CN106755279A - Nutrient solution, kit and detection method for detection bacterium vaginitis - Google Patents
Nutrient solution, kit and detection method for detection bacterium vaginitis Download PDFInfo
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- CN106755279A CN106755279A CN201710177758.6A CN201710177758A CN106755279A CN 106755279 A CN106755279 A CN 106755279A CN 201710177758 A CN201710177758 A CN 201710177758A CN 106755279 A CN106755279 A CN 106755279A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
The invention discloses a kind of nutrient solution for detection bacterium vaginitis, contain in every 100ml nutrient solutions：0.1~1g of OX-heart powder；0.5~5g of tryptone；0.1~2g of glucose；0.1~1g of sodium chloride；0.1~1g of bromocresol purple sodium salt；5~20ml of horse serum；0.0001~0.001g of neomycinsulphate；0.0001~0.001g of nystatin.The invention also discloses the kit and detection method of detection bacterium vaginitis.Detection kit steady quality of the invention, detection method is simple to operate, result is accurate.Kit of the present invention possesses targeting culture and the simultaneously sensitiveness of synchronous detection medicine, a step is completed in 6 24 hours, success rate is high, drug sensitivity tests reliability, disclosure satisfy that clinical precisely diagnosing bacterial vaginitis and medicament sensitivity test, it is that the timely Precise Diagnosis of clinician and reasonable employment medicine provide reference frame, it is to avoid clinical abuse of antibiotics.
Description
Technical field
The invention belongs to microbe diagnosis technical field, and in particular to a kind of culture for detection bacterium vaginitis
Liquid, kit and detection method.
Background technology
At present to based on the detection in the conventional way of bacterial vaginitis, commonly using microscopy observation, goldstandard method be culture,
Identification is separated, but step complexity, detection cycle are long, and separation rate is not high, whole process about 4-7 days, cumbersome, can not expire
The need for sufficient quick, convenient detection, and the identification of real target bacterium can be influenceed when miscellaneous bacteria is more in sample.
Conventional medium is essentially all that, using CDC Anaerobic Agar culture mediums, it is solid that it uses agar to be adapted to as coagulator
Body flat board culture, but its nutritional ingredient is complicated, incubation time is long, it is necessary to 24~48 hours, and Clinical practice inconvenience is not suitable for bacterium
The anaerobic bacteria cultures such as the Gartner bacterium of property vaginosis infection；And do not possess targeting culture function, often there is more varied bacteria growing, under
One step bacterium colony screening operation causes larger obstacle, and repurity is identified after bacterium colony needs hand picking culture, will to operator quality
Ask higher, step is cumbersome.Using preceding needing sterilization to change into plate plate again, step is complicated, and finished product plate can only preserve temperature
Degree is at 2-8 DEG C, and the term of validity only has 7 days；Meanwhile, traditional susceptibility detection method must could be distinguished after pathogen isolation identification
Every kind of medicament sensitivity test is done, the quality to culture medium there are certain requirements, such as thickness, surface moisture hardness can influence medicine
Thing is migrated, and inhibition zone measurement operating accuracy is relevant with operating personnel's subjective consciousness, and determination range is larger, is also easy to produce human error,
Whole experiment process is complicated, and the time is long, it is necessary to 48-72 hour, and qualification process requires that peopleware is high.
The content of the invention
High it is an object of the invention to provide a kind of nutritional ingredient, incubation time is short, with targeting culture function, and is not required to
Pathogen isolation identification can be used for nutrient solution, kit and the detection side of bacterial vaginitis detection and susceptibility detection simultaneously
Method.
A kind of nutrient solution for detection bacterium vaginitis of the invention, wherein in per 100ml nutrient solutions, containing as follows
The component of weight proportion：
0.1~1g of OX-heart powder
0.5~5g of tryptone
0.1~2g of glucose
0.1~1g of sodium chloride
0.1~1g of bromocresol purple sodium salt
5~20ml of horse serum
0.0001~0.001g of neomycinsulphate
0.0001~0.001g of nystatin.
