CN116814734A - Method for high-flux screening of sensitive phage in culture solution - Google Patents
Method for high-flux screening of sensitive phage in culture solution Download PDFInfo
- Publication number
- CN116814734A CN116814734A CN202310739967.0A CN202310739967A CN116814734A CN 116814734 A CN116814734 A CN 116814734A CN 202310739967 A CN202310739967 A CN 202310739967A CN 116814734 A CN116814734 A CN 116814734A
- Authority
- CN
- China
- Prior art keywords
- phage
- bacterial
- bacteria
- liquid
- bacterial liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 74
- 238000012216 screening Methods 0.000 title abstract description 27
- 230000001580 bacterial effect Effects 0.000 claims abstract description 109
- 241000894006 Bacteria Species 0.000 claims abstract description 93
- 239000007788 liquid Substances 0.000 claims abstract description 60
- 238000012544 monitoring process Methods 0.000 claims abstract description 23
- 230000001954 sterilising effect Effects 0.000 claims abstract description 20
- 238000012258 culturing Methods 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- 238000013537 high throughput screening Methods 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 15
- 238000001514 detection method Methods 0.000 abstract description 10
- 230000004907 flux Effects 0.000 abstract description 10
- 230000008901 benefit Effects 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 8
- 230000008859 change Effects 0.000 abstract description 4
- 238000009630 liquid culture Methods 0.000 abstract description 2
- 241000588626 Acinetobacter baumannii Species 0.000 description 22
- 239000000243 solution Substances 0.000 description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 208000015181 infectious disease Diseases 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 239000007787 solid Substances 0.000 description 12
- 229960004821 amikacin Drugs 0.000 description 8
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical group O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 241000588747 Klebsiella pneumoniae Species 0.000 description 7
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 6
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 6
- 230000002101 lytic effect Effects 0.000 description 6
- 230000010355 oscillation Effects 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241001515965 unidentified phage Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000001066 phage therapy Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000003614 tolerogenic effect Effects 0.000 description 2
- 102100039377 28 kDa heat- and acid-stable phosphoprotein Human genes 0.000 description 1
- 101710176122 28 kDa heat- and acid-stable phosphoprotein Proteins 0.000 description 1
- 102000002281 Adenylate kinase Human genes 0.000 description 1
- 108020000543 Adenylate kinase Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001374 post-anti-biotic effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to a method for screening sensitive phage in culture solution at high flux, belonging to the technical field of biological detection. The method comprises the following steps: s1, preparing a phage library and culturing bacterial liquid; s2, mixing the phage library with bacterial liquid, co-incubating, and monitoring bacterial turbidity OD 600 The method comprises the steps of carrying out a first treatment on the surface of the At bacterial turbidity OD 600 The time point at which 0.5 was reached was the time of tolerance of the bacteria to phage. The method has the advantages that the method monitors the bacterial growth change in the liquid culture medium so as to judge the phage sterilization effectiveness and the time for generating phage resistance by the bacteria, and then the phage which has good sterilization effect and is not easy to generate tolerance or the combination thereof is selected from the phage library for intervention on target bacteria.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a method for screening sensitive phage in a culture solution at high flux.
Background
In the related art, no medicine is available due to the acquired medicine resistance of bacteria, the death rate of the infected person is improved, the course of disease and treatment time are prolonged, and the treatment cost is increased; the research and development speed of the new antibiotic medicine is far lower than that of bacterial medicine resistance, so that the new antibiotic therapy such as phage therapy is newly developed for solving the problems of bacterial medicine resistance and the like.
Phage is a virus which only infects bacteria, is rich in variety and wide in distribution, does not infect eukaryotic cells and is easy to genetically modify, and is an important force for resisting superbacteria in the 'post-antibiotic' era; tolerance by bacteria to phage infection is one of the major problems impeding successful phage treatment and is unavoidable during treatment. The production of phage-tolerant bacteria can be circumvented by different methods, most often by a combination of multiple phages. Selection of phages with different host ranges, commonly referred to as "phage cocktails", against different receptors of bacteria in a monotherapy formulation is effective in preventing the emergence of phage-resistant bacteria. Theoretically, the longer the phage inhibits bacterial growth, the lower the probability that phage tolerance will appear, the phage will clear the pathogen in a short period of time, and how to select the optimal combination among the many effective phages is the most important link before treatment. The traditional double-layer agar method for screening phage can sometimes take up to 48 hours to know the screening result due to different bacterial growth rates, and takes longer (more than 24 hours) to wait until phage resistant bacteria grow. The phage has the advantages of specific sterilization and no infection of human cells and normal flora, but also faces the bottleneck of narrow sterilization spectrum and easy tolerance of bacteria, and the prior phage matching technology cannot effectively solve the two bottlenecks at the same time.
Matching of appropriate phages is required prior to phage treatment. The commonly used method for matching phage is phage spotting, which involves spotting phage on a plate full of target bacteria by double-layer agar method, and selecting appropriate phage according to phage spotting. Phage with large plaques and clear are generally selected for treatment, if any, within the plaques, indicating that the phage is easily tolerated by bacteria, but the process is time consuming; plaque turbidity indicates that the target bacteria have some defense system. The method is suitable for matching a plurality of candidate phages and a plurality of target bacteria, and meanwhile, the titer of the phages can be easily judged through plaque forming units by continuously diluting and spotting phage liquid; in addition to plaque assays, phages can also be screened by liquid sterilization assays.
The most commonly used phage typing method at present is double-layer agar (DLA), namely, different phage spots are on a target bacterial plate, and the lytic power of phage at corresponding positions is judged by observing plaques. The method has the disadvantages of difficult high flux, difficult observation of bacterial tolerance, difficult observation of plaque and high cost on slow-growing bacteria, and the like. The real-time quantitative PCR technology is used for identifying infection by detecting the change of the copy number of phage nucleic acid, and has the defects of incapacity of judging tolerance, difficulty in high throughput, time consumption, high cost and the like because a primer probe is required to be designed and conditions are optimized for (almost) each phage. There are also methods for indirectly detecting the proliferation of phage, such as adenylate kinase and 5' Adenosine Triphosphate (ATP), by measuring the enzyme released in the supernatant of the culture after the bacteria are lysed by the phage, which have high sensitivity and are also easy to high throughput for liquid culture methods. However, the method has the defects that the operation is complex, the real-time monitoring is difficult, the detection time window is narrow, the difference exists in different bacteria-phage combinations and the like.
The detection board is used for screening phage of zoonotic pathogenic bacteria according to the cracking effect of the phage obtained by screening on the zoonotic pathogenic bacteria to form a phage library of the zoonotic pathogenic bacteria; the detection plate also judges the effectiveness of the phage by crisscrossing the phage with the bacteria and observing whether bacteria are lysed at the intersection of the bacteria and the phage; however, the detection plate has the advantages of insignificant bacterial lysis observation and poor flux.
The related art also discloses a method for rapidly screening virulent phage, which comprises the steps of sample acquisition and phage acquisition, test bacterial activation and single colony preparation, and then cross streak inoculation is carried out on single bacterial colonies and phage, so as to screen phage with a lytic test strain. The method provided by the application can conveniently, rapidly and accurately screen the specific phage of the test bacteria, avoids the influence of adverse factors such as bacterial mutation and the like on screening of virulent phage, and is favorable for fully researching and excavating phage resources in nature. The method is prone to slipping off during operation resulting in contamination or mispositioning. These updates have attempted to improve solid screening methods to achieve or partially achieve high throughput, but the effect of achieving is still not ideal and still cannot break through the bottleneck that solid screening methods are not easily observable for bacterial resistance and are greatly affected by bacterial growth rate.
The related technology also discloses a phage PAP1 with high specificity to pseudomonas aeruginosa, which is separated from hospital sewage, is functionalized by utilizing tosyl activated magnetic beads, and establishes phage affinity strategy for separating and detecting the living pseudomonas aeruginosa. Target bacteria are identified through the tail fibers and the substrate of phage, and pseudomonas aeruginosa is captured on the magnetic beads. After a replication cycle of about 100 minutes, the progeny phage lyse the target bacteria and release intracellular adenosine triphosphate. And then, quantitatively detecting the pseudomonas aeruginosa by adopting a firefly luciferase-adenosine triphosphate bioluminescence system. However, this method has the following disadvantages: (1) complex operation: since ATP released by phage-lysed bacterial cells is detected, interference from uncleaved bacteria needs to be eliminated, and therefore, each detection requires centrifugation of the culture broth, mixing with substrate in another dedicated well plate, and performing luciferase luminescence detection. Therefore, the operation is complex, a certain volume is consumed for each detection, and real-time monitoring is not easy. (2) The detection time window is narrow, tolerance is difficult to judge, and standardization is difficult to realize: theoretically, the more obvious the phage sterilization effect is, the higher the ATP release amount is, but when bacteria in the culture solution are greatly reduced due to phage sterilization, the ATP level is reduced due to the reduction of the number of newly infected bacteria. Thus, the method requires detection before this time, with a narrow time window. And missing this window of time does not determine whether the lysis is poor, too good, or the bacteria are tolerating. Furthermore, even for the same bacteria, the time window is not uniform due to the different patterns of a part of the growth curves of different phage-bacterial strain interactions, and thus the method is difficult to standardize.
The above mentioned prior art has certain drawbacks, especially in view of the large size of phage library and difficulty in fast, accurate and efficient screening of the most suitable phage in situations where bacteria are easily tolerant to phage.
Disclosure of Invention
The application aims at overcoming the defects in the prior art, and provides a method for screening sensitive phage in a culture solution with high flux, so as to solve the problems of slower phage screening, small flux, incapability of assessing phage tolerance and the like in the related art.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows: a method for high throughput screening of sensitive phage in culture broth comprising the steps of:
s1, preparing a phage library and culturing bacterial liquid;
s2, mixing the phage library with the bacterial liquid, incubating, and monitoring bacterial turbidity OD 600 ；
At bacterial turbidity OD 600 The time point at which 0.5 was reached was the time of tolerance of the bacteria to phage.
According to one of the technical schemes of the method, the method at least has the following beneficial effects:
the application monitors the bacterial turbidity OD in real time in the culture solution 600 By bacterial turbidity OD 600 Reflecting the bactericidal effect of the phage and the tolerance of the bacteria to the phage, thereby screening out the phage which has the most obvious inhibition on the bacterial growth and does not generate tolerance for a long time; the method is also suitable for a bacterial growth monitoring enzyme-labeled instrument commonly used in clinic, and the matched phage can simultaneously meet the two targets of strong cracking performance and difficult tolerance of target bacteria, and is closest to the effect of the phage on the bacteria in a real scene, thereby having wide application prospect.
According to some embodiments of the application, the OD of the bacterial fluid in step S1 600 0.04 to 0.06.
According to some embodiments of the application, the OD of the bacterial fluid in step S1 600 0.04 to 0.05.
According to some embodiments of the application, the volume ratio of the phage library to the bacterial liquid is 1:60-80.
According to some embodiments of the application, the monitored temperature in step S2 is between 35 ℃ and 40 ℃.
According to some embodiments of the application, the monitored temperature in step S2 is between 35 ℃ and 37 ℃.
According to some embodiments of the application, the period of monitoring in step S2 is 8min to 12min.
According to some embodiments of the application, the period of monitoring in step S2 is 8min to 10min.
According to some embodiments of the application, the monitoring in step S2 requires shaking and standing in a single cycle.
According to some embodiments of the application, the time of oscillation in the single cycle of monitoring in step S2 is 4min to 6min.
According to some embodiments of the application, the time of rest in the single cycle of monitoring in step S2 is 4min to 6min.
According to some embodiments of the application, the time of oscillation in the single cycle of monitoring in step S2 is 4min to 5min.
According to some embodiments of the application, the time of rest in the single cycle of monitoring in step S2 is 4min to 5min.
According to some embodiments of the application, the preparation of the phage library in step S1 comprises the steps of:
s01, culturing phage and a host bacterial liquid by using a double-layer agar plate method until a plurality of phage spots are formed;
s02, releasing phage in a single plaque, and purifying to obtain purified phage;
s03, mixing the purified phage obtained in the step S02 with a host bacterial liquid, amplifying, and sterilizing to obtain a high-titer phage mixed system;
s04, numbering the high-titer phage mixture system in the step S03, and adding the high-titer phage mixture system into a pore plate to obtain a phage library.
According to some embodiments of the application, the culturing in step S01 is performed using LB medium.
According to some embodiments of the application, the temperature of the culturing in step S01 is between 35 ℃ and 40 ℃.
According to some embodiments of the application, the temperature of the culturing in step S01 is between 35 ℃ and 37 ℃.
According to some embodiments of the application, the releasing in step S02 is: the plaques were mixed with normal saline and shaken.
According to some embodiments of the application, the rate of oscillation during the release is 200rpm to 300rpm.
According to some embodiments of the application, the time of oscillation during the release is 3-4 hours.
According to some embodiments of the application, the release process is post-shaking sterilization.
According to some embodiments of the application, the sterilization uses 4% to 5% chloroform.
According to some embodiments of the application, the temperature of the amplification is between 35 ℃ and 40 ℃.
According to some embodiments of the application, the rate of oscillation during the amplification is 200rpm to 300rpm.
According to some embodiments of the application, the time of oscillation during the amplification is 12-18 h.
According to some embodiments of the application, the sterilization in step S03 uses 4% to 5% chloroform.
According to some embodiments of the application, the method for culturing bacterial liquid in step S1 comprises the following steps:
s001, inoculating bacterial strains to a culture medium, and culturing until the bacterial strains reach a logarithmic phase to obtain a bacterial solution in the logarithmic phase;
s002, diluting the log phase bacterial liquid in the step S001.
According to some embodiments of the application, the OD of the log phase bacterial liquid 600 0.4 to 0.8.
According to some embodiments of the application, the bacterial fluid in step S1 comprises at least one of any fast-growing bacterial fluid comprising a corresponding phage library.
According to some embodiments of the application, the fast-growing bacteria are other bacteria than slow-growing bacteria.
According to some embodiments of the application, the slow-growing bacteria comprise mycobacteria.
According to some embodiments of the application, the bacterial fluid in step S1 includes at least one of klebsiella pneumoniae fluid, acinetobacter baumannii fluid, and escherichia coli fluid.
According to some embodiments of the application, the medium in step S001 is LB medium.
According to some embodiments of the application, the bacterial tolerance time to phage defines:
(1) Less than 6 hours is very tolerant (F, fast);
(2) Between 6h and 12h is a tolerogenic (M, medium);
(3) Between 12h and 18h is intolerable (S, slow);
(4) Greater than 18h is Extremely intolerant (ES).
Drawings
FIG. 1 is a flow chart of phage selection in an embodiment of the application.
FIG. 2 shows plaque results using a double-layer agar plate method in example 1 of the present application.
FIG. 3 is a graph showing the change in turbidity OD600 with time after infection of bacteria with Acinetobacter baumannii phage in example 1 of the present application.
FIG. 4 is a schematic diagram of a liquid-based high throughput screening of phage in example 1 of the present application.
FIG. 5 is a graph showing the time point determination of phage (Acinetobacter baumannii) screening by the liquid method in example 1 of the present application.
FIG. 6 is a graph showing the growth of bacteria after addition of different Acinetobacter baumannii phages in example 1 of the present application.
FIG. 7 is a graph showing the growth of bacteria after phage cocktail in example 2 of the present application.
FIG. 8 is a graph showing bacterial growth after phage interaction with antibiotics in example 3 of the present application.
Detailed Description
The present application will be described and illustrated with reference to the accompanying drawings and examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. All other embodiments, which can be made by a person of ordinary skill in the art based on the embodiments provided by the present application without making any inventive effort, are intended to fall within the scope of the present application.
It is apparent that the drawings in the following description are only some examples or embodiments of the present application, and it is possible for those of ordinary skill in the art to apply the present application to other similar situations according to these drawings without inventive effort. Moreover, it should be appreciated that while such a development effort might be complex and lengthy, it would nevertheless be a routine undertaking of design, fabrication, or manufacture for those of ordinary skill having the benefit of this disclosure, and thus should not be construed as having the benefit of this disclosure.
Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is to be expressly and implicitly understood by those of ordinary skill in the art that the described embodiments of the application can be combined with other embodiments without conflict.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs. The terms "a," "an," "the," and similar referents in the context of the application are not to be construed as limiting the quantity, but rather as singular or plural. The terms "comprising," "including," "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, or apparatus that comprises a series of steps or elements (elements) is not limited to the steps or elements listed and may include steps or elements not listed or may include additional steps or elements inherent to such process, method, or apparatus. The term "plurality"/"a plurality" as used herein means two or more. "and/or" describes an association relationship of an association object, meaning that there may be three relationships, e.g., "a and/or B" may mean: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship. The terms "first," "second," "third," and the like, as used herein, are merely distinguishing between similar objects and not representing a particular ordering of objects.
The screening principle of the sensitive phage in the embodiment of the application is as follows:
phages are a class of viruses that infect bacteria, and a typical infection process can be divided into three phases: infection stage, proliferation stage and maturation stage.
Infection stage: the first step in phage infection of a host bacterium is "adsorption", i.e., the tail of the phage adheres to the cell wall of the bacterium, and then the genetic material (nucleic acid) of the head is injected into the bacterium.
Proliferation stage: after the nucleic acid of the phage enters the bacterial cell, a series of changes are caused: phage gradually control the metabolism of bacterial cells. Phages inhibit on the one hand the synthesis of substances by the host bacteria themselves and on the other hand, by the enzymes and other substances of the host cells, replicate in large amounts the nucleic acids and proteins necessary for the composition of the phage and assemble into complete phage particles.
Maturation stage: after the progeny phage are assembled in a large quantity and mature, the lysozyme of the cell wall of the lytic bacteria encoded by the phage gradually increases to promote cell lysis, so that the progeny phage is released. After the progeny phage is released, neighboring bacterial cells are again infected, and next-generation phage is generated, and the process is repeated. Thus, by taking advantage of the properties of phage to infect, kill bacteria and replicate themselves, it can be applied to the treatment of bacterial infections.
In the embodiment of the application, throughPhage were incubated with bacteria and bacteria were detected at an optical density of 600nm (OD 600 ) Is shown in FIG. 1. In the embodiment of the application, the enzyme-labeled instrument is used for monitoring the phage-bacteria interaction in real time, so that various conditions, such as phage cocktail, phage-antibiotic combination and the like, can be comprehensively evaluated.
In addition, the condition that each phage is tolerant to bacteria can be distinguished in a short time (4-8 h) (compared with a double-layer agar method, more than 24h is generally required for generating tolerant bacteria in plaques on a flat plate), so that phages with long bacterial tolerance time can be rapidly selected for treatment, pathogenic bacteria can be killed in a short time, and phage tolerance bacteria are not generated.
In the embodiment of the application, the liquid method is used for screening effective phage, and for most of fast-growing bacteria (such as klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, staphylococcus aureus and the like), the sterilization capability of phage can be observed in a short time (2-8 h, and the time is shorter than that of a double-layer agar method), and whether a certain phage is tolerated by the bacteria or not can be judged in 18h. The above criteria facilitate selection of the optimal therapeutic phage combination.
Example 1
The embodiment is a method for screening sensitive phage in a culture solution with high flux, which comprises the following steps:
s1, preparing a phage library:
phage (Acinetobacter baumannii phage) and original host bacteria liquid (Acinetobacter baumannii) are mixed in an LB culture medium with the volume fraction of 0.75% of agar (semi-solid) by a double-layer agar plate method, spread on a solid LB plate, and the upper semi-solid LB culture medium is placed in a 37 ℃ incubator after solidification.
After appearance of plaques as shown in FIG. 2, individual plaques were picked up in physiological saline and placed on a thermostatic shaker at 37℃for 4h with shaking at 200rpm, releasing phages. After the shaking, 5% chloroform was added to the phage-containing physiological saline, and the mixture was left to stand for 10 minutes after the shaking was completed, followed by sterilization. Centrifuging 10000g for 5min at 4deg.C, and sucking supernatant to obtain purified phage.
The purified phage was mixed with the corresponding host bacterial liquid in LB medium at a multiplicity of infection of 0.1 (multiplicity of infection, MOI), and cultured overnight (12 h) at a constant temperature of 37℃in a shaker at 200rpm, whereby the bacterial liquid was observed to be cloudy to clear and then cloudy (FIG. 3). Adding 5% chloroform into the amplified phage mixture, and sterilizing in the same manner to obtain phage solution with high titer>10 9 PFU/mL)。
And sequentially numbering the amplified phage liquid, and adding the phage liquid into a 96-well plate to obtain the prepared phage library.
S2, resuscitating bacteria and culturing bacterial liquid:
the stored strain to be detected (Acinetobacter baumannii) was inoculated on a plate by a three-zone streak method, and placed in an incubator at 37℃for resuscitative culture (shown in FIG. 4). After bacterial colonies grow up, the monoclonal colonies are picked up and inoculated into 5mL of LB medium, and cultured overnight at a constant temperature of 37 ℃ by shaking table at 200 rpm/min. The overnight bacterial liquid was transferred to 5mL of LB medium at a volume ratio of 1:100, and cultured until the logarithmic phase (OD) 600 0.4 to 0.8). Diluting the log phase bacterial liquid to OD by using fresh LB culture medium 600 The diluted bacterial solution was added to a 96-well plate at 200. Mu.L per well at 0.05.
S3, high-flux phage screening by a liquid method:
and (2) transferring 2.5 mu L/hole of the prepared phage library into the 96-well plate containing the bacterial liquid prepared in the step (S1) by a gun, additionally arranging a control group without adding phage, and placing the 96-well plate into an enzyme-labeled instrument after the operation is finished.
The conditions for the microplate reader were as follows: shaking for 5min at 37deg.C, standing for 5min as a period, and reading bacterial turbidity OD at the end time point every period 600 And (5) continuously monitoring.
By bacterial OD 600 To 0.5 is a criterion for determining the tolerance of the bacterium to the phage, and the time point is the tolerance time of the bacterium to the phage.
According to the data (only a part of representative data is shown in FIG. 5, A in FIG. 5 is a bacterial growth curve after phage addition of different Acinetobacter baumannii, B in FIG. 5 is a time ratio of tolerance of different Acinetobacter baumannii to different phages), the tolerance time of the same bacterium to different phages is different, and the tolerance time is mainly concentrated in 6h, 12h and 18h, and a very small part of the bacteria is after 24h, so that the monitoring end point of phage screening by a liquid method is 18h.
The time-to-tolerance ratios of the different Acinetobacter baumannii to their different phages are shown in Table 1. The bacteria used are from clinical samples of the patient.
TABLE 1 time to tolerance ratio of different Acinetobacter baumannii to their different phages
Definition of phage tolerance time for the above bacteria:
(1) Less than 6 hours is very tolerant (F, fast);
(2) Between 6h and 12h is a tolerogenic (M, medium);
(3) Between 12h and 18h is intolerable (S, slow);
(4) Greater than 18h is Extremely intolerant (ES).
S4, data analysis:
according to the absorbance continuously monitored by the enzyme labeling instrument, a growth curve of each hole bacterial liquid is drawn, the growth curve of an experimental group added with phage is superimposed and compared with that of a control group without phage, whether the phage added in each hole is effective or not and the time of the phage being tolerated by bacteria are analyzed (for example, figure 6), and the above information is counted and recorded.
In this example, the growth curves of bacteria after adding different Acinetobacter baumannii phages are shown in fig. 6, and the growth curves of bacteria can be inhibited within about 8 hours after adding Acinetobacter baumannii phage #1 (phase 1, green in color mode) and Acinetobacter baumannii phage #2 (phase 2, blue in color mode) to a control group (no phase, red in color mode) without adding phages, but after 8 hours, the bacteria of an experimental group added with phage #1 start to grow, which indicates that phage tolerance bacteria appear; while the experimental group added with phage #2 had no apparent bacterial growth at 8-48 hours, indicating that the phage was not easily tolerated by bacteria.
Example 2
The embodiment is a method for screening sensitive phage in a culture solution with high flux, which comprises the following steps:
s1, preparing a phage library:
mixing each bacteriophage (Acinetobacter baumannii bacteriophage) with original host bacteria liquid (Acinetobacter baumannii) by using a double-layer agar plate method, putting the mixed bacteriophage and the original host bacteria liquid (Acinetobacter baumannii) in an agar (semi-solid) LB culture medium with the volume fraction of 0.75%, spreading the mixed bacteriophage on a solid LB plate, solidifying an upper semi-solid LB culture medium, and placing the solidified upper semi-solid LB culture medium in a 37 ℃ incubator.
After appearance of plaques as shown in FIG. 2, individual plaques were picked up in physiological saline and placed on a thermostatic shaker at 37℃for 4h with shaking at 200rpm, releasing phages. After the shaking, 5% chloroform was added to the phage-containing physiological saline, and the mixture was left to stand for 10 minutes after the shaking was completed, followed by sterilization. Centrifuging 10000g for 5min at 4deg.C, and sucking supernatant to obtain purified phage.
The purified phage was mixed with the corresponding host bacterial liquid in LB medium at a multiplicity of infection of 0.1 (multiplicity of infection, MOI), and cultured overnight (12 h) at a constant temperature of 37℃in a shaker at 200rpm, so that the bacterial liquid was observed to be clear from turbidity to turbidity. Adding 5% chloroform into the amplified phage mixture, and sterilizing in the same manner to obtain phage solution with high titer>10 9 PFU/mL)。
And sequentially numbering the amplified phage liquid, and adding the phage liquid into a 96-well plate to obtain the prepared phage library.
S2, resuscitating bacteria and culturing bacterial liquid:
inoculating the strain to be detected (Acinetobacter baumannii) on a plate by a three-area streak method, and placing the plate in a 37 ℃ incubator for resuscitating culture. After bacterial colonies grow up, the monoclonal colonies are picked up and inoculated into 5mL of LB medium, and cultured overnight at a constant temperature of 37 ℃ by shaking table at 200 rpm/min. The overnight bacterial liquid was transferred to 5mL of LB medium at a volume ratio of 1:100, and cultured until the logarithmic phase (OD) 600 0.4 to 0.8). Fresh LB culture for log phase bacterial liquidDilution of the base to OD 600 The diluted bacterial solution was added to a 96-well plate at 200. Mu.L per well at 0.05.
S3, high-throughput screening phage combination by liquid method:
and (3) transferring 2.5 mu L/hole of the prepared phage library to a 96-well plate containing the bacterial liquid prepared in the step (S1) by a gun, wherein the ratio of different phages in the phage combination is 1:1, setting single phages as a control group (the example is shown in FIG. 7, the acinetobacter baumannii phages #1 and #2 are phage combinations), and placing the 96-well plate into an enzyme-labeled instrument after the operation is finished.
The conditions for the microplate reader were as follows: shaking for 5min at 37deg.C, standing for 5min as a period, and reading bacterial turbidity OD at the end time point every period 600 And (5) continuously monitoring.
By bacterial OD 600 To 0.5 is a criterion for determining the tolerance of the bacterium to the phage, and the time point is the tolerance time of the bacterium to the phage.
S4, data analysis:
according to the absorbance continuously monitored by the enzyme labeling instrument, the growth curve of each hole bacterial liquid is drawn, the growth curve of the experimental group added with phage combination is superimposed and compared with the growth curve of the control group added with single phage, whether each hole added phage combination is more effective than single phage or not is analyzed, the time of the phage combination being tolerated by bacteria is counted and recorded (see figure 7).
In the embodiment, the growth curve of the bacteria after the combination of the Acinetobacter baumannii phages is shown in fig. 7, and after about 10 hours, the bacteria start to grow in a single phage control group phage#2, which indicates that phage tolerance bacteria appear; control phage #1 tolerated more rapidly and bacteria began to grow after about 2 hours. The addition of the acinetobacter baumannii phage combination (phage #1+ # 2) was effective in inhibiting bacterial growth within 24 hours, indicating that the phage combination was less tolerant to bacteria.
Example 3
The embodiment is a method for screening sensitive phage in a culture solution with high flux, which comprises the following steps:
s1, preparing a phage library:
phage (Klebsiella pneumoniae phage) and original host bacterial liquid (Klebsiella pneumoniae) are mixed in an LB culture medium with the volume fraction of 0.75% of agar (semi-solid) by a double-layer agar plate method, spread on a solid LB plate, and the upper semi-solid LB culture medium is solidified and then placed in a 37 ℃ incubator.
After appearance of plaques, individual plaques were picked up in physiological saline and placed on a shaking table at a constant temperature of 37℃for 4h with 200rpm shaking, releasing phages. After the shaking, 5% chloroform was added to the phage-containing physiological saline, and the mixture was left to stand for 10 minutes after the shaking was completed, followed by sterilization. Centrifuging 10000g for 5min at 4deg.C, and sucking supernatant to obtain purified phage.
The purified phage was mixed with the corresponding host bacterial liquid in LB medium at a multiplicity of infection of 0.1 (multiplicity of infection, MOI), and cultured overnight (12 h) at a constant temperature of 37℃in a shaker at 200rpm, so that the bacterial liquid was observed to be clear from turbidity to turbidity. Adding 5% chloroform into the amplified phage mixture, and sterilizing in the same manner to obtain phage solution with high titer>10 9 PFU/mL)。
And sequentially numbering the amplified phage liquid, and adding the phage liquid into a 96-well plate to obtain the prepared phage library.
S2, resuscitating bacteria and culturing bacterial liquid:
inoculating the strain to be detected (Klebsiella pneumoniae) on a plate by a three-area streak method, and placing the plate in a 37 ℃ incubator for resuscitating culture. After bacterial colonies grow up, the monoclonal colonies are picked up and inoculated into 5mL of LB medium, and cultured overnight at a constant temperature of 37 ℃ by shaking table at 200 rpm/min. The overnight bacterial liquid was transferred to 5mL of LB medium at a volume ratio of 1:100, and cultured until the logarithmic phase (OD) 600 0.4 to 0.8). Diluting the log phase bacterial liquid to OD by using fresh LB culture medium 600 The diluted bacterial solution was added to a 96-well plate at 200. Mu.L per well at 0.05.
S3, high-flux phage screening by a liquid method:
and (2) transferring 2.5 mu L/hole of the prepared phage library into the 96-well plate containing the bacterial liquid prepared in the step (S1) by a gun, additionally arranging a control group without adding phage and antibiotics, and placing the 96-well plate into an enzyme-labeled instrument after the operation is finished.
The conditions for the microplate reader were as follows: shaking for 5min at 37deg.C, standing for 5min as a period, and reading bacterial turbidity OD at the end time point every period 600 And (5) continuously monitoring.
By bacterial OD 600 To 0.5 is a criterion for determining the tolerance of the bacterium to the phage, and the time point is the tolerance time of the bacterium to the phage.
S4, data analysis:
according to the absorbance continuously monitored by an enzyme-labeled instrument, a growth curve of each pore fungus liquid is drawn, an experimental group growth curve added with phage and antibiotics simultaneously is superimposed and compared with a control group growth curve added with phage or antibiotics only, whether the phage added in each pore is effective or not and the time of the phage being tolerated by bacteria are analyzed (see fig. 8, control group in fig. 8 is no phage and amikacin, phase group is phage added group, amikacin is amikacin added (concentration is 20 mug/mL), phase+amikacin group is phage added and amikacin added (concentration is 20 mug/mL)), and the above information is counted and recorded. In the embodiment, the growth curve of the bacteria after the Klebsiella pneumoniae phage and amikacin are added for combined use is shown in figure 8, and phage tolerance bacteria and bacteria start to grow after about 5 hours of single Klebsiella pneumoniae phage; amikacin alone also began to appear as resistant bacteria after about 9 hours. However, the addition of phage and amikacin can effectively inhibit the growth of bacteria within 24 hours, which proves that the combination of phage and antibiotics can well solve the problem of phage tolerant bacteria.
In the embodiment of the application, the aim of phage high-flux screening is realized by utilizing a plurality of enzyme-labeled instruments, the liquid method is easy to realize high flux in the embodiment of the application, the liquid method is easy to integrate into an automatic system such as a pipetting workstation and the like, and the bacterial quantity change sensitivity is far higher than that of the plaque observed by naked eyes through the OD value monitoring, so that the phage sterilization effect can be judged in the shortest 2 hours.
In the embodiment of the application, the bacteriostasis effect of the phage is easily judged by monitoring the bacterial growth curve in real time through the descending of the curve, and the time for generating tolerance to the phage and timing the generation of the tolerance is judged when the bacterial growth curve is on the way again, in theory, the later the time, the less easy the phage to induce the target bacterial tolerance can be reflected. For some lytic phages that are susceptible to bacterial tolerance (in particular to tolerance within 8 hours), use is not recommended, or when there is no other option, the phages need to be used in combination with a "two-wire" lytic phage against which bacterial tolerance is to occur, thereby excluding lytic phages that are susceptible to induction of bacterial tolerance.
The most commonly used instruments and consumables of the microbiology laboratory, clinical laboratory, etc. associated with phage therapy are used in embodiments of the present application. For example, the bacterial growth monitoring enzyme-labeled instrument can be applied to the equipment such as BioTek 800Tsi, ELx808 and the like, and has the advantages of high laboratory configuration rate, simple operation and convenient use, and is matched with an automatic growth curve analysis system. In the embodiment of the application, the conventional 96-well cell culture plate is used, the price is economical, customization is not needed, and a pipetting workstation or a manual gun can be used for filling liquid. The use and clinical application of different automation degrees are convenient.
The application has the advantage that the application monitors the bacterial turbidity OD in real time in the culture solution 600 By bacterial turbidity OD 600 Reflecting the bactericidal effect of the phage and the tolerance of the bacteria to the phage, thereby screening out the phage which has the most obvious inhibition on the bacterial growth and does not generate tolerance for a long time; the method is also suitable for a bacterial growth monitoring enzyme-labeled instrument commonly used in clinic (such as 800Tsi and ELx808 of BioTek), and the matched phage can simultaneously meet the two targets of strong cracking property and difficult tolerance of target bacteria, and is closest to the effect of the phage on bacteria in a real scene, thereby having wide application prospect.
The foregoing description is only illustrative of the preferred embodiments of the present application and is not to be construed as limiting the scope of the application, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present application, and are intended to be included within the scope of the present application.
Claims (10)
1. A method for high throughput screening of sensitive phage in culture broth comprising the steps of:
s1, preparing a phage library and culturing bacterial liquid;
s2, mixing the phage library with the bacterial liquid, incubating, and monitoring bacterial turbidity OD 600 ；
At bacterial turbidity OD 600 The time point at which 0.5 was reached was the time of tolerance of the bacteria to phage.
2. The method for high-throughput screening of sensitive phage in culture broth according to claim 1, wherein the OD of the bacterial broth in step S1 600 0.04 to 0.06.
3. The method for high-throughput screening of sensitive phage in culture broth according to claim 1, wherein the volume ratio of phage library to bacterial broth is 1:60-80.
4. The method according to claim 1, wherein the temperature monitored in step S2 is between 35℃and 40 ℃.
5. The method according to claim 1, wherein the period of monitoring in step S2 is 8-12 min.
6. The method for high throughput screening of sensitive phage in culture broth according to claim 1, wherein the preparation of phage library in step S1 comprises the steps of:
s01, culturing phage and a host bacterial liquid by using a double-layer agar plate method until a plurality of phage spots are formed;
s02, releasing phage in a single plaque, and purifying to obtain purified phage;
s03, mixing the purified phage obtained in the step S02 with a host bacterial liquid, amplifying, and sterilizing to obtain a high-titer phage mixed system;
s04, numbering the high-titer phage mixture system in the step S03, and adding the high-titer phage mixture system into a pore plate to obtain a phage library.
7. The method of claim 6, wherein the amplification temperature is 35℃to 40 ℃.
8. The method for high-throughput screening of sensitive phage in culture according to claim 1, wherein the method for culturing bacterial liquid in step S1 comprises the steps of:
s001, inoculating bacterial strains to a culture medium, and culturing until the bacterial strains reach a logarithmic phase to obtain a bacterial solution in the logarithmic phase;
s002, diluting the log phase bacterial liquid in the step S001.
9. The method for high-throughput screening of sensitive phage in culture broth according to claim 8, wherein said log phase bacterial broth has an OD 600 0.4 to 0.8.
10. The method according to claim 1, wherein the bacterial liquid in step S1 comprises at least one of any fast-growing bacterial liquid in a corresponding phage library.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310739967.0A CN116814734A (en) | 2023-06-21 | 2023-06-21 | Method for high-flux screening of sensitive phage in culture solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310739967.0A CN116814734A (en) | 2023-06-21 | 2023-06-21 | Method for high-flux screening of sensitive phage in culture solution |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116814734A true CN116814734A (en) | 2023-09-29 |
Family
ID=88142301
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310739967.0A Pending CN116814734A (en) | 2023-06-21 | 2023-06-21 | Method for high-flux screening of sensitive phage in culture solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116814734A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170368116A1 (en) * | 2016-06-22 | 2017-12-28 | United States Of America As Represented By The Secretary Of The Navy | Bacteriophage Compositions and Methods of Selection of Components Against Specific Bacteria |
| CN112725289A (en) * | 2021-01-25 | 2021-04-30 | 南京悦联生物科技有限公司 | Method for rapidly screening bacteriophage for treatment |
| CN115197919A (en) * | 2022-07-26 | 2022-10-18 | 中国科学院深圳先进技术研究院 | Vibrio phage composition and preparation method and application thereof |
-
2023
- 2023-06-21 CN CN202310739967.0A patent/CN116814734A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170368116A1 (en) * | 2016-06-22 | 2017-12-28 | United States Of America As Represented By The Secretary Of The Navy | Bacteriophage Compositions and Methods of Selection of Components Against Specific Bacteria |
| CN112725289A (en) * | 2021-01-25 | 2021-04-30 | 南京悦联生物科技有限公司 | Method for rapidly screening bacteriophage for treatment |
| CN115197919A (en) * | 2022-07-26 | 2022-10-18 | 中国科学院深圳先进技术研究院 | Vibrio phage composition and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| DENIS RAJNOVIC ET AL.: "Fast phage detection and quantification: An optical density-based approach", 《PLOS ONE》, vol. 14, no. 5, 9 May 2019 (2019-05-09), pages 1 - 14 * |
| PATRICIA E SØRENSEN ET AL.: "Classification of In Vitro Phage–Host Population Growth Dynamics", 《MICROORGANISMS》, vol. 9, no. 12, 30 November 2021 (2021-11-30), pages 2 - 3 * |
| 陈义宝 等: "沙门菌烈性噬菌体的分离鉴定及其生物学特性的研究", 《中国兽医科学》, vol. 46, no. 12, 20 December 2016 (2016-12-20), pages 1476 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wendel | Assessing viability and stress tolerance of probiotics—a review | |
| US8568970B2 (en) | Bacteriophages as selective agents for enriching target bacteria | |
| Sabour et al. | Bacteriophages in the control of food-and waterborne pathogens | |
| Tanji et al. | Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage | |
| AU680279B2 (en) | Method and test kits for detection of bacteriophage | |
| Odumeru et al. | Salmonella detection methods for food and food ingredients | |
| Hansen et al. | Absolute enumeration of probiotic strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04® via chip-based digital PCR | |
| Graziani et al. | Virulotyping of Salmonella enterica serovar Napoli strains isolated in Italy from human and nonhuman sources | |
| AU757653B2 (en) | Bacteriophage assay | |
| Van Lint et al. | Evaluation of a real-time multiplex PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica in fecal samples | |
| RU2573934C2 (en) | Method for obtaining bacteriophage strain, specific strains of bacteriophages and their application | |
| Peng et al. | Application of the VPp1 bacteriophage combined with a coupled enzyme system in the rapid detection of Vibrio parahaemolyticus | |
| Schellenberg et al. | A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture | |
| Yu et al. | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli | |
| Yang et al. | Modified PMA-qPCR method for rapid quantification of viable Lactobacillus spp. in fermented dairy products | |
| Dane et al. | Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects | |
| Huang et al. | Dual phage amplification-mediated multiplex detection strategies for the simultaneous detection of Salmonella enterica and Staphylococcus aureus | |
| CN112961805B (en) | Salmonella typhimurium with quinolone drug resistance genes gyrA and parE mutated simultaneously and application thereof | |
| Arsenieff et al. | Diatom viruses | |
| CN116814734A (en) | Method for high-flux screening of sensitive phage in culture solution | |
| Gharst et al. | Evaluation and single-laboratory verification of a proposed modification to the US Food and Drug Administration method for detection and identification of Campylobacter jejuni or Campylobacter coli from raw silo milk | |
| Zourob et al. | Bacteriophage-based biosensors | |
| Wu et al. | Study on rapid quantitative detection of total bacterial counts by the ATP‐bioluminescence and application in probiotic products | |
| Fung | Overview of rapid methods of microbiological analysis | |
| Glonti et al. | In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success. Viruses 2022, 14, 1490 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |