Rapid detection method of chicken infectious anemia virus and kit
A technology of chicken infectious anemia and detection kit, which is applied in the field of detection of chicken infectious anemia virus, can solve the problems of no detection of chicken infectious anemia virus, etc., and achieve the effect of wide application range, strong specificity and high sensitivity
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Embodiment 1
[0082] The establishment of embodiment 1 LAMP detection method
[0083] 1. pTCIAV cloning and plasmid template construction
[0084] 1.1 Experimental materials
[0085] 1.1.1 Virus strains:
[0086] The CuX-1 strain of CIAV standard virus is preserved by our laboratory and can be released to the public.
[0087] 1.1.2 Plasmid vector and recipient strain:
[0088] Plasmid vector: pGEM-T Easy Vector Kit was purchased from Invitrogen.
[0089] Recipient strain: Escherichia coli Top10 was purchased from Tiangen Biological Company.
[0090] 1.1.3 Medium:
[0091] 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.
[0092] LB solid medium: Add imported agar powder to LB liquid medium to a final concentration of 1.5%, autoclave, and keep at 4°C.
[0093] 1.1.4 Polymerase a...
Embodiment 2
[0193] Application of Example 2 LAMP Detection Method in Differential Diagnosis
[0194] 1. Test material
[0195] 1.1 Virus strains:
[0196] CIAV standard virus CuX-1 strain is preserved by our laboratory, pTCIAV positive clone strain.
[0197] 1.2 Control strains: (All are kept by the laboratory and can be distributed to the public.)
[0198] 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).
[0199] DNA virus group: chicken egg drop syndrome virus (EDS), chicken Marek's virus (MD).
[0200] Bacterial control group: Haemophilus paracoli (IC), Pasteurella subspecies A, Escherichia coli, Salmonella typhi, and Avian bacteria.
[0201] 1.3 Polymerase and other tool enzymes:
[0202] Pyrobest DNA polymerase. Trizol Reagent (Invitrogen Company), plasmid micro-extraction kit was purchased from Tianhe Co...
Embodiment 3
[0226]The reaction was carried out at 63°C, and the reaction time was 30 minutes. After the LAMP reaction, 5 μL of the product was mixed with 1 μL of 6 times bromophenol blue loading buffer, and detected by 2% agarose gel electrophoresis. The results are shown in Figure 8 (A, B) No other pathogens were positively amplified. Application of Example 3 LAMP Detection Method in Clinical Samples
[0227] 1. Test material
[0228] 1.1 Virus strains:
[0229] The tissue disease materials of CIAV infection and the serum of chickens infected with CIAV virus are all preserved by our laboratory.
[0230] 1.2 Polymerase and other tool enzymes:
[0231] Bst DNA polymerase.
[0232] 1.3 Lysis solution: including 10mM Tris-Hcl (pH8.8); 1mM EDTA; 15mM Nacl; 0.5% SDS.
[0233] 1.4 Other solutions and reagents
[0234] SYBR Green staining solution, Goldview staining solution
[0235] 2. Test method:
[0236] Processing of Clinical Samples
[0237] Take 100 μL of serum or disease materia...
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