RT-LAMP detection primer group and kit for identifying H7 subtype HPAIV and LPAIV and application thereof

A technology of RT-LAMP and detection primers, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, microorganisms, etc. It can solve the problems of long time consumption and unsuitable for grass-roots application, and achieve the effect of simple operation

Pending Publication Date: 2018-08-10
防城港市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional identification method is HA gene sequence determination,

Method used

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  • RT-LAMP detection primer group and kit for identifying H7 subtype HPAIV and LPAIV and application thereof
  • RT-LAMP detection primer group and kit for identifying H7 subtype HPAIV and LPAIV and application thereof
  • RT-LAMP detection primer group and kit for identifying H7 subtype HPAIV and LPAIV and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 :Design and synthesis of primers

[0023] Download the HA gene sequence of H7 subtype AIV in GeneBank, use MEGA6.0 software for sequence comparison, and use the online software Primer Explorer V4 to design two sets of LAMP primers respectively according to the conservative and specific sequences of H7 subtype AIV. For the detection of H7 subtype and H7 subtype LPAIV, the primer sequences are shown in Table 1.

[0024] Table 1 H7 subtype AIV and H7 subtype LPAIV detection primer sequence

[0025]

[0026]

Embodiment 2

[0027] Example 2: Establishment and optimization of RT-LAMP detection system

[0028] 2.1 Viral nucleic acid extraction and cDNA synthesis

[0029] Refer to the manual of RNA extraction reagent RNAIso plus to extract viral allantoic fluid RNA, and finally dissolve RNA with DEPC water for reverse transcription. 50μL reverse transcription system: 5× buffer 10μL, dNTP (10mmol / L) 2μL, reverse transcription primer 1.5μL (concentration), AMV reverse transcriptase 1μL (10U / μL), RNA inhibitor 0.5μL (40U / μL), 35 μL of viral RNA. After mixing, act at 42°C for 30 minutes.

[0030] 2.2 Preparation of standards

[0031] Use the two pairs of primers H7-F3, H7-B3, H7-F3, H7-B3 in Table 1 to amplify the target fragments of the H7 subtype AIV HA and NA genes, run the PCR product and cut the target fragments after electrophoresis, and connect the vector pEASY, transform DH5α E. coli competent cells, use Amp-containing LB plates to screen E. coli, pick positive colonies for PCR identification, and ...

Embodiment 3

[0035] Example 3: RT-LAMP specificity and sensitivity test

[0036] Using optimized reaction conditions, different subtypes of AIV samples were tested to test the specificity of the RT-LAMP method. The prepared standard was amplified by the established method to test its sensitivity.

[0037] Such as Figure 1-2 As shown, the established H7 subtype AIV LAMP can detect a standard of 10 copies / μL; the established H7 subtype LPAIV LAMP can detect a standard of 10 copies / μL.

[0038] Table 2 RT-LAMP specific detection

[0039]

[0040] As shown in Table 2, the RT-LAMP test results of H7 subtype AIV RNA were positive, and the test results of other HA subtypes AIV were negative.

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Abstract

The invention belongs to the technical field of avian virus detection, and particularly relates to an RT-LAMP detection primer group and kit for identifying H7 subtype HPAIV and LPAIV and an application thereof. According to the RT-LAMP detection primer group and kit for identifying the H7 subtype HPAIV and LPAIV and the application thereof, an LAMP nucleic acid detection technology serves as a basis, amino acid sequences of an H7 subtype AIV HA protein cleavage sites are fully taken into account, two detection methods of universal LAMP and identification LAMP are specifically established. Theuniversal LAMP can simultaneously detect H7 subtype HPAIV and LPAIV, the identification LAMP can only detect H7 subtype LPAIV, and a new method for preliminary typing of H7 subtype AIV is provided. By optimizing each reaction condition, the detection method for H7 subtype avian influenza and H7 subtype attenuated strains is established, and the specificity and sensitivity experimental verifications are carried out on the detection method. By reacting for two hours at 62 DEG C in a turbidity meter, the H7 subtype avian influenza and the H7 subtype attenuated strains can be specifically identified and do not have cross reaction with other subtype avian influenza, and at least 10 copies of viral nucleic acids per microliter can be detected.

Description

Technical field [0001] The invention belongs to the technical field of avian virus detection, and in particular relates to an RT-LAMP detection primer set, kit and application thereof for distinguishing H7 subtype avian influenza virus and H7 subtype avian influenza attenuated strain. Background technique [0002] Influenza virus (Avian influenza, AIB) belongs to the Orthomyxoviridae Influenza virus genus. It is a segmented single-stranded negative-strand RNA virus. It contains 8 monomeric RNA strands and can encode up to 17 proteins (HA, NA, NP, M1, M2, M42, NS1, NS2, NS3, PA, PA-X, PA-N155, PA-N182, PB1, PB1-F2, PB1-N40, PB2). According to the variation of surface antigen HA and NA proteins, influenza A viruses are currently further divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (NA1-NA11). [0003] According to the pathogenicity of the virus to chickens, avian influenza (AIV) can be divided into highly pathogenic avian influenza (HPAIV) and low pathogenic avian influen...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 熊文婕范晴曹国敏覃海曾婷婷严小东凌小英
Owner 防城港市动物疫病预防控制中心
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