The preparation method of the nutrient solution for detection bacterium vaginitis described above, it is to containing OX-heart powder, pancreas egg
In the mixture of white peptone, glucose, sodium chloride and bromocresol purple sodium salt plus sterilizing purified water is miscible, autoclaving is subsequently adding
Horse serum, neomycinsulphate and nystatin, obtain final product nutrient solution.
A kind of kit for detection bacterium vaginitis of the invention, including dehydrated medium, culture medium dilution
And drug sensitive plate, the dehydrated medium is the freeze-dried powder of nutrient solution described above of the invention, and the drug sensitive plate is provided with culture and puts
Thing platform, negative control hole C-, Positive control wells C+, some groups of drug test holes, drug test hole is divided into high concentration medicine described in every group
Thing hole and low concentration medicine hole.
Culture medium dilution described above is sterilizing purified water.
A kind of detection method of detection bacterium vaginitis of the invention, it is using present invention reagent described above
Box.
The detection method of the detection bacterium vaginitis that the present invention is provided, comprises the steps：
(1) various high-concentration and low-concentration drug solutions are coated with advance in the drug test hole of drug sensitive plate；
(2) dehydrated medium is diluted to fluid nutrient medium with culture medium dilution, draws a small amount of fluid nutrient medium to medicine
The negative control hole C of quick plate-；
(3) doubtful bacterial vaginitis patient genital secretion is taken with sterile cotton swab, after normal saline dilution, is drawn
It is a small amount of to being mixed into bacterium solution in remaining liq culture medium；
(4) a small amount of bacterium solution to the Positive control wells C of drug sensitive plate is drawn respectively+, in each drug test hole, drug sensitive plate is covered
Lid is incubated, 6-24h observation results；
(5) testing result interpretation：
Hole C+Culture medium and hole C-It is positive (+) compared to yellow is become；
Hole C+Culture medium and hole C-It is negative (-) compared to nondiscolouring；
Hole C-Negative (-), hole C+Positive (+) experiment is effective, judges remaining each hole result；
Hole C-Positive (+), represents pollution, and it is invalid to test；
Hole C-Negative (-), hole C+Negative (-) shows negative findings, remaining hole not judged result；
(6) drug sensitivity tests judge：
Hole A (-) hole B (-) is sensitive,
It is quick in hole A (-) hole B (+),
Hole A (+) hole B (+) resistance.
Preferably, a kind of nutrient solution for detection bacterium vaginitis of the invention, wherein per 100ml nutrient solutions
In, the component containing following weight proportion：
0.1~0.6g of OX-heart powder
1~4g of tryptone
0.5~1.5g of glucose
0.5~1.5g of sodium chloride
0.1~1g of bromocresol purple sodium salt
10~20ml of horse serum
0.0003~0.0006g of neomycinsulphate
0.0003~0.0006g of nystatin.
