CN110244049A - Pathogenic Candida detection and identification method - Google Patents
Pathogenic Candida detection and identification method Download PDFInfo
- Publication number
- CN110244049A CN110244049A CN201910700999.3A CN201910700999A CN110244049A CN 110244049 A CN110244049 A CN 110244049A CN 201910700999 A CN201910700999 A CN 201910700999A CN 110244049 A CN110244049 A CN 110244049A
- Authority
- CN
- China
- Prior art keywords
- candida
- detection
- pathogenic
- monoclonal antibody
- test strips
- 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.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention provides a kind of Pathogenic Candida detection and identification methods, specifically: using the specificity olefinic alcohol enzyme that Pathogenic Candida is secreted as detection target molecules, using the monoclonal antibody of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme, marker and coating carrier, detection and identification is carried out to Pathogenic Candida by double antibody sandwich method.The present invention also provides Pathogenic Candida detection and identification cards and preparation method thereof.The present invention is realized to separate Pathogenic Candida for the first time and be identified, using the universal antibody of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata enolase as marker, 4 kinds of bacteriums are enable to detected on same detection and identification card, the trouble that successively single Blind Test determines the infection of which kind of bacterial strain is saved, economy is facilitated.
Description
Technical field
The present invention relates to vitro diagnostic techniques fields, and in particular to Pathogenic Candida detection and identification method.
Background technique
Candida albicans is in oval, and it is a kind of polymorphic fungi that Grain stain is positive, is that human body is most common parasitic true
Bacterium is typically found in normal human mouth, respiratory tract, enteron aisle and vagina, is common conditioned pathogen.Common Pathogenic candida albicans
For Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata, wherein Candida albicans account for about 80%~
90%, Candida tropicalis, Candida parapsilosis and Candida glabrata account for 10%~20% altogether.At this stage, in-vitro diagnosis industry is read
The detection and identification of pearl bacterium relies primarily on sample culture, and blood infection relies primarily on blood culture but verification and measurement ratio is less than 50%, has
Report detects candida albicans with Test paper, but can not identify candida albicans, also there are no commodity production.Due to human body
Class non-pathogenic candida albicans there are many parasitic, whole detection candida albicans can cause false positive because of the interference of non-pathogenic candida albicans
Diagnosis.
Different pathogenic candida albicans is different to antimycotic sensibility, and antibiotic can only be leaned on blindly to treat when not identifying, indulged in
The accidentally state of an illness, Candida still relies on candida albicans colour developing plate culture at this stage, and this method incubation time is long, operation fiber crops
It is tired, cause clinically often to be difficult to early diagnose.Therefore there is an urgent need to a kind of quick, sensitive, efficient methods examines to carry out auxiliary
It is disconnected.
Enolase is also known as 2-phospho-D-glycerate hydrolase, is an expression on candida albicans surface and has high
The molecule of immunogenicity.Enolase encoding gene and protein structure have well-conserved, candida albicans and other several primary yeasts
The similitude of bacterium is 90% or more.Therefore detection enolase can be used to diagnose Pathogenic Candida, and by Candida albicans, heat
Band candida albicans, Candida parapsilosis and Candida glabrata are distinguished.
Summary of the invention
The technical problem to be solved by the present invention is to overcome candida albicans cultivation Testing and appraisal, time-consuming, technology easy to pollute
Defect provides a kind of detection and identification method of candida albicans enolase, utilizes the specific monoclonal of candida albicans enolase
Antibody can timely and accurately judge whether to contain candida albicans enolase in sample to be tested and then judge whether there is beads indirectly
Bacterium infection, and identification and quantitative analysis are carried out to four kinds of common Pathogenic Candidas.
The present invention provides a kind of Pathogenic Candida detection and identification methods.Specific technical solution is as follows:
A kind of Pathogenic Candida detection and identification method, specifically: the specific enol secreted with Pathogenic Candida
Change enzyme as detection target molecules, using the Dan Ke of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme
Grand antibody, marker and coating carrier carry out detection and identification to Pathogenic Candida by double antibody sandwich method.
Further, the Pathogenic Candida includes Candida albicans, Candida tropicalis, Candida parapsilosis and smooth
Candida albicans.
Further, the specificity that Candida albicans is secreted in the monoclonal antibody of the candida albicans specificity olefinic alcohol enzyme
Enolase corresponds to monoclonal antibody specific A, and the specificity olefinic alcohol enzyme of Candida tropicalis secretion corresponds to specific monoclonal
Antibody B, the specificity olefinic alcohol enzyme of Candida parapsilosis secretion correspond to monoclonal antibody specific C, Candida glabrata secretion
Specificity olefinic alcohol enzyme corresponds to monoclonal antibody specific D, single-minded monoclonal antibody and other enolase no cross reactions；
The Candida albicans, Candida tropicalis, Candida parapsilosis and the specificity olefinic alcohol enzyme of Candida glabrata secretion are all corresponding
Monoclonal antibody specific E.
