Real-time fluorescent PCR (polymerase chain reaction) kit and oligonucleotides for identifying zygosaccharomyces rouxii

An oligonucleotide and real-time fluorescence technology, applied in the field of inspection and quarantine, can solve the problems of cumbersome operation, fast customs clearance at unfavorable ports, long identification cycle, etc., to ensure food safety, optimize reaction conditions, and have good specificity and stability. Effect

Inactive Publication Date: 2014-03-19
北京市海淀区产品质量监督检验所
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

These conventional methods have played an important role in the research of Vibrio parahaemolyticus, but with the development of modern science and technology, the traditional method of identifying Saccharomyces ruckerii by relying on phenotypic characteristics is far from being able to meet the requirements for this bacterium. The need for rapid identification has exposed many shortcomings: cumbersome operation, long identification period, which is not conducive to the rapid customs clearance requirements at ports; changes, and traditional identification methods do not represent their genetic characteristics well
[0006] Using real-time fluorescent PCR technology to detect Zygomyces ruckeri, there is no relevant report at home and abroad

Method used

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  • Real-time fluorescent PCR (polymerase chain reaction) kit and oligonucleotides for identifying zygosaccharomyces rouxii
  • Real-time fluorescent PCR (polymerase chain reaction) kit and oligonucleotides for identifying zygosaccharomyces rouxii
  • Real-time fluorescent PCR (polymerase chain reaction) kit and oligonucleotides for identifying zygosaccharomyces rouxii

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Experimental program
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Embodiment 1

[0040] Design and synthesis of embodiment 1 primers and probes

[0041] In the present invention, firstly, sequence comparison and analysis are carried out on Luxie conjugative yeast and its nine similar yeasts, and the ITS1, 5.8S, and ITS2 gene sequences of these more than ten kinds of yeasts are used to establish a local database. ITS1, 5.8S, and ITS2 gene sequences were extracted using Python language scripts, and independent databases of ITS1, 5.8S, and ITS2 genes were established for ten yeast species. Use MEGA4 for sequence alignment, and use Python language scripts to find the conserved sequences in different types of yeast and the variant sequences among different types of yeast. Species-specific primers and probes were designed using Becaon software considering conserved sequences and variant sequences. Perform electronic PCR according to the obtained primers and probes to obtain the theoretical amplified sequence, and on this basis, perform BLAST on the theoreticall...

Embodiment 2

[0046] Embodiment 2 identifies the real-time fluorescent PCR kit of Ruby's binding yeast

[0047] (1) Real-time fluorescent PCR reaction solution, which includes the upstream primer shown in SEQ ID No.1 in the sequence table, the downstream primer shown in SEQ ID No.2 in the sequence table, and the fluorescent probe shown in SEQ ID No.3 in the sequence table , the 5' end of the probe is labeled with the reporter fluorophore FAM, and the 3' end is labeled with the quencher fluorophore BHQ1;

[0048] (2) Negative control: sterile double distilled water;

[0049] (3) Positive control: Zygosaccharomyces rouxii CGMCC2.1915.

[0050] Further, the real-time fluorescent PCR reaction solution also includes reagents and enzymes required for conventional real-time fluorescent PCR reactions. Preferably, the commercially purchased TaqMan Universal PCR Master Mix (2×) used in the present invention is purchased from Beijing Dongfang Jiacheng Science and Trade Co., Ltd.

Embodiment 3

[0051] Embodiment 3 specificity test

[0052] The present invention selects Saccharomyces cerevisiae CGMCC2.1882, Candida parasilosis ATCC22019, Pichia carsonii CGMCC2.1908, Debaryomyces hansenii CGMCC2.1595 , Dekkera bruxellensis JCM11407, Rhodotorula glutinis CGMCC2.0703, Pichia membranifaciens CGMCC2.1705, Bayer combined yeast Zygosaccharomyces bailii (CGMCC2.1452), Dispora combined yeast (Zygosaccharomyces bisporus) CGMCC2.1529, Escherichia coli (E.coli) ATCC25922, Staphylococcus aureus (Staphylococcus aureus) CMCC26001 were used as samples to be tested. ), the Japanese Culture Collection (JCM) and the American Type Culture Collection (ATCC). Zygosaccharomyces rouxii CGMCC2.1915 was used as a positive control.

[0053] The genomic DNA of the above-mentioned strains to be tested and the positive control were extracted respectively, the DNA concentration was adjusted to 30 ng / μL, and real-time fluorescent PCR amplification reactions were carried out according to the follow...

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Abstract

The invention discloses a real-time fluorescent PCR (polymerase chain reaction) kit and oligonucleotides for identifying zygosaccharomyces rouxii. Specifically, the invention provides a group of oligonucleotides for identifying the zygosaccharomyces rouxii. The roup of oligonucleotides are shown in sequence tables from SEQ ID No:1 to SEQ ID No:3, wherein the sequences of SEQ ID NO:1 and SEQ ID NO:2 respectively are an upstream primer and a downstream primer for identifying the zygosaccharomyces rouxii, and the sequence of SEQ ID NO:3 is a fluorescent probe for identifying the zygosaccharomyces rouxii. The invention also provides the real-time fluorescent PCR (polymerase chain reaction) kit containing the primers and the probe and a real-time fluorescent PCR identification method of the zygosaccharomyces rouxii in foods. The identification sensitivity of the kit and oligonucleotides disclosed by the invention is within 10-4ng / microliter.

Description

technical field [0001] The invention relates to a real-time fluorescent PCR kit and oligonucleotide for identifying Zygomyces rouxii, and belongs to the field of inspection and quarantine. Background technique [0002] Zygosaccharomyces rouxii is the most common hyperosmotic yeast. It is not only the main production bacteria of traditional fermented food, but also the spoilage bacteria of certain low water activity food and raw materials. Zygomyces rouckeri is an important strain for the production of fermented products such as soy sauce, Japanese miso and Thai fermented fish products. Spoilage bacteria are found in juices, jams, soft drinks, salad dressings, and ketchups. [0003] The traditional detection method of Zygomyces rouckeri is based on phenotypic characteristics such as morphological characteristics, staining characteristics, and biochemical characteristics. These conventional methods have played an important role in the research of Vibrio parahaemolyticus, bu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 饶红蔡雪凤付溥博陈世琼逄波郭铮蕾
Owner 北京市海淀区产品质量监督检验所
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