Detection kit steady quality for detection bacterium vaginitis of the invention, simple to operate, result precisely, medicine
Quick reliability.Nutrient solution for detection bacterium vaginitis of the invention, it is fluid nutrient medium, rather than solid plate culture
Base, and culture medium is processed into dehydrated medium by freeze-drying, dehydrated medium is dissolved into liquid using preceding use sterilizing purified water
Body culture medium, fluid nutrient medium culture increases bacterium speed faster, and dehydrated medium steady quality, can normal temperature storage and transport, have
The effect phase is up to 2 years.General, inorganic salts such as phosphate, sulfate etc. can be added in fluid nutrient medium, on the one hand it be mainly
Such as make the cell traffic function normal operation of microorganism for needed for growth of microorganism by adjusting culture medium osmotic pressure, by culture medium
In peptone or other nutritional ingredients be transported into microbial cell, while inorganic ion can also participate in microbial cell
The normal operation of interior other functions, the pH value that on the other hand it as pH buffer compositions, can adjust fluid nutrient medium is to give bacterium
The living environment that group adapts to, but if such inorganic salts excessive addition, it is easily caused that osmotic pressure is excessive, the culture to anaerobic bacteria
Totally unfavorable, while the excessive addition of such inorganic salts can also influence the color change of culture medium, influence is sentenced to final result
It is disconnected, it is not easy to the identification culture of the pathogenic anaerobic bacterias such as vagina Gartner bacterium.Component OX-heart powder in culture medium of the invention is training
One of basic ingredient of base is supported, it provides most basic nutritional ingredient includes necessary amino acid, the dimension of multiple-microorganism culture
Raw element, growth factor etc., necessary to these compositions are also anaerobism bacteria growing, compound proportion is only close to human internal environment,
There can be preferable culture effect；Tryptone is a kind of high-quality protein peptone, and it is digested through pancreatin with fresh beef and ox bone, than
General proteins peptone contains more rich nitrogen source, amino acid, is more suitable for the growth of microorganism, and tryptone is compared with other peptones
Its amino acid is more complete, and the comparision contents of particularly tryptophan are high, is most appropriate to do sugar fermentating test, and microorganism utilizes pancreas
Peptone carry out sugar fermentation produce acidic materials, be also product colour change one of the main reasons, ratio it is too high it is too low not
Beneficial to growth of microorganism；Horse serum mainly provides some growth of microorganism factors, and anaerobic bacteria growth factor includes the V factors, if
It is not added with, microorganism will not grow or grow very slowly, does not reach product effect, horse serum is added in culture medium, it is necessary to by removing
Bacterium process, but the present invention does not select commercially available horse serum freeze-dried powder because its had in production process is dried some growth because
Son is destroyed, therefore horse serum freeze-dried powder is applied to some less demanding microcultures, and is unsuitable for anaerobic bacteria culture.This
OX-heart powder, tryptone, horse serum, the adequate nutrition proportioning of glucose are conducive to bacterium of the present invention in invention culture medium
The growth of (anaerobic bacteria), and the addition of the dual anti-bacteriostatic agent being made up of neomycinsulphate, nystatin makes culture medium of the invention
More targeting culture function (bacteriostatic agent in the present invention is acted on anaerobic bacteria unrestraint, at the same to most of clinical bacterias and
Fungi has stronger inhibitory action, and optimum is used as the Selective agar medium of anaerobic bacteria, former also in compliance with compatibility of drugs in addition
Then, have synergy between medicine, strengthen fungistatic effect) so that in the detection to bacterial vaginitis of the present invention
Shi Wuxu is further purified identification.Meanwhile, used as color change indicator, its excursion best suits anaerobism to bromocresol purple sodium salt
Bacterium cultivates the pH value excursion of wild Oryza species, and ratio is too high or too low all to cause color change inaccurate, and influence result is sentenced
Read, therefore its color change becomes apparent from compared to other indicator discolorations, it is easier to visually observe identification.
Susceptibility detection basis in the present invention《Clinical examination operational procedure》With《Clinical Antibiogics usage guide》It is selected
The medicine that clinic is currently in use, need to only select 2 kinds of concentration to measure medicaments insensitive by passing through color change with reference to international MIC standards
Property.This kit possesses targeting culture and the simultaneously sensitiveness of synchronous detection multi-medicament, and a step is completed in 6~24 hours, inspection
Extracting rate is high, disclosure satisfy that clinical precisely diagnosing bacterial vaginitis and medicament sensitivity test, is that clinician accurately examines in time
Disconnected and reasonable employment medicine provides reference frame, it is to avoid clinical abuse of antibiotics.
Brief description of the drawings
Fig. 1 is susceptibility plate structure schematic diagram.
Specific embodiment
Following embodiments are further illustrating as the explaination to the technology of the present invention content for present invention, but
Substance of the invention is not limited in described in following embodiments, one of ordinary skill in the art can with and should know appoint
What simple change or replacement based on true spirit all should belong to protection domain of the presently claimed invention.