A kind of Pathogenic Candida detection and identification card, including get stuck and test strips, it gets stuck and is divided into two panels up and down, for solid
Determine test strips, upper piece have the observation window and well of hollow out, be bonded on the inside of the well with non-textile mulch；Test strips include
Sample process pad, labeling pad, nitrocellulose filter and blotting paper.
Further, the test strips sample pad is all-glass paper, is dried after being infiltrated with PBST-EDTA buffer
It arrives.
Further, the test strips labeling pad, using marker labeled monoclonal antibody E, even application is in nitric acid
On cellulose membrane, drying is obtained；The marker includes but is not limited to colloidal gold, peroxidase, acridinium ester, alkaline phosphatase
Enzyme, latex beads or fluorescent material.
Further, it is coated with monoclonal antibody A, B, C, D and sheep anti mouse matter respectively on the test strips nitrocellulose filter
Control antibody；It is coated on carrier such as nitrocellulose filter and the pigment different from labelled antibody color is added to coated antibody, make to be coated with
Line on pernitric acid cellulose membrane is clear and easy to see.
A kind of preparation method of above-mentioned Pathogenic Candida detection and identification card, the specific steps are as follows:
Step 1: being with the PBS buffer solution diluted concentration containing 30%BSA respectively by the monoclonal antibody E for marking substance markers
Marking fluid is made in 0.1~1mg/mL, is sprayed on all-glass paper as labeling pad；
Step 2: by monoclonal antibody A, B, C and D respectively with the PBS buffer solution diluted concentration containing 30%BSA be 0.1~
Coating buffer is made in 1mg/mL, and is separately added into the sunset yellow of 0.1mg/mL, and with film instrument is drawn, successively coating is arrived from left to right in order
Nitrocellulose filter is as 4 detection lines.Sheep anti-mouse antibody is diluted to 1mg/mL with same PBS buffer solution, and is added
The sunset yellow of 0.1mg/mL is used and is drawn as nature controlling line in film instrument coating to the nitrocellulose filter on the right side of D line, and nitric acid is fine after drying
It ties up and has 5 lurid bands on plain film；
Step 3: the all-glass paper PBST-EDTA buffer (pH=7.2) of 0.01~0.1mol/mL is infiltrated, leaching
All-glass paper after profit is dried using constant temperature blast drying oven, as sample pad；
Step 4: test strips assemble, sample pad, labeling pad, nitrocellulose filter and blotting paper are successively assembled into PVC
It is cut again on bottom plate；
Step 5: being filtered on the inside of well with the non-woven fabrics that waterproof Instant cement is stained with one layer of 100~300 mesh in the upper cover that gets stuck
Well can be completely covered in cloth, non-woven fabrics；
It gets stuck Step 6: test strips are put into, well is directed at sample pad, and observation window is directed at nitrocellulose in test strips
Film, the compression that gets stuck up and down.
Using the method for above-mentioned Pathogenic Candida detection and identification card identification Pathogenic Candida: taking 50~200 μ L samples
This, is added drop-wise in sample pad, waits 5~15 minutes, and after the completion of sample chromatography, nature controlling line becomes red from yellow, it was demonstrated that this
Detection and identification card is effective, observes tetra- detection line discolorations of ABCD, which detection line, which becomes red, then proves to contain in sample
There are which kind of bacterium or its secretion, A corresponds to Candida albicans, and B corresponds to Candida tropicalis, and C corresponds to Candida parapsilosis, and D is corresponding
Candida glabrata.
Using the method for above-mentioned Pathogenic Candida detection and identification card quantitative detection Pathogenic Candida: using dilution
The enolase of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata is diluted to different concentration, is added
It is read after entering onto reagent strip by marker analyzer, then makes calibration curve, sample to be tested is added and is analyzed in marker
It is back-calculated after being read on instrument.
Other existing detection methods and detection card detection disparity items are all a plurality of using a card, and what the present invention used
In double antibody, one group of specific antibody for candida albicans, another group of monospecific antibody for four kinds of candida albicans hypotypes makes four kinds of bacteriums
Secretion can detected in same test strips, it is convenient and efficient, reduce testing cost.
Due to liquid component complexity of clinically carrying disease germs, such as secretion pH is guided there was only 4 or so, directly detection easily causes
Mistaken diagnosis, in addition vaginal fluid, blood culture liquid and running sore liquid contain a large amount of cell fragment polymer, clot, rotten shape object, without place
Reason is difficult to detect, and enolase concentration, which is lower, after Sterile dilution be easy to cause false negative, Sample pretreatment filtering of the invention and
Buffer system keeps reagent detection sample range wider, is conducive to the pattern detection under varying environment.This method has specificity
Good, the advantages of sensitivity is high, simple and quick and strong antijamming capability.
