Primer, probe and reagent kit for identifying Brucella S2 vaccine strains in aerocolloid

A technology for Brucella and Brucella, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of lack of primers and probes, and achieve strong specificity , Simple identification and detection, easy to use

Inactive Publication Date: 2015-08-26
何洪彬
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are currently no specific rules for the design of primers and probes for the RPA-nfo reaction. Only after the RPA reaction can the lateral flow chromatography test strip (LFD) be used to screen and obtain the combination of primers and probes that can be used in clinical testing.

Method used

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  • Primer, probe and reagent kit for identifying Brucella S2 vaccine strains in aerocolloid
  • Primer, probe and reagent kit for identifying Brucella S2 vaccine strains in aerocolloid
  • Primer, probe and reagent kit for identifying Brucella S2 vaccine strains in aerocolloid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0042] Design and Screening of Example 1 Primers and Probes

[0043] At present, there are no specific operating rules for the design of RPA-LFD primers and probes. Only after RPA reaction and lateral flow chromatography (LFD) detection can the primers and probes that can be used for clinical detection be screened. The length of the RPA primer is generally 30-35nt, and if the primer is too short, it will seriously affect the activity of the recombinase. Longer primers do not necessarily improve amplification performance, but rather increase the likelihood of secondary structure formation. The length of the LF probe is generally 46-52nt, and the nfo ribozyme is about 30 bases away from the 5' end of the probe, and about 16-22 bases away from the 3' end.

[0044] In the experiment, it is necessary to design multiple pairs of primers and probes from both ends of the target sequence for optimization and screening, and the substitution or increase or decrease of individual bases w...

Embodiment 2

[0052] The establishment of embodiment two Brucella S2 vaccine strain differential diagnosis RPA-LFD detection method

[0053] 1. Experimental steps

[0054] (1) Extraction of bacterial genomic DNA

[0055] Take 1ml of the bacterial liquid, and extract the total bacterial DNA according to the instructions of the Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0056] (2) Establishment of RPA-LFD reaction system for differential diagnosis of Brucella S2 vaccine strains

[0057] The RPA reaction system is 50 μL:

[0058]

[0059]

[0060] Sample DNA or crude lysate and ddH to be tested 2 O 13.2 μL

[0061] Add the above mixture into the TwistAmp nfo reaction tube, mix well and dissolve. Then add 2.5 μL of magnesium acetate solution, and place the reaction tube at 38°C for 20 minutes. After the reaction, mix 2 μL with 98 μL LFD detection buffer, immerse LFD in the above mixed buffer, and observe the results within 5 minutes. band, th...

Embodiment 3

[0068] Differential diagnosis of clinical samples Brucella S2 vaccine strains such as embodiment three aerosols

[0069] 1. Experimental steps

[0070] (1) Collection of aerosol samples

[0071] An international standard all-glass-impinger (AGI) was used, with 10 mL of phosphate buffered saline (PBS) with a pH value of 7.0 as the sampling medium, and a sampling flow rate of 12.5 L / min for 20 minutes. Collect aerosol samples from the farm environment. Centrifuge at 12000r / min for 10min at room temperature, discard the supernatant, and precipitate for direct lysis.

[0072] (2) On-site preparation of clinical samples such as aerosol

[0073] Dilute aerosol and other clinical samples with 200μL TE buffer (1.0M Tris-HCl (pH8.0) 10mL, 0.5M Na 2 EDTA·2H 2 (2mL (pH8.0), add distilled water to 1000mL) to resuspend, take 200μL of liquid sample directly, then add 30μL of 10% (w / v) SDS and 3μL of 2% (w / v) proteinase K, mix for 37 Incubate at ℃ for 1h.

[0074] (3) Identification a...

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Abstract

The invention relates to a primer, probe and reagent kit for identifying Brucella S2 vaccine strains in aerocolloid. The forward primer sequence of Brucella-RPA-LFD is shown as SEQIDNo.1, the backward primer sequence of Brucella-RPA-LFD is shown as SEQIDNo.2, and the probe sequence of Brucella-RPA-LFD is shown as SEQIDNo.3; the forward primer sequence of S2-RPA-LFD is shown as SEQIDNo.4, the backward primer sequence of S2-RPA-LFD is shown as SEQIDNo.5, and the probe sequence of S2-RPA-LFD is shown as SEQIDNo.6. The combination of the RPA-LFD primer and the probe, or the reagent kit, for identifying and diagnosing the Brucella S2 vaccine strains, provided by the invention are high in sensitivity and specificity, and can at least detect 2.5*100 cfu of Brucella or the Brucella S2 vaccine strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a probe and a kit for identifying Brucella S2 vaccine strains in aerosol. Background technique [0002] Brucellosis is a zoonotic infectious disease caused by bacteria of the genus Brucella (cattle, sheep, pig and canine Brucella, etc.), and it is also an important pathogen for statutory quarantine and purification in my country . In recent years, the incidence rate has shown an increasing trend year by year, especially for dairy animals. The abortion rate is as high as 50%-80%, and the output of milk and meat is reduced by 10%-20%. According to the "Veterinary Bulletin" statistics, in 2013, there were 3,620 cases of brucellosis nationwide, involving 42,720 cattle and sheep. Therefore, brucellosis poses a serious threat to social and economic development and stability, and national public health security. [0003] The source of infection of brucellosis is main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2521/507C12Q2537/1376C12Q2522/101
Inventor 王洪梅赵贵民何洪彬
Owner 何洪彬
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