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Primer set for quickly, qualitatively and quantitatively detecting six intestinal microorganisms and application thereof

A technology for the quantitative detection of intestinal microbes, applied in the field of microbes, to achieve the effect of broad application prospects

Inactive Publication Date: 2019-04-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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After searching, there is no patent related to the specific primer set and its application that can quickly qualitatively and quantitatively detect Bacteroides, Lactobacillus, Escherichia coli, Enterococcus, Streptococcus and Bifidobacterium in the intestinal tract to report

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  • Primer set for quickly, qualitatively and quantitatively detecting six intestinal microorganisms and application thereof

Examples

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Embodiment 1

[0042] Example 1: Qualitative analysis of six intestinal microorganisms (Bacteroides, Lactobacillus, Escherichia coli, Enterococcus, Streptococcus and Bifidobacterium) in stool samples

[0043] Based on the similarity comparison of the 16S rRNA gene sequences of Bacteroides, Lactobacillus, Escherichia coli, Enterococcus, Streptococcus, Bifidobacterium, and other bacteria that are closely related to them, the target bacteria are different from other bacteria. The specific conserved sequences of five species of bacteria and similar bacteria were obtained, and six sets of specific primer pairs were designed according to the conserved sequences. Wherein: the upstream primer Bacter F5 of the Bacteroides specific detection primer pair: 5'-AAGACAACGATGGATAGGGGTT-3', the downstream primer Bacter R6: 5'-TTCACGCTACTTGGCTGGT-3', the pair of primers amplified to obtain a 135bp Bacteroides genomic DNA fragment; The upstream primer Lacter F5 of the Lactobacillus family-specific detection pr...

Embodiment 2

[0061] Example 2: Quantitative detection of six intestinal microorganisms (Bacteroides, Lactobacillus, Escherichia coli, Enterococcus, Streptococcus and Bifidobacterium) in stool samples

[0062]The application of the above six sets of primer pairs in the quantitative detection of Bacteroides, Lactobacillus, Escherichia coli, Enterococcus, Streptococcus and Bifidobacteria in volunteer stool samples by fluorescent quantitative PCR.

[0063] Among them, the steps and conditions involved in the specific application method are:

[0064] (1) Preparation of genomic DNA: Take fresh stool samples from volunteers, divide 200 μL or 200 mg each into 2 mL centrifuge tubes, use the QIAampDNA Stool Mini Kit to extract total DNA from the stool samples according to the instructions, and store them at -20°C for later use .

[0065] (2) Fluorescent quantitative PCR reaction system preparation: accurately pipette 10 μL TB Green Premix Ex Taq, 0.8 μL upstream primer, 0.8 μL downstream primer, 1 ...

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Abstract

The invention discloses a primer set for quickly, qualitatively and quantitatively detecting six common intestinal microorganisms, which consists of six groups of specific primer pairs respectively used for detecting pseudomonas, lactobacillus, escherichia coli, enterococcus, streptococcus and bifidobacterium. PCR detection by using the primer set combined with agarose gel electrophoresis can carry out quantitative analysis on a sample; the primer set is used for fluorescence quantitative PCR detection to carry out quantitative detection on the sample. The primer set can be carried out under the same PCR reaction program, can carry out qualitative and quantitative detection on six common microorganisms in the intestinal tract within four hours, and has wide application prospect in the fields of medical detection, food sanitation and the like.

Description

technical field [0001] The invention belongs to the technical field of microbes, in particular to a set of primers for rapid qualitative and quantitative detection of six common intestinal microorganisms and applications thereof. Background technique [0002] The gut is the most important digestive organ in the human body, and it is also a diverse and vibrant microbial ecosystem, housing trillions of microorganisms. A large number of studies have shown that intestinal microorganisms not only participate in the host's food transformation, but also play an important role in the body's physiological functions such as nerves and immunity. Metagenome sequencing found that the dominant bacteria in the gut are mainly distributed in Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria, including Bacteroides, Lactobacillus, Escherichia coli, Enterococcus Common intestinal microorganisms such as genus Streptococcus, Bifidobacterium and Bifidobacterium; the composition and qua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/10C12Q1/06C12Q1/04C12R1/225C12R1/19C12R1/46C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16
Inventor 孔健郭婷婷顾心怡
Owner SHANDONG UNIV
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