Lamp detection primers and Lamp detection method for discrimination of equine herpesvirus type 1/4

A technique for equine herpesvirus and detection method, applied in the field of equine herpesvirus type 1/4 typing, detection and identification

Inactive Publication Date: 2014-04-30
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a simple, rapid, specific, simple operation and easy-to-determine Lam

Method used

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  • Lamp detection primers and Lamp detection method for discrimination of equine herpesvirus type 1/4
  • Lamp detection primers and Lamp detection method for discrimination of equine herpesvirus type 1/4
  • Lamp detection primers and Lamp detection method for discrimination of equine herpesvirus type 1/4

Examples

Experimental program
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Effect test

example 1

[0028] Example 1. Using the established LAMP method to detect horse nasal swabs collected in situ

[0029] The collected 10 horse nasal swabs were put into 1 mL of sterile phosphate buffer (containing 100 U / mL penicillin and 100 μg / mL streptomycin) for 30 minutes, and then sterilized by 0.22 μm filtration. Viral DNA from Omega Company in the United States was used. Extraction kit to extract viral DNA.

[0030]65°C water bath; about 300μL of ethanol per sample; keep both samples and Operate Buffer at room temperature; preheat Elution Buffer (500μL / sample) at 65°C, add EHV1 / 4 virus solution (1-250μL) to a sterile centrifuge To the tube, add 250 μL of pre-warmed Elution Buffer; add 10 μL Operate Buffer, 250 μL Buffer BL, 4 μL Linear A, respectively, and vortex for 15 s at the maximum speed; incubate at 65°C for 10 min; during incubation, vortex appropriately; Add 260 μL of absolute ethanol and vortex for 20 s at maximum revolutions. After fully mixing, it was instantly detached...

example 2

[0032] Example 2. Detection of equine tissue collected in situ using the established LAMP method

[0033] Using 10 brain tissue samples collected on site, take 0.1 g of disease material, add 100 μL of tissue extract, then add 5 μL of proteinase K, 55 °C for 12 h, then centrifuge at 12000 r for 3 min, aspirate the supernatant, add 500 μL of saturated phenol to extract and shake, Centrifuge at 12000r for 5min, transfer the supernatant to another centrifuge tube, add twice the volume of absolute ethanol, and place at -20°C for 20min. Upside down several times, centrifuge at 12000r for 15min, discard the supernatant, add 1ml of 70% ethanol to wash once, centrifuge at 12000r for 15min, discard the supernatant, dry, add 30μL TE to dissolve, and store at -20°C. Using the extracted viral DNA as a template, the Lamp method was used to detect the EHV1Lamp reaction system as follows: FIP (4 μM), BIP (4 μM), F3 (0.5 μM), B3 (0.5 μM), dNTP (7.5 μM), MgCl 2 (125 μM), 10×buffer (2.5 μM), Bs...

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Abstract

The invention discloses Lamp (loop-mediated isothermal amplification) detection primers and a Lamp detection method for discrimination of equine herpesvirus type 1/4 and application thereof, belonging to the field of molecular biological diagnosis methods for animal virosis. A corresponding detection primer group is designed according to the gB gene sequence of equine herpesvirus by using Lamp technology and comprises outer primers FIP and BIP and inner primers F3 and B3, and the Lamp detection method for typing equine herpesvirus type 1 and equine herpesvirus type 4 is invented with the detection primer group as the detection primers. According to the invention, equine herpesvirus infection and typing and other functions at the level of DNA can be realized by carrying out extraction of total DNA and Lamp amplification on equine nose swab, equine brain tissue and lung tissue samples, and the characteristics of easiness, convenience, fastness, good specificity, easy determination of results and the like are obtained.

Description

technical field [0001] The invention relates to a Lamp primer of equine herpesvirus and a rapid detection method thereof, in particular to equine herpesvirus type 1 / 4 typing detection and identification, and belongs to the field of animal virus disease detection, identification and prevention. Background technique [0002] Equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) are two closely related alphaherpesviruses that cause Equine rhinopneumonitis (ER). In addition to causing respiratory diseases, EHV-1 can mainly cause abortion in pregnant mares, and occasionally cause neurological diseases in horses; EHV-4 mainly causes respiratory symptoms, and can occasionally cause abortion in pregnant mares. EHV-1 / 4 are closely related in heredity, antigenicity and pathogenicity, and have many common epidemiological, clinical and pathological features. Sequence analysis data also confirmed that the genomes of the two types of viruses are closely related but also different...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/70C12Q1/6844C12Q2531/119
Inventor 曲娟娟胡晓亮于敏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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