CN110029142A - A kind of two Methods for Fungi Detection based on liquid-based tabletting technology - Google Patents
A kind of two Methods for Fungi Detection based on liquid-based tabletting technology Download PDFInfo
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Abstract
The embodiment of the present application discloses a kind of two Methods for Fungi Detection based on liquid-based tabletting technology, to contain the viscous substance in the cell-preservation liquid neutralizing sample to its digestion ingredient of leukorrhea, impurity is filtered out by filter screen, and target detection thing is retained by mwco membrane, to, target detection thing can be enriched with and be transferred on glass slide and carry out film-making, realize the accurate detection to fungi, pass through the technical solution proposed using the embodiment of the present application, realization that can the be simple and efficient impurity screening in sample to be detected, the fungi that being enriched with need to detect carries out film-making, in order to observe and detect, it is cumbersome in the presence of the effective solution prior art, time-consuming for process, problem more than required reagent.
Description
Technical field
This application involves medicine to chemically examine field, in particular to a kind of two Methods for Fungi Detection based on liquid-based tabletting technology.
Background technique
Fungi belongs to eucaryote, and without plastid, nutritional mode is to absorb, no phagocytosis.Cell wall contains chitin
And beta glucan.It can cause the mankind, the disease of animal, referred to as nosomycosis can also cause disease, the mankind's anaphylactia of plant
And mycotoxicosis.Fungi invades human body often secondary to other illness, such as the chronic debilitating disease of malignant tumour, diabetes
Disease；Big drug amount roentgen radiation x；Immunosuppressor etc. leads to immune function and lower for the resistance；Widely apply cortex hormone of aadrenaline
Patient, how suppressed inflammatory reaction is, also be easy infection fungi；In addition the application such as organ transplant, intubation catheter, radiotherapy causes
Local tissue damage provides condition for the invasion of fungi.In recent years due to the emergence of immunodeficiency virus infection person, make
Originally non-pathogenic fungi switchs to pathomycete.Therefore, fungal infection has become an increasingly serious problem.
Fungi is divided into superficial mycosis and deep fungal.Superficial mycosis: main infringement body skin, it is parasitic and saprophytic in table
In the collenchyme on skin, hair and deck, cause superficial mycoses, abbreviation tinea disease.Clinical most common superficial mycoses has: body
Tinea, jock itch, the tinea manuum and tinea pedis.
Fungal infection common detection method include: direct Microscopical Method For Detection (such as KOH smear or lactophenol cotton blue smear) and
Dyeing microscopic examination method.Wherein, direct Microscopical Method For Detection disadvantage is that positive rate is lower, and negative findings can not exclude to diagnose, and background is mixed and disorderly,
Sensitivity is low, and omission factor is high, requires operator's profession recognition capability high.General dyeing microscopic examination method include: Grain stain,
Giemsa staining, PAS method, hexamine silver method (GMS), silver liquid ammonia dip method (G-S), Gridley decoration method, mucicarmine
(Mayer ' s Mucicarmin stain) decoration method, Papanicolaou's vaginal smear technique etc..
The advantages of these types of special staining, is: under the premise of controlling dyeing details, can dye beautiful fungi
Form, it is recognizable.
Applicant has found that above-mentioned existing processing scheme at least has the following problems during realizing the application:
1, relatively high to operator's professional skill requirement.
2, cumbersome, with duration (1 hour or so).
3, mating required reagent is many kinds of, and stock management is troublesome.
Summary of the invention
The embodiment of the present application provides a kind of two Methods for Fungi Detection based on liquid-based tabletting technology, realization that can be simple and efficient
The impurity screening in sample to be detected, the fungi that being enriched with need to detect carry out film-making, in order to observe and detect, effective solution
Time-consuming for cumbersome, process in the presence of the prior art, the problem more than required reagent.
