Primer set and kit for triple RT-PCR detection of H10N8 subtype avian influenza viruses and application of primer set and kit

A technology of RT-PCR and avian influenza virus, which is applied in the field of poultry virus detection, can solve the problems of lack of correction function and achieve the effect of high sensitivity, low cost and strong specificity

Inactive Publication Date: 2016-03-09
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the genome of avian influenza virus is replicated in segments, the replication is completed by relying on RNA polymerase, and RNA polymerase lacks the correction function, so the frequency of genetic mutation of avian influenza virus is extremely high

Method used

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  • Primer set and kit for triple RT-PCR detection of H10N8 subtype avian influenza viruses and application of primer set and kit
  • Primer set and kit for triple RT-PCR detection of H10N8 subtype avian influenza viruses and application of primer set and kit
  • Primer set and kit for triple RT-PCR detection of H10N8 subtype avian influenza viruses and application of primer set and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 : Primer design and kit preparation for avian influenza virus triple RT-PCR

[0028] (1) Design of primers

[0029] According to the conserved sequences of 3 genes including the HA gene of H10 subtype AIV, the NA gene of N8 subtype AIV and the M gene of all subtype AIV published in GenBank, three pairs of specificity were designed and synthesized through NCBI Blast verification Primers (see Table 1) for the detection of H10 subtype AIV, N8 subtype AIV and all subtypes of AIV.

[0030] Table 1 Primer information

[0031]

[0032] Note: AIVH10-1 and H10-2 primers are used to detect whether it contains H10 subtype avian influenza virus; AIVN8-1 and N8-2 primers are used to detect whether it contains N8 subtype avian influenza virus; AIVM-1 and M- 2 primer pairs for universal detection of all subtypes of avian influenza virus.

[0033] (2) Sample preparation

[0034] According to the instructions of the MinibestviralRNA / DNAextraction kit ver.4.0 extraction ...

Embodiment 2

[0042] Example 2: Specificity test of triple RT-PCR detection kit

[0043] Avian influenza virus (H1N1, H2N3, H3N2, H4N6, H5N1, H6N8, H7N2, H8N4, H9N2, H10N3, H10N7, H11, H12N5, H13N5, H14N5, H15N9, H16N3), Newcastle disease virus, infectious bronchitis virus and The RNA / DNA of infectious laryngotracheitis virus was added to the reverse transcription reagent and PCR reagent in the triple RT-PCR detection kit, and the optimal reaction conditions were applied to detect avian influenza virus, Newcastle disease virus, and infectious bronchitis virus. and infectious laryngotracheitis virus nucleic acid for RT-PCR amplification to detect its specificity.

[0044] The result is as figure 1 As shown, viral nucleic acid templates containing H10+N8 subtype AIV, H10 subtype AIV, N8 subtype AIV, and other subtypes of AIV can amplify amplified bands that match the size of the experimental design, while other common respiratory birds Viruses at the same position without any amplificatio...

Embodiment 3

[0046] Example 3: Sensitivity test of triple RT-PCR detection kit

[0047] The H10 subtype avian influenza virus HA gene, N8 subtype avian influenza virus NA gene and avian influenza virus M gene full-length primers were used for PCR amplification with the corresponding cDNA templates, and the positive products were recovered and purified by gel, and connected to pGEM- TEasy vector, extract positive cloned plasmids, send them to Dalian Bao Biological Co., Ltd. for sequencing, calculate the copy number of each plasmid according to the molecular weight and concentration; dilute the plasmids of AIV-H10, AIV-N8, and AIV-M by 10 times as a gradient ratio At the same time, it was added to the above-mentioned best RT-PCR kit for amplification, and its sensitivity was tested.

[0048] Sensitivity assay (results see figure 2 ), the RT-PCR method established by the present invention can detect H10 subtype AIV, N8 subtype AIV at least, and the M gene of universal detection avian infl...

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Abstract

The invention belongs to the technical field of avian virus detection and discloses a primer set and a kit for triple RT-PCR detection of H10N8 subtype avian influenza viruses and application of the primer set and the kit. Three pairs of specific primers are designed and combined into the kit for triple RT-PCR detection of H10N8 avian influenza viruses targeting conserved sequences of HA genes of H10 subtype avian influenza viruses, NA genes of N8 subtype avian influenza viruses and M genes of all avian influenza viruses. Through detection with one tube, H10N8 subtype avian influenza virus infection can be determined. By the application of the primer pairs or the primer pair set or the kit, whether a sample to be tested is infected by the H10N8 subtype avian influenza viruses or the H10 subtype avian influenza viruses or the N8 subtype avian influenza viruses or other subtype avian influenza viruses can be detected, great significance is achieved in effective prevention and control of avian influenza and cut-off of the propagation path of avian influenza, and application prospect is broad.

Description

technical field [0001] The invention belongs to the technical field of poultry virus detection, in particular to a triple RT-PCR detection primer set and kit for H10N8 subtype avian influenza virus, identification of H10 subtype avian influenza virus, N8 subtype avian influenza virus, general detection of all subtypes A primer pair or a primer pair set and a kit thereof for type avian influenza virus. Background technique [0002] Avian Influenza Virus (AIV) is divided into different subtypes according to the surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) antigenic differences, and 18 HA subtypes (H1-H18) have been found so far. ) and 11 NA subtypes (N1-N11). AIV is an enveloped, single-stranded negative-strand, segmented RNA virus, and the viral genome consists of eight segmented negative-strand RNAs. Because the genome of avian influenza virus is replicated in segments, the replication is completed by relying on RNA polymerase, and RNA pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/16
Inventor 谢芝勋罗思思谢志勤黄莉谢丽基邓显文范晴黄娇玲张艳芳王盛曾婷婷
Owner GUANGXI VETERINARY RES INST
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