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Rapid detection method of chicken infectious anemia virus and kit

A technology of chicken infectious anemia and detection method, which is applied to the detection kit for detecting chicken infectious anemia virus and the field of rapid detection of chicken infectious anemia virus, which can solve the problems of no detection of chicken infectious anemia virus, etc. Broad, high sensitivity, strong specificity

Active Publication Date: 2009-02-04
北京翎羽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no detection method and detection kit for chicken infectious anemia virus using the loop-mediated isothermal amplification method

Method used

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  • Rapid detection method of chicken infectious anemia virus and kit
  • Rapid detection method of chicken infectious anemia virus and kit
  • Rapid detection method of chicken infectious anemia virus and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Establishment of LAMP detection method

[0085] 1. pTCIAV cloning and plasmid template construction

[0086] 1.1 Experimental materials

[0087] 1.1.1 Virus strains:

[0088] The CuX-1 strain of CIAV standard virus is preserved by our laboratory and can be released to the public.

[0089] 1.1.2 Plasmid vector and recipient strain:

[0090] Plasmid vector: pGEM-T Easy Vector Kit was purchased from Invitrogen.

[0091] Recipient strain: Escherichia coli Top10 was purchased from Tiangen Biological Company.

[0092] 1.1.3 Medium:

[0093] LB liquid medium: every 1000ml contains tryptone 10g; yeast extract 5g; NaCl 10g, prepared according to conventional methods and used for the cultivation of E.coli TOP10. Ampicillin (Ampicillin, Amp) was purchased from Sigma at a concentration of 100 μg / mL.

[0094] LB solid medium: Add imported agar powder to LB liquid medium to a final concentration of 1.5%, autoclave, and keep at 4°C.

[0095] 1.1.4 Polymerase and othe...

Embodiment 2

[0197] Example 2 Application of LAMP Detection Method in Differential Diagnosis

[0198] 1. Test material

[0199] 1.1 Virus strains:

[0200] CIAV standard virus CuX-1 strain is preserved by our laboratory, pTCIAV positive clone strain.

[0201] 1.2 Control strains: (All are kept by the laboratory and can be distributed to the public.)

[0202] RNA virus group: avian influenza antigen, chicken infectious bursal virus (IBD), avian encephalomyelitis virus (REV), chicken Newcastle disease virus (ND), chicken infectious bronchial virus (IB), avian leukosis virus (ALV).

[0203] DNA virus group: chicken egg drop syndrome virus (EDS), chicken Marek's virus (MD).

[0204] Bacterial control group: Haemophilus parainfectious rhinitis (IC), Pasteurella subspecies A, Escherichia coli, Salmonella typhi, and Avian bacteria.

[0205] 1.3 Polymerase and other tool enzymes:

[0206] Pyrobest DNA polymerase. Trizol Reagent (Invitrogen Company), plasmid micro-extraction kit was purchased...

Embodiment 3

[0231] Example 3 Application of LAMP detection method in clinical samples

[0232] 1. Test material

[0233] 1.1 Virus strains:

[0234] The tissue disease materials of CIAV infection and the serum of chickens infected with CIAV virus are all preserved by our laboratory.

[0235] 1.2 Polymerase and other tool enzymes:

[0236] Bst DNA polymerase.

[0237] 1.3 Lysis solution: including 10mM Tris-Hcl (pH8.8); 1mM EDTA; 15mM Nacl; 0.5% SDS.

[0238] 1.4 Other solutions and reagents

[0239] SYBR Green staining solution, Goldview staining solution

[0240] 2. Test method:

[0241] Processing of Clinical Samples

[0242] Take 100 μL of serum or disease material (add appropriate amount of normal saline to grind and dilute), add 20 μL of lysate, shake and mix on the shaker, boil in a water bath for 10 minutes, place on ice for more than 5 minutes, then centrifuge at 4°C, 12000rpm for 10 minutes, and take the supernatant for later use .

[0243] LAMP reaction:

[0244] The L...

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Abstract

The invention relates to a method for rapidly testing chicken infectious anemia virus and a kit, which relate to the technical field of biology. The target region of chicken infectious anemia virus genes is amplified by using the specific LAMP primer group selectivity through the LAMP method to determine whether amplification products exist. The method of the invention reasonably avoids the inconvenience brought by the special requirements for temperature circulation of a conventional PCR; SYBR Green I stain can be added in the LAMP final product in the experiment for directly observing the results without the need of observation through gel electrophoresis or the turbidity monitoring method of the by-product-magnesium cell pyrophosphate. The method is simple and easy to be operated. A novel, rapid and easily-operated chicken infectious anemia virus identification and diagnosis method is provided by utilizing the method of the invention, further establishes the working foundation for controlling chicken infectious anemia, and has good economic benefits and market prospects.

Description

technical field [0001] The invention discloses a method for rapidly detecting chicken infectious anemia virus. The invention proposes for the first time that a loop-mediated isothermal nucleic acid amplification LAMP (loop-mediated isothermal amplification) detection method is applied to the detection of chicken infectious anemia virus . In particular, it relates to a primer and a primer set specific to a specific gene fragment of chicken infectious anemia virus; it also relates to a detection kit for detecting chicken infectious anemia virus by using a loop-mediated isothermal amplification method to detect the primer and primer set. Background technique [0002] Chicken infectious anemia virus (CIAV) is the pathogen that causes chicken infectious anemia (CIA). CIAV infection of chickens can cause immune dysfunction, resulting in immunosuppression, and then increased susceptibility to other pathogens and immune responses to certain vaccines decline, leading to secondary in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 杨兵夏永恒周宏专陈小玲徐福洲徐菁王昌青
Owner 北京翎羽生物科技有限公司
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