Bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof

A detection kit, the technology of Brucella bovis, applied in the field of biotechnology detection, can solve the problems of affecting the epidemiological investigation and disease prevention of brucellosis, cumbersome experimental process, inability to type, etc., so as to reduce cross-contamination and environmental problems. Risk of contamination, improved detection efficiency, and the effect of easy operation

Active Publication Date: 2016-06-01
广东省疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no uniform standard, and many methods have cumbersome and time-consuming experimental procedures, and most methods have some atypical strains that cannot be typed by existing methods
Seriously affected the epidemiological investigation and disease prevention of brucellosis

Method used

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  • Bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof
  • Bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof
  • Bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation and use method of embodiment 1 kit

[0048] Design and synthesize primers and probes for Brucella bovis, primers and probes for Brucella ovis, primers and probes for Brucella suis, primers and probes for Brucella canis, respectively. The nucleotide sequence is shown in Table 1 below:

[0049] Table 1

[0050]

[0051] The above-mentioned sets of primer pairs and probes can be packaged individually, or can be combined to make a multiple fluorescent PCR detection mixture. The amount of each of the above-mentioned primers and probes contained in the multiplex fluorescent PCR detection mixture can be the conventional amount known to those skilled in the art.

[0052] That is to say, the kit of the present invention may contain the aforementioned independently packaged sets of primer pairs and probes, or may contain a configured multiplex fluorescent PCR detection mixture containing each set of primer pairs and probes.

[0053] Further, the kit can also...

Embodiment 2

[0057] The sensitivity analysis of embodiment 2 kit

[0058] 1. Experimental method

[0059] 1.1 Preparation of multiple fluorescent PCR detection mixture:

[0060] According to the conventional dosage in this field, configuration contains four sets of primer pairs and probe, dNTP, ddH in Example 1 2 O, Mg 2 +, 2xPCR buffer multiplex fluorescent PCR detection mixture, a total of 36 μl.

[0061] 1.2 Preparation of the positive control substance for Brucella typing:

[0062] The method for constructing the positive control plasmid of Brucella bovis: the amplicon sequence of Brucella bovis was chemically synthesized and constructed on the vector pGH. The cloning site is SmaI. The amplicon sequence length of the inserted Brucella bovis is 171bp, and the nucleotide sequence is as shown in SEQ ID NO.13, specifically:

[0063] GCAACAATTCACGTCATGATGGCCAATACGGCGATTTCAGCGATGATCGTGATGTCGCTGATGGCGGCGTAAGCACCGGCAAGATCGCCTACACCTTCACCGGCGGAAACGGCTTCTCGCTGTGATCGCTCTCGAACAGGGTGGCGAAGACGT...

Embodiment 3

[0086] Application of embodiment 3 kit

[0087] 1. Experimental method

[0088] 1.1 Experimental objects

[0089] Adopt the following samples of kit of the present invention to carry out fluorescence detection: 2 examples of Brucella bovis whole blood leukocyte samples (numbering is SA-1, SA-2), 1 example of human whole blood leukocyte samples (numbering is SA-3) and 1 case of milk (coded SA-4); 2 cases of whole blood leukocyte samples of Brucella sheep (coded SM-1, SM-2), 2 cases of human whole blood leukocyte samples (coded SM-3, SM- 4); 3 cases of Brucella suis whole blood leukocyte samples (numbered SS-1, SS-2, SS-3), 1 case of vaginal secretions (numbered SS-4); whole blood samples of Brucella canis 3 examples of white blood cell samples (numbered as SC-1, SC-2, SC-3), 1 example of vaginal secretions (numbered as SC-4), and a positive control (SA Yangshen is prepared in Example 2 with a concentration of 4x10^ 7 The brucella bovis positive control plasmid of copies / m, ...

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Abstract

The invention belongs to the field of biotechnical detection, and particularly relates to a bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof. The bovine, goat, porcine and canine brucella typing fluorescent PCR reagent kit contains bovine brucella detection primers and probes, goat brucella detection primers and probes, porcine brucella detection primers and probes and canine brucella detection primers and probes. The bovine, goat, porcine and canine brucella typing fluorescent PCR detection reagent kit, the preparation and the application have the advantages that the bovine, goat, porcine and canine brucella typing fluorescent PCR detection reagent kit is high in detection sensitivity, the lowest detection limit is 1*10<3> copy/ml, and the accuracy and the positive rate can reach 100%.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a fluorescent PCR detection kit for typing Brucella in cattle, sheep, pigs and dogs and its preparation and application. Background technique [0002] Brucella is a Gram-negative, non-spore-forming, facultative intracellular bacterium that is the causative agent of the zoonotic infectious disease brucellosis. Infection of livestock can cause abortion in female animals and orchitis in male animals, leading to a decline in the reproductive capacity and production performance of sick livestock, affecting the quality and safety of livestock products, and causing serious economic losses; infection in humans can cause wave fever, arthritis and testicular Inflammation, epididymitis, pregnant women can cause miscarriage, etc., and severe cases can lose their ability to work. Seriously affect the development of my country's animal husbandry and human health. [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2600/158C12Q2600/16C12Q2531/113C12Q2563/107C12Q2537/143C12Q2547/101
Inventor 柯昌文陈经雕肖红邵俊斌朱勤玮王凯熊磊刘燕张捷
Owner 广东省疾病预防控制中心
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