CN110244049B - Pathogenic candida detection and identification method - Google Patents
Pathogenic candida detection and identification method Download PDFInfo
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Abstract
The invention provides a method for detecting and identifying pathogenic candida, which specifically comprises the following steps: specific enolase secreted by pathogenic candida is used as a detection target molecule, a monoclonal antibody, a marker and a coating carrier of the candida specific enolase are produced by adopting genetic engineering and cell engineering technologies, and the pathogenic candida is detected and identified by a double-antibody sandwich method. The invention also provides a pathogenic candida detection and identification card and a preparation method thereof. The invention realizes the separation and identification of pathogenic candida for the first time, and adopts the common antibodies of candida albicans, candida tropicalis, candida parapsilosis and candida glabrata enolase as markers, so that 4 bacteria can be detected on the same detection and identification card, the trouble of determining which strain is infected by single blind detection in sequence is saved, and the method is convenient and economic.
Description
Technical Field
The invention relates to the technical field of in vitro diagnosis, in particular to a method for detecting and identifying pathogenic candida.
Background
The candida is oval and gram-positive, is a polymorphic fungus, is the most common parasitic fungus of a human body, is usually present in the oral cavity, the respiratory tract, the intestinal tract and the vagina of a normal human body, and is a common conditional pathogen. The common pathogenic candida is candida albicans, candida tropicalis, candida parapsilosis and candida glabrata, wherein the candida albicans accounts for about 80-90%, and the candida tropicalis, candida parapsilosis and candida glabrata account for 10-20%. At the present stage, the detection and identification of candida in the in vitro diagnosis industry mainly depend on sample culture, blood infection mainly depends on blood culture but the detection rate is less than 50%, and the candida is detected by using a test paper in some reports, but the candida cannot be identified, and commercial production is not seen. Because the human body is parasitized with a plurality of non-pathogenic candida, the overall detection of the candida can cause false positive diagnosis due to the interference of the non-pathogenic candida.
The susceptibility of different pathogenic candida to epiphyte is different, blind treatment can only be carried out by antibiotics when the candida are not identified, the disease condition is delayed, the candida identification at the present stage still depends on candida chromogenic plate culture, the method has long culture time and troublesome operation, and the clinical early diagnosis is often difficult. Therefore, a fast, sensitive and efficient method for auxiliary diagnosis is urgently needed.
Enolase, also known as 2-phospho-D-glycerate hydrolase, is a molecule expressed on the surface of Candida and has very high immunogenicity. The enolase coding gene and the protein structure have high conservation, and the similarity of candida and other yeasts is more than 90%. Thus, detection of enolase can be used to diagnose pathogenic candida and distinguish candida albicans, candida tropicalis, candida parapsilosis, and candida glabrata.
Disclosure of Invention
The invention aims to solve the technical problems of long time consumption and easy pollution in detection and identification by a candida culture method, and provides a method for detecting and identifying candida enolase.
The invention provides a method for detecting and identifying pathogenic candida. The specific technical scheme is as follows:
a method for detecting and identifying pathogenic candida comprises the following steps: specific enolase secreted by pathogenic candida is used as a detection target molecule, a monoclonal antibody, a marker and a coating carrier of the candida specific enolase are produced by adopting genetic engineering and cell engineering technologies, and the pathogenic candida is detected and identified by a double-antibody sandwich method.
Further, the pathogenic candida includes candida albicans, candida tropicalis, candida parapsilosis, and candida glabrata.
Furthermore, in the candida specific enolase monoclonal antibodies, the specificity enolase secreted by candida albicans corresponds to the specificity monoclonal antibody A, the specificity enolase secreted by candida tropicalis corresponds to the specificity monoclonal antibody B, the specificity enolase secreted by candida parapsilosis corresponds to the specificity monoclonal antibody C, the specificity enolase secreted by candida glabrata corresponds to the specificity monoclonal antibody D, and the specific monoclonal antibody has no cross reaction with other enolases; the specificity enolase secreted by the candida albicans, candida tropicalis, candida parapsilosis and candida glabrata corresponds to the specificity monoclonal antibody E.
A pathogenic candida detection and identification card comprises a card shell and a test strip, wherein the card shell is divided into an upper piece and a lower piece and used for fixing the test strip, the upper piece is provided with a hollowed observation window and a sample adding hole, and the inner side of the sample adding hole is covered and adhered by non-woven fabric; the test strip comprises a sample treatment pad, a marker pad, a nitrocellulose membrane and absorbent paper.
