GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of primer group and kit

A bird flu virus and primer set technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, microorganisms, etc., can solve the problems of nucleic acid detection technology that have not been reported yet, and achieve healthy and sustainable development, rapid sensitivity, and high sensitivity Effect

Active Publication Date: 2016-02-10
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the nucleic acid detection methods for detecting H5 and H9 subtypes of avian influenza virus are at most aimed at different HA subtypes, and one reaction simultaneously amplifies five genes of H5, N2, N1, H9 and M to detect and distinguish H5N1 and H9N2 subtypes. Nucleic acid detection technology of type avian influenza virus has not been reported yet

Method used

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  • GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of primer group and kit
  • GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of primer group and kit
  • GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 : Multiplex RT-PCR Primer Design for Avian Influenza Virus

[0026] Referring to related literature, download the sequences of M, H5, H9, N1 and N25 genes of avian influenza virus from the GenBank database, use DNAStar to analyze and compare the nucleotide sequences of each gene, and find out the conserved regions suitable for designing specific primers , Utilize the GeXPexpressprofiler tool to design specific primers for 5 genes of avian influenza virus (see Table 1), the designed primers are analyzed and screened using PrimerPremier5.0, NCBIPrimerBlast and Oligo7.0, then in all forward primers and reverse A non-homologous unique sequence was added to the 5' ends of the primers as a universal primer (Uni-Primer), and the 5' end of the upstream universal primer was labeled with a fluorescent dye Cy5, namely Cy5-Tag-F, which was synthesized by Shanghai Invitrogen Company and purified by HPLC.

[0027] Table 1 Primer information

[0028]

[0029] In Table ...

Embodiment 2

[0030] Example 2: Establishment of multiplex PCR detection system

[0031] 2.1 Preparation of template and monoclonal plasmid standard containing target gene

[0032] According to the instructions of TaKaRa Company MiniBESTViralRNA / DNAExtractionKitVer.5.0 (catalogue number DV819A), extract nucleic acids of different subtypes (H1-16 and N1-9) of avian influenza virus and other poultry viruses, obtain 50 μL nucleic acid samples, and divide them into devices at -80°C save. The RT reaction system was carried out according to the instructions of TaKaRa reverse transcriptase (catalogue number D2639A), and the obtained RNA samples were reverse-transcribed according to the following reaction system and reaction conditions to obtain cDNA; DEPC water was used as the control of total RNA.

[0033] The reverse transcription RT reaction solution is 9 μL per tube: 5 μL of 5×ReverseTranscriptase Buffer, 1 μL of 50 pmol Random Primer (12mer), 2 μL of 10 mM dNTPMixture, 0.5 μL of 40URibonuc...

Embodiment 3

[0049] Example 3: GeXPsystem multiple detection system specific detection

[0050] Extract HA (1-16) and NA (1-9) subtype avian influenza virus nucleic acids from allantoic fluid according to MiniBESTViralRNA / DNAExtraction KitVer. In the GeXP multiple detection system established in Example 2, the specificity of the method was tested. After the multiplex PCR was completed, the PCR products were analyzed by GeXP capillary electrophoresis, and the results showed that only specific signals appeared in each reaction, and there was no cross-reaction. HA gene except H5 subtype, H9 subtype, NA gene other NA subtypes except N1 and N2 subtypes, as well as NDV, IBV, ILTV and the blank control had no reaction signal, suggesting that the established method had strong specificity, and was comparable to There was no cross-reaction with other test objects.

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Abstract

The invention belongs to the technical field of poultry virus detection and discloses a GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of the primer group and kit. According to the GeXP rapid detection primer group and kit and the application of the primer group and kit, based on a GeXP system, five pairs of specific primers and one pair of universal primers are researched and designed, and accordingly, the GeXP rapid detection kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously is established. By means of the GeXP rapid detection primer group and kit and application of the primer group and kit, the H5N1 and H9N2 subtype avian influenza viruses can be identified synchronously, and the sensitivity is 102 copy/micron L. The GeXP rapid detection primer group and kit and application of the primer group and kit have the advantages of being high in flux, high in specificity, high in sensitivity, high in speed and the like and have significance to epidemiological investigation and differential diagnosis of the H5N1 and H9N2 subtype avian influenza viruses.

Description

technical field [0001] The invention belongs to the technical field of poultry virus detection, and in particular relates to a GeXP rapid detection primer set, a kit and an application thereof for simultaneously identifying H5N1 and H9N2 subtype avian influenza viruses. Background technique [0002] Avian influenza virus (Avian influenza virus, AIV) has many subtypes and frequent mutations. 16 different hemagglutinin subtypes (HA) and 9 different neuraminidase subtypes (NA) have been found. According to different pathogenicity, avian influenza can be divided into high pathogenicity and low pathogenicity. The H5 and H7 subtypes of AIV can cause the occurrence of highly pathogenic avian influenza. In 1996, the H5N1HPAI outbreak first broke out in a goose farm in Guangdong, China. Since then, the virus has become endemic in poultry in Southeast Asia. At present, H5N1AIV has spread to more than 60 countries around the world, and each outbreak can cause large-scale of the poultr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 谢芝勋李孟谢志勤罗思思谢丽基黄莉邓显文黄娇玲范晴张艳芳曾婷婷王盛
Owner GUANGXI VETERINARY RES INST
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