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LAMP kit for detecting PRV and preparation method thereof

A porcine pseudorabies virus and a kit technology are applied in the field of animal health inspection, which can solve the problems of unfavorable basic-level laboratory promotion and application, long amplification reaction time, easy contamination of target genes, etc., to improve the level of immune control and low cost. , high specificity and sensitivity

Active Publication Date: 2012-04-25
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there have been quite a lot of researches on PCR technology, but in the application process, it has been found that it has many shortcomings: (1) the target gene is easily contaminated
(2) often cause non-specific amplification
(3) The taq DNase required for the reaction is often affected by components such as tissue, blood, and IgG, which reduces the activity, thereby inhibiting the amplification reaction
(4) Requires more expensive PCR instrument
(5) The amplification reaction takes a long time, usually several hours, which is not conducive to the promotion and application in grassroots laboratories, etc.
However, there is no precedent for the detection of pseudorabies virus using the LAMP method (loop-mediated isothermal amplification, loop-mediated isothermal amplification)

Method used

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  • LAMP kit for detecting PRV and preparation method thereof
  • LAMP kit for detecting PRV and preparation method thereof
  • LAMP kit for detecting PRV and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Extraction of porcine pseudorabies virus PK-15 cell culture DNA:

[0051] Cells with typical lesions were harvested, frozen and thawed once, centrifuged at 4000r / min to obtain 445 μL of supernatant, and 12.5 μL of proteinase K (20 mg / mL) and 25 μL of 10% SDS were added. Water bath at 50°C for 2 hours, extract phenol, phenol:chloroform, and chloroform once each, and absorb the water phase. 2 times the volume of absolute ethanol was precipitated at -20°C for 1 hour, centrifuged at 10,000 r / min for 10 minutes, the precipitate was washed with 70% ethanol, dried, dissolved in 20 μL TE, and frozen at -20°C for future use.

[0052] 2. Amplification reaction

[0053] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND) and 2 times Buffer solution; pseudorabies virus gE gene LAMP primer; dNTPs

[0054] (2) Amplification reaction system: the total volume of the amplification reaction is 25 μL, and its various components and final concentrations a...

Embodiment 2

[0058] 1. Extraction of porcine pseudorabies virus PK-15 cell culture DNA:

[0059] Boil method. Take 200 μL of virus cell culture, bathe in water at 100°C for 10 minutes, bath in ice for 5 minutes, centrifuge at 1000 rpm, and use the supernatant for detection.

[0060] 2. Amplification:

[0061] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND) and 2 times Buffer solution; pseudorabies virus gE gene LAMP primer; dNTPs

[0062] (2) Amplification reaction system: the total volume of the amplification reaction is 25 μL, and its various components and final concentrations are: 2×Buffer (containing Mg 2+ ) 2.5 μL; gE3-F3 0.5 μL; gE3-B3 0.5 μL; gE3-FIP 4 μL; gE3-BIP 4 μL; dNTPs 3.5 μL; 8 U / μL Bst enzyme 1 μL; water 7 μL; DNA sample 2 μL.

[0063] (3) Amplification reaction process: denature at 95°C for 5 minutes, proceed at 63°C for 45 minutes, and continue at 80°C for 2 minutes, store at 4°C;

[0064] (4) After the amplification reaction is over...

Embodiment 3

[0066] 1. Extract DNA from diseased piglets infected with porcine pseudorabies virus: Cut the collected tonsils and brain tissues into pieces in a sterilized mortar, grind them thoroughly, and wash them with a solution containing 100 U / mL penicillin and 100 mg / L streptomycin. Hank's solution was diluted into a 5-fold suspension, centrifuged at 4000r / min for 30min at 4°C, and the supernatant was taken, and the virus DNA in the tissue was extracted according to the method of Example 1.

[0067] 2. Amplification reaction:

[0068] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND) and 2 times Buffer solution; pseudorabies virus gE gene LAMP primer; dNTPs

[0069] (2) Amplification reaction system: the total volume of the amplification reaction is 25 μL, and its various components and final concentrations are: 2×Buffer (containing Mg 2+ ) 2.5 μL; gE3-F3 0.5 μL; gE3-B3 0.5 μL; gE3-FIP 4 μL; gE3-BIP 4 μL; dNTPs 3.5 μL; 8 U / μL Bst enzyme 1 μL; water 7 ...

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Abstract

The invention relates to a LAMP kit for detecting PRV, an establishment method and an application thereof. The kit comprises a detection system consisting of LAMP reaction solution of four primers. Detection has verified that the kit can quickly and conveniently detect the PRV with high efficiency, high specificity and high sensitivity under 60 to 65DEG C isothermal condition, does not need any complicated instrument, can well meet the requirement on the field detection to the PRV, can distinguish PRV gE gene deletion vaccine and field strains, provides a novel technical platform for the safety detection of food, can well meet the urgent need on the field detection of the PRV, is used for the field detection of import and export quarantine, food health departments, livestock farms and thelike, and is easy to be popularized and applied in a large scope.

Description

technical field [0001] The invention belongs to the technical field of animal health inspection, in particular to a novel method for rapidly detecting the gE gene of porcine pseudorabies virus. The amplification is judged by observing the turbidity and color change of the reaction tube, or observing the agarose gel electrophoresis image under ultraviolet light situation to judge the outcome. Background technique [0002] Pseudorabies (Pseudorabies, PR) is also known as pruritus, Ouyeji's disease, and its pathogen is pseudorabies virus (Pseudorabies Virus, PRV) of the herpesviridae family. Clinically, it is characterized by fever and neurological symptoms. By 2007, there were reports of this disease in 44 countries and regions in the world, and this disease has become one of the most serious diseases that endanger the swine industry in the world. Breeding pigs infected with pseudorabies virus cause infertility; pregnant sows have abortion, stillbirth and mummified fetuses; ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 张莉鄢明华李秀丽王东任卫科韩伟孙英峰
Owner 天津市农业科学院
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