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Primer group for analyzing intestinal tract microbes and application thereof

A technology of gut microbes and primer sets, applied in the field of molecular biology, can solve the problems of time-consuming, laborious operation, and unstable research results of microorganisms, and achieve the effect of increasing the complexity and increasing the quality of sequencing.

Inactive Publication Date: 2018-04-20
SUZHOU PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Many classic methods have been used in the study of intestinal flora, such as the study of colony characteristics, bacterial morphology, physiological and biochemical characteristics, metabolites and other indicators. These traditional methods are mainly based on pure culture techniques, but there are many in the intestinal tract. Microorganisms cannot achieve pure culture; microorganisms that can be purely cultured have problems such as time-consuming, laborious and complicated operations, and are largely restricted by culture conditions, which often leads to instability of research results, resulting in incomplete and objective results. reflect the real situation of intestinal flora

Method used

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  • Primer group for analyzing intestinal tract microbes and application thereof
  • Primer group for analyzing intestinal tract microbes and application thereof
  • Primer group for analyzing intestinal tract microbes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The preparation of embodiment 1 detection kit

[0074] (1) Preparation of primer set

[0075] The primer set includes a first forward primer and a second reverse primer, and its nucleic acid sequence is as follows:

[0076] First forward primer: AATGATACGGCGACCACCGAGATCTACACTCT-[i5]-TATGGTAATTNNCCTACGGRRBGCASCAGKVRVGAAT;

[0077] Among them, i5 is the P5 tag sequence, N is any base in A\T\G\C, R is selected from any base in A / G, and B is selected from any base in T / C / G base, S is selected from any base in C / G, K is selected from any base in T / G, and V is selected from any base in A / C / G;

[0078] First reverse primer: CAAGCAGAAGACGGCATACGAGAT-[i7]-AGTCAGCCAGNNGGACTACNVGGGTWTCTAATCC;

[0079] Among them, i7 is the P7 tag sequence, N is any base in A\T\G\C, V is selected from any base in A / C / G, W is selected from any base in A / T base.

[0080] The first tag sequence is a P5 tag sequence, its nucleic acid sequence is shown in SEQ ID NO.5-14, and the nucleic acid sequen...

Embodiment 2

[0087] The extraction of embodiment 2 sample DNA

[0088]Select 3 stool samples: A, B, and C, divide them into 2 parts on average, and mark them as: A1 / A2, B1 / B2, C1 / C2, and process the three samples of A1, B1, and C1 with the method described in this patent and obtain data , A2, B2, and C2 samples are directly sent to Jinweizhi Company for processing. The specific DNA extraction method includes the following steps:

[0089] (1) Take 700-800μl of buffer A and 250mg of glass beads into a 2ml centrifuge tube;

[0090] (2) Add 200mg sample to the above 2ml centrifuge tube, vortex and mix for 10-30s;

[0091] (3) Add 50 μl of lysate B to the sample, vortex for 10 minutes to mix the sample, centrifuge at 12,000 rpm (~13,400×g) for 30 s, transfer the supernatant (400 μl) to a new 2ml centrifuge tube;

[0092] (4) Add 200μl suspension C, vortex for 10s, place at room temperature for 2min, centrifuge at 12,000rpm (~13,400×g) for 60s, and precipitate the sample particles;

[0093] (...

Embodiment 3

[0102] Example 3 library construction

[0103] (1) Prepare the following reaction solution on ice, except DNA extraction solution and H 2 Other than O, according to the following components, first prepare the amplification premix Mix according to the amount of reaction number + α, and take the prepared premix Mix and distribute it into PCR reaction tubes. The primer sequences are as follows:

[0104] Upstream primer (SEQ ID NO.27): AATGATACGGCGACCACCGAGATCTACACTCT-TATCCTCT-TATGGTAATTAGCCTACGGGACGCAGCAGTCGGGAAT;

[0105] Downstream primer (SEQ ID NO.28): CAAGCAGAAGACGGCATACGAGAT-TTCTGCCT-AGTCAGCCAGTGGGACTACACGGGTATCTAATCC;

[0106] Concrete reaction system is as follows:

[0107] Reagent

Usage amount

2×PCR Mixture (Mg 2+ ,dNTPs)

12.5μl

Upstream primer (1 μM)

5μl

Downstream primer (1 μM)

5μl

Sample DNA 5ng / μl (or ddH 2 O)

2.5μl

Total

25μl

[0108] Set up a set of negative controls with water as the s...

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Abstract

The invention belongs to the field of molecular biology, and relates to a primer group for analyzing intestinal tract microbes and application thereof. The primer group comprises a first forward primer and a first reverse primer, wherein the first forward primer comprises five sequentially connected parts from the 5' end to 3' end: a P5 connector sequence, a first label sequence, a first intervalsequence, a 2bp random basic group sequence and an intestinal tract microbe 16S rRNA V3-V4 region specific forward primer; the first reverse primer comprises five sequentially connected parts from the5' end to 3' end: a P7 connector sequence, a second label sequence, a second interval sequence, a 2bp random basic group sequence and an intestinal tract microbe 16S rRNA V3-V4 region specific reverse primer. The primer composition provided by the invention uses a one-step method for amplification so as to obtain a sequencing library; the random basic group is included; the library sequence complexity is increased; the phix proportion is reduced; higher sequencing quality can be ensured; the library coverage degree is improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a primer set for analyzing intestinal microorganisms and its application, in particular to a primer set for constructing a library through a round of PCR, analyzing and classifying intestinal microorganisms, a detection kit composed of it and its application . Background technique [0002] The human intestinal tract co-exists with a complex structure and a large number of microorganisms. Through long-term co-evolution with humans, these microorganisms have become important "functional organs" that cannot be ignored in maintaining human health. More and more research evidence shows that intestinal flora imbalance is closely related to obesity, diabetes, non-alcoholic fatty liver, inflammatory bowel disease, gastrointestinal tumors and other diseases. Therefore, it is of great significance to explore the relationship between intestinal flora and the occurrence and development of dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/04C12N15/11C40B50/06
CPCC12Q1/6869C12Q1/689C40B50/06
Inventor 周燕朱永亮穆延召
Owner SUZHOU PRECISION BIOTECH CO LTD