Gexp rapid detection primer set, kit and application for simultaneous identification of three h5 subtype avian influenza virus combinations infecting humans

A technology of avian influenza virus and detection kit, which is applied in the direction of microbial determination/inspection, recombinant DNA technology, biochemical equipment and methods, etc., and can solve problems such as difficult typing and determination, unfavorable prevention and control, and formulation of emergency measures , to achieve the effect of ensuring healthy and sustainable development, fast sensitivity and high sensitivity

Active Publication Date: 2018-09-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since H5N1, H5N2, and H5N6 combined with H5 subtypes of avian influenza viruses infecting humans have similar clinical symptoms, it is difficult to accurately type and judge them in a short period of time with existing diagnostic techniques. It is not conducive to the formulation of its prevention and control and emergency measures
At present, most of the nucleic acid detection methods for detecting H5 subtype AIV are aimed at its HA gene, and the technology of simultaneously detecting and typing the H5 subtype avian influenza virus combinations of H5N1, H5N2, and H5N6 infecting humans has not been reported yet.

Method used

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  • Gexp rapid detection primer set, kit and application for simultaneous identification of three h5 subtype avian influenza virus combinations infecting humans
  • Gexp rapid detection primer set, kit and application for simultaneous identification of three h5 subtype avian influenza virus combinations infecting humans
  • Gexp rapid detection primer set, kit and application for simultaneous identification of three h5 subtype avian influenza virus combinations infecting humans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0026] Refer to relevant literature to download the sequences of five genes of avian influenza virus M, H5, N1, N2 and N6 from the GenBank database, use DNAStar to analyze and compare the nucleotide sequences of each gene, and find out the conserved genes suitable for designing specific primers. region, utilize the GeXP express profiler tool to design specific primers for 5 genes of avian influenza virus (see Table 1), the designed primers are analyzed and screened using Primer Premier 5.0, NCBI PrimerBlast and Oligo7.0, and then in all forward A non-homologous unique sequence was added to the 5' ends of the primer and reverse primer respectively 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 , HPLC purification.

[0027] Table 1 Primer Information

...

Embodiment 2

[0031] Example 2: Establishment of multiplex PCR detection system

[0032] 2.1 Preparation of template and monoclonal plasmid standard containing target gene

[0033] According to the instructions of TaKaRa Company MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (catalogue number DV819A), extract nucleic acids of different subtypes (H 1-16 and N 1-9) of avian influenza virus and other poultry viruses, and obtain 50 μL of nucleic acid samples , and stored at -80°C. 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.

[0034] The reverse transcription RT reaction solution is 9 μL per tube: 5 μL of 5×Reverse Transcriptase Buffer, 1 μL of 50 pmol Random Primer (12mer), 2 μL of 10 mM dNTP Mixture, 0.5 μL of 40U Ribonuclease...

Embodiment 3

[0050] Example 3: GeXP system multiple detection system specific detection

[0051] According to the instructions of MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0, extract HA(1-16) and NA(1-9) subtype avian influenza virus nucleic acid from allantoic fluid, and simultaneously extract IBV, NDV and ILTV and other common poultry viruses Nucleic acids were added to the GeXP multiple detection system established in Example 2 to detect the specificity of the method. 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, NA gene except N1, N2 and N6 subtypes, and other NA subtypes, as well as NDV, IBV, ILTV and blank control had no reaction signal, suggesting that the established method has strong specificity and is different from other detection methods. Subjects had no cross-reactivity.

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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 combination of three types of infectious H5 subtype avian influenza viruses synchronously and application of the primer group and kit. According to the GeXP rapid detection primer group and kit and application of the primer group, five pairs of specific primers and one pair of universal primer are researched and designed based on a GeXP system, and accordingly the GeXP rapid detection kit for identifying combination of three types of infectious H5 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, three types of infectious H5 subtype H5N1, H5N2 and H5N6 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 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 three types of H5 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, kit and application thereof for simultaneously identifying combinations of three H5 subtype avian influenza viruses that infect humans. Background technique [0002] Avian influenza is an acute infectious disease of poultry caused by avian influenza virus, and some subtypes of avian influenza virus can also infect humans. Avian influenza viruses can be divided into 16 HA subtypes and 9 NA subtypes according to the differences in the antigenic relationship between their surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). According to its pathogenicity, it can be divided into highly pathogenic avian influenza and low pathogenic avian influenza. Highly pathogenic avian influenza includes H5 and H7 subtypes. Especially in recent years, the H5 subtype avian influenza outbreaks have caused huge economic losses...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
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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