In order to reach above-mentioned technical purpose, the embodiment of the present application provides a kind of fungal detection based on liquid-based tabletting technology
Method, the method specifically include:
Step 1, collecting sample, and put samples into containing in the cell-preservation liquid to its digestion ingredient of leukorrhea, change
Solve viscous substance；
Step 2 is filtered the cell-preservation liquid comprising sample by filter membrane；
Step 3 retains the target detection thing in filtered fluid by mwco membrane；
The target detection thing that the mwco membrane retains is transferred to glass slide by step 4, carries out film-making；
Step 5, the covered on the glass slide that film-making is completed, and fungal detection is carried out by microscope.
Preferably, the step 1, specifically includes:
It puts samples into sample reagent box or test tube equipped with cell-preservation liquid；
Sample reagent box or test tube are placed in shake on oscillator and mixed, thick substances are sufficiently dissolved.
Preferably, step 2 is completed to the processing of step 4 especially by TCT pelleter；
Wherein, the treatment process of step 2 to step 4 is executed by the TCT pelleter, specifically:
(1) specimen filter being added in fungi sample solution, the specimen filter is made of two bodies up and down being connected,
Upper body is mounted with filter screen, and fungi needed for guaranteeing can install mwco membrane by filter screen, lower body, the fungi quilt observed needed for guaranteeing
It is blocked on mwco membrane；
(2) sample filter is installed in the TCT pelleter, the bottom of the sample filter is by described
Vacuum pump built in TCT pelleter generates negative pressure, and sample solution suction and pumping are passed through the filter screen and the mwco membrane, wherein
For impurity in the fungi sample solution by the filter screen filtration, the fungi of required observation is trapped film retention；
(3) the TCT pelleter separates the bi-body structure up and down of the specimen filter, and glass slide push-in is separated
It is described up and down bi-body structure centre；
(4) the TCT pelleter by containing required observation fungi mwco membrane from the bottom to top be covered on the load glass
On piece stops 10~20s of sample transfer time, the fungi of required observation is transferred on the glass slide；
(5) the TCT pelleter releases the glass slide that film-making is completed, and film-making terminates.
Preferably,
The filter opening diameter of the filter screen is 0.75-1.5mm.
Preferably,
The filter opening diameter of the mwco membrane is 5-20 μm.
Preferably, before the step 5, further includes:
Fluorescent staining liquid is added dropwise on the glass slide, target detection thing is dyed.
Preferably, the fluorescent staining liquid is mixed by formula as below according to weight percent:
Fluorescent whitening agent 0.001%, potassium hydroxide 10%, dimethyl sulfoxide 4%, sodium chloride 3.6%, glycerol 10%, she
Train of thought indigo plant 0.01%, surplus are purified water；
Preferably, the configuration method of the fluorescent staining liquid are as follows:
(1) potassium hydroxide for weighing corresponding weight proportion is added part water and carries out that dissolution heat dissipation is sufficiently stirred, and is cooled to
After room temperature, dimethyl sulfoxide, sodium chloride and glycerol are sequentially added, is evenly stirred until no lamination, course of dissolution exotherm,
Cooled to room temperature is spare, obtains clear, colorless clear liquid, is named as solution A；
(2) fluorescent whitening agent for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains clear, colorless
Or faint yellow clear liquid, it is named as second liquid；
(3) Evans blue for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains bluish violet without analysis
Composition granule clear liquid out is named as the third liquid；
(4) second liquid is added to solution A and is mixed well, it is ensured that without solid precipitating or floccule in container, obtain clear, colorless
Or faint yellow clear liquid, it is named as fourth liquid；
(5) finally the third liquid is mixed with fourth liquid.
Preferably, described that fluorescent staining liquid is added dropwise on the glass slide, after being dyed to target detection thing, also wrap
It includes:
The glass slide that fluorescent staining liquid has been added dropwise is stood 30 seconds.
Preferably, the amount for the fluorescent staining liquid being added dropwise on the glass slide, specially 50ul.
