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Pigeon chlamydia PCR diagnosis kit and detection method thereof

A diagnostic kit and technology for chlamydia pigeons are applied in the field of molecular biology diagnosis of animal diseases in veterinary medicine, which can solve the problems of long detection cycle, inability to diagnose chlamydia pigeons quickly, and high cost, saving diagnostic reagents, shortening diagnostic time, and making it easy to effect of operation

Inactive Publication Date: 2018-04-20
XIANYANG VOCATIONAL TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] PCR technology has been widely used in the rapid diagnosis of animal diseases. At present, there is no PCR diagnostic kit for the diagnosis of pigeon chlamydia. It is impossible to quickly diagnose pigeon chlamydia clinically, and the detection period is long and the cost is high.

Method used

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  • Pigeon chlamydia PCR diagnosis kit and detection method thereof
  • Pigeon chlamydia PCR diagnosis kit and detection method thereof
  • Pigeon chlamydia PCR diagnosis kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] A pigeon chlamydia PCR diagnostic kit, comprising a lysing solution, an amplification reaction mixture, a negative control and a positive control, as follows:

[0039] 1) Lysis solution: the composition is DNAiso Reagent, 20 reactions totaling 20mL, packed into 1 bottle;

[0040] 2) Amplification reaction mixture: sterilized three-distilled water, 10× PCR Buffer, 2.5 mmol·L -1 dNTP, 25μmol·L -1 CPS-F upstream primer, 25 μmol·L -1CPS-R downstream primers and 5U / μL rTaq DNA polymerase, the volume ratio of each reaction is 17.0:2.5:2:0.5:0.5:0.5, a total of 23μL, 20 reactions totaling 460μL, packed into 1 tube; the primers are designed and prepared as follows:

[0041] Referring to the genome sequence of pigeon chlamydia published on GenBank, after comprehensive analysis and comparison, two primers were designed for the pathogenic gene of pigeon chlamydia. The sequences of the primers are as follows:

[0042] The sequence of the upstream primer of CPS-F is: 5'-ATTTTCTG...

Embodiment 2

[0071] 1. Processing of tissue samples

[0072] Take 3-5 g of tissue materials from suspected cases, such as larynx, trachea and lung, cut into a paste, add 5 times sterile PBS buffer, fully grind with a tissue grinder, collect the suspension, and centrifuge at 12000 r / min at 4°C 10min, aspirate the supernatant and set aside;

[0073] 2. DNA extraction

[0074] 2.1 DNA extraction from tissue disease material samples: Pipette 100 μL of the obtained supernatant into a 1.5 mL sterile EP tube, add 800 μL of lysis solution, invert and mix, let stand for lysis for 10 min, add 600 μL of ice-cold absolute ethanol, invert and mix well, and let stand for lysis. Set the precipitation for 10min, centrifuge at 12000r / min for 10min at 4°C, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube, dry naturally in the air, add 40 μL of 8 mmol·L -1 Dissolve with NaOH to obtain DNA extraction solution of tissue disease material;

[0075] 2.2 Extraction of...

Embodiment 3

[0088] 1. Treatment of serum samples

[0089] Collect blood from the fin vein of the pigeons of the suspected case, and coagulate it naturally. After the serum is separated out, the serum is separated for use;

[0090] 2. DNA extraction, PCR amplification reaction and electrophoresis detection

[0091] The specific detection method is the same as that in Example 2, except that the tissue disease material sample in Example 2 is replaced with a serum sample.

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Abstract

The invention aims to provide a pigeon chlamydia PCR diagnosis kit which comprises a lysate, an amplification reaction mixture, a negative control and a positive control, wherein a component in the lysate is a DNAiso Reagent; the amplification reaction mixture contains sterilized tri-distilled water, PCR Buffer, dNTP, a CPS-F upstream primer, a CPS-R downstream primer and rTaq DNA polymerase; thenegative control is sterilized tri-distilled water; the positive control is a pigeon Chlamydia positive plasmid. The pigeon chlamydia PCR diagnosis kit provided by the invention is high in sensitivity, good in specificity, high in stability and intuitive in results; a detection method of the pigeon chlamydia PCR diagnosis kit is easy to operate, convenient and quick, and the detection time can begreatly shortened. Therefore, a powerful technical means is provided for clinical control on pigeon chlamydia infection.

Description

technical field [0001] The invention relates to the field of molecular biology diagnosis of animal diseases in veterinary medicine, in particular to a PCR diagnostic kit for chlamydia in pigeons and a detection method thereof. Background technique [0002] Pigeon chlamydia is a contact infectious disease with different syndromes caused by the infection of pigeons with Chlamydophila psittaci (Cps). It is a serious infectious disease that endangers the pigeon industry. Chlamydia persists in the flock due to the 'hen-to-young' transmission cycle. Chlamydia is excreted with the nasal secretions, feces, tears, mucus of beak and throat, crop milk and other secretions of sick parent pigeons. Young pigeons are infected by inhaling, eating or drinking contaminated food and drinking water. The disease can also be infected through the bite of blood-sucking insects, resulting in transmission from host to host. The best way to control pigeon chlamydia is early diagnosis, early treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 朱小甫吴旭锦
Owner XIANYANG VOCATIONAL TECHN COLLEGE
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