CN112725289A - Method for rapidly screening bacteriophage for treatment - Google Patents
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- CN112725289A CN112725289A CN202110099068.XA CN202110099068A CN112725289A CN 112725289 A CN112725289 A CN 112725289A CN 202110099068 A CN202110099068 A CN 202110099068A CN 112725289 A CN112725289 A CN 112725289A
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000012216 screening Methods 0.000 title claims abstract description 16
- 241001515965 unidentified phage Species 0.000 title claims description 25
- 238000012360 testing method Methods 0.000 claims abstract description 27
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 241001465754 Metazoa Species 0.000 claims abstract description 19
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 13
- 230000005764 inhibitory process Effects 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 210000001519 tissue Anatomy 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000010255 intramuscular injection Methods 0.000 claims description 3
- 239000007927 intramuscular injection Substances 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 210000004556 brain Anatomy 0.000 claims description 2
- 210000003205 muscle Anatomy 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 210000000056 organ Anatomy 0.000 claims description 2
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- 238000011534 incubation Methods 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 7
- 241000282414 Homo sapiens Species 0.000 abstract description 6
- 208000035143 Bacterial infection Diseases 0.000 abstract description 4
- 208000022362 bacterial infectious disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000003242 anti bacterial agent Substances 0.000 description 7
- 229940088710 antibiotic agent Drugs 0.000 description 7
- 241000272517 Anseriformes Species 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 4
- 238000001066 phage therapy Methods 0.000 description 4
- 241000272525 Anas platyrhynchos Species 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- 210000003128 head Anatomy 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 241000282412 Homo Species 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000424 optical density measurement Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12N2795/00—Bacteriophages
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- C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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- C12N2795/00011—Details
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00071—Demonstrated in vivo effect
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Abstract
The invention provides a method for rapidly screening therapeutic phage, aiming at the problem of screening therapeutic phage in vitro, the invention uses in vitro bacteria inhibition test, and can rapidly screen phage which can be used for treating bacterial infection diseases of human and animals by naked eye observation or spectrophotometer detection after culture, thereby saving cost.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for rapidly screening therapeutic phage.
Background
Since the discovery of antibiotics by human beings, the wide application of the antibiotics is accompanied with the history of drug resistance of bacteria, the problem of drug resistance of bacteria is increasingly serious nowadays, and various superbacteria resistant to corresponding antibiotics continuously appear and gradually become a great hidden danger of healthy life of human beings. China is a big country for producing and using antibacterial drugs. In the animal husbandry, the use of antibiotics solves the harm of bacterial infectious diseases of livestock and poultry to a certain extent and improves the productivity, but the residue of the antibiotics is transmitted to human beings through a food chain after long-term and large-scale use, thereby indirectly influencing the health of the human beings. Related ministerial committees in China set' action plan for inhibiting bacterial drug resistance in countries (2016-.
Under the background, governments and researchers around the world pay attention to the treatment effect of the Phage again, the Phage therapy becomes a potential alternative by virtue of various advantages such as no residue, low cost and the like, and under the condition that the problem of drug-resistant bacteria is increasingly severe, the Phage therapy (Phage therapy) has wider application space.
Bacteriophages (bacteriophages/phage) were discovered in 1915 by British bacteriologists Tport (1877-1950) and Canadian biologists F lilix D' H relle, and are a generic term for viruses that infect microorganisms such as bacteria, fungi, actinomycetes, and spirochetes. The phage can specifically kill pathogenic bacteria, has extremely high accuracy and very obvious treatment effect. In general, pathogenic bacteria have a very low rate of resistant mutations to bacteriophages, with a frequency of about 10-7And the frequency of resistance mutation to antibiotics is 10-6The phage may effectively kill pathogenic bacteria before they develop resistance. In addition, the bacteriophage therapy has no drug residue problem and does not pollute livestock and poultry products. Bacteriophages are also a ubiquitous organism and are often associated with bacteria. Usually in some locations full of bacterial flora, such as: the trace of the phage can be found in the soil and the internal organs of animals. By virtue of its many advantages, bacteriophage becomes an ideal substitute for antibiotics. Phage therapy has begun to find application in animal husbandry, aquaculture, and in the treatment of bacterial infectious diseases in humans.