Embodiment 1
It is prepared by one, dehydrated mediums：
Nutrient solution for detection bacterium vaginitis of the invention, wherein in per 100ml nutrient solutions, containing following weight
The component of proportioning：OX-heart powder 0.3g, tryptone 3g, glucose 1g, sodium chloride 1g, bromocresol purple sodium salt 0.1g, horse serum
15ml, neomycinsulphate 0.0005g, nystatin 0.0005g.To OX-heart powder, tryptone, grape containing said ratio
In sugar, the mixture of sodium chloride and bromocresol purple sodium salt plus sterilizing purified water is miscible, 121 DEG C of autoclavings add horse after cooling
Serum, neomycinsulphate and nystatin, obtain final product nutrient solution, are then distributed into 3ml/ bottles, and dry powder culture is turned into after freeze-drying
Base.
It is prepared by two, dilutions：Every bottle of packing 3mL sterilizing purified water.
Three, drug sensitive plates make：Drug sensitive plate can use commercially available prod, its totally 20 hole, as shown in figure 1, drug sensitive plate is provided with training
Support articles holding table, negative control hole C-, Positive control wells C+, some groups of drug test holes, drug test hole is divided into highly concentrated described in every group
Degree medicine hole A and low concentration medicine hole B.Wherein：A1 is C-Hole (negative control hole), B1 is C+Hole (Positive control wells), A2~
A10, B2~B10 are coated with the high concentration and low concentration of 9 kinds of medicines respectively, and specific composition is shown in Table one：
Table one
Note：A2~A10：Represent high concentration (ug/mL)；B2~B10：Represent low concentration (ug/mL)；C-：Negative control hole；
C+：Positive control wells；The corresponding drug concentration of digitized representation.
Four, detection methods
1st, the drug solution of various high-concentration and low-concentrations is coated with the drug test hole of drug sensitive plate in advance；
2nd, one bottle of 3mL diluted of a dehydrated medium draws 100uL into fluid nutrient medium with micropipettor
It is added to drug sensitive plate C-Hole (negative control hole)；
3rd, doubtful bacterial vaginitis patient genital secretion is taken with sterile cotton swab, it is dilute with 1mL SPSSs
Release mixing, then draw 100uL to being mixed into bacterium solution in remaining liq culture medium；
4th, 100uL bacterium solutions to 19 holes of drug sensitive plate residue are drawn respectively with micropipettor, drug sensitive plate is closed the lid and is put into
10%CO235 ± 1 DEG C of insulating box cultures, 6~24 hours observation results.
Five, result interpretations
1st, positive and negative criterion
1)C+Hole culture medium and C-Hole, compared to yellow is become, is positive (+)；
2)C+Hole culture medium and C-Nondiscolouring (blue-green) is compared in hole, is negative (-).
2、C-Hole feminine gender (-), C+Positive (+) experiment in hole is effective, judges remaining each hole result.
3、C-The hole positive (+), represents pollution, and it is invalid to test.
4、C-Hole feminine gender (-), C+Hole feminine gender (-) shows negative findings, remaining hole not judged result.
5th, drug sensitivity tests judge：A holes (-) B holes (-) are sensitive；It is quick in A holes (-) B holes (+)；A holes (+) B holes (+) resistance.
Six, clinical statisticses：
1st, healthcare hospital for women ＆ children of Anhui Province clinic 203 patients for being suspected to be vaginosis infection of total number of cases, clinic group diagnosis are picked up from
Using classical culture protocols, product group diagnosis uses kit of the present invention and method；Classical culture protocols CDC culture mediums are selected
CDC Anaerobic Agar HB0310。
2nd, 21 (positive rates in clinical group bacterial vaginitis 6 (positive rate 2.96%) of the positive, product group 24 hours
10.34%), including 6 of clinic group detection, recall rate is higher by 7.38%；This product sensitivity minimization in 10000cfu,
Result detection not only for severe infection is accurate and still has preferable detection sensitivity for mild or moderate infection；
3rd, drug sensitivity tests are normal, and product group is consistent with the drug sensitivity tests of clinical group.
