Freeze-dried microchip for identifying porcine reproductive and respiratory syndrome virus classic strain and NADC30-Like strain, kit and method thereof

A technology of porcine blue ear virus and classic strains, applied in the field of virus molecular biology detection, can solve the problems of easy pollution, cumbersome operation of common PCR, long detection time, etc.

Active Publication Date: 2020-03-17
CHINA ANIMAL DISEASE CONTROL CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary PCR still has disadvantages such as cumbersome operation, long detection time and easy contamination.

Method used

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  • Freeze-dried microchip for identifying porcine reproductive and respiratory syndrome virus classic strain and NADC30-Like strain, kit and method thereof
  • Freeze-dried microchip for identifying porcine reproductive and respiratory syndrome virus classic strain and NADC30-Like strain, kit and method thereof
  • Freeze-dried microchip for identifying porcine reproductive and respiratory syndrome virus classic strain and NADC30-Like strain, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0126] Experimental Example 1 Fluorescent RT-PCR detection kit for pig blue ear classic strain and NADC30-Like strain virus freeze-dried microchip specificity verification

[0127] 1. Design primers and Taqman-MGB probes

[0128] According to the domestic detection of classic strain of pig blue ear and NADC30-Like virus, find out the specific conserved sequence of NSP2 gene of classic pig blue ear strain and NADC30-Like strain virus, and design multiple pairs of primers and probes. After comparison and screening, a set of optimal primers and a Taqman-MGB probe were finally determined.

[0129] Upstream primer of porcine blue ear classic strain: 5'-TGTTGTGACTTGAGCGTCGAT-3'

[0130] Downstream primer of porcine blue ear classic strain: 5'-CAAAAGGCCAGGAACCGTAA-3'

[0131] Taqman probe for classic strain of pig blue ear: 5'FAM-TTTGTGATGCTCGTCAGG-MGB-3';

[0132] NADC30-Like strain upstream primer: 5'-GCCTCGCTCAGAACTTCCT-3'

[0133] NADC30-Like strain downstream primer: 5'-...

experiment example 2

[0148] Experimental example 2. Fluorescent RT-PCR detection kit for lyophilized microchip virus of pig blue ear classic strain and NADC30-Like strain Sensitivity verification and comparison with conventional fluorescent RT-PCR reagents

[0149] Set the concentration to 1 x 10 7 TCID 50 / mL of the classic strain of porcine blue-eared virus liquid was subjected to 10-fold serial dilution. Extract 1×10 6 TCID 50 / mL~1×10 0 TCID 50 / mL Genomic RNA of classic strain of porcine blue ear virus at each gradient concentration is used as a template, and nuclease-free water is used as a negative control, and the freeze-dried microchip fluorescent RT-PCR reagent and conventional fluorescent RT-PCR reagent are used to detect classic strain of porcine blue ear virus. Sensitivity testing.

[0150] The freeze-dried microchip fluorescent RT-PCR system was prepared according to Example 1. Add 10× buffer dilution to 24 μL of nuclease-free water for shaking and mixing, then add 0.6 μL to ...

experiment example 3

[0156] Experimental Example 3: Fluorescent RT-PCR Detection Kit for Freeze-dried Microchip Fluorescence RT-PCR of Porcine Blue Ear Classic Strain and NADC30-Like Strain Virus preparation and testing

[0157] 1. Preparation of the kit:

[0158] Preparation of lyophilized microchips:

[0159] Prepare the freeze-dried microchip RT-PCR system according to the following reaction system:

[0160]

[0161] The whole freeze-dried microchip sample hole of the AriaYSB microchip (Beijing Yisenbao Biotechnology Co., Ltd.) of fluorescent quantitative PCR is 30, and the above-mentioned fluorescent quantitative PCR system (comprising 2 sets of pig blue ear classic strain and NADC30-Like strain primer probe), 1.2 μL was added to each microchip well.

[0162] The microchips coated with PCR reagents were frozen at -80°C for 1 h. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the clapboard drops to -55°C, and the holding time...

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Abstract

The invention discloses a freeze-dried microchip for identifying a porcine reproductive and respiratory syndrome virus classic strain and an NADC30-Like strain, a kit and a method thereof, which belong to the field of molecular detection. The freeze-dried microchip is characterized in that a fluorescent PCR reaction system used for identifying the porcine reproductive and respiratory syndrome virus classic strain and the NADC30-Like strain is fixed on the microchip through freeze-drying; wherein the fluorescent PCR reaction system comprises the following primers and probes: a porcine reproductive and respiratory syndrome virus classic strain upstream primer, a porcine reproductive and respiratory syndrome virus classic strain downstream primer and a porcine reproductive and respiratory syndrome virus classic strain Taqman probe sequence; an NADC30-Like strain upstream primer, an NADC30-Like strain downstream primer, and an NADC30-Like strain Taqman probe. According to the present invention, the porcine reproductive and respiratory syndrome classical strain virus and the NADC30-Like strain virus are simultaneously detected, and the detection kit has characteristics of high accuracy,high specificity, high sensitivity and short detection time.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of viruses, in particular to a freeze-dried microchip, a kit and a method for distinguishing classic strains of porcine PRRS virus and NADC30-Like strains. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is commonly known as "pig blue ear disease". The disease is an important disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) that produces reproductive and respiratory disorders in pigs. PRRSV mainly infects pigs, especially sows. The disease seriously affects their reproductive function. The main clinical features are miscarriage, stillbirth, mummified fetus, weak fetus, dyspnea, and transient skin cyanosis on both ears during the course of the disease. Therefore, it is also called blue ear disease. It is the number ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2561/101C12Q2563/107C12Q2531/113C12Q2545/113
Inventor 韩焘周智倪建强刘玉良王传彬杨林王新杰高姗姗孙晓明胡祥钰
Owner CHINA ANIMAL DISEASE CONTROL CENT
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