Pathogenic Candida detection and identification method provided by the invention has below compared with prior art beneficial to effect
Fruit:
(1) present invention realize for the first time to Pathogenic Candida separate identify, using Candida albicans, Candida tropicalis,
The universal antibody of Candida parapsilosis and Candida glabrata enolase as marker, enable 4 kinds of bacteriums in same detection and
It detected on identification card, save the trouble that successively single Blind Test determines the infection of which kind of bacterial strain, facilitate economy.
(2) the drawbacks of being unable to parting the present invention overcomes original enolase polyclonal antibody, using Candida albicans, heat
Monoclonal antibody specific with candida albicans, Candida parapsilosis and Candida glabrata enolase is used as capture coated antibody,
Specifically every kind of bacterium all can detected by double-antibody method detection, and used in patent document CN 105004865A
Competition law compare, accuracy rate is higher.
(3) present invention keeps 4 detection lines and 1 nature controlling line discoloration clear and easy to see, no using the method for coating premix pigment
Easily obscure, and traditional colloidal gold technique only has when developing the color just it is observed that band, is also easy to miss because pillar location deviates
It examines.
(4) present invention passes through the indirect detection bacterium of bacterial secretory object and is aided with the high sensitivity of immune diagnostic technique, can
Viable bacteria infection is detected under the conditions of amount of bacteria is extremely low, this is that traditional isolated culture method cannot achieve.
(5) present invention covers bonding non-woven fabrics in well, can filter the granule foreign that sample contains, be conducive to bacterium
Culture solution, the detections of running sore equal samples, make it be not easy to influence to be influenced chromatographic effect by the sample of particle contamination, and use sample
Pad buffering adjustment sample pH, avoid the pH exceptional sample such as vaginal fluid from impacting testing result, to serum, blood plasma,
Vaginal fluid, urine, sputum, cerebrospinal fluid, skin swab sample, throat swab sample, hospital environment monitor sample or bacterium
Culture solution equal samples have preferable detection effect, improve Detection accuracy.
Detailed description of the invention
Fig. 1 is Pathogenic Candida detection and identification card structure schematic diagram of the present invention, wherein the sample application zone 1-, 2-PVC bottom plate,
3- sample pad, 4- labeling pad, 5- nitrocellulose filter, 6- detection line A, 7- detection line B, 8- detection line C, 9- detection line D,
10- nature controlling line, 11- blotting paper.
Fig. 2 is the upper cover full face that gets stuck.
Fig. 3 is the upper cover reverse side photo that gets stuck.
Fig. 4 is the lower cover photo that gets stuck.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with preferred implementation of the invention
Example, and attached drawing is cooperated to be described in detail.
Embodiment 1
A kind of Pathogenic Candida detection and identification method, specifically: the specific enol secreted with Pathogenic Candida
Change enzyme as detection target molecules, using the Dan Ke of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme
Grand antibody, marker and coating carrier carry out detection and identification to Pathogenic Candida by double antibody sandwich method.
Further, the Pathogenic Candida includes Candida albicans, Candida tropicalis, Candida parapsilosis and smooth
Candida albicans.
Further, the specificity that Candida albicans is secreted in the monoclonal antibody of the candida albicans specificity olefinic alcohol enzyme
Enolase corresponds to monoclonal antibody specific A, and the specificity olefinic alcohol enzyme of Candida tropicalis secretion corresponds to specific monoclonal
Antibody B, the specificity olefinic alcohol enzyme of Candida parapsilosis secretion correspond to monoclonal antibody specific C, Candida glabrata secretion
Specificity olefinic alcohol enzyme corresponds to monoclonal antibody specific D, single-minded monoclonal antibody and other enolase no cross reactions；
The Candida albicans, Candida tropicalis, Candida parapsilosis and the specificity olefinic alcohol enzyme of Candida glabrata secretion are all corresponding
Monoclonal antibody specific E.
A kind of Pathogenic Candida detection and identification card, including get stuck and test strips, it gets stuck and is divided into two panels up and down, for solid
Determine test strips, upper piece have the observation window and well of hollow out, be bonded on the inside of the well with non-textile mulch；Test strips include
Sample process pad, labeling pad, nitrocellulose filter and blotting paper.
Further, the test strips sample pad is all-glass paper, is dried after being infiltrated with PBST-EDTA buffer
It arrives.
Further, the test strips labeling pad, using colloid gold label monoclonal antibody E, even application is in nitric acid
On cellulose membrane, drying is obtained；
Further, it is coated with monoclonal antibody A, B, C, D and sheep anti mouse matter respectively on the test strips nitrocellulose filter
Control antibody；It is coated on carrier such as nitrocellulose filter and the pigment different from labelled antibody color is added to coated antibody, make to be coated with
Line on pernitric acid cellulose membrane is clear and easy to see.
