Rapid micro-droplet type digital PCR detection method of Escherichia hermannii

A technology of Escherichia hertzii and a detection method is applied in the field of rapid detection of Escherichia hertzii by droplet type digital PCR to achieve the effects of reducing detection cost, improving sensitivity and high sensitivity

Inactive Publication Date: 2020-01-10
LINYI UNIVERSITY
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However, so far, there have been no reports in China that are conducive to

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[0020] First, take 1 g of the sample to be tested by aseptic operation, add it into a sterile container filled with 9 ml of YM medium, and incubate under aerobic conditions at 30°C±1°C for 24h. The cultured bacterial solution was added to a centrifuge tube, centrifuged at 12,000 rpm for 1 min, the supernatant was discarded, and genomic DNA was extracted by bacterial precipitation and diluted for later use.

[0021] Then carry out micro-droplet digital PCR reaction, the total volume of each micro-droplet digital PCR reaction is 25 μl, including 2 μl of extracted genomic DNA; PCR premix (2×ddPCR Master Mix) 12.5 μl; 1 μl (10 pmol / μl) of each downstream primer, 0.5 μl (10 pmol / μl) of the probe; finally add ddH 2 0 to 25 μl. Transfer the well-mixed PCR system to the droplet generator card, add 70 μl of special oil for droplet generator to the droplet generator card, and put the droplet generator card into the droplet generator for reaction. All the generated micro-droplets were ...

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Abstract

The invention discloses a rapid micro-droplet type digital PCR detection method of Escherichia hermannii. The method includes the steps of extracting the genomic DNA of a to-be-detected enrichment broth sample and performing dilution for later use; designing a specific primer and probe by utilizing the conserved DNA sequence of the genome of Escherichia hermannii; and using the primer and probe toperform micro-droplet type digital PCR amplification and detection on the genomic DNA of the to-be-detected enrichment broth sample. After the PCR amplification, each micro-droplet is detected one byone by adopting a micro-droplet analyzer, the micro-droplets having fluorescence signals are interpreted as 1, and the micro-droplets which have no fluorescence signals are interpreted as 0, and ultimately, the concentrations or copy numbers of to-be-detected target molecules can be calculated according to a Poisson distribution principle and the proportion of positive micro-droplets. Micro-droplet type digital PCR can be used to directly obtain the number of DNA molecules, so that absolute quantification can be performed on the Escherichia hermannii in the sample. The method has the advantages of high sensitivity, precise quantification, wide linear range of detection, good specificity and moderate costs.

Description

technical field [0001] The present invention relates to a kind of Escherichia hertzii ( Escherichia hermannii ) droplet digital PCR rapid detection method. Background technique [0002] Escherichia Hertzii ( Escherichia hermannii ) is a species of Enterobacteriaceae and Escherichia, and is an opportunistic pathogen. Escherichia Hertzii is a bacillus, 1.1~1.5×6μm. Perinate flagella motile or immobile, non-spore-forming, Gram-negative bacteria. Facultatively anaerobic, grows rapidly on common media. It can ferment lactose to produce gas, produce indole, methyl red reaction (+), VP reaction (-). Escherichia hertzii is not only a food spoilage microorganism, but also can cause primary and secondary infections in humans, such as diarrhea, septic arthritis, cephalohematoma, wound infection and osteomyelitis. Studies have found that Escherichia Hertzii also has the function of fermenting and producing exopolysaccharides (Feng Xueping. Screening of strains producing high-visco...

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/10C12Q1/686C12R1/185
CPCC12Q1/689C12Q1/686
Inventor 刘云国扈晓杰王芳芳彭善丽康大成
Owner LINYI UNIVERSITY
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