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Brucella detection kit and Brucella detection method

A detection kit, technology for Brucella, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Simple operation, low cost, rapid detection effect

Active Publication Date: 2021-02-05
江苏博嘉生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nucleic acid hybridization technology has the characteristics of high sensitivity, strong specificity, and good practicability. It is a routine method for the diagnosis of various animal diseases, but the operation is complicated and the cost is high. Ordinary laboratories do not have corresponding equipment, and false positives are prone to occur
Gene chips and high-throughput sequencing technologies are fast and simple, with strong specificity, high sensitivity and high-throughput quantification, but they are costly and require high levels of operators

Method used

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  • Brucella detection kit and Brucella detection method
  • Brucella detection kit and Brucella detection method
  • Brucella detection kit and Brucella detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 is used to detect the design of the crRNA of Brucella and obtain

[0038] 1. According to the specific sequence of the brucellosis Omp25 gene as shown in SEQ ID NO.1, design 4 specific crDNA sequences with T7 promoter, the sequences of which are shown in SEQ ID NO.2-5 respectively .

[0039] 2. Design of crRNA targeting gene mutation site

[0040] (1) Design principles of crRNA targeting gene mutation sites

[0041]Since the CRISPR-Cas12a system is a novel targeted DNA gene editing system, in which Cas12a combines with crRNA to form a monitoring complex, the guide region of crRNA recognizes the target DNA with a complementary sequence, and Cas12a degrades the target DNA strand. Wherein the crRNA design requirement: crRNA includes protein anchor sequence and guide sequence, sequence format is: 5`-anchor sequence combined with Cas12a protein-crRNA guide sequence-3`, protein anchor sequence needs to be determined according to Cas12a protein , so that it can...

Embodiment 2

[0049] Detection kit and detection method of embodiment 2 Brucella

[0050] 1. The composition of the kit

[0051] This kit includes 4 crRNAs (shown in SEQ ID NO.6-9) or 4 crDNAs (shown in SEQ ID NO.2-5) for detecting Brucella, when it is crDNA in the kit, it needs The operator first generates RNA from the crDNA fragments under the action of T7 RNA polymerase, recovers and purifies the crRNA, see Example 1 for details), a specific fluorescent probe (selected from Table 2), Cas12a protein (this example uses FnCas12a protein), enzyme-free water, DNase inhibitor;

[0052] Table 2 Fluorescent probe sequences

[0053] fluorescent probe Sequence (5'-3') Probe 1 FAM-TTTTTTTT-BHQ1 Probe 2 FAM-TTTTTTTTTT-BHQ1 Probe 3 FAM-TTTTTTTTTTTT-BHQ1

[0054] 2. Detection method of Brucella

[0055] (1) Take 50-100ng of sample DNA to be tested and add it to the isothermal amplification system (RPA isothermal amplification system). The RPA isothermal amplifi...

Embodiment 3

[0062] Embodiment 3 specific detection

[0063] 1. Screening and detection of crRNA

[0064] Synthesize the target sequence of the brucella Omp25 gene, use the 4 crRNAs designed in Example 1 to construct the CRISPR-Cas12a system, and carry out detection and screening, use the fluorescent quantitative PCR instrument to detect the change of the fluorescent signal of each system, and the detection results are as follows figure 1 shown. The results show that different crRNAs designed for the same target gene have different detection effects. Compared with the negative control, the change in fluorescence value in the system containing the target gene is more significant, that is, the crRNA with higher sensitivity and better effect is selected, that is, crRNA shown in SEQ ID NO.6. Wherein the result that the fluorescence value of crRNA shown in SEQ ID NO.6 changes with time when detecting is as follows figure 2 As shown, the results show that the system (brucellosis) containing ...

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Abstract

The invention discloses crRNA, a kit and a detection method for detecting brucella. The crRNA is selected from the following sequences: crRNA1, the sequence of which is shown as SEQ ID NO. 6; crRNA2,the sequence of which is as shown in SEQ ID NO. 7; crRNA3, the sequence of which is as shown in SEQ ID NO. 8; and crRNA4, the sequence of which is as shown in SEQ ID NO. 9. According to the present invention, crRNA is designed and screened based on the Brucella Omp25 gene, meanwhile, a CRISPR-Cas12a system and an RPA isothermal amplification technology are combined, whether Brucella exists in a to-be-detected sample or not can be detected within a short time, the operation is easy, the detection speed is high, the cost is low, repeated detection can be achieved, at the same time, the detectionsensitivity and the detection specificity are remarkably improved, wherein the lowest detection limit can reach 10 copies per microliter.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection kit and a detection method for Brucella. Background technique [0002] Brucellosis is a disease caused by Brucella infection. It is a zoonotic systemic infectious disease, referred to as "brucellosis". There are also Mediterranean relaxation fever, Malta fever, and wave fever. and so on. The World Organization for Animal Health (OIE) lists it as a statutory animal report disease, and my country has classified it as a Class B infectious disease in the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases", which is more familiar with "SARS", " "Swine flu", anthrax, AIDS, rabies, hepatitis B, etc. belong to the first class of infectious diseases. Brucella can invade the body through skin and mucous membranes, respiratory tract, digestive tract, etc., causing clinical symptoms such as fever, miscarriage, inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/113C12Q1/04C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2521/327C12Q2525/161C12Q2563/107C12Q2565/625Y02A50/30
Inventor 姚杰赵洪友程诚
Owner 江苏博嘉生物医学科技有限公司
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