Specific primer, kit and method for qualitatively and/or quantitatively detecting lactobacillus rhamnosus

A technology for quantitative detection of Lactobacillus rhamnosus, applied in the field of genetic engineering, to achieve high sensitivity, fast and accurate qualitative, and good stability

Pending Publication Date: 2019-06-28
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no efficient detection method to realize the relative content and changes of M9 in different samples

Method used

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  • Specific primer, kit and method for qualitatively and/or quantitatively detecting lactobacillus rhamnosus
  • Specific primer, kit and method for qualitatively and/or quantitatively detecting lactobacillus rhamnosus
  • Specific primer, kit and method for qualitatively and/or quantitatively detecting lactobacillus rhamnosus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Metagenomic DNA Extraction from Fecal Samples

[0055] Take 0.5g of the sample and place it in a 2mL centrifuge tube, add 1000μL Inhibit EX lyase, shake for 10min; put it in an 80°C water bath for 10min after shaking, vortex for 15s, and centrifuge at 12000×g for 5min at room temperature; collect the supernatant and transfer to another Add 25 μL proK to a centrifuge tube, preheat AL buffer 300 μL at 70°C, shake for 5 minutes; bathe in water at 70°C for 10 minutes; add 300 μL of absolute ethanol, absorb all the liquid to the spin column after shaking, centrifuge at 12000×g for 2 minutes; add 500 μL AW1, let stand 1min, centrifuge at 12000×g for 2min; add 500μLAW2, let it stand for 1min, centrifuge at 12000×g for 2min; spin for 2min, change the collection tube and redissolve for later use. The DNA extraction kit is QIAamp FastDNA Stool Mini Kit (50), Cat.No.51604.

Embodiment 2

[0057] Qualitative detection of specific primers for Lactobacillus rhamnosus M9

[0058] Based on a specific fragment (SEQ ID NO.3) in the whole genome sequence of Lactobacillus rhamnosus M9, the following primer sequences were designed:

[0059] Upstream primer M9F: 5'-GTAATGTAAATGGGGTTCCTGTG-3' (SEQ ID NO.1);

[0060] Downstream primer M9R: 5'-TGGTTTCCCCTATAATCGTTGTCC-3' (SEQ ID NO.2).

[0061] After designing the specific primers for Lactobacillus rhamnosus M9, the specificity of the primers was verified, and four strains of rhamnosus with higher homology to Lactobacillus rhamnosus M9 in taxonomic status were selected. Lactobacillus was amplified by PCR as a control.

[0062] A total of 12 strains of lactic acid bacteria were used in this test, including 5 strains of Lactobacillus rhamnosus including M9, 1 strain of Lactobacillus casei and 6 strains of Lactobacillus plantarum. The above strains were obtained from the Lactic Acid Bacteria Resource Bank (LACBB) of Inner Mo...

Embodiment 3

[0073] Quantitative detection of specific primers for Lactobacillus rhamnosus M9

[0074] Amplification system: 20μL: 10×PCR supermix 10μL, 10mol / L upstream and downstream primers 0.2μL each, DNA template 10ng 2μL, ddH 2 O supplemented to 20 μL;

[0075] The amplification conditions were: denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 54°C for 1 min, and 30 cycles; extension at 4°C for 5 min, extension at 90°C for 5 min, and storage at 4°C.

[0076] Put the PCR-amplified droplets into the droplet analyzer. After the droplet analyzer absorbs all the droplets, they are divided into positive droplets and negative droplets according to the fluorescence intensity; among them, the positive droplets are Lactobacillus rhamnosus The amplified fragment of M9 strain DNA, the negative micro-droplet is non-M9 DNA fragment, according to the percentage of positive micro-droplets in the total micro-droplets, according to the formula (copy number × 20) ÷ 2 × retu...

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Abstract

The invention relates to a specific primer, kit and method for qualitatively and/or quantitatively detecting lactobacillus rhamnosus, and belongs to the technical field of genetic engineering. The specific primer comprises an upstream primer M9F and a downstream primer M9R; the sequence of the upstream primer M9F is shown in SEQ ID NO. 1; the sequence of the downstream primer M9R is shown in SEQ ID NO. 2. The specific primer can be used for quickly, accurately, qualitatively and quantitatively detecting the lactobacillus rhamnosus M9, and has the advantages of good specificity, high sensitivity, good stability and good repeatability.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a specific primer, a kit and a method for qualitatively and / or quantitatively detecting Lactobacillus rhamnosus M9. Background technique [0002] Lactobacillus rhamnosus (Lactobacillus rhamnosus) belongs to the genus Lactobacillus, is a Gram-positive bacterium, has no plasmid, and is one of the normal flora of the human body. It has a high intestinal adhesion rate and strong colonization ability, can promote the growth of Bifidobacteria and Lactobacillus acidophilus, and can effectively reduce cholesterol and promote cell division, which can regulate intestinal flora, prevent and treat diarrhea, eliminate toxins, Prevent dental caries, improve the body's immunity and anti-cancer and other important physiological health functions. With the development of technology, more and more lactobacilli have been discovered, making the field of lactic acid bacteria gradually exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12Q1/04C12N15/11C12R1/225
Inventor 钟智张和平孙志宏李伟程高旭
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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