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Detection primer set for Rickettsia beinii and its detection reagent, real-time fluorescent quantitative PCR method

A technology of real-time fluorescence quantification of Rickettsia bainierii, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problem of difficult early diagnosis of specific antibody detection results, time-consuming isolation of Rickettsia bainiette It is used to detect problems such as samples to achieve the effect of good gradient repeatability, stable repeatability and small error

Inactive Publication Date: 2018-05-04
吉林出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] The most reliable basis for the detection of Rickettsia bainieri is to isolate Rickettsia bainierii from the sample, which can be separated from animals, chicken embryos, and cells. Poor sensitivity, difficult to perform in general laboratories
At present, the most commonly used detection method for Rickettsia basilica is serum-specific antibody detection. Because high-level specific antibodies appear late, the detection results of specific antibodies are difficult to be used for the early diagnosis of Q fever, and cannot be used. To detect whether the sample carries the pathogen

Method used

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  • Detection primer set for Rickettsia beinii and its detection reagent, real-time fluorescent quantitative PCR method
  • Detection primer set for Rickettsia beinii and its detection reagent, real-time fluorescent quantitative PCR method
  • Detection primer set for Rickettsia beinii and its detection reagent, real-time fluorescent quantitative PCR method

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Experimental program
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Effect test

preparation example Construction

[0035] The preparation method of Rickettsia bainiferi standard substance is:

[0036] First, the target gene was synthesized according to the Rickettsia beinii IS111a gene sequence published by NCBI (see SEQID NO: 4 for the sequence, synthesized by Shanghai Bioengineering Co., Ltd.) and the PMD18T vector (TAKARA Company) was ligated and transferred into E. coli, and directly extracted Plasmid DNA;

[0037] Plasmids were used as Rickettsia bainiferi standards for experiments. The plasmid had a 260nm / 280nm ratio of 2.0 and a concentration of 56.00 ng / μL.

[0038] Buffer ATL, Buffer AL, Buffer AW1, Buffer AW2, Buffer AE, and QIA&Mini spin column collection tubes used for extracting DNA from samples to be tested were from DNA extraction kits purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Embodiment 1

[0040] The detection method of embodiment 1 Rickettsia bainiferi

[0041] Taking the nucleotide sequence shown in SEQ ID NO:1 as an upstream primer; using the nucleotide sequence shown in SEQ ID NO:2 as a downstream primer; using the nucleotide sequence shown in SEQ ID NO:3 as a probe to detect shellfish Rickettsiae.

[0042] According to the orthogonal verification of the matrix method, the optimal concentration and dosage of primers and probes were determined. Using the plasmid containing the Rickettsia bainiferi gene as a template, a fluorescent PCR reaction system was established, and the total reaction system was 25 μL. Under the condition that the template concentration is the same, use 5, 10, 15, 20 pM primer concentration and 5, 10, 20 pM probe concentration for primer and probe respectively, and use matrix method to screen the optimal concentration of primer and probe. Results The optimal primer concentration was 10pM, and the probe concentration was 10pM. The best...

Embodiment 2

[0051] Embodiment 2 specific detection

[0052] According to the reaction system and the conditions of Example 1, Rickettsia bainii, Rickettsia siberia, Yersinia pestis, Tularemia, Brucella, Escherichia coli, Listeria were detected respectively, and the results were as follows figure 1 . Through the amplification, only Rickettsia bainiferi showed an amplification curve, and the others did not have an S-shaped curve amplification, and the negative control was established, indicating good specificity. The results of the study showed that the established detection method had strong specificity and could be used for the specific identification of Rickettsia bainiferi.

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Abstract

The invention relates to the field of biology, in particular to a detection primer set for Rickettsia basilica, a detection reagent, and a real-time fluorescent quantitative PCR method. The primer set includes an upstream primer with the nucleotide sequence shown in SEQ ID NO:1 and a downstream primer with the nucleotide sequence shown in SEQ ID NO:2. The real-time fluorescent quantitative PCR method using the primer set provided by the invention has high amplification efficiency, small error, objective and accurate results, and good specificity, repeatability and sensitivity.

Description

technical field [0001] The invention relates to the field of biology, in particular to a detection primer set for Rickettsia basilica, a detection reagent, and a real-time fluorescent quantitative PCR method. Background technique [0002] Q fever (Q fever) is a disease that can infect humans and various animals and produce fever. The course of the disease can be divided into acute phase and chronic phase. Symptoms of acute infection include: high fever, shivering, muscle pain, headache, etc. More severe cases can lead to pneumonia or hepatitis. When infected during pregnancy, it can result in miscarriage of the fetus or premature birth of the newborn. About 1% of infections eventually turn into chronic disease. In 1950, the first case of Q fever was discovered in my country, and then there were many outbreaks in slaughterhouses, farms and pastures, and the pathogens were isolated from more than 50 kinds of wild animals. At present, the disease has spread all over the worl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/689
Inventor 王伟利孟庆峰姚贵哲于钦垒李颖
Owner 吉林出入境检验检疫局检验检疫技术中心
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