Polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV)

A technology of duck plague virus and detection method, which is applied in the field of scientific research and production of animal medicine, can solve problems such as inability to judge duck farms, and achieves the effect of simple judgment method and broad application prospect.

Inactive Publication Date: 2012-08-22
SICHUAN AGRI UNIV
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Problems solved by technology

Therefore, if the diagnosis and monitoring of duck plague virus in a farm that has been immunized against duck plague virus is positive, the current detection

Method used

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  • Polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV)
  • Polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV)
  • Polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Differentiate the PCR detection method of duck plague virus virulence and vaccine attenuation

[0028] 1 test material

[0029] The 10-day-old duck embryo was negative for DPV and antibody of the breeding duck.

[0030] 2× Dumb PCR Mixtrue, purchased from Bao Biology.

[0031] Virulent duck plague virus: Duck plague virus CH virulent strain (DPV CHv), referred to as CHv strain, was isolated and preserved in this laboratory. Center (CCTCC), deposit number: CCTCC NO: V201209, classified as duck plague virus CH virulent strain (DPV CHv).

[0032] Duck plague virus attenuated vaccine virus: purchased from Chengdu Tianbang Biological Products Co., Ltd., veterinary drug production license number: (2006) veterinary drug production certificate number 07022; approval number: veterinary doctor word (2006) 070222023.

[0033] 2 Experimental methods

[0034] 2.1 Preparation method of normal duck embryo fibroblasts (DEF)

[0035] Take 10-day-old healthy duck embryo...

Embodiment 2

[0052] Embodiment 2 PCR specificity evaluation test

[0053] Duck viral hepatitis virus (DHV) ATCC VR1313, infectious laryngotracheitis virus (ILTV) ATCC VR-783, Marek's virus (MDV) ATCC VR-585, Muscovy duck parvovirus (MDPV) CVCC AV238, Avian Influenza (AIV) CCTCC-V200312, Riemerella Anatipestifer (RA) ATCC 11845, Salmonella Duck (Sa1.A) CMCC 50083, Pasteurella (PM) CAU0085 and Duck Escherichia coli (E.coli) CVCC 83003 by The DNA extracted by the conventional method was subjected to PCR, and at the same time, a positive control of the attenuated vaccine strain, a positive control of the strong virulence of the CHv strain, and a blank control were set up, and the reaction system and parameters were the same. Similarly, 6 μL of the PCR product was electrophoresed on a 1% agarose gel, and DL2000 was set, and the presence or absence of amplified fragments was observed by ultraviolet light irradiation.

[0054] For the test results of the specificity test, see figure 2 , in the...

Embodiment 3

[0055] Sensitivity test of embodiment three PCR detection

[0056] Method 1: TCID 50 sensitivity

[0057] Use 1.5*10 8 TCID 50 / ml of CHv strain duck plague virulent virus to extract DNA, also use 2*10 5.5 TCID 50 / ml of duck plague attenuated vaccine strain virus extraction DNA, the templates were diluted to 10 times the concentration gradient by 10 times. 8 Carry out PCR amplification, and set up a blank control at the same time. The reaction system and parameters are the same. Take 6 μL of PCR products and electrophoresis on 1% agarose gel.

[0058] TCID 50 For the results of the sensitivity test, see image 3 ;In the figure: Lane 10 is undiluted DNA, 1.5*10 8 TCID 50 / ml; Lane 9: 1.5*10 7 TCID 50 / ml; Lane 8: 1.5*10 6 TCID 50 / ml; Lane 7: 1.5*10 5 TCID 50 / ml; Lane 6: 1.5*10 4 TCID 50 / ml; Lane 5: 1.5*10 3 TCID 50 / ml; Lane 4: 1.5*10 2 TCID 50 / ml; Lane 3: 1.5*10TCID 50 / ml; Lane 2: 1.5TCID 50 / ml; lane 1: blank control; lane M: DL2000. It can be se...

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Abstract

The invention discloses a polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV). The method comprises the steps of: designing a specific primer sequence according to the gene difference between the virulent strains and the weak vaccine strains UL2 of the DPV, extracting a deoxyribonucleic acid (DNA) of a sample to be detected to be used as a template, and carrying out PCR; and analyzing the result of the PCR, wherein if a PCR amplification band is 1018bp, the sample to be detected contains the virulent strains of the DPV, and if the PCR amplification band is 490bp, the sample to be detected contains the weak vaccine strains of the DPV. The detection method for distinguishing the virulent strains and the weak vaccine strains of the DPV has the advantages of being simple, convenient, rapid, specific, sensitive and economical, and the result judgment method is simple and convenient, so that the detection method is suitable for scientific research, clinical application and a basic laboratory, and has wide application prospect.

Description

technical field [0001] The invention relates to the field of scientific research and production of veterinary medicine, in particular to a PCR detection method based on the UL2 gene difference between a strong duck plague virus strain and an attenuated vaccine strain Background technique [0002] Duck plague (DP), also known as duck viral enteritis (DVE), is caused by duck plague virus (Duck plague virus, DPV) infection. An acute contact infectious disease characterized by vascular injury, tissue hemorrhage, and damage to the digestive tract and lymphatic organs. The disease can lead to the decline in egg production and death of commercial waterfowl, and it is distributed in all duck-raising areas in the world. Its prevention and control are directly related to the sustainable and stable development of waterfowl farming. The prevention of duck plague in my country mainly adopts inoculation of attenuated vaccine, and accurate diagnosis and monitoring are the basis and premis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 程安春尹雪琴汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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