Multiple rpa detection primer sets and kits for bovine, ovine and suis brucella

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

Active Publication Date: 2021-07-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few reports on the interspecies identification of Brucella using this technology.

Method used

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  • Multiple rpa detection primer sets and kits for bovine, ovine and suis brucella
  • Multiple rpa detection primer sets and kits for bovine, ovine and suis brucella
  • Multiple rpa detection primer sets and kits for bovine, ovine and suis brucella

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Experimental program
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Effect test

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 multiple RPA detection primer sets and kits for cattle, sheep and suis Brucella, belonging to the technical field of Brucella detection; the primer set includes B 107 F.B. 107 R.B. 192 F.B. 192 R.B. 285 F and B 285 R; the B 107 F.B. 107 R.B. 192 F.B. 192 R.B. 285 F and B 285 The nucleotide sequence of R is shown in SEQ ID No.1 to SEQ ID No.6 in turn. The present invention performs multiple RPA detection on samples through a specific primer set, and the detection sensitivity to Brucella bovis is 10 pg / μL, and the detection sensitivity to Brucella melis and Brucella suis is 1 pg / μL; The primer set and kit provided by the present invention detect three kinds of spiked samples of artificially set serum, beef and milk respectively, and compare with the national standard "GB / T 18646-2018 Animal Brucellosis Diagnostic Technology" Brucella Brucella ‑Ladder detection method was used as a control, and the results were 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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Patent Type & Authority Patents(China)
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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