At present, there are some problems to be solved by bacteriophage in the treatment of bacterial diseases, the most important of which are: although phages specifically kill pathogenic bacteria, it is not easy to screen in vitro for specific phages. The method generally adopted comprises the following steps: a "cocktail" method, i.e., a mixture of multiple bacteriophages is used for treatment; and secondly, directly carrying out animal experiments by using the separated phage. The method has high cost, long period, blindness and low success rate.
Disclosure of Invention
The invention aims to provide a method for rapidly screening therapeutic phages, aiming at the problem of screening therapeutic phages in vitro, the method rapidly screens the phages with targeted antibacterial effect by using an in vitro bacterial inhibition test and observing by naked eyes or detecting by a spectrophotometer after culture, thereby being applied to the treatment of bacterial diseases.
The invention is realized by adopting the following technical scheme:
a method for rapid screening of therapeutic phages comprising the steps of:
(1) aseptically shearing 0.1-0.5 g to 1.5-2 ml of diseased tissue into a small centrifuge tube (EP tube), adding 0.2-0.8 ml of Phosphate Buffer Solution (PBS) until the tissue is completely covered, whirling for 30-90 seconds, centrifuging for 1-3 minutes at 1000g, and taking the supernatant to obtain a supernatant;
(2) adding the supernatant obtained in the step (1) into 5-20 ml of a bacterial culture medium, culturing for 1 hour in a shaking table, and observing whether bacteria grow or not by naked eyes or detecting OD (optical density) by using a spectrophotometer600Repeating the steps for 2-3 times to obtain an average value;
(3) adding 1-2 ml of the bacterial culture medium treated in the step (2) into 2-20 small centrifuge tubes (EP tubes) with 1.5-2 ml, adding 10 mul of SM buffer solution into the 1 st tube as a control group, and adding 10 mul of different phages (with titer of 1 x 10) into the other tubes7PFU/ml) as a test group, marking, and then carrying out shake culture;
(4) in the culture time, the turbidity condition of the culture solution of each tube of the test group and the control group is visually observed every 1 hour, if the culture solution of the test group is obviously clearer than that of the control group, the tube of the bacteriophage can be judged to have the inhibition effect on the bacteria; or detecting the OD of the culture solution in each tube of the test group and the control group by a spectrophotometer every 1 hour during the culture time600Value, repeat 2-3 times to get the average value, control group culture solution OD600Values such as OD of test group600If the value is higher than 0.05, the phage in the test tube can be judged to have the inhibition effect on the bacteria;
(5) enriching the bacteriophage with the inhibiting effect, respectively preparing a single preparation or a mixed preparation, taking the diseased animal, carrying out intramuscular injection with the dose of 100-500 mu l for each animal, and observing the recovery condition, wherein if the injected animal is obviously more obvious than the non-injected animal, the bacteriophage or the mixed bacteriophage has the treatment effect on the diseased animal.
Further, the bacterial culture medium in the step (1) is an LB culture medium.
Further, the diseased tissue in the step (1) is a blood, organ, brain, skin and muscle sample of a suspected bacterium-infected diseased animal.
Further, the phage in step (3) may be a single phage or a mixture of phage.
Further, in the step (2) and the step (3), the temperature of shaking table culture is 36-38 ℃.
Further, the culture time of the phage mixed with the culture medium in the step (3) is 3-6 hours.
Further, in the step (4), the basis for determining that the tube phage has an inhibitory effect on the bacteria is as follows: detecting OD of each tube culture solution of the test group and the control group by a spectrophotometer every 1 hour during the culture time600Value, repeat 2-3 times to get the average value, control group culture solution OD600Values such as OD of test group600If the value is higher than 0.05, the phage in the test tube can be judged to have the inhibition effect on the bacteria.
The principle of the invention is as follows:
in an LB bacterial culture medium, the culture medium becomes more turbid along with the growth of bacteria, and the OD value rises continuously; if the specific phage inhibits, the culture medium is relatively clear and the OD value is slowly increased, so that the phage with specific therapeutic action can be screened out and verified through animal experiments.
The beneficial technical effects of the invention are as follows:
the invention provides a method for rapidly screening therapeutic phage, aiming at the problem of screening therapeutic phage in vitro, the invention uses in vitro bacteria inhibition test, and can rapidly screen phage which can be used for treating bacterial infection diseases of human and animals by naked eye observation or spectrophotometer detection after culture, thereby saving cost.
