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Modified CrRNA fragment and African swine fever virus kit

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

Active Publication Date: 2022-01-25
厦门腾基医疗科技有限责任公司
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  • 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] Embodiment 1, construction is used to detect the CrRNA of ASFV

[0040] Taking the T5 site and T8 site of the P72 gene of ASFV as the detection target, design guide sequences with lengths of 23bp, 20bp, and 17bp, respectively, and combine the guide sequence and the anchor sequence for the Cas12a protein into CrRNA in order to obtain better results. Cutting efficiency and reduce off-target rate. The constructed CrRNA is shown in Table 3:

[0041] Table 3 CrRNA sequences for testing

[0042] .

[0043] Construct a CRISPR / DX detection system (20 μl) for detecting ASFV, including: LBcas12a (1 μM) 1 μl; CrRNA (1 μM) 2 μl; probe (10 μM) 1 μl; RNase inhibitor 1 μl; Buffer2 2 μl; plasma 1 μl (7×10 9 copy / μl); water 10μl; MgCl 2 2 μl (17.5 mM); the reaction temperature is 47°C, and the reaction time is 20 minutes. The sequence of the fluorescent reporter molecule (probe) is 5'-FAM-CCCCCC-BHQ1-3'.

[0044] According to Table 3, CrRNA sequences of different lengths were syn...

Embodiment 2

[0045] Embodiment 2, CrRNA-DNA structural optimization design

[0046] (2-1) Select the T5 site of the P72 gene of ASFV to verify the effect of adding a DNA modification fragment to the 3' end of CrRNA in the CRISPR / DX detection system on the cutting efficiency. Construct detection system: LBcas12a (1μM) 1μl; CrRNA (1μM) 2μl; probe (10μM) 1μl; RNase inhibitor 1μl; Buffer2 2μl; plasma 0.5μl (7×10 9 copy / ul); water 10.5μl; MgCl 2 2 μl (17.5 mM); the reaction temperature is 47°C, and the reaction time is 20 minutes. The CrRNA is based on the RNA sequence targeting the T5 site, with T5-20 as the No. 1 CrRNA, and the No. 2 CrRNA to No. 8 CrRNA sequences are respectively modified with 5'-TATTATT-3' at the 3' end of T5-20 DNA sequence of 5'-AAATAAA-3', 5'-TTTATTT-3', 5'-TCCCGCC-3', 5'-TGGGGGG-3', 5'-TGGGCGGG-3' or 5'-TCCCCCC-3' .

[0047] The test results of each test system sample are as follows: figure 2 As shown, in the same positive plasmid detection, compared with the bas...

Embodiment 3

[0050] Example 3, Condition optimization of CRISPR / DX detection system

[0051] The reaction rate of the CRISPR / DX detection system is also related to multiple reaction conditions, including magnesium ion concentration, reaction temperature, target pairing and other factors. Therefore, a higher reaction rate can be obtained by adjusting various conditions. Based on the CRISPR / DX detection system in Example 2 (2-1), the CrRNA used was T8-23, and its magnesium ion concentration, reaction temperature and target selection were optimized respectively.

[0052] (3-1) Optimization of magnesium ion concentration.

[0053] Magnesium chloride solutions with concentrations of 17.5mM, 16.4mM, 15mM, 13mM, and 10mM were respectively prepared for the CRISPR / DX detection system to detect the same ASFV positive plasmid, and the reaction temperature was set at 37°C, which is commonly used in the CRISPR / DX detection system. Test results such as Figure 4 As shown, the RFU data of the detection...

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Abstract

The invention discloses a modified CrRNA fragment and an African swine fever virus kit. The modified CrRNA fragment is formed by adding a DNA modification fragment at the 3' end of the CrRNA fragment. The modified CrRNA fragment can further improve the reaction rate of a CRISPR / DX detection system, so that the detection time is shortened. According to the CRISPR / DX detection system, unique double-target matching is adopted, two modified CrRNAs aiming at different target spots are in cooperation in one reaction system, and the overall reaction rate can be increased. After the CRISPR / DX detection system is combined with an RPA amplification system, the CcMFD African swine fever virus kit can be constructed. Particularly, the cutting efficiency of an LBcas12a protein can be improved, and the target specificity can be improved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a modified CrRNA fragment and an African swine fever virus kit. Background technique [0002] African swine fever is a severe and highly contagious animal disease caused by African swine fever virus (ASFV). Once the pig is infected, the mortality rate can reach 100%. The World Organization for Animal Health (OIE) lists it as As an animal disease that must be reported, my country also lists African swine fever as a first-class animal disease. The African swine fever virus genome is a double-connected closed linear DNA molecule with a size of about 170–194kb, containing 150–160 open reading frames (ORFs), and encoding a total of 54 structural proteins and more than 100 nonstructural proteins. The African swine fever virus genome is mainly composed of three parts: the hairpin loop structure at the end, the relatively stable gene region in the middle part, and the variable reg...

Claims

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Application Information

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Patent Type & Authority Applications(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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