Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof

A technology for respiratory syndrome and pig reproduction, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to identify and detect PRRSV mutant strains at the same time, achieve good application prospects, and improve prevention and control. Control level, reduce detection cost and detection time effect

Inactive Publication Date: 2015-08-12
TECON BIOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0028] This method can detect whether the pathogenic PRRSV Tianjin strain (TJ strain) and PRRSV epidemic virus (including highly pathogenic PRRSV Jiangxi strain, Hunan strain and PRRSV classic strain) are contained in the sample in one reaction, but this method cannot further Differentiate PRRSV mutant strains (including highly pathogenic PRRSV Jiangxi strain, Hunan strain and their similar strains) and PRRSV classic strains
[0029] In summary, none of the above four patented methods can simultaneously detect whether the sample contains the classic PRRSV strain, TJM-F92 strain and other highly pathogenic pig breeding and variant strains through one reaction, and identify the three

Method used

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  • Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof
  • Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof
  • Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Kit sensitivity test

[0064] 1. RNA extraction of PRRSV TJM-F92 strain (commercially available):

[0065] The cultured PRRSV TJM-F92 strain was diluted to 10 5 TCID 50 、10 4 TCID 5 、10 3 TCID 50 、10 2 TCID 50 、10 1 TCID 50 、10 0 TCID 50 . Viral RNA was extracted by the Trizol method,

[0066] Methods as below.

[0067] (1) Add 600 μL Trizol Reagent to every 200 μL sample solution, vortex for 15 seconds, and let stand at room temperature for 5 minutes.

[0068] (2) Add 200 μL of chloroform to the centrifuge tube, mix it upside down, and let it stand at room temperature for 5 minutes.

[0069] (3) Centrifuge at 12,000rpm at 4°C for 10 minutes, take the upper liquid and put it in a new centrifuge tube (be careful not to suck up the protein on the layered surface by mistake, try to stick to the wall as much as possible, and change the pipette tip every time). Add 800 μL of isopropanol (containing 0.01% DEPC), cap and turn it upside down 1 or 2 times,...

Embodiment 2

[0083] Kit specificity test:

[0084] 1. Table 2 of the control strains, including:

[0085]

[0086]

[0087] 2. Virus and bacterial DNA extraction: Use the cell / tissue DNA extraction kit from Tiangen Biological Company, and refer to the instruction manual for the method.

[0088] (1) Take 1ml of the bacterial solution and centrifuge at 12,000rpm for 5 minutes, remove the supernatant, add 200μL GA solution to the pellet, resuspend and mix well.

[0089] (2) Add 20 μL proteinase K solution and mix well. The samples were mixed by inversion 2-3 times in a water bath at 65°C for 1 hour. Remove from the water bath and centrifuge briefly to remove water droplets from the inside of the cap.

[0090] (3) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. The solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0091] (4) Add 200 μL of absolute ethanol, shake and mix well for 1...

Embodiment 3

[0116] RT-PCR Amplification Test to Differentiate PRRSV American Classic Strain, Variant Strain and TJM-F92 Strain

[0117] 1. Table 3 of the control strains, including:

[0118]

[0119] 2. Extraction of porcine reproductive and respiratory syndrome virus RNA: according to the Trizol method, refer to Example 1.

[0120] 3. cDNA synthesis

[0121] Use 20.0 μL cDNA synthesis system, that is, PRRSV RNA template 11 μL, 5×RT Buffer 5.0 μL, Random Primers (50pmol / μL) 1.0 μL, dNTP Mixture 2.0 μL, RNase Inhibitor (40 U / μL) 0.5 μL, MLV Reverse Transcriptase XL (5U / μL) 0.5 μL.

[0122] Reaction program: 10 min at 25°C, 60 min at 42°C, 5 min at 99°C, 5 min at 4°C. After cDNA synthesis, perform PCR amplification or store at -20°C for later use.

[0123] 4. PCR reaction amplification: use the porcine reproductive and respiratory syndrome virus identification and detection kit for PCR amplification.

[0124] The total volume of the amplification reaction is 25 μL, and its various co...

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PUM

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Abstract

The invention relates to a detection kit for identifying a porcine reproductive and respiratory syndrome virus and an application thereof. The kit comprises a pair of PRRSV specific detection primers and a detection system. The detection verifies that the kit is capable of rapidly, conveniently, effectively, high-specifically and high-sensitively detecting the PRRSV; the PRRSV TJM-F92 strain, the American classic type strain and the variant strain (highly pathogenic porcine reproductive and respiratory syndrome virus) can be distinguished through one pair of primers; and the reagent is low in cost, convenient to operate and is suitable for identifying the PRRSV by the import and export quarantine and professional laboratory.

Description

technical field [0001] The invention belongs to the technical field of animal virus molecular biology detection, and relates to a primer and a kit for differentially diagnosing PRRSV TJM-F92 strain, American type classic strain and variant strain and application thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "pig blue ear disease", is a disease caused by PRRSV (Porcine reproductive and respiratory syndrome virus, PRRSV), characterized by reproductive disorders in pregnant sows and respiratory symptoms in piglets. It is one of the main diseases that cause reproductive disorders in pig farms. The disease first broke out in the United States, and then spread to all parts of the world, causing huge economic losses to the world's pig industry. In China, Guo Baoqing and others first isolated PRRSV from aborted fetuses in Beijing in 1996, thus confirming the existence of the disease in my country. [0003] In recent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 周绪斌鄢明华万春燕孙英峰黎作华王利丽张莉李秀丽路超
Owner TECON BIOLOGY CO LTD
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