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Double PCR detection primer combination, detection kit and method for chicken parvoviruses and H9 subtype avian influenza viruses

A technology of chicken parvovirus and avian influenza virus, applied in the field of molecular biology, can solve the problems of mixed infection, difficult identification and diagnosis, etc., and achieve the effect of fast and simple operation, high sensitivity and easy operation

Pending Publication Date: 2020-02-28
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because AIV-infected animals have similar clinical symptoms, and the phenomenon of mixed infection is also common, it is difficult to make a timely and accurate differential diagnosis in daily diagnosis

Method used

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  • Double PCR detection primer combination, detection kit and method for chicken parvoviruses and H9 subtype avian influenza viruses
  • Double PCR detection primer combination, detection kit and method for chicken parvoviruses and H9 subtype avian influenza viruses
  • Double PCR detection primer combination, detection kit and method for chicken parvoviruses and H9 subtype avian influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer Development and Synthesis

[0027] According to the HA gene sequence of the H9 subtype AIV of the avian influenza virus and the specific conserved region of the ChPV NS gene, two pairs of specific primers for the amplification of the H9 subtype AIV HA gene and the ChPV NS gene were designed and screened, including Primer H9-F, Primer H9-R, Primer ChPV-F and Primer ChPV-R. The sequence of primer H9-F is shown in SEQ ID NO.1, the sequence of primer H9-R is shown in SEQ ID NO.2, the sequence of primer ChPV-F is shown in SEQ ID NO.3, and the sequence of primer ChPV-R is shown in SEQ ID NO.3. The sequence is shown in SEQ ID NO.4. Primers were synthesized by Thermo Fisher Guangzhou Branch.

Embodiment 2 2

[0028] Establishment and optimization and specificity test of embodiment 2 double PCR reaction system

[0029] 1. Strains: AIV strains (H1N2, H3N2, H6N2, H9N2, H9N6, NDV, IBV, ARV, ILTV, ChPV, Adv4, RTV, aMPV, CAV, AHEV and MDV) were all produced by the Guangxi Key Laboratory of Veterinary Biotechnology Preservation; AIV (H7N2) cDNA templates were donated by Pennsylvania State University; AIV strain (H5N1) cDNA templates were donated by Connecticut State University; other AIV (H2N3) strains or cDNA templates were donated by Hong Kong University;

[0030] 2. Main reagents and instruments: PCR instrument was purchased from Bio-Rad Laboratories of the United States; ultra-micro spectrophotometer was purchased from Thermo Company of the United States; 2×PCR Mix, DNA / RNA co-extraction kit, small amount of plasmid extraction kit, condensation Glue recovery kit, competent cells, PCR product quick ligation carrier and DNA Marker were purchased from Beijing Quanshijin Biotechnology Co....

Embodiment 3

[0036] Embodiment 3 sensitivity test

[0037] 1. Preparation of standard products: refer to [Li Dan, Xie Zhixun, Song Degui, et al. The establishment of a double RT-PCR detection method for H9 and H6 subtype avian influenza viruses [J]. China Animal Husbandry and Veterinary Medicine, 2016, 43(12): 3101-3106.], using the primers of ChPV NS gene and H9N2 HA full-length gene, and using the cDNA of H9N2 and ChPV as templates to amplify RT-PCR respectively to obtain the full-length target fragments of HA and NS genes, and These two gene fragments were respectively linked to the vector of pMD18-T. The recombinant vectors containing the correct sequences of the HA gene and ChPV NS gene fragments of H9 subtype AIV were named H9-T and NS-T, respectively. Use a plasmid extraction kit to extract the plasmids of H9-T and NS-T, and use a micro-nucleic acid detector to measure the nucleic acid concentration. Calculate the corresponding copy number according to the molecular mass and nuclei...

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Abstract

The invention discloses a double PCR detection primer combination, detection kit and method for chicken parvoviruses and H9 subtype avian influenza viruses. Specific primers targeting the chicken parvoviruses and the H9 subtype avian influenza viruses are designed, different primer combinations are tested and screened to obtain the primer combination good in amplification effect, and primer concentration, primer proportion, annealing temperature and amplification time in a reaction system are further optimized to finally build the method capable of simultaneously detecting the chicken parvoviruses and the H9 subtype avian influenza viruses. The primer combination and the detection method have the advantages that the mixed infection or single infection condition of the chicken parvovirusesand the H9 subtype avian influenza viruses in a sample can be determined at the same time through one PCR reaction; the method is high in specificity and sensitivity, fast and simple, easy to operate,quite suitable for being applied and popularized in grassroots units, capable of providing technical support for the monitoring and control of the chicken parvoviruses and the H9 subtype avian influenza viruses and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a double PCR detection primer set, kit and method for chicken parvovirus and H9 subtype avian influenza virus. Background technique [0002] Chicken parvovirus (Chincken parvovirus, ChPV) is one of the important viruses that cause enteric diseases in chickens. Clinically, it is characterized by diarrhea, mental depression, growth retardation and increased feed-to-meat ratio. ChPV mainly affects young chickens, mainly causing diarrhea and stunting in sick chickens. The virus is prevalent in some chicken flocks. Studies have found that ChPV DNA can be detected in the serum of chickens with developmental disabilities and dwarf syndrome. Epidemiological surveys showed that the infection rate was higher in commercial broiler chickens, followed by laying hens or breeders. In recent years, studies have shown that ChPV is common in chicken flocks in many countries around the wor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 谢芝勋李丹李孟谢丽基罗思思张艳芳张民秀黄娇玲范晴王盛谢志勤邓显文曾婷婷
Owner GUANGXI VETERINARY RES INST
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