Universal primers and probe for on-site rapid detection of Brucella and kit

A general-purpose primer and Brucella technology, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve problems such as low success rate, time-consuming and laborious, and judgment errors, and achieve strong specificity, The effect of high sensitivity and rapid detection

Active Publication Date: 2015-09-30
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the isolation and culture of bacteria requires P3 laboratories, and it is time-consuming and laborious, the success rate is low
Molecular biology methods such as conventional PCR and fluorescent quantitative PCR have been favored by people in recent years, but they have high requirements for equipment and conditions, and are prone to aerosol cross-contamination and false positives.
Although the constant isothermal amplification technology (LAMP) can be used for on-site detection, its reaction time is generally about 1 hour, and there are still problems such as human judgment errors in the judgment of the results.

Method used

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  • Universal primers and probe for on-site rapid detection of Brucella and kit
  • Universal primers and probe for on-site rapid detection of Brucella and kit
  • Universal primers and probe for on-site rapid detection of Brucella and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1. Design and screening of primers and probes

[0041] 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.

[0042] 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. In the experiment, it is necessary to design multiple pairs of primers from both ends of the target sequence for optimization, screening, and the replacement or increase or decrease of individual bases will have an important impact on the experimental results. But there are still some points for attention: (1) The 3-5 nucleotides at the 5' end should avoid polyguanine (G) and cy...

Embodiment 2

[0047] Embodiment two, the establishment of Brucella RPA-LFD detection method and the detection of clinical samples

[0048] 1. Experimental steps

[0049] (1) Extraction of bacterial genomic DNA and preparation of DNA templates for on-site detection of clinical samples

[0050] 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.

[0051] The clinical tissue samples were washed with 200 μL TE buffer (1.0M Tris-HCl (pH8.0) 10mL, 0.5M Na 2 EDTA·2H 2 (pH8.0) 2mL, add distilled water to 1000mL) to resuspend, take 200μL of the liquid sample directly, then add 30μL 10% (w / v) SDS and 3μL 2% (w / v) proteinase K, mix and warm at 37°C Breed for 1h.

[0052] (2) Establishment of Brucella RPA-LFD reaction system

[0053] The RPA reaction system is 50 μL:

[0054]

[0055] Sample DNA or crude lysate and ddH to be tested 2 O 13.2 μL

[0056] Add t...

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Abstract

The invention discloses universal primers and a probe for on-site rapid detection of Brucella and a kit. The forward primer sequence is shown as SEQIDNo.1, the reverse primer sequence is shown as SEQIDNo.2, and the probe sequence is shown as SEQIDNo.3. The universal primers, the probe and the kit provided by the invention for detection of Brucella have high sensitivity and strong specificity, minimumly can detect 6.0*10<0>cfu Brucella, and have no cross reaction with Escherichia coli O157:H7, yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus and other bacteria. The universal primers, the probe and the kit provided by the invention not only can be used for detection of Brucella strains, but also can be used for detection of clinical samples, which mainly include blood, milk samples, aerosol samples, and tissue samples, etc. The kit provided by the invention is convenient to use, has no need for special equipment, can carry out sensitive, specific and rapid detection of Brucella on the crude split product of a to-be-detected sample in 25min just at 38DEG C, and is suitable for field or grassroots brucellosis quarantine work.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to general primers and probes for identifying Brucella using recombinase polymerase amplification-lateral flow chromatography test strip detection technology (Recombinase Polymerase Amplification and Lateral Flow Dipstick, RPA-LFD). needles and test kits. 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%. Human infection with brucellosis is mainly caused by contact with polluted environment, secretions, body fluids, corpses of sick animals, or ingestion of products from contamina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/686C12Q1/689C12Q2521/507C12Q2522/101C12Q2531/119C12Q2565/625
Inventor 何洪彬赵贵民王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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