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Fluorescent quantitative detection kit for typing identification of avian metapneumovirus

A fluorescent quantitative detection technology for avian metapneumovirus, applied in the field of genetic engineering, can solve the problems of inability to identify different subtypes of avian metapneumovirus, low sensitivity of avian metapneumovirus, lack of kits, etc.

Inactive Publication Date: 2018-06-08
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a fluorescent quantitative detection kit for typing and identification of avian metapneumovirus, so as to solve the technical problem of lacking such a kit in the prior art
[0006] Another technical problem to be solved by the present invention is that the serological detection methods for avian metapneumovirus in the prior art cannot realize the typing and identification of different subtypes of avian metapneumovirus
[0007] Another technical problem to be solved by the present invention is that the detection method for avian metapneumovirus in the prior art has low sensitivity

Method used

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  • Fluorescent quantitative detection kit for typing identification of avian metapneumovirus
  • Fluorescent quantitative detection kit for typing identification of avian metapneumovirus
  • Fluorescent quantitative detection kit for typing identification of avian metapneumovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The design of embodiment 1 aMPV identification detection kit primer and probe

[0037] 1. Design of primers and probes for aMPV TaqMan-MGB PCR identification kit:

[0038] Comprehensively compare the complete gene sequences of the four subtypes A, B, C, and D published by GeneBank, avoid the homologous regions of the four subtypes, ensure the specificity of fluorescent probes and primers, and use Primer Express 2.0 software , designed four sets of primers, respectively named as MGB-A (F / R), MGB-B (F / R), MGB-C (F / R), MGB-D (F / R). The design results of primers and probes are shown in Table 1

[0039] Table 1 Primers and probes for aMPV typing and identification

[0040]

[0041] 2. Design and screening of chicken housekeeping genes.

[0042] Based on the chicken housekeeping gene sequence published by GeneBank, two pairs of primers and probes were designed as candidate internal references, named ACTB-F1, ACTB-P1, ACTB-R1; ACTB-F2, ACTB-P2, ACTB-R2, respectively. Th...

Embodiment 2

[0057] Example 2 Optimization of the reaction system of the aMPV TaqMan-MGB PCR identification and detection kit:

[0058] All of the following optimizations are carried out using the following reference reaction system and reference cycle parameters: see Table 4 and Table 5

[0059] Table 4 reference reaction system

[0060]

[0061] 1. Optimization of upstream and downstream primer concentration of aMPV TaqMan-MGB PCR identification kit

[0062] The concentration optimization of aMPV primers adopts the gradient increasing method, and 5 gradients of 0.3, 0.4, 0.5, 0.6, and 0.7um are selected for cross-matching combination experiments. The reaction conditions are the same as in Table 5. Each concentration combination was repeated several times, the average value was taken, and the optimization results were comprehensively judged in combination with the smoothness of the curve and the fluorescence intensity.

[0063] The verified aMPV primers MGB-A (F / R), MGB-B (F / R), MGB-...

Embodiment 3

[0091] The optimization of embodiment 3 ACTB FQ-PCR reaction system

[0092] 1. Optimization of upstream and downstream primer concentrations for ACTB FQ-PCR

[0093] The verified ACTB primers ACTB-F and ACTB-R were selected to optimize the concentration combination, and the average value of 3 repetitions was taken. The concentration of ACTB primers was optimized using the gradient increasing method, and 5 gradients of 0.3, 0.4, 0.5, 0.6, and 0.7uM were selected for cross-matching combination experiments. The reaction conditions are the same as shown in Table 5. Each concentration combination was repeated times, and the average value was taken, and the optimization results were comprehensively judged in combination with the smoothness of the curve and the fluorescence intensity. It can be seen from Table 12 that when the upstream primer (ACTB-F) is 0.4uM and the downstream primer (ACTB-R) is 0.6uM, the CT value is relatively small, the amplification quality is the best, and t...

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Abstract

The invention provides a fluorescent quantitative detection kit for typing identification of avian metapneumovirus. Firstly, through design and screening of specific fluorescent primers, optimizationof an FQ-PCR reaction system and conditions, and verification of specific tests, sensitivity tests and sensibility tests, a rapid, sensitive and specific identification detection method is establishedand is applied to clinical detection. Then, an ACTB FQ-PCR reaction system is established with a chicken ACTB gene as an internal reference; with compatible optimization with aMPV TaqMan-MGB PCR conditions, a relative quantitative FQ-PCR method of aMPV nucleic acid is established. Compared with detection methods of avian metapneumovirus in the prior art, the detection kit has higher degree of precision, can realize typing identification of different serotypes at the same time, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fluorescent quantitative detection kit for typing and identifying avian metapneumovirus. Background technique [0002] Avian metapneumovirus is an acute and highly contagious disease caused by avian metapneumovirus (Avian metapneumovirus, aMPV), which is also known as Turkey rhinotracheitis virus (Turkey rhinotracheitis virus, TRTV) or avian pneumonia virus ( Avian pneumonia virus, APV). Mainly airborne, avian metapulmonosis causes severe economic losses to the poultry industry worldwide. At present, my country is the largest chicken-raising country in the world, with 9.6 billion chickens. The mortality rate due to various poultry diseases is as high as 20% to 25% every year, and tens of billions of yuan are lost. Therefore, the development of specific, sensitive and simple identification and detection kits is an urgent need for the prevention and control of epide...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114
Inventor 李守军阎旭李亚杰付旭彬郁宏伟刘海霞王艳晓
Owner TIANJIN RINGPU BIO TECH
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