Primer set for detecting American canine influenza virus subtype H3N8 and application thereof

A technology of canine influenza virus, H3N8, which is applied to the detection of North American H3N8 subtype canine influenza virus primer set and its application field, which can solve the problems of easy mutual influence, cumbersome operation, and low sensitivity, and achieve great application value and simple operation , the effect of high sensitivity

Inactive Publication Date: 2017-05-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hemagglutination inhibition test involves the separation of pathogens, the operation is relatively cumbersome, and the subtypes are easy to interact with each other, the sensitivity is low, and it is time-consuming and labor-intensive

Method used

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  • Primer set for detecting American canine influenza virus subtype H3N8 and application thereof
  • Primer set for detecting American canine influenza virus subtype H3N8 and application thereof
  • Primer set for detecting American canine influenza virus subtype H3N8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the design of primer set

[0043] After a large number of sequence analysis, multiple primer pairs were obtained, and the specificity and sensitivity of each primer pair were tested through preliminary experiments, and finally two primer pairs for identifying H3N8 subtype canine influenza virus were screened out.

[0044] Primer pair A (the target sequence is located in the HA gene, the annealing temperature is 59° C.) consists of primer F1 and primer R1.

[0045] Primer pair B (the target sequence is located in the NA gene, and the annealing temperature is 58° C.) consists of primer F2 and primer R2.

[0046] Primer F1 (sequence 1 of the sequence listing): 5'-TTGCTACCCATATGACATCCCTGAC-3';

[0047] Primer R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-GTGAATCCCTCTGCTGTGAATTCCA-3'.

[0048] Primer F2 (sequence 3 of the sequence listing): 5'-CCCCAATGAAGGGAAGGTGGAATGC-3;

[0049] Primer R2 (SEQ ID NO: 4 of the Sequence Listing): 5'-ATCCTCTCCCCTAGGGGTGTCAGTG...

Embodiment 2

[0050] Embodiment 2, establishment of method

[0051] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA.

[0052] The sample to be tested can be a tissue to be tested or a virus to be tested. The tissue to be tested can be canine liver tissue, canine brain tissue, canine lung tissue, canine nasal cavity swab, etc.

[0053] 2. Use the cDNA obtained in step 1 as a template, and use the primers designed in Example 1 to perform fluorescent quantitative PCR on A, and monitor the fluorescence intensity in real time.

[0054] Fluorescent quantitative PCR reaction system (20 μL): SYBR Premix Ex Taq (2X) 10 μL, primer F1 0.5 μL, primer R1 0.5 μL, cDNA 2 μL, add double distilled water to 20 μl.

[0055] The reaction program of fluorescent quantitative PCR: 95°C for 10min; 95°C for 15s, 59°C for 1min, 40 cycles.

[0056] 3. Using the cDNA obtained in step 1 as a template, use the primers designed in Example 1 to carry out fluorescent quantitative ...

Embodiment 3

[0062] Embodiment 3, establishment of standard curve

[0063] Gene template copy number = DNA mass concentration / DNA molecular weight.

[0064] 1. Preparation of standard quality grain A

[0065] The DNA molecule shown in sequence 5 in the sequence listing is the HA gene fragment of North American H3N8 subtype canine influenza virus. Insert the DNA molecule shown in sequence 5 in the sequence listing into the multiple cloning site of pUC57 vector to obtain standard plasmid A.

[0066] 2. Preparation of standard plasmid B

[0067] The DNA molecule shown in sequence 6 in the sequence listing is the NA gene fragment of North American H3N8 subtype canine influenza virus. The DNA molecule shown in sequence 6 of the sequence listing was inserted into the multiple cloning site of the pUC57 vector to obtain standard plasmid B.

[0068] 3. Establishment of standard curve A

[0069] 1. Gradiently dilute the standard quality Granules with double distilled water 10 times to obtain a ...

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Abstract

The invention discloses a primer set for detecting American canine influenza virus subtype H3N8 and application thereof. The primer set provided by the invention comprises a primer pair A and a primer pair B; the primer pair A is composed of a primer F1 and a primer R1; the primer pair B is composed of a primer F2 and a primer R2; the primer F1 is as shown in a sequence 1; the primer R1 is as shown in a sequence 2; the primer F2 is as shown in a sequence 3; and the primer R2 is as shown in a sequence 4. The application of the primer set comprises (c1) assistant identification of the American canine influenza virus subtype H3N8; and (c2) assistant identification of that whether a to-be-detected sample contains the American canine influenza virus subtype H3N8. The primer set has good specificity and high sensitivity when used for detection and can detect a clinical sample within 4 to 5 h; and the primer set is simple to operate, easy to promote and convenient for basic-level operation and application, and is of great application value to diagnosis and epidemiological investigation of diseases caused by the American canine influenza virus subtype H3N8.

Description

technical field [0001] The invention relates to a primer set for detecting North American H3N8 subtype canine influenza virus and its application. Background technique [0002] In recent years, reports of influenza A viruses infecting dogs have emerged continuously, including H3N8 subtype influenza viruses, H3N2 subtype influenza viruses, H5N1 subtype influenza viruses and H1N1 subtype influenza viruses. Reports on outbreaks of canine influenza virus. The H3N8 subtype canine influenza virus can effectively spread in dogs, causing flu-like symptoms such as fever, cough, and runny nose, and canine severe hemorrhagic pneumonia and death. [0003] At present, there have been no reports of dogs being infected with North American H3N8 subtype canine influenza virus in China. With the expansion of the pet market in recent years, and the increasing frequency of the introduction of police dogs and military dogs in various countries, this has increased the possibility of North Ameri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2563/107
Inventor 蒲娟宋晶伟孙洪磊王晨曦张谞霄刘金华
Owner CHINA AGRI UNIV
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