A kind of preparation method of above-mentioned Pathogenic Candida detection and identification card, the specific steps are as follows:
Step 1: being with the PBS buffer solution diluted concentration containing 30%BSA respectively by the monoclonal antibody E of colloid gold label
Golden standard liquid is made in 0.1mg/mL, and 5 μ L/cm of metal spraying amount is sprayed on all-glass paper as labeling pad；
Step 2: with the PBS buffer solution diluted concentration containing 30%BSA being respectively 0.1mg/ by monoclonal antibody A, B, C and D
Coating buffer is made in mL, and is separately added into the sunset yellow of 0.1mg/mL, and with film instrument is drawn, successively coating arrives nitric acid from left to right in order
Cellulose membrane is as 4 detection lines；Sheep anti-mouse antibody is diluted to 1mg/mL with same PBS buffer solution, and 0.1mg/ is added
The sunset yellow of mL is used and is drawn as nature controlling line in film instrument coating to the nitrocellulose filter on the right side of D line, nitrocellulose filter after drying
On have 5 lurid bands；
Step 3: the all-glass paper PBST-EDTA buffer (pH=7.2) of 0.01mol/mL is infiltrated, after infiltration
All-glass paper dried using constant temperature blast drying oven, as sample pad；
Step 4: test strips assemble, according to shown in attached drawing 1 successively by sample pad, labeling pad, nitrocellulose filter and suction
Water paper is assembled on PVC bottom plate and cuts again；
Step 5: the non-woven fabrics filter cloth of one layer of 100 mesh, nothing are stained on the inside of well with waterproof Instant cement in the upper cover that gets stuck
Well can be completely covered in woven fabric；
It gets stuck Step 6: test strips are put into, well alignment sample pad proves, it is fine that observation window is directed at nitric acid in test strips
Plain film is tieed up, the compression that gets stuck up and down.
Embodiment 2
A kind of Pathogenic Candida detection and identification method, specifically: the specific enol secreted with Pathogenic Candida
Change enzyme as detection target molecules, using the Dan Ke of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme
Grand antibody, marker and coating carrier carry out detection and identification to Pathogenic Candida by double antibody sandwich method.
Further, the Pathogenic Candida includes Candida albicans, Candida tropicalis, Candida parapsilosis and smooth
Candida albicans.
Further, the specificity that Candida albicans is secreted in the monoclonal antibody of the candida albicans specificity olefinic alcohol enzyme
Enolase corresponds to monoclonal antibody specific A, and the specificity olefinic alcohol enzyme of Candida tropicalis secretion corresponds to specific monoclonal
Antibody B, the specificity olefinic alcohol enzyme of Candida parapsilosis secretion correspond to monoclonal antibody specific C, Candida glabrata secretion
Specificity olefinic alcohol enzyme corresponds to monoclonal antibody specific D, single-minded monoclonal antibody and other enolase no cross reactions；
The Candida albicans, Candida tropicalis, Candida parapsilosis and the specificity olefinic alcohol enzyme of Candida glabrata secretion are all corresponding
Monoclonal antibody specific E.
A kind of Pathogenic Candida detection and identification card, including get stuck and test strips, it gets stuck and is divided into two panels up and down, for solid
Determine test strips, upper piece have the observation window and well of hollow out, be bonded on the inside of the well with non-textile mulch；Test strips include
Sample process pad, labeling pad, nitrocellulose filter and blotting paper.
Further, the test strips sample pad is all-glass paper, is dried after being infiltrated with PBST-EDTA buffer
It arrives.
Further, the test strips labeling pad, using marker labeled monoclonal antibody E, even application is in nitric acid
On nitrocellulose filter, drying is obtained；The marker can be, but not limited to, peroxidase, acridinium ester, alkaline phosphatase
Enzyme, latex beads or fluorescent material.
Further, it is coated with monoclonal antibody A, B, C, D and sheep anti mouse matter respectively on the test strips nitrocellulose filter
Control antibody；It is coated on carrier such as nitrocellulose filter and the pigment different from labelled antibody color is added to coated antibody, make to be coated with
Line on pernitric acid cellulose membrane is clear and easy to see.
A kind of preparation method of above-mentioned Pathogenic Candida detection and identification card, the specific steps are as follows:
Step 1: being with the PBS buffer solution diluted concentration containing 30%BSA respectively by the monoclonal antibody E of colloid gold label
Golden standard liquid is made in 0.5mg/mL, and 5 μ L/cm of metal spraying amount is sprayed on all-glass paper as labeling pad；
Step 2: with the PBS buffer solution diluted concentration containing 30%BSA being respectively 0.5mg/ by monoclonal antibody A, B, C and D
Coating buffer is made in mL, and is separately added into the sunset yellow of 0.1mg/mL, and with film instrument is drawn, successively coating arrives nitric acid from left to right in order
Cellulose membrane is as 4 detection lines；Sheep anti-mouse antibody is diluted to 1mg/mL with same PBS buffer solution, and 0.1mg/ is added
The sunset yellow of mL is used and is drawn as nature controlling line in film instrument coating to the nitrocellulose filter on the right side of D line, nitrocellulose filter after drying
On have 5 lurid bands；
Step 3: the all-glass paper PBST-EDTA buffer (pH=7.2) of 0.05mol/mL is infiltrated, after infiltration
All-glass paper dried using constant temperature blast drying oven, as sample pad；
Step 4: test strips assemble, according to shown in attached drawing 1 successively by sample pad, labeling pad, nitrocellulose filter and suction
Water paper is assembled on PVC bottom plate and cuts again；
Step 5: the non-woven fabrics filter cloth of one layer of 200 mesh, nothing are stained on the inside of well with waterproof Instant cement in the upper cover that gets stuck
Well can be completely covered in woven fabric；
It gets stuck Step 6: test strips are put into, well alignment sample pad proves, it is fine that observation window is directed at nitric acid in test strips
Plain film is tieed up, the compression that gets stuck up and down.
