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wbgz-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying shigella sonnei

A technology of real-time fluorescence and detection kits, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as low sensitivity, time-consuming, labor-intensive, and complex problems

Inactive Publication Date: 2015-10-07
内蒙古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

[0003] At present, due to the lack of accurate and reliable identification techniques in developing countries, most Shigella cannot be identified and grouped. The identification and grouping of Shigella mainly rely on conventional biochemical identification methods and immunological methods. These methods are relatively complicated and time-consuming. Time-consuming, low sensitivity
There are also some scholars who have studied and established molecular biology detection technology mainly to identify Shigella, but there are also certain limitations that lead to false detection and missed detection.

Method used

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  • wbgz-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying shigella sonnei
  • wbgz-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying shigella sonnei
  • wbgz-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying shigella sonnei

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Embodiment 1

[0042] Embodiment 1 identifies the establishment of the real-time fluorescent PCR method of Shigella sonnei

[0043] 1. Materials and methods

[0044] 1.1 Primers and probes

[0045] According to the wbgZ gene of Shigella sonnei, 3 pairs of primers and probes were designed by using http: / / bioinfo.ut.ee / primer3-0.4.0 / primer online design website to identify Shigella sonnei bacteria, and 3 pairs of primers and probes were designed according to the ompA gene as internal references. The primers and probes were synthesized by Shanghai Aoji Biotechnology Co., Ltd., and the sequences are shown in Table 1.

[0046] Table 1 Primers and Probes

[0047]

[0048] 1.2 Method

[0049] 1.2.1 Real-time fluorescent PCR specific detection

[0050] 1.2.1.1 Bacterial culture

[0051] All bacteria (Table 2) were inoculated in TSB-YE and cultured at 37°C for 24h. Take 1.5mL bacterial culture solution and centrifuge at 8000r / min for 5min, discard the supernatant, wash the pellet once with ...

Embodiment 2

[0076] Embodiment 2 identifies the real-time fluorescent PCR sensitivity test and actual sample detection of Shigella sonnei

[0077] The present invention selects primers and probes Son1-F / Son1-R / Son1-P (i.e. a pair of primers consisting of SEQ ID No.1 and SEQ ID No.2; the nucleotide sequence of the probe is SEQ ID No.3 Shown) is used for identifying Shigella sonnei, with primer and probe Ctr1-F / Ctr1-R / Ctr1-P (being the primer pair that is made up of SEQ ID No.10 and SEQ ID No.11; Probe The nucleotide sequence is shown in SEQ ID No.12) as an internal reference.

[0078] 1. Experimental method

[0079] 1.1 Real-time fluorescent PCR sensitivity test

[0080] 1.1.1 Bacterial strains

[0081] Shigella and other bacteria were purchased from China National Institute for the Control of Pharmaceutical and Biological Products and Shanghai Hanni Company. The strains are shown in Table 2.

[0082] 1.1.2 Samples

[0083] Sterilized fresh milk, ice cream, cheese, yogurt, sausage, coo...

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Abstract

The invention discloses a wbgZ-gene-based real-time fluorescent PCR (polymerase chain reaction) method and kit for identifying Shigella sonnei. The invention firstly discloses a real-time fluorescent PCR primer pair and probe for identifying Shigella sonnei, wherein the primer pair is composed of a forward primer of which the nucleotide sequence is disclosed as SEQ ID No.1 and a reverse primer of which the nucleotide sequence is disclosed as SEQ ID No.2; and the nucleotide sequence of the probe is disclosed as SEQ ID No.3. The invention also discloses a real-time fluorescent PCR method for identifying Shigella sonnei, which comprises the following steps: extracting DNA of the sample to be detected as a template, establishing a PCR reaction system by using the primer pair and probe, and carrying out PCR amplification; and if an S-shaped amplification curve appears in the result, determining that the sample to be detected contains Shigella sonnei. The real-time fluorescent PCR method has the advantages of high sensitivity and time saving, is convenient and quick, and can be used for detecting Shigella sonnei in samples.

Description

technical field [0001] The present invention relates to a method for detecting Shigella sonneui, in particular to a real-time fluorescent PCR method for identifying Shigella sonnei for the wbgZ gene, and the present invention also relates to real-time detection of Shigella sonnei. A fluorescent PCR detection primer and a kit belong to the detection field of Shigella sonnei. Background technique [0002] Shigella is still causing major health problems in many countries and regions of the world, especially in underdeveloped and developing countries due to lack of clean water, lack of necessary sanitation and medical equipment and many other factors, leading to public health problems caused by Shigella Health problems are more serious. Worldwide, shigellosis is most morbid and fatal among children 1 to 5 years of age or older. Most shigellosis is associated with three species of Shigella, Shigella flexneri, Shigella sonneii, and Shigella dysenteriae. Of the three Shigella sp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686
Inventor 王海艳敖威华赵治国张捷催强赵林立李刚潘国卿韩祎陟
Owner 内蒙古出入境检验检疫局检验检疫技术中心
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