Application of recombinant luminescent pseudomonas aeruginosa PAO1-lux in detection of bacterial adhesion of medical material
A technology of Pseudomonas aeruginosa and pao1-lux, which is applied in the field of biomedicine, can solve problems such as methodological research, and achieve the effects of accurate detection, convenient and fast detection, and strong repeatability of results
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Embodiment 1
[0042] Example 1: Application of recombinant luminescent Pseudomonas aeruginosa PAO1-lux in the detection of bacterial adhesion of medical materials
[0043] The invention provides an application of recombinant luminescent Pseudomonas aeruginosa PAO1-lux in the detection of bacterial adhesion of medical materials, specifically, the recombinant luminescent Pseudomonas aeruginosa is the bacterial chromosome that integrates the luxCD ABE gene into the standard strain PAO1 In , the recombinant luminescent strain PAO1-lux was screened by PIA selection medium.
[0044] The inoculation pH of the medium of the recombinant luminescent bacterial strain PAO1-lux is 6.2 or 7.2 or 8.2.
[0045] The inoculation medium of the recombinant luminescent strain PAO1-lux can be selected from conventional LB medium or M9 medium or PBS buffer.
[0046] It is more appropriate to set the detection window period of the luminescence value of the recombinant luminescent strain PAO1-lux to 0h-24h.
[00...
Embodiment 2
[0049] Example 2: Construction of recombinant luminescent strain PAO1-lux
[0050] The PalgU-lux promoter sequence containing restriction site was obtained by PCR, digested and connected to pMS402 plasmid, transformed into Escherichia coli, and obtained the reporter gene vector containing PalgU-lux promoter; secondly, the CTX6.1 plasmid was combined with fluorescein reporter The vector was digested, ligated, and transformed to obtain the CTX-lux strain; finally, the wild-type PAO1, CTX-lux, and PRK2013 triparental strains were hybridized to integrate the luciferase gene into the PAO1 chromosome, and the luminescent single clone was screened to obtain recombinant PAO1 -lux strains. A schematic diagram of the construction process is attached figure 1 shown.
[0051] The colony morphology and luminescent properties of PAO1-lux were detected by the Bio-Rad imaging system. The wild-type strain PAO1 and the recombinant luminescent strain PAO1-lux were both visible in the white lig...
Embodiment 3
[0053] Example 3: Study on Luminescent Stability of PAO1-lux Strain
[0054] The constructed recombinant luminescent strain PAO1-lux and the wild-type strain PAO1 were inoculated on LB and PIA plates respectively, and the inoculated plates were photographed in White light and Luminescence modes with the Bio-Rad imaging system, at intervals Continuous observation was carried out on 1d, 3d, and 7d to monitor the luminescent stability of PAO1-lux over time. Routine culture of PAO1-lux was carried out for 10 consecutive passages, and the strains of each generation were preserved; at the same time, the strains of different generations were activated, and the overnight culture solution was diluted in multiples and then added to a 96-well plate to determine the luminous properties and luminous intensity. and quantitative testing.
[0055] The PAO1-lux strain was routinely cultured to the logarithmic phase (OD=0.5-0.6), the bacterial solution was diluted 10 times, and the bacterial s...
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