Compared with prior art, the advantageous effects for the technical solution that the embodiment of the present application is proposed include:
The embodiment of the present application discloses a kind of two Methods for Fungi Detection based on liquid-based tabletting technology, containing to leukorrhea, it disappears
Change the viscous substance in the cell-preservation liquid neutralizing sample of action component, impurity is filtered out by filter screen, and pass through mwco membrane pair
Target detection thing is retained, and carries out film-making it is thus possible to which target detection thing is enriched with and is transferred on glass slide, is realized to true
The accurate detection of bacterium, by the technical solution proposed using the embodiment of the present application, realization that can be simple and efficient is to be detected
Impurity screening in sample is enriched with the fungi that need to detect and carries out film-making, in order to observe and detect, the effective solution prior art
In the presence of cumbersome, process time-consuming, the problem more than required reagent.
Detailed description of the invention
It, below will be to attached drawing needed in embodiment description in order to illustrate more clearly of the technical solution of the application
It is briefly described, it should be apparent that, the drawings in the following description are only some examples of the present application, general for this field
For logical technical staff, without creative efforts, it is also possible to obtain other drawings based on these drawings.
A kind of process for two Methods for Fungi Detection based on liquid-based tabletting technology that Fig. 1 is proposed by the embodiment of the present application is illustrated
Figure；
Fig. 2 is proposed a kind of by TCT pelleter execution step S102 to the place of step S104 by the embodiment of the present application
The flow diagram of reason process；
Being contaminated based on liquid-based tabletting technology and fluorescence under a kind of concrete application scene that Fig. 3 is proposed by the embodiment of the present application
The flow diagram of the two Methods for Fungi Detection of color technology；
Under a kind of concrete application scene that Fig. 4 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 40 times of object lens, by observed by fluorescence microscope
Candida albicans and spore imaging schematic diagram；
Under a kind of concrete application scene that Fig. 5 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 10 times of object lens, by observed by fluorescence microscope
First bits imaging schematic diagram；
Under a kind of concrete application scene that Fig. 6 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 10 times of object lens, by observed by fluorescence microscope
Scurf imaging schematic diagram；
Under a kind of concrete application scene that Fig. 7 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 10 times of object lens, by observed by fluorescence microscope
Vaginal fluid imaging schematic diagram；
Under a kind of concrete application scene that Fig. 8 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 40 times of object lens, by observed by fluorescence microscope
Candida albicans imaging schematic diagram；
Under a kind of concrete application scene that Fig. 9 is proposed by the embodiment of the present application, by based on the true of liquid-based tabletting technology
Bacterium detection method treated fungi sample, in the case where 10 times of eyepiece, 10 times of object lens, by observed by fluorescence microscope
Candida albicans imaging schematic diagram.
Specific embodiment
It is existing cumbersome, process to exist in the prior art time-consuming as the application background technique is stated, institute
The problems such as needing reagent more.
In order to solve the problems, the embodiment of the present application provides a kind of fungal detection side based on liquid-based tabletting technology
Method, realization that can the be simple and efficient impurity screening in sample to be detected, the fungi that being enriched with need to detect carries out film-making, in order to see
It examines and detects.
As shown in Figure 1, a kind of two Methods for Fungi Detection based on liquid-based tabletting technology proposed by the embodiment of the present application
Flow diagram, this method are applied to include server, data acquisition device corresponding to compensation device of respectively casting anchor, and monitoring
In the real-time monitoring system of the compensation device of casting anchor of information display platform, the method is specifically included:
Step S101, collecting sample, and put samples into containing in the cell-preservation liquid to its digestion ingredient of leukorrhea,
Dissolve viscous substance.
In specific sample collection procedure, it can be operated using the acquisition swab of disposable flocking head.
Such material release rate is up to 95%, and falls off without microfibre.Also, sampling is put into cell-preservation liquid
Afterwards, in order to avoid sample loss, can directly the bar for acquiring swab be fractureed in protecting in storage bottle, by sample when avoiding taking out swab
It takes out of, causes the loss of detected material.And the acquisition swab head being broken off, then it can be carried out by the filter operation of step S102
It filters out, subsequent operation will not be impacted.
