RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primers for specifically detecting SIV (swine influenza virus), application of primers, detection reagent and method

A 1. RT-LAMP, swine flu virus technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of weak positive judgments that are not accurate enough, difficult to detect in broad spectrum, and easy to pollute the environment and other problems, to achieve the effect of real-time observation of results, high detection sensitivity, and avoidance of pollution

Inactive Publication Date: 2019-03-15
CAPITALBIO CORP
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Problems solved by technology

Currently, there is only one patent authorized for the detection of swine influenza virus using loop-mediated isothermal amplification (LAMP). The color agent detects the amplification product by observing the color change, but this process must be opened to add dyes, which is extremely easy to pollute the environment, and the resolution of the naked eye is limited and the judgment of weak positives is not accurate enough; 54 swine influenza virus sequences published by GeneBank, and the coverage of primers is narrow as the virus mutates, making it difficult to achieve the purpose of broad-spectrum detection

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primers for specifically detecting SIV (swine influenza virus), application of primers, detection reagent and method
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primers for specifically detecting SIV (swine influenza virus), application of primers, detection reagent and method
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primers for specifically detecting SIV (swine influenza virus), application of primers, detection reagent and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1, the screening of primer combination

[0102] 1. Design LAMP primers according to the specific segment of the NP gene sequence of the swine influenza virus (MG983192.1) in GenBank, and select the optimal primer combination as follows:

[0103] The primer set SIV1 sequence is as follows:

[0104] Outer primer SIV1-F3: 5'-TCTGGAGGGGTGAAAATGGA-3'

[0105] Outer primer SIV1-B3: 5'-GTGCAACTGATCCCCTCAG-3'

[0106] Internal primer SIV1-FIP:

[0107] 5'-GCCCTCTGGGCAGCTGTTTGCGAAGGACAAGGGTTGCTTA-3'

[0108] Internal primer SIV1-BIP:

[0109] 5'-AAGTCGAAACCCAGGAAACGCTAATGAGTGCTGACCGTGC-3'

[0110] According to the specific segment of the NP gene sequence of swine influenza virus (MG836780.1) in GenBank, LAMP primers were designed, and the optimal primer combination was selected as follows:

[0111] The primer set SIV2 sequence is as follows:

[0112] Outer primer SIV2-F3: 5'-AGAAAGTGATTCCAAGAGGA-3'

[0113] Outer primer SIV2-B3: 5'-GGTTGCTCTTTTCAAAAGGGA-3'

...

Embodiment 2

[0148] Embodiment 2, specificity experiment

[0149] Swine influenza virus (SIV), swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine blue ear virus American type highly pathogenic strain (HP-PRRSV), porcine blue ear virus American type classic strain (PRRSV), Each test sample of porcine parvovirus (PPV1) and porcine circovirus (PCV2) carries out the following steps respectively:

[0150] 1. Extract the genomic DNA or RNA of the sample to be tested.

[0151] 2. Using the viral genome extracted in step 1 as a template, the primer set screened in Example 1 was used to perform reverse transcription-loop-mediated isothermal amplification.

[0152] Reaction system (20 μL): 10 μL reaction solution, 2.96 μL primer mixture, 2 μL genomic DNA / RNA (5pg-50pg), 0.5 μL AMV reverse transcriptase (added in the RNA template system), filled to 20 μL with RNase-free water. The primer mixture is the mixture of each primer in the primer set. In the reaction system, 0.12 μL each ...

Embodiment 3

[0157] Embodiment 3, sensitivity

[0158] Sample to be tested: the plasmid DNA of swine influenza virus prepared in Example 1.

[0159] 1. Extract the plasmid DNA of the sample to be tested, and perform gradient dilution with sterilized pure water to obtain each dilution.

[0160] 2. Using the dilution obtained in step 1 as a template, the primer combination prepared in Example 1 was used to perform loop-mediated isothermal amplification.

[0161] Reaction system (20 μL): 10 μL reaction solution (product of Boao Biological Group Co., Ltd., catalog number: CP.440020), 2.96 μL primer mixture, 2 μL diluent (the copy number of plasmid DNA contained in 1 μL diluent is 5× 10 2 、10 2 or 10 1 ), replenish water to 20 μL. The primer mixture is the mixture of each primer in the primer combination. In the reaction system, 0.12 μL each of 0.3 mM outer primers F3 and B3; 0.96 μL each of 2.4 mM inner primers FIP and BIP; 0.4 μL each of 1 mM loop primers LF and LB, if the primer lacks ...

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Abstract

The invention relates to the biotechnology field, in particular to RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primers for specifically detecting SIV (swine influenza virus), an application of the primers, a detection reagent and a method. The RT-LAMP detection primers are selected from one or several of the primer groups: (1) a primer group SIV1; (2) a primergroup SIV2; (3) a primer group SIV3. The RT-LAMP primer group can accurately detect the SIV and has no cross reaction with HP-PRRSV, PRRSV, PPV, PCV2, CSFV, PRV and the like. The three screened primers cover more than 300 SIV sequences in an NCBI database and have high detection sensitivity and specificity, the established detection method has the advantages of high accuracy, short detection timeand allowance of real-time observation of results, only 1 h is taken from sample treatment to result report, operation is simple, and pollution is avoided as midway opening is not required.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to RT-LAMP detection primers for specifically detecting swine influenza virus and its application, detection reagent and method. Background technique [0002] Swine influenza (SI) is an acute, highly contagious mass respiratory disease caused by swine influenza virus (SIV). Characterized by sudden onset, rapid spread, rapid recovery, and low mortality, it is a common seasonal disease in intensive and large-scale pig farms, causing serious economic losses to the pig industry. In addition, since pig respiratory epithelial cells have sialic acid receptors that recognize and bind both human influenza virus and avian influenza virus, it has been proved to be an important intermediate host for human and avian influenza viruses, playing the role of a gene "mixer". The co-infection of multiple influenza viruses causes the influenza viruses in pigs to continuously undergo genetic recombination ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107
Inventor 张岩马银平陈燕旌邢婉丽程京边素莹
Owner CAPITALBIO CORP
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