Primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application of primer probe combination

A primer probe and microorganism technology, applied in the field of microorganism detection, can solve the problems of complicated operation, complicated preparation and dyeing, etc., and achieve the effect of specific amplification

Active Publication Date: 2021-01-05
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation and staining of this method are complicated, requiring smearing and staining on slides, and the need to switch between different microscope light sources during the observation process, which is cumbersome in operation

Method used

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  • Primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application of primer probe combination
  • Primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application of primer probe combination
  • Primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application of primer probe combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This embodiment provides a primer-probe combination and a method for detecting vaginal microorganisms using it.

[0063] (1) Primer design

[0064] In order to distinguish the four species of Lactobacillus (LI, LJ, LC, LG) to be detected, the unique genes of the four species of Lactobacillus were selected in this example to design primers and probes, thereby ensuring the specificity of the primers , to avoid primers that amplify other strains.

[0065] Among them, the specific genes are the folE gene of Lactobacillus inere (LI), the gapR gene of Lactobacillus janesus (LJ), the CbiQ1 gene of Lactobacillus crispatus (LC) and the murQ gene of Lactobacillus formatiformis (LG). The sequences of the primers are as follows:

[0066] LI folE forward primer (SEQ ID NO: 1):

[0067] 5'-GTGGCAGTAGGTGAAGATCC-3';

[0068] LI folE reverse primer (SEQ ID NO:2):

[0069] 5'-TGGTCGATGATTGAGTGACG-3';

[0070] LJ gapR forward primer (SEQ ID NO:3):

[0071] 5'-GCGAATTGGAGTTTGTTCCG-3...

Embodiment 2

[0111] In the present embodiment, the primer probe combination and detection method provided in Example 1 are used to carry out the plasmid mixture of Lactobacillus inert (LI), Lactobacillus janesus (LJ), Lactobacillus crispatus (LC), and Lactobacillus formatiformis (LG). Typing.

[0112] Specific steps are as follows:

[0113] (1) Design primers for LI folE, LJ gapR, LC CbiQ1, and LG murQ genes, and use primers to amplify LI folE, LJ gapR, LC CbiQ1, and LG murQ from gDNA extracted from clinically isolated LI, LJ, LC, and LG strains, respectively The gene fragments were cloned and transformed using the pEASY-Blunt Cloning Kit;

[0114] (2) Use TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 (TaKaRa) to extract plasmids from E.coli;

[0115] (3) Mix the plasmids of LI folE, LJ gapR, LC CbiQ1, and LG murQ as the sample to be tested, and the concentration of each plasmid is 2×10 3 copies / μL; the rest of the detection steps were carried out with the steps described in the refe...

Embodiment 3

[0121] The difference from Example 2 is that in this example, ddPCR is not used for amplification, and only a general amplification method is used for amplification, that is, after mixing the primer-probe combination with the sample, the PCR reaction system is directly prepared, and Perform fluorescent quantitative PCR;

[0122] Among them, the fluorescent quantitative PCR uses a 20 μL reaction system, including a reaction system a for detecting LG and LJ and a reaction system b for detecting LC and LI.

[0123] Reaction system a is shown in Table 4 below:

[0124] Table 4

[0125]

[0126]

[0127] Wherein, after the reaction system is prepared, the concentration of each primer is 300nM; the concentration of the LJ gapR probe is 200nM, and the concentration of the LG murQ probe is 200nM.

[0128] Reaction system b is shown in Table 5 below:

[0129] table 5

[0130]

[0131] Wherein, after the reaction system is prepared, the concentration of each primer is 300nM...

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Abstract

The invention provides a primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application of the primer probe combination. The primer probe combination comprises a primer pair and a probe, wherein the primer pair and the probe are used for amplifying and detecting a polE gene of inert lactobacillus, a galR gene of lactobacillus jensenii, aCbiQ1 gene of lactobacillus crispatus and a murQ gene of lactobacillus gasseri, wherein the primer pair has relatively high specificity, can avoid primer amplification of other strains during amplification, is used in combination with quadruple droplet digital PCR, classifies and quantitatively analyzes droplets after PCR amplification, and can determine the concentration of each lactobacillus ina sample, determine the dominant microorganisms of the female vagina and realize vagina microorganism typing.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a combination of primers and probes for detecting female vaginal microorganisms based on quadruple droplet digital PCR and its application. Background technique [0002] There are a large number of microorganisms inside and outside the human body, and they are widely involved in processes such as digestion, metabolism, and immune defense. There are many microbial flora symbiotic in the female vagina, which have a natural defense function against the invasion of pathogens. The vaginal microorganisms of healthy women are usually composed of Lactobacillus inere (LI), Lactobacillus janesus (LJ), Lactobacillus crispatus (LC) and Lactobacillus format (LG) One of the four types of Lactobacillus dominates. These Lactobacilli maintain the low pH environment of the vagina by producing lactic acid and hydrogen peroxide, and also produce bacteriocin and other antibacterial su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12Q1/06C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2563/159C12Q2537/143C12Q2563/107Y02A50/30
Inventor 陈雯雯陈永欣王纪东郑佳莹黄炜塨刘晓蕾
Owner SHENZHEN UNIV
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