Embodiment 3
A kind of Pathogenic Candida detection and identification method, specifically: the specific enol secreted with Pathogenic Candida
Change enzyme as detection target molecules, using the Dan Ke of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme
Grand antibody, marker and coating carrier carry out detection and identification to Pathogenic Candida by double antibody sandwich method.
Further, the Pathogenic Candida includes Candida albicans, Candida tropicalis, Candida parapsilosis and smooth
Candida albicans.
Further, the specificity that Candida albicans is secreted in the monoclonal antibody of the candida albicans specificity olefinic alcohol enzyme
Enolase corresponds to monoclonal antibody specific A, and the specificity olefinic alcohol enzyme of Candida tropicalis secretion corresponds to specific monoclonal
Antibody B, the specificity olefinic alcohol enzyme of Candida parapsilosis secretion correspond to monoclonal antibody specific C, Candida glabrata secretion
Specificity olefinic alcohol enzyme corresponds to monoclonal antibody specific D, single-minded monoclonal antibody and other enolase no cross reactions；
The Candida albicans, Candida tropicalis, Candida parapsilosis and the specificity olefinic alcohol enzyme of Candida glabrata secretion are all corresponding
Monoclonal antibody specific E.
A kind of Pathogenic Candida detection and identification card, including get stuck and test strips, it gets stuck and is divided into two panels up and down, for solid
Determine test strips, upper piece have the observation window and well of hollow out, be bonded on the inside of the well with non-textile mulch；Test strips include
Sample process pad, labeling pad, nitrocellulose filter and blotting paper.
Further, the test strips sample pad is all-glass paper, is dried after being infiltrated with PBST-EDTA buffer
It arrives.
Further, the test strips labeling pad, using marker labeled monoclonal antibody E, even application is in nitric acid
On nitrocellulose filter, drying is obtained；The marker can be, but not limited to, peroxidase, acridinium ester, alkaline phosphatase
Enzyme, latex beads or fluorescent material.
Further, it is coated with monoclonal antibody A, B, C, D and sheep anti mouse matter respectively on the test strips nitrocellulose filter
Control antibody；It is coated on carrier such as nitrocellulose filter and the pigment different from labelled antibody color is added to coated antibody, make to be coated with
Line on pernitric acid cellulose membrane is clear and easy to see.
A kind of preparation method of above-mentioned Pathogenic Candida detection and identification card, the specific steps are as follows:
Step 1: being with the PBS buffer solution diluted concentration containing 30%BSA respectively by the monoclonal antibody E of colloid gold label
Golden standard liquid is made in 1mg/mL, and 5 μ L/cm of metal spraying amount is sprayed on all-glass paper as labeling pad；
Step 2: with the PBS buffer solution diluted concentration containing 30%BSA being respectively 1mg/mL by monoclonal antibody A, B, C and D
Coating buffer is made, and is separately added into the sunset yellow of 0.1mg/mL, successively coating is fine to nitric acid from left to right with film instrument is drawn in order
Plain film is tieed up as 4 detection lines；Sheep anti-mouse antibody is diluted to 1mg/mL with same PBS buffer solution, and 0.1mg/mL is added
Sunset yellow, use draw film instrument coating on the nitrocellulose filter on the right side of D line as nature controlling line, after drying on nitrocellulose filter
There are 5 lurid bands；
Step 3: the all-glass paper PBST-EDTA buffer (pH=7.2) of 0.1mol/mL is infiltrated, after infiltration
All-glass paper is dried using constant temperature blast drying oven, as sample pad；
Step 4: test strips assemble, according to shown in attached drawing 1 successively by sample pad, labeling pad, nitrocellulose filter and suction
Water paper is assembled on PVC bottom plate and cuts again；
Step 5: the non-woven fabrics filter cloth of one layer of 300 mesh, nothing are stained on the inside of well with waterproof Instant cement in the upper cover that gets stuck
Well can be completely covered in woven fabric；
It gets stuck Step 6: test strips are put into, well alignment sample pad proves, it is fine that observation window is directed at nitric acid in test strips
Plain film is tieed up, the compression that gets stuck up and down.
