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Chicken infectious anemia virus LAMP (loop-mediated isothermal amplification) detection kit and detection method thereof

A technology for chicken infectious anemia and a detection method, which is applied in the field of kits for rapid detection of chicken infectious anemia virus by a warm amplification method, and achieves the effects of high specificity, easy results, and result judgment.

Inactive Publication Date: 2012-02-08
王旋 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CAV infection can cause chicken bone marrow hematopoietic tissue and thymus and other lymphoid tissues to atrophy, resulting in severe anemia and immunosuppression, causing secondary infection and vaccine immunization failure

Method used

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  • Chicken infectious anemia virus LAMP (loop-mediated isothermal amplification) detection kit and detection method thereof
  • Chicken infectious anemia virus LAMP (loop-mediated isothermal amplification) detection kit and detection method thereof
  • Chicken infectious anemia virus LAMP (loop-mediated isothermal amplification) detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] 1. Cloning of pMD18-CAV and construction of plasmid template

[0092] 1.1 Experimental materials

[0093] 1.1.1 Virus strains:

[0094] CAV isolate AH10-1 was preserved by the Key Laboratory of Poultry Disease Control and Monitoring in Anhui Province.

[0095] 1.1.2 Plasmid vectors and recipient strains

[0096] Plasmid vector: pMD18-T Vector Kit was purchased from TaKaRa Company.

[0097] Recipient strain: Competent Escherichia coli DH5a was purchased from Nanjing Tianwei Times Biotechnology Co., Ltd.

[0098] 1.1.3 Medium

[0099] LB liquid medium: Weigh 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride, put them in a 1L beaker, add 800mL of deionized water, stir and dissolve; add 5mol / L NaOH (about 0.2mL) dropwise, adjust pH to 7.0; add deionized water to make up to 1L, autoclave, and store in a 4°C refrigerator for later use.

[0100] LB solid medium: Add 1.5% high-quality agar powder on the basis of LB liquid medium, after autoclaving, add...

Embodiment 2

[0216] 1. Materials and Reagents

[0217] 1.1 Virus (bacteria) strain

[0218] The CAV isolates and the CAV clone strain pMD18-CAV-2 containing the target gene are all preserved by our laboratory.

[0219] 1.2 Control (bacteria) strains

[0220] Bacterial control group: Escherichia coli, Staphylococcus, Enterococcus faecalis, Pasteurella multocida.

[0221] Virus control group: chicken Newcastle disease virus (NDV), avian influenza virus (AIV), chicken infectious bursal disease virus (IBDV), chicken Marek's disease virus (MDV-1), J subtype avian leukemia virus (ALV-J ).

[0222] 1.3 Reagents

[0223] pfu DNA polymerase and common Taq DNA polymerase were purchased from Invitrogen. Trizol and AMV reverse transcription kits were purchased from TaKaRa Company.

[0224] 2. Experimental method

[0225] 2.1 Preparation of DNA virus sample and bacterial sample DNA

[0226] DNA virus samples and bacterial samples were extracted according to Item 1.2.1 in Example 1.

[022...

Embodiment 3

[0246] 1. Materials and Reagents

[0247] 1.1 Viruses and samples

[0248] CAV isolate AH10-1 and its infected tissue disease materials and blood and other positive samples were kept by the Key Laboratory of Poultry Disease Control and Monitoring in Anhui Province.

[0249] 1.2 Reagents

[0250] Bst DNA polymerase large fragments were purchased from New England Biolabs; SYBR Green I dye was purchased from Beijing Fanbo Biochemical Co., Ltd.

[0251] 1.3 Other solutions

[0252] Buffer used in the experiment is the same as 1.2.2 in embodiment 1

[0253] 2. Test method

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Abstract

The invention discloses a chicken infectious anemia virus LAMP (loop-mediated isothermal amplification) detection kit and a detection method thereof and relates to the technical field of biology. 198 highly conserved nucleotide sequences at the positions of 532bp to 729bp of a chicken infectious anemia virus gene sequence are selected as target regions to design a specific LAMP primer group; the specific LAMP primer group comprises positive and negative outer primers F3 and B3, positive and negative inner primers FIP and BIP and positive and negative ring primers LF and LB; and the nucleotide sequences are shown in SEQNO1-6. The high efficiency amplification of the target regions can be completed by the primer group in one hour; and agarose electrophoresis is dyed by a product according to the conditions, or the magnesium pyrophosphate precipitation turbidity of a by-product is detected, or after a color-developing agent is added, the color reaction is observed to judge a result. The kit formed by the invention has the advantages of convenience, rapidness, low price and strong specificity and is suitable for clinical diagnosis and monitoring of the chicken infectious anemia.

Description

technical field [0001] The present invention relates to a biological identification method for chicken infectious anemia virus, in particular to a primer set specific for a specific gene segment of chicken infectious anemia virus, and to using the primer set to rapidly detect chicken infectious anemia virus Kit for Infectious Anemia Virus. Background technique [0002] Chicken infectious anemia virus (CAV) is the pathogen of chicken infectious anemia (CIA). CAV infection can cause chicken bone marrow hematopoietic tissue and thymus and other lymphoid tissues to atrophy, resulting in severe anemia and immunosuppression, resulting in secondary infection and vaccine immunization failure. In my country, CAV was first isolated in Heilongjiang Province in 1992. At present, the prevalence of the disease in my country is expanding and the infection rate is increasing. [0003] Chicken anemia virus is one of the smallest known animal viruses, which is a single-stranded circular DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 王旋张训海龚争赵磊王军汪小红
Owner 王旋
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