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Gexp rapid detection primer set, kit and application for simultaneous identification of main subtypes of avian influenza

A technology for detection kits and detection primers, which is applied in the determination/inspection of microorganisms, microorganisms, recombinant DNA technology, etc., can solve problems that have not been reported yet, and achieve the effects of rapid identification, rapid sensitivity, and high sensitivity

Active Publication Date: 2020-01-14
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, H7 and H9 subtypes of avian influenza virus are at most aimed at different HA subtypes, and one reaction simultaneously amplifies seven genes of H5, H7, H9, N1, N2, N9 and M to detect And the nucleic acid detection technology for distinguishing the main subtypes of avian influenza virus has not been reported yet

Method used

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  • Gexp rapid detection primer set, kit and application for simultaneous identification of main subtypes of avian influenza
  • Gexp rapid detection primer set, kit and application for simultaneous identification of main subtypes of avian influenza
  • Gexp rapid detection primer set, kit and application for simultaneous identification of main subtypes of avian influenza

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 : Multiplex RT-PCR Primer Design for Avian Influenza Virus

[0025]Refer to relevant literature to download the sequences of 7 genes of avian influenza virus M, H5, H7, H9, N1, N2 and N9 from the GenBank database, and use DNAStar to analyze and compare the nucleotide sequences of each gene to find out the suitable design specific gene sequence. Conserved region of sex primer, utilize GeXP express profiler tool to design the specific primer (seeing table 1) for 7 pairs of genes of avian influenza virus, the designed primer adopts Primer Premier 5.0, NCBI PrimerBlast and Oligo7.0 to analyze and screen, then A non-homologous unique sequence was added to the 5' ends of all forward primers and reverse 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 determined by Synthesized by Shanghai Invitrogen Company and purified by HPLC.

[0026] Table 1 Primer in...

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 MiniBEST Viral RNA / DNAExtraction Kit Ver.5.0 (catalogue number DV819A), extract nucleic acids of different subtypes (H1-16 and N1-9) of avian influenza virus and other poultry viruses, and obtain 50 μL of nucleic acid samples, and divide them into devices Store 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.

[0033] Reaction system (25μL): 5×Reverse Transcriptase Buffer 5μL, 50mmol / L RandomPrimer (9mer) 1μL, dNTP Mixture (10mM / L) 2μL, 40U Ribonuclease Inhibitor 0.5μL, 5U / μL MLV Reverse Trans...

Embodiment 3

[0048] Example 3: GeXP system multiple detection system specific detection

[0049] 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 subtypes except H5, H7, and H9, NA gene subtypes except N1, N2, and N9 subtypes, and NDV, IBV, ILTV, and blank control had no reaction signals, suggesting that the established method had strong specificity , no cross-reaction with other test objects.

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Abstract

The invention belongs to the technical field of detection of avian influenza viruses and discloses a GeXP quick detection primer set and kit for identifying simultaneously major epidemic subtypes of avian influenza and their application. Seven pairs of specific primers and one pair of universal primers are designed based on the study on a GeXP system, and the GeXP kit for identifying simultaneously the major subtypes of avian influenza is established accordingly. The primer set and kit can be used to identify the subtypes H5, H7 and H9 and different subtypes of combined avian influenza, with a sensitivity of 102 copies per MuL. The primer set and kit have the advantages of high throughput, high specificity, high sensitivity, high speed and the like and are significant to the epidemiological investigation of major epidemic subtypes of avian influenza and their prevention and control, as well as the guarantee on the healthy substantial development of aviculture.

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 main subtypes of avian influenza. Background technique [0002] There are many subtypes of avian influenza virus (AIV), and they vary frequently. So far, 16 different hemagglutinin subtypes (HA) and 9 different neuraminidase subtypes (NA) have been found. The combination of HA and NA can produce various subtypes of influenza virus. 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. At present, the low pathogenicity avian influenza widespread in poultry is mainly H9 subtype. H5, H7, and H9 are widely prevalent in poultry, and the H5 subtype HPAI has the greatest impact. This subtype has been detecte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2563/107C12Q2565/125
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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