Sensitivity and specificity test
Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata are existed using liquid culture medium respectively
Bacteria suspension is ground after culture 48 hours and is crushed by 36 DEG C of progress pure cultures, and gradient centrifugation removes impurity after filtering, uses ammonium sulfate
Method is settled out enolase, is condensed into enolase solution after sieving purification column purification process using dextran molecule, and measure it
Corresponding concentration.
The enolase solution of four kinds of candida albicans is diluted to 0.1ng/ml, 100ng/ml and 25ng/ using physiological saline
Tri- concentration of ml detect in test strips, and the Candida albicans enolase of 0.1ng/ml, 0.15ng/ml and 0.2ng/ml is added
Test strips when only have first and Article 5 band become red, it is other all non-discolouring；Be added 0.1ng/ml, 0.15ng/ml and
Only have when the test strips of the Candida tropicalis enolase of 0.2ng/ml second and Article 5 band become red, it is other all constant
Color；Be added 0.1ng/ml, 0.15ng/ml and 0.2ng/ml Candida parapsilosis enolase test strips when only have third and
Article 5 band becomes red, other all non-discolouring；The Candida glabrata alkene of 0.1ng/ml, 0.15ng/ml and 0.2ng/ml is added
Only have when the test strips of Enolase the 4th and Article 5 band become red, it is other all non-discolouring.
48 hours inoculums of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata are used
Test strips detection, test strip on the other side and Quality Control band become red；Yeast bacteria culture fluid, Escherichia coli are cultivated
Liquid, staphylococcus aureus culture solution and P. aeruginosa bacteria culture fluid are detected with test strips, in addition to Quality Control band becomes red
Other bands are all non-discolouring.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, ability
Other modifications or equivalent replacement that domain those of ordinary skill makes technical solution of the present invention, without departing from skill of the present invention
The spirit and scope of art scheme, are intended to be within the scope of the claims of the invention.
Claims (9)
1. a kind of Pathogenic Candida detection and identification method, which is characterized in that the specific alkene secreted with Pathogenic Candida
Enolase is as detection target molecules, using the list of genetic and cell engineering technology production candida albicans specificity olefinic alcohol enzyme
Clonal antibody, marker and coating carrier carry out detection and identification to Pathogenic Candida by double antibody sandwich method.
2. Pathogenic Candida detection and identification method according to claim 1, which is characterized in that the pathogenic beads
Bacterium bag includes Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata.
3. Pathogenic Candida detection and identification method according to claim 2, which is characterized in that the candida albicans is special
Property enolase monoclonal antibody in Candida albicans secretion specificity olefinic alcohol enzyme correspond to monoclonal antibody specific A,
The specificity olefinic alcohol enzyme of Candida tropicalis secretion corresponds to monoclonal antibody specific B, the specificity of Candida parapsilosis secretion
Enolase corresponds to monoclonal antibody specific C, and the specificity olefinic alcohol enzyme of Candida glabrata secretion corresponds to specific monoclonal
Antibody D, single-minded monoclonal antibody and other enolase no cross reactions；The Candida albicans, is closely put down at Candida tropicalis
The specificity olefinic alcohol enzyme of sliding candida albicans and Candida glabrata secretion all corresponds to monoclonal antibody specific E.
4. a kind of Pathogenic Candida detection of Pathogenic Candida detection and identification method any according to claim 1 ~ 3
And identification card, which is characterized in that including getting stuck and test strips, get stuck and be divided into two panels up and down, for fixing test strips, upper piece engrave
Empty observation window and well, well inside non-woven fabrics filter cloth covering bonding；Test strips include sample process pad, mark
Remember object pad, nitrocellulose filter and blotting paper.
5. Pathogenic Candida detection and identification card according to claim 4, which is characterized in that the test strips sample pad
For all-glass paper, obtained with being dried after the infiltration of PBST-EDTA buffer.
6. Pathogenic Candida detection and identification card according to claim 4, which is characterized in that the non-woven fabrics filter cloth mesh
Number is 100~300.
7. Pathogenic Candida detection and identification card according to claim 4, which is characterized in that the test strips marker
Pad, using marker labeled monoclonal antibody E, on nitrocellulose filter, drying obtains even application；The marker includes
But it is not limited to colloidal gold, peroxidase, acridinium ester, alkaline phosphatase, latex beads or fluorescent material.
8. Pathogenic Candida detection and identification card according to claim 4, which is characterized in that the test strips nitric acid is fine
It ties up and is coated with monoclonal antibody A, B, C, D and sheep anti mouse Quality Control antibody respectively on plain film；Be coated on carrier such as nitrocellulose filter to
The pigment different from labelled antibody color is added in coated antibody, keeps the line being coated on pernitric acid cellulose membrane clear and easy to see.
