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Primer set used in duck plague virus detection, and LAMP reaction system composed thereof

A reaction system, duck plague virus technology, applied in recombinant DNA technology, microbe-based methods, microbes, etc., can solve the problems of low detection rate, low sensitivity, complicated experimental operation, etc., and achieve simple steps and easy identification , the effect of high sensitivity

Inactive Publication Date: 2012-01-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The routine serological diagnostic methods suitable for duck plague virus mainly include virus isolation and neutralization test, enzyme-linked immunosorbent assay, indirect hemagglutination test and other methods, but these detection techniques have long experimental period, complicated experimental operation, Due to the defects of low sensitivity and low detection rate, coupled with the rapid development of duck plague infection, it is urgent to establish a more rapid, specific and sensitive detection method

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  • Primer set used in duck plague virus detection, and LAMP reaction system composed thereof
  • Primer set used in duck plague virus detection, and LAMP reaction system composed thereof
  • Primer set used in duck plague virus detection, and LAMP reaction system composed thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Acquisition and Identification of Duck Plague Virus

[0047] 1. Materials

[0048] 1.1 Viruses and pathogens

[0049] The duck plague attenuated vaccine strain was provided by the poultry disease room of Guangdong Wen's Food Group.

[0050] Control pathogens: avian infectious laryngotracheitis positive strain (ILTV), muscovy duck parvovirus positive strain (MDPV), H9 subtype avian influenza virus antigen (H9 AIV), duck viral hepatitis type Ⅰ strain (DHVI) and Muscovy duck reovirus strain (MDRV) was isolated and preserved in the Poultry Research Laboratory of the School of Animal Science, South China Agricultural University.

[0051] 15 visceral tissues of dead ducks and diseased ducks were provided by Zhongshan Veterinary Epidemic Prevention Supervision Institute and Putian Branch of Guangdong Wen's Food Group.

[0052] 1.2 Duck Embryo

[0053] 13-14 days old non-immune duck embryos were provided by Putian Branch of Guangdong Wens Food Group.

[0054] 1...

Embodiment 2

[0086] The establishment of embodiment 2 duck plague virus LAMP detection method

[0087] Primer design and synthesis

[0088] The conserved segment of duck plague virus UL6 protein gene was selected by Blast, MegAlign and DNAStar programs, and four primers were designed using Primer Explorer V4 software, including two inner primers (FIP and BIP) and two outer primers (F3 and B3), the internal primer FIP is composed of F1c and F2, in order to facilitate the enzyme digestion and identification of subsequent amplification products, BIP is composed of B1c, B2 and GAATTC ( Eco RI restriction site sequence) as a linker. This test also used the PCR primer pair DEV-F and DEV-R for diagnosing duck plague virus to compare the PCR detection method with the newly established LAMP detection method. All primers were synthesized by Beijing Aoke Bioengineering Company. The synthesized primers were diluted with ultrapure water to a 10 mmol / mL solution and stored at -20 °C. The primer seq...

Embodiment 3

[0113] Example 3 Detection of DEV by LAMP method

[0114] 1. Extraction of Viral Nucleic Acid

[0115] DNA virus (ILTV, MDPV) extract nucleic acid according to the method 3.1.2.1. Viral antigen (H9 AIV, DHVI and MDRV) RNA was extracted according to Invitrogen's TRIzol LS Reagent RNA extraction kit. Add 250 μL viral allantoic fluid (or tissue homogenate supernatant) and 750 μL TRIzol to a 1.5 mL EP tube, mix thoroughly, and place at room temperature for 10 min; add 200 μL chloroform, shake vigorously for 15 sec, and let stand at room temperature for 5 min Afterwards, centrifuge at 12000 rpm at 4 °C for 15 min; take the supernatant into a new sterilized 1.5 mL microcentrifuge tube, add 500 μL isopropanol, mix thoroughly, place at room temperature for 10 min, and centrifuge at 12000 rpm at 4 °C for 10 min; Discard the supernatant, use 750 μL of 70% ethanol for the pellet, mix gently, wash once, and centrifuge at 12 000 rpm for 10 min at 4 °C; discard the supernatant and air-d...

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Abstract

The invention discloses a primer set used in duck plague virus detection, and a LAMP reaction system composed thereof. The detection primer set comprises a forward outer primer F3:SEQ ID NO:1, a backward outer primer B3:SEQ ID NO:2, a forward inner primer FIP:SEQ ID NO:3, and a backward inner primer BIP:SEQ ID NO:4. The LAMP reaction system comprising the primers also comprises a reaction buffer, dNTPs, Mg<2+>, betaine, BstDNA polymerase, detection sample genome DNA, deionized water, and the like. The reaction system has advantages of simple operation method, high specificity, high sensitivity, and convenient detection result identification. The system has wide prospect to be applied in small institutes, veterinary stations and common raising households.

Description

technical field [0001] The invention relates to a primer set for duck plague virus detection and a LAMP reaction system formed thereof. Background technique [0002] Duck plague (Duck Plague, DP), also known as duck viral enteritis, is an acute septic and highly fatal disease of ducks, geese, swans and other birds of the order Anseriformes caused by duck enteritis virus (DEV). of viral infectious diseases. Duck plague virus belongs to the α-herpesvirus subfamily of the family Herpesviridae. It is spherical and enveloped, with a diameter of about 160-180 nm and a capsid of icosahedral symmetry. It is a pantropic infection virus (Kaleta, 1990). The virus is mainly composed of four parts: core, capsid, tegument, and envelope. The viral nucleic acid is linear and double-stranded DNA. The morphological structure of the capsid has the important characteristics of herpes virus, which is composed of 162 capsomers that are connected radially and have hollow shaft holes. The nucleo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢青梅冀君邹禄生孙宝丽马静云毕英佐
Owner SOUTH CHINA AGRI UNIV
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