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Primers for rapidly detecting Bartonella henselae, preparation method and kit

A kit and rapid technology, applied in the biological field, can solve the problems of long time, strict nutritional requirements, limited large-scale application, etc., to achieve the effect of rapid detection and diagnosis, fast detection speed, and low requirements for equipment and equipment

Inactive Publication Date: 2018-06-15
SHIHEZI UNIVERSITY
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Problems solved by technology

Due to the particularity of this pathogen, it has very strict nutritional requirements, and it takes a long time to isolate and culture Bh for the first time, so the pathogenic detection is difficult
[0006] Indirect immunofluorescence assay (IFA) is a commonly used serological method for detecting Bh. At present, the detection technology for Bh infection has formed a commercial kit, but there are still many problems in the practical application of IFA diagnostic method: First, IFA detection requires Expensive fluorescence microscope; secondly, the detection results of this method are completely dependent on manual interpretation, which is easily affected by the experience of detection technicians
However, the PCR method also has certain shortcomings, such as the need for specialized equipment and technology, which limits the large-scale clinical application of this method.

Method used

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  • Primers for rapidly detecting Bartonella henselae, preparation method and kit
  • Primers for rapidly detecting Bartonella henselae, preparation method and kit
  • Primers for rapidly detecting Bartonella henselae, preparation method and kit

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Embodiment Construction

[0061] 1. Preparation of Bh genomic DNA

[0062] Take 30mL of Bh logarithmic growth phase culture in a 50mL centrifuge tube, centrifuge at 6000r / min for 15min, collect the bacteria, discard the supernatant, suspend the bacteria in TE buffer, extract genomic DNA with bacterial genomic DNA extraction kit, and measure the extraction DNA concentration and purity (A260 / A280), ready for use.

[0063] 2. Design and synthesis of BhRPA primers

[0064] Download the gltA genes of different Bh isolates published by GenBank, compare the sequences of the BhgltA genes with Blast N and DNAMAN software, and screen highly conserved sequences as target sequences for amplification. Referring to the basic principles of RPA primer design, 3 pairs of specific primers (Table 1) were designed using Primer 5.0 software, and RPA detection primers with high specificity and sensitivity were screened out through experiments. The designed RPA primers amplify the target fragment with a size of about 220bp...

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Abstract

The invention discloses the establishment of a visual rapid detection method of a Bartonella henselae genome DNA, and belongs to the technical field of biology. A pair of high-specificity and strong-sensitivity RPA primers, namely an upstream primer (210)2 and a downstream primer (210)3, are screened, and an RPA (recombinase polymerase amplification) detection system of the Bh genome DNA is established. Compared with the conventional PCR method, the RPA-LFA method has the advantages of low requirements on instrument equipment (only one thermostat water bath is needed), high detection speed (about 40min), visualization of a detection result (the detection result can be directly observed by naked eyes) and the like, so that because of the advantages, the RPA-LFA method provided by the invention can be applied to rapid detection and diagnosis of cat Bh infection in a pet hospital.

Description

technical field [0001] The invention relates to the establishment of a visual rapid detection method for Bartonella henselae genome DNA, which belongs to the field of biotechnology. Background technique [0002] Bartonellosis (Bartonellosis) is caused by different species and subspecies of Bartonella (Bartonella). It is a kind of important zoonotic infectious disease with worldwide distribution. It is one of the new infectious diseases in my country in recent years. Cat-scratch disease (CSD) is a kind of Bartonellosis (Bartonellosis), which is mainly caused by Bartonella henselae (Bh). Cats are the natural host of the pathogen. When cats are infected with Bh, they can maintain bacteremia for a long time without obvious clinical symptoms, and can transmit it to humans through direct scratches or bites, or through cat flea bites. Indirect transmission. The disease exists in the United States, Germany, France, Holland, Switzerland, Italy, Australia, Japan and other countries,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12Q1/6844G01N33/569C12Q1/6806C12R1/01
CPCC12Q1/689C12Q1/6806C12Q1/6844G01N33/56911C12Q2531/119C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 孟庆玲乔军李重阳乔梦凡伍晔晖孟丹贡莎莎王熙凤李静张凯田路路张星星张再超
Owner SHIHEZI UNIVERSITY
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