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Detection method of four intestinal protection bacteria and nucleic acid detection kit

A technology for intestinal and Clostridium butyricum, applied in the field of nucleic acid detection kits, can solve the problems of inability to distinguish amplification products from non-specific amplification, poor specificity, and inability to quantify different target products separately, achieving a repeatable detection method , fast detection and high sensitivity

Active Publication Date: 2021-12-03
广东君道营养科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although fluorescent quantitative PCR has the advantages of cheap price and wide applicability, its specificity is poor, and it cannot distinguish amplification products from non-specific amplification, nor can it quantify different target products separately.
At present, there are no reports on the reagents and methods for the simultaneous detection of intestinal protective bacteria of Bifidobacterium pseudosmall chains, Clostridium butyricum, Lactobacillus casei and Ekkermansia

Method used

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  • Detection method of four intestinal protection bacteria and nucleic acid detection kit
  • Detection method of four intestinal protection bacteria and nucleic acid detection kit
  • Detection method of four intestinal protection bacteria and nucleic acid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Design and screening of embodiment 1 primers and probes

[0038] 1. Design of primers and probes

[0039] The present invention analyzes the gene information characteristics of four intestinal protective bacteria pseudosmall chain Bifidobacterium, Clostridium butyricum, Lactobacillus casei and Ekmansella, selects the 16S rRNA conserved region as the target region, and targets each 2 pairs of specific primers and 2 specific probes were designed for each of the bacteria (including 1 downstream primer shared by Bifidobacterium pseudosmallstrand, Clostridium butyricum and Ekmansia spp.), and the corresponding gene accession numbers were NZ_CABHOD010000015. 1. CABHIF010000001.1, CP006690.1 and CP036293.1.

[0040] At the same time, the present invention constructs recombinant plasmids respectively containing the target fragments of Pseudomonas Bifidobacterium, Clostridium butyricum, Lactobacillus casei and Ekmansia, and connects the synthetic target fragments of the four ki...

Embodiment 2

[0054] Embodiment 2 detection method and nucleic acid detection kit

[0055] In order to determine the detection system of primers and probes, the present invention also tested the effects of different final concentrations of primers and probes on the fluorescent PCR reaction.

[0056] (1) Optimization of the amount of primers and probes

[0057] The primers and probes confirmed by screening in Example 1 were formulated into a solution with a certain concentration, and then the amount of primers and probes was adjusted, combined to prepare different PCR reaction systems, and the synthetic recombinant plasmid was diluted 10 times into 4 The concentration gradient is used as a positive template for amplification, and the influence of the amount of different primers and probes on the detection effect is detected, and the appropriate concentration and amount of primers and probes are selected.

[0058] Optimization of the amount of primers and probes in the detection system in ta...

Embodiment 3

[0082] Embodiment 3 Sensitivity detection

[0083] The recombinant plasmids containing 4 kinds of target genes for protecting intestinal bacteria were mixed as the initial sample, and diluted to a concentration of 10 6 copies / mL, and then diluted to 10 5 、10 4 、10 3 And 500copies / mL as the sample to be tested, detect the sensitivity of primer and probe, reaction system and condition are the same as embodiment 2.

[0084] In order to avoid the influence of overlapping curves on viewing, the present invention observes the results through the fluorescent channel corresponding to the detection probe, and the results are as follows Figure 1~4 shown, where figure 1 It is the detection result of the sensitivity of the detection primers and probes for pseudo-small chain bifidobacteria, figure 2 Sensitivity detection results of detection primers and probes for Clostridium butyricum, image 3 Sensitivity test results for Lactobacillus casei detection primers and probes, Figure...

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Abstract

The invention discloses a detection method of four intestinal protection bacteria and a nucleic acid detection kit. The four intestinal protection bacteria are bifidobacterium pseudocatenulatum, clostridium butyicum, lactobacillus casei and Akkermansia Muciniphila. According to the invention, highly conserved regions of 16S rRNA of the four intestinal protecting bacteria are taken as target regions, and specific primers and probes are designed and screened, so that a detection method and a detection kit capable of simultaneously detecting the four intestinal protecting bacteria are established. Compared with a traditional bacterial identification method, the detection method of the four intestinal protection bacteria has the advantages of being high in specificity, high in sensitivity, convenient to operate, rapid and accurate in detection and the like, and can be used for qualitative detection of the intestinal protecting bacteria in an actual sample.

Description

technical field [0001] The invention belongs to the technical field of intestinal microorganism detection. More specifically, it relates to a detection composition, a detection method and a nucleic acid detection kit (PCR-fluorescent probe method) for four intestinal protective bacteria. Background technique [0002] Gut microbes play an important role in regulating the material metabolism, energy flow, and signal transduction of organisms, and have important effects on animal diet, nutrition, immunity, neuromodulation, and the occurrence of chronic diseases. In-depth and detailed research on gut microbes is an important way to understand the pathogenesis of various chronic diseases and immune and pathogenic microbial diseases. [0003] Type 2 Diabetes Mellitus (T2DM) is a common metabolic disease in clinical practice. Glucolipid metabolism disorder often occurs in the body, which leads to a high level of blood sugar in patients for a long time, which seriously affects thei...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/145C12R1/245C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2600/158C12Q2600/178C12Q2537/143C12Q2563/107C12Q2521/531Y02A50/30
Inventor 方鹏宇郭红辉
Owner 广东君道营养科技有限公司