Claims (6)
1. a kind of nutrient solution for detection bacterium vaginitis, wherein in per 100ml nutrient solutions, containing following weight proportion
Component：
0.1~1g of OX-heart powder
0.5~5g of tryptone
0.1~2g of glucose
0.1~1g of sodium chloride
0.1~1g of bromocresol purple sodium salt
5~20ml of horse serum
0.0001~0.001g of neomycinsulphate
0.0001~0.001g of nystatin.
2. the preparation method of the nutrient solution of detection bacterium vaginitis is used for described in claim 1, it is characterised in that to containing ox
Add sterilizing purified water miscible in the mixture of heart powder, tryptone, glucose, sodium chloride and bromocresol purple sodium salt, autoclaving,
Horse serum, neomycinsulphate and nystatin are subsequently adding, nutrient solution is obtained final product.
3. a kind of kit for detection bacterium vaginitis, including dehydrated medium, culture medium dilution and drug sensitive plate, institute
The freeze-dried powder that dehydrated medium is nutrient solution that is described in claim 1 or being obtained by claim 2 preparation method is stated, it is described
Drug sensitive plate is provided with culture articles holding table, negative control hole C-, Positive control wells C+, some groups of drug test holes, medicine described in every group
Test hole is divided into high concentration medicine hole and low concentration medicine hole.
4. kit as claimed in claim 3, it is characterised in that the culture medium dilution is sterilizing purified water.
5. a kind of detection method of detection bacterium vaginitis, it is characterised in that using the kit described in claim 3 or 4.
6. detection method as claimed in claim 5, it is characterised in that comprise the steps：
(1) various high-concentration and low-concentration drug solutions are coated with advance in the drug test hole of drug sensitive plate；
(2) dehydrated medium is diluted to fluid nutrient medium with culture medium dilution, draws a small amount of fluid nutrient medium to drug sensitive plate
Negative control hole C-；
(3) doubtful bacterial vaginitis patient genital secretion is taken with sterile cotton swab, after normal saline dilution, draws a small amount of
Bacterium solution is mixed into in remaining liq culture medium；
(4) a small amount of bacterium solution to the Positive control wells C of drug sensitive plate is drawn respectively+, in each drug test hole, drug sensitive plate is closed the lid
It is incubated, 6-24h observation results；
(5) testing result interpretation：
Hole C+Culture medium and hole C-It is positive (+) compared to yellow is become；
Hole C+Culture medium and hole C-It is negative (-) compared to nondiscolouring；
Hole C-Negative (-), hole C+Positive (+) experiment is effective, judges remaining each hole result；
Hole C-Positive (+), represents pollution, and it is invalid to test；
Hole C-Negative (-), hole C+Negative (-) shows negative findings, remaining hole not judged result；
(6) drug sensitivity tests judge：
Hole A (-) hole B (-) is sensitive,
It is quick in hole A (-) hole B (+),
Hole A (+) hole B (+) resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710177758.6A CN106755279A (en) | 2017-03-23 | 2017-03-23 | Nutrient solution, kit and detection method for detection bacterium vaginitis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710177758.6A CN106755279A (en) | 2017-03-23 | 2017-03-23 | Nutrient solution, kit and detection method for detection bacterium vaginitis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106939329A (en) * | 2017-03-23 | 2017-07-11 | 合肥百盛园生物药业有限公司 | A kind of nutrient solution, kit and detection method for being used to detect colpomycosis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106939329A (en) * | 2017-03-23 | 2017-07-11 | 合肥百盛园生物药业有限公司 | A kind of nutrient solution, kit and detection method for being used to detect colpomycosis |
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