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A multiple PCR detection kit for diagnosing poultry tuberculosis and its detection method

A technology for detection kits and kits, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., and can solve the problems of cumbersome operation, large influence of subjective judgment, and long time

Active Publication Date: 2020-06-16
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is mainly aimed at the shortcomings of the current traditional method for diagnosing poultry tuberculosis, such as poor sensitivity (clinical symptoms and staining method), complicated operation and long time (cultivation and biochemical test method), many influencing factors, and great influence of subjective judgment (allergic reaction) method), poor specificity (conventional single-gene PCR method), invented a set of multiple PCR method detection kits and detection methods for rapid diagnosis of poultry tuberculosis, so as to realize rapid diagnosis of poultry tuberculosis, timely prevention and control

Method used

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  • A multiple PCR detection kit for diagnosing poultry tuberculosis and its detection method
  • A multiple PCR detection kit for diagnosing poultry tuberculosis and its detection method
  • A multiple PCR detection kit for diagnosing poultry tuberculosis and its detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0047] ——Multiplex PCR establishment and optimization of annealing temperature

[0048] Using the extracted whole genome DNA of the avian tuberculosis reference strain (CVCC 68201) as a template, and referring to the sequences of DnaJ, IS1245 and IS901 published by NCBI, 3 pairs of primers were designed for amplification, and the annealing temperature was designed to be different temperature gradients. The reaction system is 20 μL: Genome 1 μL, DnaJ primer (20 pmol / μL), IS1245 primer (10 pmol / μL), IS900 (10 pmol / μL) and IS901 (10 pmol / μL) upstream and downstream primers 1 μL each, 2×PCR reaction solution Premix Taq 10 μL, sterile double distilled water 1 μL. Reaction program: first step, 96°C for 2min; second step, 96°C for 10s, 50°C (52°C, 54°C, 56°C, 58°C, 60°C and 62°C) for 10s, 72°C for 1min, 35 cycles; The third step, 2min at 72°C. After the PCR reaction, 10 μL of the product was taken for 1% agarose gel electrophoresis, and the results were observed on an ultraviolet g...

Embodiment 2

[0058] ——Determination of multiplex PCR kit sensitivity (minimum detection amount of genomic DNA)

[0059] After measuring the concentration of the extracted genomic DNA of Mycobacterium avium tuberculosis reference strain (CVCC 68201) using the Nanodrop micrometer, it was diluted with double distilled water to 1.5ng / μL, 0.75ng / μL, 0.38ng / μL, 0.19ng / μL μL, 0.09ng / μL and 0.05ng / μL, use the established multiplex PCR kit for detection and electrophoresis to determine the minimum detection amount of DNA.

[0060] Depend on figure 2 It can be seen that as the template concentration in the reaction system decreases, the minimum detection limit of the multiplex PCR kit is 0.38 ng / μL, that is, 0.38 ng in a 20 μL reaction system.

Embodiment 3

[0062] ——Multiplex PCR kit specific detection

[0063] Extract the genomic DNA of representative strains CVCC 68201 (avian subspecies), CVCC 323 (paratuberculosis subspecies), CVCC 280 (human porcine subspecies) of each subspecies of Mycobacterium avium, and select the standard strains of Mycobacterium bovis CVCC 68001, The genomic DNA of CVCC 68002 and CVCC 291 was used as a control, and the established multiplex PCR reaction system was used for detection and electrophoresis to determine the specificity detection results. Depend on image 3 It can be seen that Mycobacterium bovis and Paratuberculosis subspecies can only amplify 142bp bands, and representative strains of Mycobacterium avium subspecies can amplify 142bp bands, among which avian subspecies amplified 142bp and 387bp bands. , 579bp bands, 142bp, 387bp bands were amplified from human-pig subspecies.

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Abstract

The invention relates to a multi-PCR (Polymerase Chain Reaction) detection kit for diagnosing fowl tuberculosis and a detection method thereof. The kit comprises a multi-PCR primer group, 2*PCR core reagent premixed materials (Premix Taq reaction liquids), positive control (fowl tuberculosis reference strain DNA), negative control (ddH2O) and a DL1000 molecular weight standard solution (Marker). The detection method of the kit comprises the following steps of (1) providing DNA of a suspected clinic sample to be tested;(2) amplifying the DNA of the sample according to a conventional PCR method, detecting an amplified result through agarose gel electrophoresis, and determining according to an amplified band result. The method overcomes the defects of poor acid-fast staining sensitivity, more biochemical test method procedures, long time and poor common single-gene PCR method specificity in a traditional diagnostic method. The detection kit and the detection method provided by the invention have the advantages of specificity in method, simplicity and convenience in operation, and favorable sensitivity, and are suitable for directly diagnosing the DNA of the suspected clinic poultry tissue sample.

Description

[0001] Technical field The present invention relates to a multiplex PCR detection kit for diagnosing poultry tuberculosis and its detection method, belonging to the field of veterinary microbiology diagnosis. Background technique [0002] Avian tuberculosis is a chronic infectious disease of poultry caused by Mycobacterium avium infection. The disease is distributed worldwide, mostly sporadic. The disease mainly passes through the digestive tract and respiratory tract infection, and it is a chronic process, with progressive weight loss, anemia, comb shrinkage, lameness, and reduced or stopped egg production. The incubation period is about 2 months to 1 year, and the sick birds die suddenly due to failure or liver rupture, without obvious seasonality and regionality. The main feature of the disease is the formation of granulomas and caseous nodules in poultry liver, spleen and intestinal tract. Once introduced to poultry farms, it exists for a long time and is difficult to er...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2545/113
Inventor 朱良全张阁丁家波刘国权彭永蒋卉杨劲松毛开荣
Owner CHINA INST OF VETERINARY DRUG CONTROL
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