Certainly, in the case where not influencing testing result, other sample acquisition modes also can be applied to the application implementation
The technical solution that example is proposed, it is such to change the protection scope that will not influence the application.
In specific application scenarios, this step can also further increase the operation that stirring or oscillation shake up, so as to reality
Existing abundant dissolution of the sample in cell-preservation liquid, as far as possible dissolves viscous substance.Such variation also belongs to
The protection scope of the application.
Specifically, the oscillation in practical operation shakes up process can assist completing by oscillator, convenient and efficient, specifically
Operating process are as follows:
It puts samples into sample reagent box or test tube equipped with cell-preservation liquid；
Sample reagent box or test tube are placed in shake on oscillator and mixed, thick substances are sufficiently dissolved.
Step S102, the cell-preservation liquid comprising sample is filtered by filter membrane.
In specific application scenarios, the filter opening diameter of the filter screen can be 0.75-1.5mm, reach and let off target
Detectable substance filters out the purpose of large-scale impurity.
Specific filter opening diameters can be adjusted according to actual needs, and such variation will not influence the application
Protection scope.
Step S103, the target detection thing in filtered fluid is retained by mwco membrane.
In specific application scenarios, the filter opening diameter of the mwco membrane is 5-20 μm.Reach retention target detection thing, puts
Fine particle is crossed, the purpose of checked for impurities is filtered out.
Specific filter opening diameters can be adjusted according to actual needs, and such variation will not influence the application
Protection scope.
Step S104, the target detection thing that the mwco membrane retains is transferred to glass slide, carries out film-making.
So far, film-making process based on liquid-based tabletting technology is completed, and the step S102 to step S104 during this can be with
It operates one by one stage by stage, TCT (Thinprep Cytologic Test, ThinPrep cytologic test) pelleter can also be passed through
It is automatically performed, further increases efficiency, reduce manually-operated fault and interfere, and improve accuracy in blocks.
Specifically, executing flow diagram such as Fig. 2 of the treatment process of step S102 to step S104 by TCT pelleter
It is shown, specifically:
Step S201, fungi sample solution is added specimen filter, the specimen filter by be connected up and down two
Body is constituted, and upper body is mounted with filter screen, and fungi needed for guaranteeing can install mwco membrane by filter screen, lower body, observation needed for guaranteeing
Fungi be blocked on mwco membrane.
It is similar with the explanation in aforementioned step S102 and step S103, the filter of filter screen and mwco membrane in this step
Bore dia can equally refer to above-mentioned dimensional values, reach identical filter effect.
Step S202, the sample filter is installed in the TCT pelleter, the bottom of the sample filter by
Vacuum pump built in the TCT pelleter generates negative pressure, and sample solution suction and pumping are passed through the filter screen and the mwco membrane.
Wherein, for the impurity in the fungi sample solution by the filter screen filtration, the fungi of required observation is trapped film
Retention.
By Negative pressure, the rate of filtration is improved, and filter effect can be enhanced.
Step S203, the described TCT pelleter separates the bi-body structure up and down of the specimen filter, and glass slide is pushed into
The centre of the bi-body structure up and down separated.
Step S204, the described TCT pelleter by containing required observation fungi mwco membrane from the bottom to top be covered on institute
It states on glass slide, stops 10~20s of sample transfer time, the fungi of required observation is transferred on the glass slide.
Step S205, the described TCT pelleter releases the glass slide that film-making is completed, and film-making terminates.
Certainly, the processing for above-mentioned step S102 to step S104 being executed by TCT pelleter is to further increase film-making
Speed improves the processing behave of film-making precision effect, but in the case where reaching same technique effect, if uses TCT film-making
Machine replaces artificial or other instruments operations to carry out above-mentioned processing, will not influence the protection scope of the application.
Step S105, the covered on the glass slide that film-making is completed, and fungal detection is carried out by microscope.
So far, a kind of processing scheme for two Methods for Fungi Detection based on liquid-based tabletting technology that the embodiment of the present application is proposed
It completes, is operated according to above-mentioned fungal detection, testing result generated has filtered out the interference of impurity, and preparation process is convenient.
It is further noted that in order to further increase detection convenience, by the clearer progress of target detection thing
It shows, the development effect of target detection thing can be further promoted by way of adding fluorescent staining liquid, it is specific to operate
Mode is that fluorescent staining liquid is added dropwise on the glass slide, dyes to target detection thing before step S105 execution.
Further, further include stewing process below to more fully colour after dyeing is completed:
The glass slide that fluorescent staining liquid has been added dropwise is stood 30 seconds.Certainly, if stood and the length of time of repose
It is short to be adjusted according to actual needs, it is such to change the protection scope that will not influence the application.
In actual application, the amount for the fluorescent staining liquid being added dropwise on the glass slide, specially 50ul.Certainly,
The dosage for the fluorescent staining liquid being added dropwise can be adjusted according to actual needs, and such variation will not influence the application's
Protection scope.
In actual application, the weight percent and effect being respectively formulated in above-mentioned fluorescent staining liquid are described as follows:
Fluorescent whitening agent, 0.001%, and the combination of fungal cell wall β-polysaccharose substance specificity, thus mark fluorescent
It is detected, under the specific exciting light wave band of fluorescence microscope (340~400nm), mycelia or spore issue bright bluish-green
Color fluorescence.
Potassium hydroxide, 10%, cutin is dissolved, tissue permeabilization is made.
Dimethyl sulfoxide, 4%, promote fluorescent whitening agent to dissolve in potassium hydroxide solution, while helping fluorescent whitening agent
It penetrates into sample.
Sodium chloride, 3.6%, the bond strength of fluorescent whitening agent and fungal cell's wall construction is improved, is excited in Ultraluminescence
In the case where, being more clear of can allowing phage structure highlights is visible, three-dimensional, is more convenient identification fungi.
Glycerol, 10%, moisturizing keeps the humidity for having dripped the sample certain time of dyeing liquor, convenient under the microscope.
Evans blue, 0.01%, mixed and disorderly background is weakened, compares background and fungi obvious, convenient for identification.
It is purified water with ingredient, plays the role of dissolving each formula.
In specific application scenarios, according to above-mentioned formula, the configuration method of fluorescent staining liquid specifically:
(1) potassium hydroxide for weighing corresponding weight proportion is added part water and carries out that dissolution heat dissipation is sufficiently stirred, and is cooled to
After room temperature, dimethyl sulfoxide, sodium chloride and glycerol are sequentially added, is evenly stirred until no lamination, course of dissolution exotherm,
Cooled to room temperature is spare, obtains clear, colorless clear liquid, is named as solution A.
(2) fluorescent whitening agent for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains clear, colorless
Or faint yellow clear liquid, it is named as second liquid.
(3) Evans blue for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains bluish violet without analysis
Composition granule clear liquid out is named as the third liquid.
(4) second liquid is added to solution A and is mixed well, it is ensured that without solid precipitating or floccule in container, obtain clear, colorless
Or faint yellow clear liquid, it is named as fourth liquid.
(5) finally the third liquid is mixed with fourth liquid.
Compared with prior art, the advantageous effects for the technical solution that the embodiment of the present application is proposed include:
The embodiment of the present application discloses a kind of two Methods for Fungi Detection based on liquid-based tabletting technology, containing to leukorrhea, it disappears
Change the viscous substance in the cell-preservation liquid neutralizing sample of action component, impurity is filtered out by filter screen, and pass through mwco membrane pair
Target detection thing is retained, and carries out film-making it is thus possible to which target detection thing is enriched with and is transferred on glass slide, is realized to true
The accurate detection of bacterium, by the technical solution proposed using the embodiment of the present application, realization that can be simple and efficient is to be detected
Impurity screening in sample is enriched with the fungi that need to detect and carries out film-making, in order to observe and detect, the effective solution prior art
In the presence of cumbersome, process time-consuming, the problem more than required reagent.
