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Method and kit for detecting type 2 diabetes mellitus susceptible intestinal bacteria

A technology for type 2 diabetes and intestinal bacteria, applied in the field of microbial detection, to achieve the effect of strong detection specificity, good stability and saving consumables

Inactive Publication Date: 2021-11-12
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, there is no multiplex fluorescent PCR method for simultaneous detection of type 2 diabetes-susceptible enterobacteria, namely Bacteroides polymorpha, Clostridium tenebilis, Clostridium sphaericus and Lachnospira, which are four types of type 2 diabetes-susceptible enterobacteria. Therefore, providing a multiplex qPCR method for detecting the above four bacteria is of great significance to the research of type 2 diabetes

Method used

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  • Method and kit for detecting type 2 diabetes mellitus susceptible intestinal bacteria
  • Method and kit for detecting type 2 diabetes mellitus susceptible intestinal bacteria
  • Method and kit for detecting type 2 diabetes mellitus susceptible intestinal bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Design and screening of embodiment 1 multiplex qPCR primers and probes

[0045] The present invention utilizes multiple fluorescent PCR technology to target the highly conserved region of 16S rRNA of four types of intestinal bacteria susceptible to type 2 diabetes patients, namely, Bacteroides polymorpha, Clostridium tenabilis, Clostridium sphaericus and Lachnospira. Three pairs of specific primers were designed, and two Taqman probes were designed for each strain. The gene accession numbers of the genes used are: CP012937.1, CAJJWU010000001.1, CAJTKH010000001.1, PEDL01000001.1. Through the single-channel primer-probe NTC test and the four-channel primer-probe test, a set of qPCR primers and probes that can be used to simultaneously detect the above four intestinal bacteria and have good detection effects were screened out. The screening process is as follows:

[0046] (1) Single channel primer probe NTC test

[0047] Arrange and combine the designed and synthesized ...

Embodiment 2

[0057] Embodiment 2 Establishment of Multiplex qPCR Detection Method and Detection Kit

[0058] Using the constructed recombinant plasmids of Bacteroides polymorpha, Clostridium tenabilis, Clostridium sphaericus and Lachnospira as templates, the primers and probes screened in Example 1 were used to construct a multiplex qPCR detection method and optimize it.

[0059] Optimization of the reaction system:

[0060] (1) Optimizing the amount of primer probe

[0061] Prepare the primer-probe combination confirmed by screening into a PCR reaction system, adjust the amount of primer probe, prepare multiple systems, and dilute the synthesized plasmid 10 times into 4 gradient concentrations as positive templates for amplification detection , select the appropriate amount of primer probe.

[0062] Table 2 Optimization of the amount of primers and probes for multiplex qPCR

[0063] Material name System 1 System 2 System 3 System 4 System 5 5×DNA PCR Buffer 5...

Embodiment 3

[0083] Sensitivity detection of embodiment 3 multiplex qPCR primers and probes

[0084] Mix the recombinant plasmids described in Example 2 that have been rated as the initial sample, and dilute to a concentration of 10 6 copies / mL, serially diluted to 10 5 、10 4 、10 3 And 500copies / mL as the sample to be tested, test the sensitivity of multiple qPCR primers and probes, the reaction system and reaction conditions are the same as in Example 2.

[0085]In order to avoid the influence of the overlapping curves on viewing, the present invention observes the results separately through the fluorescent channels corresponding to the detection probes. The sensitivity test results of qPCR primers and probes are as follows: Figure 1~4 as shown, Figure 1~4 The sensitivity detection results of the detection primers and probes of Bacteroides polymorpha, Clostridium tenabilis, Clostridium sphaericus and Lachnospira are shown in turn. It can be seen from the figure that the detection r...

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Abstract

The invention discloses a method and a kit for detecting type 2 diabetes mellitus susceptible intestinal bacteria. According to the method and the kit, a multiplex fluorescent PCR (polymerase chain reaction) technology is utilized, a detection method for the type 2 diabetes mellitus susceptible intestinal bacteria, namely bacteroides thetaiotaomicron, clostridium leptum, clostridium coccoides and lachnospiraceae bacterium, is established by designing and screening primers and probes and optimizing a reaction system, one-tube multiplex detection of the four bacteria is realized, and the kit has the advantages of strong detection specificity, high sensitivity and good repeatability; the kit is simple, convenient and rapid to operate, consumables are saved, the cost is low, the kit can be used as a qualitative detection reagent for bacteroides thetaiotaomicron, clostridium leptum, clostridium coccoides and lachnospiraceae bacterium, and a rapid, specific and repeatable detection means is provided for research on type 2 diabetes mellitus.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection. More specifically, it relates to a multiplex qPCR primer and probe, a detection method and a kit for detecting type 2 diabetes susceptible intestinal bacteria. Background technique [0002] With the changes in my country's economy, environment, and living standards, the prevalence of diabetes is increasing year by year, and the prevalence of type 2 diabetes mellitus (T2DM) is even higher. Type 2 diabetes is considered to be caused by a series of risk factors, such as genetics, age, obesity, and unhealthy lifestyle, and more and more evidence shows that type 2 diabetes is related to the development of the number of intestinal flora Sex, through the detection of intestinal flora, can provide auxiliary evidence for the diagnosis of type 2 diabetes. For example, Chinese patent CN109797190A discloses a microbial marker and its application for assessing the risk of type II diabetes, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/145C12R1/01
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 方鹏宇郭红辉
Owner 广东君道营养科技有限公司
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