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Multiple-fluorescence quantitative PCR primers, kit and detection method for detection of multiple types of bacteria

A technology of multiple fluorescence quantification and detection kits, applied in the field of microbial detection, can solve the problems of low detection efficiency and poor specificity of multiple strains, and achieve the effect of good specificity, high throughput and accurate detection results

Active Publication Date: 2020-03-24
知几未来(成都)临床医学检验有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] For this reason, the embodiments of the present invention provide a multiplex fluorescent quantitative PCR primer, kit and detection method for detecting various bacteria, so as to solve the problem of low detection efficiency of multiple bacterial species in the prior art due to poor specificity of detection. The problem

Method used

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  • Multiple-fluorescence quantitative PCR primers, kit and detection method for detection of multiple types of bacteria
  • Multiple-fluorescence quantitative PCR primers, kit and detection method for detection of multiple types of bacteria

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Embodiment 1

[0036] The embodiment of the present invention provides multiple fluorescent quantitative PCR primers and probes for detecting various bacteria, specifically, the PCR primers and probes are used to detect Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis in human intestinal tract , Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri and Akkermansia, etc., design primers and probes according to the specific 16SrDNA sequences of these 8 kinds of bacteria, and test it. By continuously designing and optimizing PCR primers and probes based on the detection results, and adjusting the PCR reaction system in a targeted manner. Design a group of combinations of fluorescent quantitative PCR primers and probes that are relatively ideal in terms of specificity, mutual interference and amplification efficiency of primers and probes, as shown in Table 1, for the detection of multiple Multiplex fluorescent quantitative PCR...

Embodiment 2

[0041] The PCR primers and probes designed and prepared in Example 1 are divided into the first component, the second component and the third component, as shown in Table 2-Table 4; wherein, the first component includes the long Bifidobacterium, Bifidobacterium breve and Bifidobacterium animalis primers and probes and PCR reaction system required buffer and enzyme; the second component includes the Lactobacillus rhamnosus and Lactobacillus reuteri primers and probe PCR reaction system required buffer and enzyme; the third component includes Akkermansia, Lactobacillus acidophilus and Lactobacillus plantarum primers and probes and PCR reaction system required buffer fluids and enzymes.

[0042] Table 2

[0043]

[0044] table 3

[0045]

[0046] Table 4

[0047]

[0048]

[0049] The reaction program of the multiplex fluorescent quantitative PCR method in the embodiment of the present invention is shown in Table 5.

[0050] table 5

[0051]

[0052] In the em...

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Abstract

The invention discloses multiple-fluorescence quantitative PCR primers, a kit and detection method for detection of multiple types of bacteria. The primers include 16SrDNA designed primer pairs and probes which target Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri and Akkemansia, wherein the nucleotide sequences of the primer pairs of Bifidobacterium longum, animalis Bifidobacterium and Bifidobacterium breve are shown in SEQ ID NO:1 and SEQ ID NO:2. The invention also discloses a multiple-fluorescence quantitative PCR detection kit which comprises the PCR primers and probes. The multiple-fluorescent quantitative PCR primers and probes for detection of multiple typesof bacteria are specific-sequence primers and probes which are designed and synthesized according to multiple experiments, and through the optimized specific primers and probes, it is ensured that good specificity and reduction of mutual interference among primer probes in the same reaction system can be achieved during amplification.

Description

technical field [0001] The embodiments of the present invention relate to the technical field of microbial detection, and in particular to a multiplex fluorescent quantitative PCR primer, kit and detection method for detecting various bacteria. Background technique [0002] Human intestinal microbes are closely related to human health, and the proportion of some important types of bacteria has an important impact on human health. By detecting the content and proportion of some important bacterial species in intestinal microorganisms, it has certain significance for judging certain health conditions and adjuvant treatment of subjects. At present, the common detection methods for the number and type of bacteria are: 1. Traditional selective medium culture: This method mainly uses a specific medium to cultivate specific bacteria in the sample, and the number of colonies reflects the species in the sample. the number of bacteria. This method is a classic and commonly used meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01C12R1/225C12R1/23C12R1/25
CPCC12Q1/689C12Q1/686C12Q2563/107C12Q2545/114C12Q2537/143Y02A50/30
Inventor 夏广亮
Owner 知几未来(成都)临床医学检验有限公司
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