Below in conjunction with the attached drawing in the application, clear, complete description is carried out to the technical solution in the application, is shown
So, described embodiment is a part of the embodiment of the application, instead of all the embodiments.Based on the implementation in the application
Example, those of ordinary skill in the art's every other embodiment obtained without making creative work, all belongs to
In the range of the application protection.
In order to overcome the drawbacks of the prior art, a kind of fungi based on liquid-based tabletting technology that the embodiment of the present application is proposed
Detection method, realization that can the be simple and efficient impurity screening in sample to be detected, the fungi that being enriched with need to detect carry out film-making, with
Convenient for observing and detecting.
In order to further embody more preferably detection effect, the present embodiment is based on " goldstandard " method and fungi culture, and addition
The concrete application scene of fluorescent dye, the two Methods for Fungi Detection based on liquid-based tabletting technology proposed to the application are said
It is bright.
Why the fluorescent staining liquid of aforementioned formula is added dropwise, and being is eucaryote because of are as follows: fungi, containing more in cell wall
β-the polysaccharose substance such as chitin and cellulose.And fluorescent whitening agent can be with human body various organization or sample (including scurf, first
Bits, hair, urine, body fluid and vaginal fluid etc.) in cell wall β-polysaccharose substance specificity that may be present combination,
To which mark fluorescent is detected.Various filamentous fungis and saccharomycete can be by fluorescent stainings, including Mycotoruloides, trichophyta
Category, Epidermophyton, Microsporon, Histoplasma, aspergillus etc..The different developmental phases of hypha,hyphae can be glimmering
Light dyeing, facilitates pathological diagnosis.Fluorescence colour affinity is high, and the reaction time only needs several tens of seconds complete, greatly improves
Clinical fungi detection sensitivity and efficiency.(340~400nm), mycelia or spore under the specific exciting light wave band of fluorescence microscope
Son issues bright blue-green fluorescent, and fungi profile and dark background can form sharp contrast contrast, and fungal morphology structure is compared with KOH
Humidity strip method is relatively sharp, is highly convenient for looking for and identify.And fungi fluorescent staining liquid also contains 10%KOH, dissolves cutin, makes group
Knit permeabilization.It is worth noting that, fluorescence colour also can labeled plant cellulose, a small amount of fiber present on air and slide,
It fluoresces under fluorescence microscope.But fibre morphology is generally irregular, and size, thickness, brightness etc. all have very big difference with fungi,
There is no typical fungi tabula and budding structure, by rule of thumb generally can be to avoid false positive the occurrence of.This product is single dose
Type dyeing liquor, it is not only operationally consistent with KOH method step, only need tens of seconds can observation under mirror (first bits need to extend 1~
2min).And it is clear in structure after mycelia, spore fluorescent marker in sample, significantly improve thallus identification.
As shown in figure 3, under a kind of concrete application scene proposed by the embodiment of the present application based on liquid-based tabletting technology
And the flow diagram of the two Methods for Fungi Detection of fluorescent staining technique, this method specifically include two processes:
1, " goldstandard " method and fungi culture.
(1) shift to an earlier date half an hour open superclean bench ultraviolet lamp disinfection sterilizing, the clothing and hand of operator, carry out cleaning and
Disinfection.
(2) configured culture medium is taken out, it is corresponding with gynaecology's sample to be numbered.
(3) it according to reference numeral, opens plate and first drips sterilized physiological saline on it, sampling swab is dipped in
It is uniformly coated on plate after physiological saline, covers culture ware lid, sampling swab is put back in former pipe.
(4) it is inverted culture dish and cultivates 24-72h in 33 DEG C of biochemical cultivation cases, during which observe culture dish colony and grow feelings
Condition.