9. a kind of preparation method according to any Pathogenic Candida detection and identification card of claim 4 ~ 8, feature
It is, the specific steps are as follows:
Step 1: will mark the monoclonal antibody E of substance markers is respectively 0.1~1 with the PBS buffer solution diluted concentration containing 30%BSA
Marking fluid is made in mg/mL, is sprayed on all-glass paper as labeling pad；
Step 2: by monoclonal antibody A, B, C and D respectively with the PBS buffer solution diluted concentration containing 30%BSA be 0.1~1 mg/
Coating buffer is made in mL, and is separately added into the sunset yellow of 0.1 mg/mL, and with film instrument is drawn, successively coating arrives nitric acid from left to right in order
Cellulose membrane is as 4 detection lines；Sheep anti-mouse antibody is diluted to 1mg/mL with same PBS buffer solution, and 0.1 mg/ is added
The sunset yellow of mL is used and is drawn as nature controlling line in film instrument coating to the nitrocellulose filter on the right side of D line, nitrocellulose filter after drying
On have 5 lurid bands；
Step 3: the all-glass paper PBST-EDTA buffer (pH=7.2) of 0.01~0.1 mol/mL is infiltrated, after infiltration
All-glass paper dried using constant temperature blast drying oven, as sample pad；
Step 4: test strips assemble, sample pad, labeling pad, nitrocellulose filter and blotting paper are successively assembled into PVC bottom plate
On cut again；
Step 5: being stained with layer of non-woven fabric filter cloth with waterproof Instant cement on the inside of well, non-woven fabrics can be complete in the upper cover that gets stuck
Cover well；
It getting stuck Step 6: test strips are put into, well is directed at sample pad, and observation window is directed at nitrocellulose filter in test strips,
The compression that gets stuck up and down.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910700999.3A CN110244049B (en) | 2019-07-31 | 2019-07-31 | Pathogenic candida detection and identification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910700999.3A CN110244049B (en) | 2019-07-31 | 2019-07-31 | Pathogenic candida detection and identification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110244049A true CN110244049A (en) | 2019-09-17 |
| CN110244049B CN110244049B (en) | 2022-05-06 |
Family
ID=67893903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910700999.3A Active CN110244049B (en) | 2019-07-31 | 2019-07-31 | Pathogenic candida detection and identification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110244049B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
| CN112730830A (en) * | 2021-01-27 | 2021-04-30 | 成都微瑞生物科技有限公司 | African swine fever antigen double-hole rapid detection card and detection method |
| CN113791216A (en) * | 2021-08-31 | 2021-12-14 | 石家庄和亚生物技术有限公司 | ELISA detection method for detecting human serum candida glabrata enolase IgG antibody |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0940678A1 (en) * | 1998-03-02 | 1999-09-08 | Akzo Nobel N.V. | Analytical test device |
| JP2000121640A (en) * | 1998-10-09 | 2000-04-28 | Nitto Denko Corp | Testing method and testing piece for plural tested substance |
| CN1945336A (en) * | 2006-10-26 | 2007-04-11 | 兰州大学 | Detecting test paper for Welch's clostridium disease and its pathogenic strain |
| US20070134747A1 (en) * | 2005-12-14 | 2007-06-14 | Kimberly-Clark Worldwide, Inc. | Detection of secreted lipase proteins from Candida species |
| CN101638688A (en) * | 2009-09-04 | 2010-02-03 | 刘伟 | Special probe and gene chip for identifying pathogenic Candida |
| CN103204929A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for candida albicans polyclonal antibody |
| US20140004538A1 (en) * | 2012-06-29 | 2014-01-02 | Bio-Rad Innovations | Method for diagnosing invasive candida infections |
| CN103616514A (en) * | 2013-12-11 | 2014-03-05 | 广西大学 | Rapid diagnosis test strip of cow mastitis candida albicans |
| CN104991060A (en) * | 2015-06-01 | 2015-10-21 | 上海凯创生物技术有限公司 | Canidia albicans antigen colloidal gold detection kit |
| CN105004865A (en) * | 2015-07-21 | 2015-10-28 | 贺政新 | Candida albicans enolase antibody test paper |
| CN106124775A (en) * | 2016-06-30 | 2016-11-16 | 广州瑞博奥生物科技有限公司 | Mouse antibodies hypotype fast typing test kit and preparation method thereof |
| CN107389940A (en) * | 2017-08-23 | 2017-11-24 | 潍坊市康华生物技术有限公司 | A kind of vaginitis multi-link detection reagent kit and preparation method thereof |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
-
2019
- 2019-07-31 CN CN201910700999.3A patent/CN110244049B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0940678A1 (en) * | 1998-03-02 | 1999-09-08 | Akzo Nobel N.V. | Analytical test device |
| JP2000121640A (en) * | 1998-10-09 | 2000-04-28 | Nitto Denko Corp | Testing method and testing piece for plural tested substance |
