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Triplex PCR detection primer group, kit and method for chicken parvovirus (ChPV) and H9 subtype avian influenza virus (AIV)

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

Pending Publication Date: 2020-02-11
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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  • Triplex PCR detection primer group, kit and method for chicken parvovirus (ChPV) and H9 subtype avian influenza virus (AIV)
  • Triplex PCR detection primer group, kit and method for chicken parvovirus (ChPV) and H9 subtype avian influenza virus (AIV)
  • Triplex PCR detection primer group, kit and method for chicken parvovirus (ChPV) and H9 subtype avian influenza virus (AIV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer Design and Synthesis

[0027] According to the M gene sequence of different subtypes of avian influenza virus, the HA gene sequence of H9 subtype AIV and the specific conserved region of ChPV NS gene, 3 pairs of M gene, H9 subtype AIV HA gene and ChPV were designed and screened through verification. The specific primers for NS gene amplification include primers M-F, primers M-R, primers H9-F, primers H9-R, primers ChPV-F and primers ChPV-R. The sequence of primer M-F is shown in SEQ.ID.NO.1, the sequence of primer M-R is shown in SEQ.ID.NO.2, the sequence of primer H9-F is shown in SEQ.ID.NO.3, and the sequence of primer H9- The sequence of R is shown in SEQ.ID.NO.4, the sequence of primer ChPV-F is shown in SEQ.ID.NO.5, and the sequence of primer ChPV-R is shown in SEQ.ID.NO.6. Primers were synthesized by Thermo Fisher Guangzhou Branch.

Embodiment 2 3

[0028] Establishment and optimization and specificity test of embodiment 2 triple 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) are preserved by Guangxi Key Laboratory of Veterinary Biotechnology The cDNA template of AIV strain (H7N2) was donated by Pennsylvania State University; the cDNA template of AIV strain (H5N1) was donated by Connecticut State University; other AIV strains (H2N3, H8N4, H10N3 and H11N3) or cDNA templates were provided by Gift from the University of Hong Kong;

[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 Super Mix, DNA / RNA co-extraction kit, small amount of plasmid extraction kit, Gel recovery kit, competent cells, PCR product quick ligation carrier and DNA Marker were purchased from Beijing...

Embodiment 3

[0036] Embodiment 3 sensitivity test

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

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Abstract

The invention discloses a triplex PCR detection primer group, kit and method for chicken parvovirus (ChPV) and H9 subtype avian influenza virus (AIV). Specific primers for the chicken ChPV and the H9subtype AIV are designed, a primer combination with a better amplification effect is obtained through testing and screening of different primer combinations, then, the primer concentration in a reaction system, ratios of primers, annealing temperature and amplification time are continuously optimized, and finally, the method capable of detecting the chicken ChPV and the H9 subtype AIV simultaneously is established. The primers and the detection method can determine mixed infection and single infection conditions of the chicken ChPV and the H9 subtype AIV in samples at the same time by one-timePCR reaction, and the method has the advantages of being high in specificity, high in sensitivity, rapid, convenient, easy to operate and the like, is quite suitable for application and promotion ingrassroots units and provides technical support for monitoring, preventing and controlling the chicken ChPV and the H9 subtype AIV.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a triple 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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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2537/143
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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