Primer sets and kits for multiple RPA detection of brucella abortus, brucella melitensis and brucella suis

A technology of Brucella suis and primer set, applied in the direction of recombinant DNA technology, microbe-based methods, DNA/RNA fragments, etc., to achieve high sensitivity, specificity and good repeatability

Active Publication Date: 2020-05-29
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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However, there are few reports on the interspec...

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  • Primer sets and kits for multiple RPA detection of brucella abortus, brucella melitensis and brucella suis
  • Primer sets and kits for multiple RPA detection of brucella abortus, brucella melitensis and brucella suis
  • Primer sets and kits for multiple RPA detection of brucella abortus, brucella melitensis and brucella suis

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Embodiment 1

[0048] Experimental strains and cells Brucella bovis 2308, Brucella melis 16M are preserved by Changchun Military Veterinary Research Institute; Brucella suis attenuated strain S2, type strain of Escherichia coli, Salmonella, Y. enterocolitica , Vibrio parahaemolyticus and Shigella flexneri were preserved by the Laboratory of Bacterial Diseases, Institute of Zoonoses, Jilin University.

[0049] Main instruments and reagents The gel imager was purchased from UVP Company; the program-controlled metal bath was purchased from China Kayoudi Biotechnology Co., Ltd.; the bacterial genome DNA extraction kit and common DNA product purification kit were purchased from Tiangen Biochemical Technology Co., Ltd.; Twist Basic Kit was purchased from TwistDx Inc, UK; Brucella Broth Medium was purchased from Qingdao Rishui Biotechnology Co., Ltd.

[0050] The primers were designed and synthesized according to the genomes of Brucella bovis (GenBank: AM040265.1), Brucella melis (GenBank: AE00891...

Embodiment 2

[0067] The multiple RPA detection method detects the spiked sample in order to evaluate the feasibility of the method of the present invention, the cultured Brucella suis S2 is counted, the concentration of the bacterial solution is calculated, and the bacterial solution is manually added to the serum sample, beef sample, and milk sample , using the boiling method to crudely extract the genome for spike detection.

[0068] Preparation of artificially spiked serum samples: Count the Brucella S2 bacterial liquid on a plate to obtain the concentration of the bacterial liquid, mix the bacterial liquid with the serum, and make the final concentration of Brucella in the serum 2.14×10 9 CFU / mL, centrifuge at 12000 rpm for 1 min to collect the bacteria, wash twice with sterile normal saline, finally add 100 μL of normal saline to resuspend the bacteria, boil for 30 min, centrifuge to get the supernatant genome solution, and then carry out 10 times The dilution was used as a template t...

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Abstract

The invention provides primer sets and kits for the multiple RPA detection of brucella abortus, brucella melitensis and brucella suis, and belongs to the technical field of brucella detection. The primer sets include B107F, B107R, B192F, B192R, B285F and B285R; and the nucleotide sequences of the B107F, B107R, B192F, B192R, B285F and B285R are successively shown as SEQ ID No. 1- SEQ ID No. 6. Multiple RPA detection can be performed on samples through the specific primer sets, the detection sensitivity of the brucella abortus is 10 pg/[mu]L, and the detection sensitivities of the brucella melitensis and brucella suis are 1 pg/[mu]L; and the provided primer sets and kits can respectively detect three spiked samples which are manually set serum, beef and milk, and compared with a brucella Bruce-Ladder detection method of national standard GB/T 18646-2018 diagnostic techniques for animal brucellosis, results are consistent.

Description

technical field [0001] The invention belongs to the technical field of Brucella detection, and in particular relates to a multiple RPA detection primer set and kit for bovine, ovine and suicine Brucella. Background technique [0002] Brucellosis is a zoonotic infectious disease caused by Brucella spp., which has a wide range of hosts and can not only infect cattle, sheep, pigs and other livestock, but also It can infect humans through direct or indirect contact, seriously endangering the development of animal husbandry and human health in my country (Ren Honglin, Lu Shiying, Zhou Yu, Li Zhaohui, Liu Zengshan. Progress in research and prevention and control of brucellosis[J]. China Animal Husbandry and Veterinary Medicine. 2009, 36(9):139-43.). The disease is widespread, and brucellosis outbreaks have occurred in more than 150 countries, and it has been listed as a B-type infectious disease by the World Organization for Animal Health, and at the same time, it is also listed as...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2521/507C12Q2522/101C12Q2537/143
Inventor 任洪林张士军柳溪林胡盼张英祝万菊卢士英柳增善李岩松闫守庆
Owner JILIN UNIV
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