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Method for accurately determining bacteria in special condition on basis of microfluidic visualization technology as well as selecting and enriching bacteria in special condition

A special state and microfluidic technology, applied in the direction of microorganism-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem that the number of bacteria in special states cannot be accurately quantified, and achieve high efficiency and high dosage. Less, precise process control effect

Inactive Publication Date: 2020-05-12
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] One of the purposes of the present invention is to overcome the problem that the number of special-state bacteria cannot be accurately quantified in the prior art, and to provide a method for accurately measuring special-state bacteria and its sorting and enrichment based on microfluidic visualization technology

Method used

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  • Method for accurately determining bacteria in special condition on basis of microfluidic visualization technology as well as selecting and enriching bacteria in special condition
  • Method for accurately determining bacteria in special condition on basis of microfluidic visualization technology as well as selecting and enriching bacteria in special condition
  • Method for accurately determining bacteria in special condition on basis of microfluidic visualization technology as well as selecting and enriching bacteria in special condition

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Effect test

Embodiment 1

[0036] The specific operation steps for obtaining a single droplet encapsulated single cell in the present invention are as follows:

[0037] 1. Streak the bacteria stored in the -80°C glycerol tube on the LB plate, and place it at 37°C for constant temperature incubation for 18-24 hours. Pick a single colony on the plate, inoculate it in LB broth, and cultivate at 37°C for 12h.

[0038] The bacterial solution was diluted with sterile physiological saline, and the appropriate gradient bacterial solution was selected as the water phase. Microfluidic devices were used to form microdroplets, and a digital PCR instrument was used to measure the copy number of the initial sample. like figure 1 As shown, the measured initial bacterial concentration was 5.4545×10 4 CFU / mL. Take 880 μL of the bacterial solution and resuspend it in 1 mL of normal saline to obtain a concentration of 4.8×10 4 CFU / mL sample solution, and resuspended in LB broth, so that the number of bacteria in the s...

Embodiment 2

[0043] Taking Cronobacter sakazakii in VBNC state as an example - counting, sorting and enrichment of bacteria in special state:

[0044] 1. Streak the bacteria stored in the -80°C glycerol tube on the LB plate, and place it at 37°C for constant temperature incubation for 18-24 hours. Pick a single colony on the plate, inoculate it in LB broth, and cultivate at 37°C for 6-7h.

[0045] 2. Take 30mL logarithmic phase bacterial solution (about 10 8 CFU / mL) was centrifuged, and the pellet was transferred to 300 mL of sterile normal saline, and 120 mg of ampicillin was added to the normal saline for induction at room temperature.

[0046] 3. Take 1 mL of the bacterial solution during the induction process, add 5 μL PMAxx dye, and incubate it in the dark for 15 min, then expose it in a nucleic acid exposure device for 15 min, and use the DNA extraction kit to extract DNA for digital PCR detection. Use the copy number to determine the bacterial concentration of the viable bacterial...

Embodiment 3

[0054] Take Staphylococcus aureus as an example—screening for antibiotic-resistant strains.

[0055] 1. Streak the golden yellow grape balls stored in a glycerol tube at -80°C on the LB plate, and place them at 37°C for incubation for 18-24 hours. Pick a single colony on the plate, inoculate it in LB broth, and cultivate at 37°C for 12h.

[0056] 2. Gradient dilution of the bacterial solution with sterile normal saline, use digital PCR to determine the bacterial concentration in the sample, and adjust the bacterial concentration to 4.8×10 according to the copy number. 4 CFU / mL.

[0057] 4, will be 4.8 × 10 4 The bacterial liquid of CFU / mL was resuspended in LB liquid medium containing penicillin as the water phase to generate droplets, which were transferred to an eight-connected tube and incubated at 37°C for 18h at a constant temperature, which can be resistant to antibiotics. The Staphylococcus aureus can reproduce during the culture process.

[0058] 5. After 18 hours ...

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Abstract

The invention belongs to the technical field of bacteria detection, and specifically discloses a method for accurately determining bacteria in special condition on basis of microfluidic visualizationtechnology as well as selecting and enriching the bacteria in the special condition. The method for accurately determining the bacteria in the special condition on basis of the microfluidic visualization technology as well as selecting and enriching the bacteria in the special condition comprises the following steps: preparing bacterium fluid into microdroplets by adopting a microfluidic technology; and then, culturing the prepared microdroplets, taking photos of the cultured microdroplets, and carrying out counting by performing manual counting or in virtue of a computer technology and the like so as to obtain the number of the bacteria in the special condition. Moreover, the method also enables selection and enrichment of the bacteria in the special condition. Compared with existing counting mode adopting plate counting, or only counting living bacteria with complete membranes, the method for accurately determining the bacteria in the special condition on basis of the microfluidic visualization technology as well as selecting and enriching the bacteria in the special condition has greatly improved accuracy, and breaks through the existing counting mode of the bacteria in the special condition, so that food hygiene status can be timely and effectively judged; and thus, the method is an effective means for eliminating foodborne diseases caused by recessive remains of foodbornepathogens. In addition, the method is capable of performing selection and enrichment on the bacteria in the special condition, so that result error caused by cell heterogeneity in research process canbe reduced; and thus, the method is of relatively great value to studies on mechanisms of cell changes and bacteria growth activities.

Description

technical field [0001] The invention belongs to the technical field of bacteria detection, and more specifically relates to a method for accurately measuring special-state bacteria and sorting and enriching them based on microfluidic visualization technology. Background technique [0002] The special state of bacteria is a reaction that bacteria produce in order to survive when they are affected by the external environment. Such as bacterial "viable but non-culturable state" (viable but non-culturable state, VBNC) and drug-resistant bacteria produced under the action of antibiotics. [0003] The VBNC state is a self-protection state that bacteria enter by adjusting their own metabolic pathways when they are threatened by an adverse external environment. During this process, the morphology of the bacteria gradually changes and cannot grow and reproduce on the plate. Therefore, the bacteria in the VBNC state cannot be detected by the conventional plate detection method. Howe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06C12Q1/04C12Q1/14C12Q1/6851C12Q1/689C12R1/01
CPCC12Q1/04C12Q1/06C12Q1/14C12Q1/6851C12Q1/689C12Q2531/113C12Q2563/159
Inventor 赵力超吕新瑞王丽古晓奎张竟丰马宇昊
Owner SOUTH CHINA AGRI UNIV
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