Nucleic acid detection kit and detection method for firmicutes and bacteroides

A technology of Firmicutes and Bacteroidetes, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Concise, fast detection effect

Inactive Publication Date: 2022-01-28
广东君道营养科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the detection of the four bacteria separately, simultaneous detection cannot be achieved
[0005] At present, there are no reports on the method and related reagents for the simultaneous detection of Firmicutes and Bacteroidetes. In order to better study the relationship be

Method used

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  • Nucleic acid detection kit and detection method for firmicutes and bacteroides
  • Nucleic acid detection kit and detection method for firmicutes and bacteroides
  • Nucleic acid detection kit and detection method for firmicutes and bacteroides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Design and screening of primers

[0048]1. Design of primers

[0049] After analyzing the gene information characteristics of Firmicutes and Bacteroidetes, the conserved region was selected as the target region, and a series of specific primers were designed for Firmicutes and Bacteroidetes.

[0050] 2. Screening of primers and probes

[0051] In order to select the best primers from the primers designed above, the present invention carried out primer tests respectively.

[0052] Template construction: The recombinant plasmids of the target fragments of Firmicutes and Bacteroidetes were constructed, and the synthetic target fragments of Firmicutes and Bacteroidetes were connected to the constructed PUC57 vector and transformed to express Escherichia coli DH5α Competent cells, the bacteria with recombinant plasmids were obtained by blue-white screening, and the recombinant plasmids were extracted for PCR amplification and sequenced identification.

[0053] P...

Embodiment 2

[0063] Example 2 Optimization of Primer Consumption

[0064] In order to determine the best detection system, the present invention also separately detects the effects of different final concentrations of primers on the fluorescent quantitative PCR reaction.

[0065] Optimization of the amount of primers: Prepare the primers confirmed by screening in Example 1 into a solution, adjust the amount of primers, combine and prepare different PCR reaction systems, and dilute the synthetic recombinant plasmid 10 times into 3 concentration gradients as positive templates Carry out amplification, detect the influence of different primer dosages on the detection effect, and select the appropriate primer concentration and dosage.

[0066] The fluorescent PCR reaction was carried out on the ABI7500 fluorescent PCR instrument, and the final reaction conditions are shown in Table 2:

[0067] Table 2 Fluorescent PCR reaction conditions

[0068]

[0069]

[0070] During the detection, ...

Embodiment 3

[0074] Example 3 Detection of Fluorescent Quantitative PCR Sensitivity

[0075] The recombinant plasmids containing the target genes of Firmicutes and Bacteroidetes were mixed as the initial sample, and diluted to a concentration of 10 6 copies / mL, and then diluted to 10 5 、10 4 and 10 3 As the sample to be tested, the sensitivity of the detection primer, the reaction system and the conditions are the same as in Example 2.

[0076] 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 12 shown, where Figure 12 Sensitivity detection results of Firmicutes and Bacteroidetes detection primers (the detection results of Firmicutes and Bacteroidetes are displayed separately), as can be seen from the figure, the detection results of samples after gradient dilution of Firmicutes and Bacteroides The linearity is good, t...

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Abstract

The invention discloses a nucleic acid detection kit and detection method for firmicutes and bacteroides. According to the nucleic acid detection kit and detection method of the invention, highly conserved regions of the firmicutes and bacteroides are taken as target regions, and the detection method capable of simultaneously detecting the firmicutes and bacteroides is established by designing and optimizing specific primers and probes. Compared with a traditional bacterial identification method, the method has the advantages of being high in specificity, high in sensitivity, convenient to operate, rapid and accurate in detection and the like; the method can be used for relative quantitative detection of the firmicutes and bacteroides of actual sample intestinal flora; the method can also be used for quantitative detection of the ratio of the firmicutes to the bacteroides; the method is helpful for research on obesity inflammation and type 2 diabetes mellitus, and has important significance for regulating intestinal flora and promoting intestinal health through research on the intestinal flora.

Description

technical field [0001] The invention belongs to the technical field of microbiological detection field. More specifically, it relates to a nucleic acid detection kit and detection method of Firmicutes and Bacteroidetes. Background technique [0002] The incidence of obesity and diabetes is increasing year by year, but its pathogenesis has not yet been elucidated. Diet is an important environmental factor, and its metabolism is closely related to gut microbes. Gut dysbiosis (altered symbiotic relationship between host and gut microbiota) is increasingly linked to a variety of diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome, obesity, allergies , autoimmune and brain disorders. Therefore, many researchers believe that intestinal health can be promoted by regulating the intestinal flora. [0003] The human intestinal flora is mainly composed of Firmicutes and Bacteroidetes, and the rest are mostly Actinobacteria, Proteobacteria and Verrucomicro...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2563/107C12Q2531/113
Inventor 方鹏宇郭红辉
Owner 广东君道营养科技有限公司
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