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Pigeon variola, pigeon chlamydia and pigeon herpes virus triple PCR diagnostic kit and detection method thereof
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A pigeon herpes virus and diagnostic kit technology is applied in the field of molecular biology diagnosis of animal diseases in veterinary medicine.
Inactive Publication Date: 2018-06-05
XIANYANG VOCATIONAL TECHN COLLEGE
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At present, there are no kits for diagnosing pigeon pox virus and pigeon chlamydia, as well as triple PCR diagnostic kits for pigeon pox virus, pigeon chlamydia and pigeon herpes virus. It is impossible to quickly diagnose the above three viruses in clinical practice. high
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Embodiment 1
[0040] A triple PCR diagnostic kit for pigeon pox, pigeon chlamydia and pigeon herpes virus, comprising lysate, amplification reaction mixture, negative control and positive control, specifically as follows:
[0041] 1) Lysis solution: the composition is DNAiso Reagent, 20 reactions total 20mL, packed into 1 bottle;
[0042] 2) Amplification reaction mixture: composed of sterilized triple distilled water, 10×PCR Buffer, 2.5mmol·L -1 dNTP, 25μmol·L -1 POX-F upstream primer, 25 μmol L -1 POX-R downstream primer, 25 μmol L -1 CPS-F upstream primer, 25 μmol L - 1 CPS-R downstream primer, 25 μmol L -1 PiHV-F upstream primer, 25 μmol L -1 PiHV-R downstream primer and 5U / μL rTaqDNA polymerase, the volume ratio of each reaction is 12.5:2.5:2:1.0:1.0:0.5:0.5:1.0:1.0:1.0, a total of 23 μL, 20 reactions totaling 460 μL, packed in 1 tube; the design and preparation of the primers are as follows:
[0043] Referring to the genome sequences of pigeon pox, pigeon chlamydia, and pigeon...
[0079] Take 3~5g of tissue disease materials from suspected cases, such as larynx, trachea, and lungs, cut them into paste, add 5 times of sterile PBS buffer, grind them thoroughly with a tissue grinder, collect the suspension, and centrifuge at 12000r / min at 4°C 10min, absorb the supernatant, set aside;
[0081] 2.1 DNA extraction from tissue disease samples: draw 100 μL of the obtained supernatant into a 1.5 mL sterile EP tube, add 800 μL of lysate, mix evenly by inversion, let stand for lysis for 10 min, add 600 μL of ice-cold absolute ethanol, mix upside down, Place the pellet for 10 min, centrifuge at 12,000 r / min for 10 min at 4°C, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube, dry naturally in the air, and use 40 μL of 8 mmol L -1 NaOH was dissolved to obtain DNA extraction solution of tissue disease material;
[0096] Take blood from the wing vein of the suspected case pigeons, coagulate naturally, and separate the serum after the serum is separated out for use;
[0098] The specific detection method is the same as that in Example 2, except that the tissue disease sample in Example 2 is replaced by a serum sample.
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Abstract
The invention provides a pigeon variola, pigeon chlamydia and pigeon herpes virus triple PCR diagnostic kit. The kit comprises a lysate, an amplifying reaction mixed liquid, negative control and positive control, wherein the component of the lysate is DNAiso Reagent; the amplifying reaction mixed liquid comprises sterilizing tri-distilled water, PCR Buffer, dNTP, a POX-F upstream primer, a POX-R downstream primer, a CPS-F upstream primer, a CPS-R downstream primer, a PiHV-F upstream primer, a PiHV-R downstream primer and rTaq DNApolymerase; negative control is sterilizing tri-distilled water;positive control is a mixture of pigeon variola, pigeon chlamydia and pigeon herpes virus positive plasmids. The kit is high in sensitivity, good in specificity, high in stability and intuitive in result, and a detection method of the kit is easily operated, can shorten the detection time greatly, thereby providing a powerful technical means for clinically controlling infection of pigeon variola,pigeon chlamydia and pigeon herpes virus.
Description
technical field [0001] The invention relates to the field of molecular biology diagnosis of animal diseases in veterinary medicine, in particular to a triple PCR diagnostic kit for pigeon pox, pigeon chlamydia and pigeon herpes virus and a detection method thereof. Background technique [0002] Pigeon pox (Pigeonpox, POX) is the viral infectious disease of the pigeon that is caused by pigeon pox virus. Clinically divided into skin type and mucous membrane type, the virus mainly invades the body through skin or mucous membrane wounds, and the vector is some blood-sucking insects, especially mosquitoes. It is characterized by scattered, nodular pox eruptions on the skin of the non-feathered parts of the body (cutaneous type), or the formation of a yellowish-white caseous pseudomembrane on the upper respiratory tract, howling horns, oral cavity, throat, and esophagus (mucosa) type), thereby affecting movement, swallowing, and breathing, which can easily cause sick pigeons to d...
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