Fungi sample based on aforesaid operations culture further executes subsequent detection operation.
2, it is operated based on the fungal detection of liquid-based tabletting technology and fluorescent staining technique.
Step S301, collecting sample, and put samples into the sample reagent box equipped with cell-preservation liquid.
Step S302, sample reagent box is placed in shake on oscillator and is mixed.
Step S303, specimen filter and glass slide are installed on liquid-based fungi picture producer.
Step S304, sample liquid all in sample reagent box are added in the specimen filter after having installed.
Step S305, starting liquid-based fungi picture producer carries out automatic film-making.
Step S306, film-making takes out slide after the completion, the dropwise addition 50ul fluorescent staining liquid on slide, quiet after covered
Set 30s.
Step S307, microscopy is carried out under fluorescence microscope.
By the above method treated sample, under fluorescence microscope observed by fungi image such as Fig. 4 to Fig. 9 institute
Show, be described as follows:
As shown in figure 4, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 40 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram of the candida albicans and spore that observe；
As shown in figure 5, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 10 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram for the first bits observed；
As shown in fig. 6, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 10 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram for the scurf observed；
As shown in fig. 7, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 10 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram for the vaginal fluid observed；
As shown in figure 8, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 40 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram of the candida albicans observed；
As shown in figure 9, under a kind of concrete application scene proposed by the embodiment of the present application, by being based on liquid-based film-making skill
Treated that fungi sample passes through fluorescence microscope institute in the case where 10 times of eyepiece, 10 times of object lens for the two Methods for Fungi Detection of art
The imaging schematic diagram of the candida albicans observed.
By applying above-mentioned processing scheme, applicant is after having accumulated the fungal detection data of more than 200 examples, obtained effect
Fruit statistics is as follows:
| Sensitivity | Specificity | Positive predictive value | Negative predictive value | Accuracy | Omission factor |
| 75.00% | 99.30% | 97.83% | 90.38% | 92.08% | 9.62% |
By comparing with detection data of the same sample in other schemes, above-mentioned statistical data has a clear superiority.
Compared with prior art, the advantageous effects for the technical solution that the embodiment of the present application is proposed include:
1, most samples can be enriched with film-making, greatly improves positive rate.
2, tableting operation is extremely simple.Specimen filter and glass slide need to be only installed to liquid-based fungi film-making by operator
On instrument, and tested sample liquid is added, liquid-based fungi picture producer will be automatically performed sample filtering within tens of seconds, waste liquid excludes, sample
The process of this collection and slide film-making improves production effect and working efficiency, reduces manual operation complexity.
3, readily identified fungi, high sensitivity improve the accuracy rate and efficiency of coloration result interpretation.
4, recall rate is high, applied widely.
It will be appreciated by those skilled in the art that the accompanying drawings are only schematic diagrams of a preferred implementation scenario, module in attached drawing or
Process is not necessarily implemented necessary to the embodiment of the present invention.
It will be appreciated by those skilled in the art that the module in device in implement scene can be described according to implement scene into
Row is distributed in the device of implement scene, can also be carried out corresponding change and is located at the one or more dresses for being different from this implement scene
In setting.The module of above-mentioned implement scene can be merged into a module, can also be further split into multiple submodule.
Disclosed above is only the specific implementation scene of the embodiment of the present invention, and still, the embodiment of the present application is not limited to
This, the changes that any person skilled in the art can think of should all fall into the business limitation range of the embodiment of the present application.
Claims (10)
1. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology, which is characterized in that the method specifically includes:
Step 1, collecting sample, and put samples into containing in the cell-preservation liquid to its digestion ingredient of leukorrhea, neutralizing is glutinous
Thick substance；
Step 2 is filtered the cell-preservation liquid comprising sample by filter membrane；
Step 3 retains the target detection thing in filtered fluid by mwco membrane；
The target detection thing that the mwco membrane retains is transferred to glass slide by step 4, carries out film-making；
Step 5, the covered on the glass slide that film-making is completed, and fungal detection is carried out by microscope.
2. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as described in claim 1, which is characterized in that the step
1, it specifically includes:
It puts samples into sample reagent box or test tube equipped with cell-preservation liquid；
Sample reagent box or test tube are placed in shake on oscillator and mixed, thick substances are sufficiently dissolved.
3. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as described in claim 1, which is characterized in that step 2 to
The processing of step 4 is completed especially by ThinPrep cytologic test TCT pelleter；
Wherein, the treatment process of step 2 to step 4 is executed by the TCT pelleter, specifically:
(1) specimen filter is added in fungi sample solution, the specimen filter is made of two bodies up and down being connected, upper body
It is mounted with filter screen, fungi needed for guaranteeing can install mwco membrane by filter screen, lower body, and the fungi observed needed for guaranteeing is blocked
On mwco membrane；
(2) sample filter is installed in the TCT pelleter, the bottom of the sample filter is by the TCT system
Vacuum pump built in piece machine generates negative pressure, and sample solution suction and pumping are passed through the filter screen and the mwco membrane, wherein described true
For impurity in bacterium sample solution by the filter screen filtration, the fungi of required observation is trapped film retention；
(3) the TCT pelleter separates the bi-body structure up and down of the specimen filter, and glass slide is pushed by isolated institute
State the centre of upper and lower bi-body structure；
(4) the TCT pelleter is by mwco membrane being covered on the glass slide from the bottom to top of the fungi containing required observation,
10~20s of sample transfer time is stopped, the fungi of required observation is transferred on the glass slide；
(5) the TCT pelleter releases the glass slide that film-making is completed, and film-making terminates.
4. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claim 1 or 3, which is characterized in that
The filter opening diameter of the filter screen is 0.75-1.5mm.
5. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claim 1 or 3, which is characterized in that
The filter opening diameter of the mwco membrane is 5-20 μm.
6. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as described in claim 1, which is characterized in that the step
Before 5, further includes:
Fluorescent staining liquid is added dropwise on the glass slide, target detection thing is dyed.
7. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claim 6, which is characterized in that the fluorescence
Dyeing liquor is mixed by formula as below according to weight percent:
Fluorescent whitening agent 0.001%, potassium hydroxide 10%, dimethyl sulfoxide 4%, sodium chloride 3.6%, glycerol 10%, ivens
Indigo plant 0.01%, surplus is purified water.
8. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claims 6 or 7, which is characterized in that described
The configuration method of fluorescent staining liquid are as follows:
(1) potassium hydroxide for weighing corresponding weight proportion is added part water and carries out that dissolution heat dissipation is sufficiently stirred, and is cooled to room temperature
Afterwards, dimethyl sulfoxide, sodium chloride and glycerol are sequentially added, is evenly stirred until no lamination, course of dissolution exotherm is natural
It is cooled to room temperature spare, obtains clear, colorless clear liquid, be named as solution A；
(2) fluorescent whitening agent for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains clear, colorless or light
Yellow clear liquid is named as second liquid；
(3) Evans blue for weighing corresponding weight, which is added in the water of part, carries out that dissolution is sufficiently stirred, and obtains bluish violet without precipitate
Particle clear liquid is named as the third liquid；
(4) second liquid is added to solution A and is mixed well, it is ensured that without solid precipitating or floccule in container, obtain clear, colorless or light
Yellow clear liquid is named as fourth liquid；
(5) finally the third liquid is mixed with fourth liquid.
9. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claim 6, which is characterized in that described in institute
Dropwise addition fluorescent staining liquid on glass slide is stated, after dyeing to target detection thing, further includes:
The glass slide that fluorescent staining liquid has been added dropwise is stood 30 seconds.
10. a kind of two Methods for Fungi Detection based on liquid-based tabletting technology as claimed in claim 6, which is characterized in that described
The amount for the fluorescent staining liquid being added dropwise on glass slide, specially 50ul.
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