fever virus kit
  • Modified crrna fragment and African swine fever virus kit
  • Modified crrna fragment and African swine fever virus kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, a crRNA for detecting ASFV is constructed

[0040] T5-bit and T8 sites in ASFV P72 genes are detected targets, respectively design the length of 23bp, 20bp, 17 bp, and the guide sequence is combined with anchor sequence for the CAS12A protein into crRNA, for obtaining better Cutting efficiency and reduced the deraid rate. The constructed crrna is shown in Table 3:

[0041] Table 3 CRRNA sequence for testing

[0042] .

[0043] The CrisPr / DX detection system (20 μL) for detecting ASFV, including 1 μl of lbcas12a (1 μm); CRRNA (1 μm) 2 μL; ProBe (10 μm) 1 μL; RNase Inhibitor 1 μL; buffer2 2 μL; PLASMID 1 μL (7 × 10 9 COPY / μL); Water 10μL; MGCL 2 2 μL (17.5 mm); the reaction temperature was 47 ° C, and the reaction was 20 minutes. The sequence of the fluorescent reporting molecule (probe) is 5'-Fam-CCCCCC-BHQ1-3 '.

[0044] The CrRNA sequences of different lengths according to Table 3 are synthesized to form different CRISPR / DX detection systems, and their det...

Embodiment 2

[0045] Example 2, CrRNA-DNA Structure Optimization Design

[0046] (2-1) The T5 site of the P72 gene of ASFV is selected, and the effect of adding DNA modified fragment on cutting efficiency in the 3 'end of Crispr / DX detection system. Construction of the test system: lbcas12a (1 μm) 1 μL; CrNA (1 μm) 2 μL; probe (10 μm) 1 μL; RNase Inhibitor 1 μL; buffer2 2 μL; PLASMID 0.5 μL (7 × 10 9 COPY / UL); Water 10.5μL; MGCL 2 2 μL (17.5 mm); the reaction temperature was 47 ° C, and the reaction was 20 minutes. CRRNA is based on the RNA sequence targeted T5 site, with T5-20 as No. 1 CrRNA, No. 2 CrRNA to No. 8 crrna, respectively, T5-20, 3 'end modifications, respectively, 5'-Tattatt-3', respectively. 5'-aaataaa-3 ', 5'-TTTATTT-3', 5'-TGGGGGGGGGGGGGGGGGGGGGGGGGG-3 ', 5'-TGGGCGGG-3' or 5'-TCCCCCC-3 'DNA sequence .

[0047] The test results of each test system sample are figure 2 As shown, in the same positive quality particle detection, a high relative fluorescence unit (RFU) was detecte...

Embodiment 3

[0050] Example 3, CRISPR / DX Detection System Condition Optimization

[0051] The reaction rate of the CRISPR / DX detection system is also related to a plurality of reaction conditions, including magnesium ion concentration, reaction temperature, target pairing, and other factors, so that the higher reaction rate can be obtained by adjustment of many conditions. The CRRNA used in Example 2 (2-1) is based on the CrRNA to T8-23, which optimizes its magnesium ion concentration, reaction temperature, and target selection.

[0052] (3-1) Optimization of Magnesium Ion Concentration.

[0053] A magnesium chloride solution having a concentration of 17.5 mm, 16.4 mm, 15 mm, 13 mm, 10 mm was formulated, and the same ASFV-yang-yasin was detected in the CRISPR / DX detection system, and the reaction temperature was set to the CRISPR / DX detection system 37 ° C. Detection results Figure 4 As shown, the RFU data of the detection system of the magnesium ion concentration is 17.5 mm is optimal...

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Abstract

The invention discloses a modified CrRNA fragment and an African swine fever virus kit. The modified CrRNA fragment is that a DNA modification fragment has been added at the 3' end of the CrRNA fragment. The modified CrRNA fragment can further increase the reaction rate of the CRISPR / DX detection system, thereby shortening the detection time. The CRISPR / DX detection system adopts a unique dual-target combination, combining two modified CrRNAs targeting different targets in one reaction system, which can improve the overall reaction rate. After the CRISPR / DX detection system is combined with the RPA amplification system, a CcMFD African swine fever virus kit can be constructed. In particular, it can improve the cleavage efficiency of LBcas12a protein, and can improve target specificity.

Description

Technical field [0001] The present invention relates to the field of gene detection, and in particular, to a modified crRNA fragment and a African swine fever virus kit. Background technique [0002] African swine fever is a strong, highly contactless animal infectious disease caused by Afric Swine Fever Virus, ASFV, and once the mortality rate can reach 100% once infected, the World Animal Health Organization (OIE) will It is listed as an animal diseases that must be reported, and my country will also list African swineons as a class of animal diseases. The African swine fever virus genome is a double-linked linear DNA molecule, a size of about 170-194 kB, containing 150-160 open reading frames (ORFs), coding 54 structural proteins and more than 100 non-structural proteins. The African swine fever virus genome mainly consists of the end of the card, the intermediate portion is a relatively stable genometric region and the variable region of the series repeat sequence and the mul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/70C12Q1/6844C12Q1/6816C12N15/11C12R1/93
CPCC12N15/1131C12Q1/701C12Q1/6844C12Q1/6816C12N2310/20C12N2310/3519C12Q2521/327C12Q2525/151C12Q2525/161C12Q2525/121C12Q2563/107
Inventor 祝海宝黄郁媚籍雁竹魏炽炬黄戴纯邓荣浩胥立群
Owner 厦门腾基医疗科技有限责任公司
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