Detailed Description
The invention will be better understood by the following description of embodiments thereof, but the applicant's specific embodiments are not intended to limit the invention to the particular embodiments shown, and any changes in the definition of parts or features and/or in the overall structure, not essential changes, are intended to define the scope of the invention.
This example
1 materials and methods
1.1 pathological material: duck heads of diseased ducks are 2.
1.2 phage: phages P777, P779, P780 and P stored in the laboratory were mixed (before use, the phages were re-enriched by a double-layer plate method, and the titer was 1 x 107PFU/ml or more).
1.3 consumable: phosphate buffer solution, SM solution, 2ml and 15ml of EP tubes, pipettors, gun heads and the like.
1.4 Instrument: spectrophotometer, vortex apparatus, centrifuge, microbalance, alcohol burner, tweezers, scissors, etc.
1.5 method: shearing duck head tissue 0.1g to 2ml EP tube with sterilized scissors and forceps in super clean bench, adding 0.8ml PBS to completely cover the tissue after staining, vortexing for 60 s, centrifuging at 1000g for 1 min with centrifuge, collecting supernatant 120 μ l to 12ml LB culture solution, shake culturing at 37 deg.C for 1 hr, observing the presence or absence of bacteria, and detecting OD600The value is repeated for 3 times, and the average value is calculated; adding 1.6ml of the LB culture solution processed above into 5 EP tubes of 2ml, adding 10. mu.l of SM buffer solution into the 1 st tube as a reference mark as group 1, adding 10. mu.l of l P777, P779, P780 and P into the other tubes respectively to mix them as test groups, respectively as group 2, group 3, group 4 and group 5, and performing shake culture at 37 ℃; detecting the OD of each tube of culture solution of the test group and the control group by a spectrophotometer every 1 hour600Recording the value, repeating twice, and taking an average value; the test was continued for 3 hours, the results are shown in Table 1, and the OD of group 4 and group 5 was determined by comparing the control group with the test group600The difference of the value is more than 0.05 compared with the control groupIt is presumed that phages P780 and P mix of groups 3 and 4 have inhibitory effects on the target bacteria.
| Time (hours) | Group 1 (control) | Group 2(P777) | Group 3(P779) | Group 4(P780) | Group 5(P mix) |
| 0 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
| 1 | 0.138 | 0.135 | 0.135 | 0.143 | 0.138 |
| 2 | 0.343 | 0.378 | 0.322 | 0.189 | 0.256 |
| 3 | 0.545 | 0.514 | 0.521 | 0.35 | 0.384 |
TABLE 1 OD measurement of different phages and cultures at different times600Value of
Mixing and enriching phages P780 and P with inhibitory effect, taking 5 diseased ducks, performing intramuscular injection in a dosage of 200 mu l per duck, and taking 5 non-injected ducks as a control to observe the rehabilitation condition.
2 results
The injection of phage (P780 mixed with P) of groups 4 and 5 into the affected ducks improved significantly 2 days after injection, while the control ducks still suffered from disease or died.
3 analysis of
1h after the test group and the control group are subjected to transfer culture, the culture solution OD600No significant difference in value; 2h, culture OD of group 4 and group 5600The values were significantly different from the control group; by 3h, the difference increases, greater than 0.05. The detection values of the groups 2 and 3 at the respective times are not obviously different from those of the control group. The phages of groups 4 and 5 are considered to have inhibitory effects on the bacteria in the disease, and therapeutic effects were also demonstrated by injection tests.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and modifications as fall within the true spirit and scope of the invention be considered as within the following claims.