| US20070134747A1 (en) * | 2005-12-14 | 2007-06-14 | Kimberly-Clark Worldwide, Inc. | Detection of secreted lipase proteins from Candida species |
| CN1945336A (en) * | 2006-10-26 | 2007-04-11 | 兰州大学 | Detecting test paper for Welch's clostridium disease and its pathogenic strain |
| CN101638688A (en) * | 2009-09-04 | 2010-02-03 | 刘伟 | Special probe and gene chip for identifying pathogenic Candida |
| CN103204929A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for candida albicans polyclonal antibody |
| US20140004538A1 (en) * | 2012-06-29 | 2014-01-02 | Bio-Rad Innovations | Method for diagnosing invasive candida infections |
| CN103616514A (en) * | 2013-12-11 | 2014-03-05 | 广西大学 | Rapid diagnosis test strip of cow mastitis candida albicans |
| CN104991060A (en) * | 2015-06-01 | 2015-10-21 | 上海凯创生物技术有限公司 | Canidia albicans antigen colloidal gold detection kit |
| CN105004865A (en) * | 2015-07-21 | 2015-10-28 | 贺政新 | Candida albicans enolase antibody test paper |
| CN106124775A (en) * | 2016-06-30 | 2016-11-16 | 广州瑞博奥生物科技有限公司 | Mouse antibodies hypotype fast typing test kit and preparation method thereof |
| CN107389940A (en) * | 2017-08-23 | 2017-11-24 | 潍坊市康华生物技术有限公司 | A kind of vaginitis multi-link detection reagent kit and preparation method thereof |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
Non-Patent Citations (2)
| Title |
|---|
| 王廷廷等: "白念珠菌烯醇化酶的研究进展", 《临床检验杂志》 * |
| 胡毓安等: "白念珠菌烯醇化酶双抗体夹心ELISA检测方法的建立", 《临床检验杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
| CN112730830A (en) * | 2021-01-27 | 2021-04-30 | 成都微瑞生物科技有限公司 | African swine fever antigen double-hole rapid detection card and detection method |
| CN112730830B (en) * | 2021-01-27 | 2023-12-29 | 杭州大瑞科技有限公司 | African swine fever antigen double-hole rapid detection card and detection method |
| CN113791216A (en) * | 2021-08-31 | 2021-12-14 | 石家庄和亚生物技术有限公司 | ELISA detection method for detecting human serum candida glabrata enolase IgG antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110244049B (en) | 2022-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110244049A (en) | Pathogenic Candida detection and identification method | |
| CN101925819B (en) | Immunodetection assay for mycobacterium tuberculosis complex | |
| CN101587043A (en) | Integrated method for enriching and detecting rare cell in biological fluid sample | |
| CN111273001A (en) | System for rapidly detecting new coronavirus 2019-nCoV in blood sample and preparation method thereof | |
| CN107765002B (en) | Colloidal gold immunochromatography test strip and preparation method and application thereof | |
| CN108169492A (en) | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application | |
| CN106053794A (en) | Reagent card for accurately detecting test object, kit and application | |
| CN112014565B (en) | Test strip and kit for detecting trichophyton rubrum and preparation method of test strip | |
| CN111398593A (en) | Rapid combined detection card and preparation method and application thereof | |
| AU672138B2 (en) | Method and apparatus for determining the sensitivity of MAI (mycobacterium avium-intracellulare) to different antibiotics and dosages thereof | |
| CN110308274A (en) | A kind of kit and preparation method thereof detecting listeria monocytogenes | |
| CN104076140A (en) | Construction and application of chemiluminiscence-colloidal gold immunochromatography test paper | |
| CN109030823A (en) | A kind of colloidal gold strip and its preparation method and application detecting tobacco ralstonia solanacearum | |
| CN102043058A (en) | Detection kit of acetyl amantadine for predicting tumors | |
| CN110133255A (en) | A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection | |
| CN107132353A (en) | A kind of streptococcic detection kit of B races and preparation method thereof | |
| CN110988365A (en) | Quantitative immune colloidal gold detection card and kit for cystatin C, microalbuminuria and urinary creatinine | |
| CN101666802A (en) | Colloidal gold immuno-chromatographic assay for quantitatively detecting staphylococcal enterotixn B and gold-immuochromatography assay test paper | |
| CN105092856A (en) | Clinic detection kit of multiple biomarkers in ischemic cerebral apoplexy | |
| EP3640644B1 (en) | Target marker gp73 for detecting steatohepatitis and detection application method | |
| CN106526166A (en) | Rapid detection of lean meat powder in pork | |
| CN101949936A (en) | Colloidal gold test strip for detecting antibodies against akabane virus and preparation method thereof | |
| US7241626B2 (en) | Isolation and confirmation of analytes from test devices | |
| CN1963518A (en) | Test paper bar for testing colloidal gold of 0157: H7 bacillus coli antigen | |
| Gray et al. | Laboratory diagnosis of systemic fungal diseases |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |