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h10 subtype avian influenza virus rt-lamp visual detection kit

A bird flu virus and kit technology, applied in the field of H10 subtype bird flu virus RT-LAMP visual detection kit, can solve the problems of unsuitable for grass-roots popularization, time-consuming, high cost, etc., easy to achieve results, equipment and operation Simple, highly sensitive effects

Active Publication Date: 2016-01-20
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional virus chicken embryo isolation method is accurate and simple, but it takes too long, usually two days to a week; IFA and ELISA are fast but require special reagents; RT-PCR, RRT-PCR, Genechips detection technology is fast and sensitive, But all require special instruments and technical personnel, and the cost is relatively high, so it is not suitable for promotion and use at the grassroots level

Method used

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  • h10 subtype avian influenza virus rt-lamp visual detection kit
  • h10 subtype avian influenza virus rt-lamp visual detection kit
  • h10 subtype avian influenza virus rt-lamp visual detection kit

Examples

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Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Preparation and use of the loop-mediated isothermal amplification kit for detecting H10 subtype avian influenza virus

[0046] 1. Preparation of loop-mediated isothermal amplification primer set for detection of H10 subtype avian influenza virus

[0047] The loop-mediated isothermal amplification primer set for detection of H10 subtype avian influenza virus in this embodiment consists of inner primer FIP-H10, inner primer BIP-H10, outer primer F3-H10, outer primer B3-H10, and loop primer LF- H10 and loop primer LB-H10. These six primers were designed by the inventors of the present invention by comparing and analyzing the HA gene sequences of a large number of H10 subtype avian influenza viruses. Their nucleotide sequences are respectively sequence 1, sequence 2, sequence 3, Sequence 4, Sequence 5 and Sequence 6 (Table 1).

[0048] Table 1 RT-LAMP primer sequence

[0049]

[0050]

[0051] 2. Optimization of reverse transcription loop-mediated isothe...

Embodiment 2

[0067] The specificity analysis of the RT-LAMP kit of embodiment 2, H10 subtype avian influenza virus

[0068] 1. Analyze the specificity of RT-LAMP kit with each subtype of avian influenza virus

[0069] Strains to be tested: Avian influenza virus strain A / duck / HK / 876 / 80 (H10N3), and the following other important subtypes of avian influenza viruses: H1N1, H3N2, H5N3, H6N8, H7N2, H9N2, H11 (see Table 2 ).

[0070] Using the RT-LAMP kit of the H10 subtype avian influenza virus obtained in Example 1, the RNA of each strain to be tested as described above is used as the sample to be tested, and the RT-LAMP is carried out according to the optimized conditions in Step 2 of Example 1. LAMP, thereby analyzing the specificity of the RT-LAMP kit of the H10 subtype avian influenza virus obtained in Example 1. The experimental setup used an equal amount of sterilized water instead of RNA as the negative control of the template. Experiments were repeated three times.

[0071] The resu...

Embodiment 3

[0084] The sensitivity analysis of the RT-LAMP kit of embodiment 3, H10 subtype avian influenza virus

[0085] One, the preparation of the plasmid of H10 subtype avian influenza virus HA gene target fragment

[0086] The RNA of the H10 subtype avian influenza virus was extracted and reverse transcribed to obtain cDNA; PCR amplification was performed with primers F3-H10 and B3-H10 to obtain the target fragment of the HA gene of the H10 subtype avian influenza virus. After the PCR products were recovered and purified by gel, they were connected to the pGEM-TEasy vector (purchased from Promega, USA), and the plasmids of positive clones were extracted, and sent to Dalian Bao Biology Co., Ltd. for sequencing. Sequencing showed that the sequence fragment at position 634-913 of GenBank: JQ924786.1 was positively connected to the pGEM-TEasy vector, and the recombinant plasmid was named H10HA-pGEM-T.

[0087] 2. Sensitivity analysis of RT-LAMP kit for H10 subtype avian influenza virus...

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Abstract

The invention discloses an RT-LAMP visual detection kit for H10 subtype avian influenza viruses. The invention provides a LAMP primer group for detection of H10 subtype avian influenza viruses, namely, a primer group A composed of a primer 1, a primer 2, a primer 3, a primer 4, a primer 5 and a primer 6, or a primer group B composed of the primer 1, the primer 2, the primer 3 and the primer 4; and the nucleotide sequences of the primer 1, the primer 2, the primer 3, the primer 4 , the primer 5 and the primer 6 are respectively represented by a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5 and a sequence 6. According to experimental results, the RT-LAMP visual detection kit has the advantages of good specificity, high sensitivity, simple instrument and equipment, easiness in operation and convenience in result observation, and is particularly suitable for quick diagnosis and early screening of the H10 subtype avian influenza viruses in grassroots veterinary stations, livestock farms and frontier ports.

Description

technical field [0001] The invention relates to an RT-LAMP visual detection kit for H10 subtype avian influenza virus. Background technique [0002] Avian influenza (Avian Influenza, AI) is the abbreviation of avian influenza, which is an infectious disease of poultry (poultry and wild birds) caused by the Orthomyxoviridae influenza virus type A avian influenza virus. According to the antigenicity of the surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) of type A influenza virus, avian influenza virus (AIV) can be further divided into different subtypes, and 16 types of HA have been found so far. subtype and nine NA subtypes. AIV is an enveloped, single-stranded negative-strand, segmented RNA virus, and the viral genome consists of eight segmented negative-strand RNAs. The segmented nature of the AIV genome makes it possible that when two or more different subtypes of influenza viruses co-infect a cell, the nucleic acid fragments of the progeny...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 谢芝勋罗思思刘加波庞耀珊邓显文谢志勤谢丽基范晴
Owner GUANGXI VETERINARY RES INST
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