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Application and method of pslg protein or its coding sequence in detecting the number of microorganisms

A microbial and protein technology, applied in the field of extracellular polymer degrading enzymes, can solve the problem of not being able to accurately reflect the real bacterial amount of the sample, and achieve the effect of accurate measurement

Active Publication Date: 2020-10-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the package of EPS, the bacterial cells in the bacterial mass or biofilm population are difficult to be broken up into free single-cell bacterial suspensions. Therefore, direct measurement using conventional cell counting methods cannot accurately reflect the true bacteria of the sample. Quantitative

Method used

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  • Application and method of pslg protein or its coding sequence in detecting the number of microorganisms
  • Application and method of pslg protein or its coding sequence in detecting the number of microorganisms
  • Application and method of pslg protein or its coding sequence in detecting the number of microorganisms

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Experimental program
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Effect test

preparation example Construction

[0046] The preparation of LBNS solid medium used 5g yeast extract, 10g tryptone, 15g agar powder and 1000ml distilled water.

[0047] LBNS liquid medium was prepared using 5 g of yeast extract, 10 g of tryptone and 1000 ml of distilled water.

[0048] The preparation of LD solid medium used 5g yeast extract, 10g tryptone, 2.5g NaCl, 15g agar powder and 1000ml distilled water.

[0049] 5 g of yeast extract, 10 g of tryptone, 2.5 g of NaCl and 1000 ml of distilled water were used for the preparation of LD liquid medium.

[0050] Jensen broth (pH 7.3) was prepared using 5 g NaCl, 2.51 g K 2 HPO 4 , 15.56g L-glutamic acid monosodium salt, 2.81g valine, 1.32g phenylalanine, 13.87g glucose, 0.165g MgSO 4 ·7H 2 O, 0.105 mg CaCl 2 2H 2 O, 5.5 μg FeSO 4 ·7H 2 O, 12 μg ZnSO 4 ·7H 2 O and 1000ml distilled water.

[0051] KLG liquid medium (pH6.8) contains 0.87g / L KH 2 PO 4 , 1.67g / L K 2 HPO 4 , 0.48g / L NaCl, 0.29g / L MgSO 4 ·7H 2 O, 0.005g / L Na 2 MoO 4 ·H 2 O, 0.072g / L...

Embodiment 1

[0052] Embodiment 1, polysaccharide hydrolase PslG can be used for the quantitative calculation of living bacteria in biofilm

[0053]Pseudomonas aeruginosa PAO1 and PaAP deletion mutant strain ΔpaaP were inoculated on LB solid medium plate, and cultured overnight at 37°C; single clones of PAO1 and ΔpaaP were picked from the plate that completed the above steps and inoculated to LB liquid culture medium, shaking culture at 200rpm at 37°C overnight; take 10μl of PAO1 and ΔpaaP overnight bacterial solution to inoculate 1ml into Jensen medium, add to 1×1×4cm four-hole glass chamber (Chambered#1.5 German CoverglassSystem , Nunc Inc.) at 30°C for 24h, 36h and 48h.

[0054] The steps for staining PAO1 and ΔpaaP biofilm dead bacteria are as follows: Before staining, gently suck out the culture solution in the chamber to make the biofilm slowly fall on the cover glass at the bottom of the chamber, and wash the biofilm once with phosphate buffered saline (PBS); LIVE / DEAD TM BacLight...

Embodiment 2

[0059] Embodiment 2, PslG processing and the comparison of counting biofilm live bacteria number after tissue homogenate processing

[0060] Inoculate Pseudomonas aeruginosa PAO1 on the LBNS solid medium plate and culture it overnight at 37°C; pick a single clone of PAO1 from the plate that has completed the above steps, inoculate it into the LBNS liquid medium, and culture it overnight at 37°C Shake culture at 200rpm overnight; wash the overnight culture medium once with PBS, then inoculate it in Jensen liquid medium at an inoculum size of 1:100 (V:V), and culture it statically at 30°C for 24 hours in a 24-well culture plate To form a biofilm at the gas-liquid interface; use a pipette to suck up the bacterial solution under the biofilm, wash the biofilm twice with PBS, remove the bacteria in the floating state, and obtain a sample of biofilm bacteria; resuspend the biofilm in In PBS buffer, they were divided into control group, homogenate group and PslG treatment group. The ...

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Abstract

The invention discloses an application and method of PslG protein or coding sequence of PslG protein in detecting microbial quantity, specifically, the invention provides extracellular polymeric substances degrading enzyme, particularly the application and method of polysaccharide hydrolytic enzyme Ps1G in detecting the microbial quantity of biofilms or bacteria solution containing cenobium. The method includes the steps that a composition is used for inhibiting, dispersing, or disintegrating the biofilms or the cenobium formed by microorganisms, thereby detecting the number of the microorganisms. The composition comprises the PslG protein. The invention also provides amino acid sequence and the coding gene sequence of the PslG protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application and method of an extracellular polymer degrading enzyme, in particular polysaccharide hydrolase PslG, in detecting the number of microorganisms in a biofilm or a bacterial liquid containing a bacterial mass. Background technique [0002] Biofilm (biofilm) refers to the highly organized and systematic multicellular population structure formed by microorganisms growing by adhesion on the surface of objects and wrapping them with extracellular polymeric substances (EPS) secreted by themselves. , life is closely related. Bacterial aggregate refers to the microbial aggregate formed when the microorganisms are shaken and cultured in the liquid because the cells of the bacteria are wrapped by EPS, which usually manifests as agglomeration or flocculation of the bacterial liquid. Some clinical strains, such as the rugose small colony variants (RSCV) of Pseudomonas aeruginosa,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12Q1/06C12R1/385C12R1/38
CPCC12N9/24C12Q1/06G01N2333/21
Inventor 马旅雁王迪张妙坤赵天湖王世伟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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