Claims (7)
1. A method for rapid screening of therapeutic phages comprising the steps of:
(1) aseptically shearing 0.1-0.5 g to 1.5-2 ml of diseased tissue into a small centrifuge tube (EP tube), adding 0.2-0.8 ml of Phosphate Buffer Solution (PBS) until the tissue is completely covered, whirling for 30-90 seconds, centrifuging for 1-3 minutes at 1000g, and taking the supernatant to obtain a supernatant;
(2) adding the supernatant obtained in the step (1) into 5-20 ml of a bacterial culture medium, culturing for 1 hour in a shaking table, and observing whether bacteria grow or not by naked eyes or detecting OD (optical density) by using a spectrophotometer600Repeating the steps for 2-3 times to obtain an average value;
(3) adding 1-2 ml of the bacterial culture medium treated in the step (2) into 2-20 small centrifuge tubes (EP tubes) with 1.5-2 ml, adding 10 mul of SM buffer solution into the 1 st tube as a control group, and adding 10 mul of different phages (with titer of 1 x 10) into the other tubes7PFU/ml) as a test group, marking, and then carrying out shake culture;
(4) in the culture time, the turbidity condition of the culture solution of each tube of the test group and the control group is visually observed every 1 hour, if the culture solution of the test group is obviously clearer than that of the control group, the tube of the bacteriophage can be judged to have the inhibition effect on the bacteria; or detecting the OD of the culture solution in each tube of the test group and the control group by a spectrophotometer every 1 hour during the culture time600Value, repeat 2-3 times to get the average value, control group culture solution OD600Values such as OD of test group600If the value is higher than 0.05, the phage in the test tube can be judged to have the inhibition effect on the bacteria;
(5) enriching the bacteriophage with the inhibiting effect, respectively preparing a single preparation or a mixed preparation, taking the diseased animal, carrying out intramuscular injection with the dose of 100-500 mu l for each animal, and observing the recovery condition, wherein if the injected animal is obviously more obvious than the non-injected animal, the bacteriophage or the mixed bacteriophage has the treatment effect on the diseased animal.
2. The method for rapidly screening bacteriophage for treatment according to claim 1, wherein the bacterial culture medium in the step (1) is LB culture medium.
3. The method according to claim 1, wherein the diseased tissue in step (1) is a blood, organ, brain, skin and muscle sample of a suspected bacterium infected diseased animal.
4. The method for rapidly screening bacteriophage for treatment according to claim 1, wherein the bacteriophage in step (3) may be a single bacteriophage or a mixture of bacteriophages.
5. The method for rapidly screening therapeutic phages according to claim 1, characterized in that the temperature of shaking culture in step (2) and step (3) is 36-38 ℃.
6. The method for rapidly screening bacteriophage for treatment according to claim 1, wherein the incubation time after mixing the bacteriophage with the culture medium in the step (3) is 3-6 hours.
7. The method for rapidly screening therapeutic phage according to claim 1, wherein in step (4), the tube phage is determined to have inhibitory effect on the bacteria according to the following formula: detecting OD of each tube culture solution of the test group and the control group by a spectrophotometer every 1 hour during the culture time600Value, repeat 2-3 times to get the average value, control group culture solution OD600Values such as OD of test group600If the value is higher than 0.05, the phage in the test tube can be judged to have the inhibition effect on the bacteria.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116814734A (en) * | 2023-06-21 | 2023-09-29 | 创噬纪(上海)生物技术有限公司 | Method for high-flux screening of sensitive phage in culture solution |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013027146A1 (en) * | 2011-08-25 | 2013-02-28 | Proteon Pharmaceuticals S.A. | The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof |
| CN103555673A (en) * | 2013-10-23 | 2014-02-05 | 靳静 | Method for in vitro screening in-vivo antibacterial activity bacteriophage |
| CN110982793A (en) * | 2020-01-02 | 2020-04-10 | 瑞科盟(青岛)生物工程有限公司 | Method for in vitro screening of in vivo antibacterial activity phage |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013027146A1 (en) * | 2011-08-25 | 2013-02-28 | Proteon Pharmaceuticals S.A. | The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof |
| US20140220659A1 (en) * | 2011-08-25 | 2014-08-07 | Proteon Pharmaceuticals S.A. | The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof |
| CN103555673A (en) * | 2013-10-23 | 2014-02-05 | 靳静 | Method for in vitro screening in-vivo antibacterial activity bacteriophage |
| CN110982793A (en) * | 2020-01-02 | 2020-04-10 | 瑞科盟(青岛)生物工程有限公司 | Method for in vitro screening of in vivo antibacterial activity phage |
Non-Patent Citations (2)
| Title |
|---|
| 伍尚忠: "《水稻白叶枯病及其防治》", 30 June 1983, 上海科学技术出版社 * |
| 路甬样: "《科学改变人类生活的119个伟大瞬间》", 30 November 2012, 浙江少年儿童出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116814734A (en) * | 2023-06-21 | 2023-09-29 | 创噬纪(上海)生物技术有限公司 | Method for high-flux screening of sensitive phage in culture solution |
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Application publication date: 20210430 |