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RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination

A primer composition and canine influenza virus technology, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve problems such as unsuitable for rapid detection, long detection cycle, and low specificity, and reduce detection costs , low cost, simple operation

Pending Publication Date: 2016-08-03
NANJING POLICE DOG RES INST OF MINIST OF PUBLIC SECURITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods have long detection cycle, low sensitivity, low specificity, or require special equipment, so they are not suitable for rapid detection in grassroots farms and disease sites

Method used

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  • RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination
  • RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination
  • RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Canine influenza virus RT-LAMP primer screening and reaction system optimization

[0071] 1. Extract nucleic acid: extract the nucleic acid of the sample to be tested according to the conventional method.

[0072] 2. Primer design and screening: According to the CIVM gene and H gene gene sequences published by GenBank, 3 sets of primers were designed through http: / / primerexplorer.jp / e / PrimerExplorer4.0, including inner primers, outer primers and loop primers. The three sets of primers designed are numbered [1], [2], [3], wherein the [1] set of primers is the H gene primers, and the [2] and [3] sets are the M gene primers. Three sets of primers were used for LAMP amplification of CIV RNA, and the amplification efficiency of the three sets of primers was detected with a LAMPRealTimeTurbidimeter LA-320C to screen the optimal set of primers. The sequences of the three sets of primers screened are shown in Table 1.

[0073] Table 1 [1], [2], [3] primer sequences ...

Embodiment 2

[0095] The specificity test of embodiment 2 canine influenza virus LAMP

[0096] Using the CIVLAMP primers designed and screened in this study, canine distemper virus (Canine distempervirus, CDV), canine parainfluenza virus (Canine Para-influenza Virus, CPIV), canine adenovirus (Canineadenovirus, CAV), H9 subtype poultry Influenza virus, canine parvovirus (Canine Parvovirus, CPV), Bordetella bronchus ATCC31124 strain, Pasteurella, Escherichia coli, and Staphylococcus aureus were samples to be tested, with CIV virus RNA as a positive control, and double distilled water as a negative control, According to the established CIVLAMP detection method, the specificity of the method was tested.

[0097] The LAMP reaction system in this example was carried out according to the optimized reaction system and conditions in Example 1.

[0098] Results Among all the measured samples, the CIV virus RNA samples were positive, and the other samples were negative, and the negative control was e...

Embodiment 3

[0099] The sensitivity test of embodiment 3 canine influenza virus LAMP

[0100] First, use the total RNA of CIV extracted by TaKaRaMiniBESTViralRNA / DNAExtractionKit, and then use eppendorfRS-232C to detect the RNA concentration, perform 10-fold gradient dilution with double distilled water, and select 10 -1 to 10 -7 The 7 different gradients were used as templates, and distilled water was used as blank control, and the sensitivity test was carried out according to the established CIVLAMP detection method.

[0101] At the same time, the same RNA samples were used for RT-PCR verification with M gene universal primers, and the products were subjected to agarose gel electrophoresis, and the results were compared with the results of the LAMP test.

[0102] The concentration of the extracted RNA was detected to be 4.3 mg / L, which was serially diluted 10 times with double distilled water, and amplified according to the optimized reaction system and conditions in Example 1. Use the...

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Abstract

The invention discloses an RT-LAMP primer combination and kit for detecting canine influenza virus as well as an application of the RT-LAMP primer combination. The LAMP primer combination consists of a forward outer primer F3 as shown in SEQ ID NO.1, a reverse outer primer B3 as shown in SEQ ID NO.2, a forward inner primer FIP as shown in SEQ ID NO.3, a reverse inner primer BIP as shown in SEQ ID NO.4 and a loop primer LB as shown in SEQ ID NO.5. The LAMP primer combination is applied to the detection of canine influenza virus. With the adoption of the screened LAMP primer combination for detecting canine influenza virus provided by the invention, a method for detecting canine influenza virus through LAMP is established, and the method has the advantages of being high in specificity, high in sensitivity, quick, simple, convenient and free of special instruments.

Description

technical field [0001] The invention relates to a primer and a kit for detecting canine influenza virus, in particular to an RT-LAMP primer set for detecting canine influenza virus H3N2 subtype and an RT-LAMP kit for detecting canine influenza virus H3N2 subtype. The invention belongs to the technical field of biological detection. Background technique [0002] Influenzavirus (Influenzavirus) belongs to Orthomyxoviridae, and its genome is composed of 8 negative-strand RNA segments, encoding 11 or 12 proteins. The virus is pathogenic to various animals including birds and mammals. It has become an important infectious pathogen threatening human and animal health. Canine influenza (Canine influenza, CI) is a kind of canine animal contact respiratory infectious disease caused by canine influenza virus (Canine influenza virus, CIV). , dyspnea, etc., the epidemic spreads rapidly, and often leads to mass epidemics and death in large-scale farms. Studies have confirmed that infl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 秦海斌温海朱骞强京宁张汇东宋珍华刘永杰贺星亮高一龙
Owner NANJING POLICE DOG RES INST OF MINIST OF PUBLIC SECURITY
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