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Detection primer set of bainite rickettsia and detection reagent of detection primer set and real-time fluorescent quantitative PCR method

A real-time fluorescence quantitative technology for Rickettsia bezieri, which is applied in the fields of biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc. Time-consuming secondary body separation, can not be used to detect samples and other problems, to achieve the effect of good gradient repeatability, stable repeatability, and small error

Inactive Publication Date: 2016-01-20
吉林出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 of bainite rickettsia and detection reagent of detection primer set and real-time fluorescent quantitative PCR method
  • Detection primer set of bainite rickettsia and detection reagent of detection primer set and real-time fluorescent quantitative PCR method
  • Detection primer set of bainite rickettsia and detection reagent of detection primer set and real-time fluorescent quantitative PCR method

Examples

Experimental program
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Effect test

preparation example Construction

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

[0036] First, synthesize the target gene according to the Rickettsia beinii IS111a gene sequence published by NCBI (sequence see SEQ ID NO: 4, synthesized by Shanghai Bioengineering Co., Ltd.) and PMD18T vector (TAKARA company) and transfer it into Escherichia coli, and directly extract the 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&Minispincolumn collection tubes used to extract 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] The nucleotide sequence shown in SEQ ID NO: 1 is used as an upstream primer; the nucleotide sequence shown in SEQ ID NO: 2 is used as a downstream primer; the nucleotide sequence shown in SEQ ID NO: 3 is used as a probe to detect Rickettsia bezieri.

[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 ...

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 relates to a detection primer set of bainite rickettsia and a detection reagent of the detection primer set and a real-time fluorescent quantitative PCR method. The primer set comprises an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2. The method for carrying out real-time fluorescent quantitative PCR by the primer set provided by the invention is high in amplification efficiency, small in error, and objective and accurate in result, and has 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 (QFever) 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 world...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
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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