Furthermore, the test strip sample pad is made of glass fiber paper, and is obtained by soaking the test strip sample pad in PBST-EDTA buffer solution and then drying the test strip sample pad.
Further, the test paper strip marker pad is obtained by uniformly spraying a marker-labeled monoclonal antibody E on a nitrocellulose membrane and drying; the label includes, but is not limited to, colloidal gold, peroxidase, acridinium ester, alkaline phosphatase, latex microspheres, or fluorescent substance.
Further, the test strip cellulose nitrate membrane is respectively coated with a monoclonal antibody A, B, C, D and a goat anti-mouse quality control antibody; the pigment with the color different from that of the marked antibody is added into the antibody to be coated on the coating carrier, such as the nitrocellulose membrane, so that the lines on the coated nitrocellulose membrane are clear and easy to see.
A preparation method of the pathogenic candida detection and identification card comprises the following specific steps:
step one, respectively diluting a monoclonal antibody E marked by a marker with PBS (phosphate buffer solution) containing 30% BSA (bovine serum albumin) to 0.1-1 mg/mL to prepare a marking solution, and spraying the marking solution on glass fiber paper to serve as a marker pad;
and step two, respectively diluting the monoclonal antibodies A, B, C and D with PBS (phosphate buffer solution) containing 30% BSA (bovine serum albumin) to 0.1-1 mg/mL to prepare coating solutions, respectively adding 0.1mg/mL sunset yellow, and sequentially coating the coating solutions on the nitrocellulose membrane from left to right by using a membrane scribing instrument to serve as 4 detection lines. Diluting goat anti-mouse antibody to 1mg/mL with the same PBS buffer solution, adding 0.1mg/mL sunset yellow, coating on the nitrocellulose membrane on the right side of the D line by using a membrane scribing instrument to serve as a quality control line, and drying to obtain 5 light yellow strips on the nitrocellulose membrane;
step three, soaking the glass fiber paper with 0.01-0.1 mol/mL PBST-EDTA buffer solution (pH is 7.2), and drying the soaked glass fiber paper by using a constant-temperature blast drying oven to obtain a sample pad;
step four, assembling the test strip, namely assembling the sample pad, the marker pad, the nitrocellulose membrane and the absorbent paper on a PVC (polyvinyl chloride) base plate in sequence and then cutting the test strip;
step five, covering the upper cover of the clamping shell, and adhering a layer of 100-300-mesh non-woven fabric filter cloth on the inner side of the sample adding hole by using waterproof quick-drying adhesive, wherein the non-woven fabric can completely cover the sample adding hole;
and step six, placing the test strip into the card shell, aligning the sample adding hole with the sample pad, aligning the observation window with the nitrocellulose membrane on the test strip, and pressing the upper card shell and the lower card shell tightly.
The method for identifying the pathogenic candida by applying the pathogenic candida detection and identification card comprises the following steps: and (3) dropwise adding a 50-200 mu L sample onto the sample pad, waiting for 5-15 minutes, changing the quality control line from yellow to red after the sample chromatography is finished, proving that the detection and identification card is effective, observing the color change condition of the four detection lines of the ABCD, and proving that the sample contains any bacteria or secretion thereof when the detection line is red, wherein A corresponds to Candida albicans, B corresponds to Candida tropicalis, C corresponds to Candida parapsilosis, and D corresponds to Candida glabrata.
The method for quantitatively detecting the pathogenic candida by applying the pathogenic candida detection and identification card comprises the following steps: diluting enolase of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata to different concentrations by using a diluent, adding the diluted enolase to a reagent strip, reading by a marker analyzer, making a calibration curve, adding a sample to be detected, reading on the marker analyzer, and calculating.
In the double antibodies adopted by the invention, one group is a specificity antibody of candida, and the other group is a single antibody of four candida subtypes, so that secretions of four bacteria can be detected on the same test strip, the detection is convenient and quick, and the detection cost is reduced.
Because the bacteria-carrying liquid has complex components clinically, if the pH of the induced secretion is only about 4, misdiagnosis is easily caused by direct detection, in addition, the vaginal secretion, blood culture fluid and abscess fluid contain a large amount of cell debris polymers, blood clots and chyliform substances, the detection is difficult without treatment, and the low concentration of the enolase after aseptic dilution easily causes false negative. The method has the advantages of good specificity, high sensitivity, simple and rapid operation and strong anti-interference capability.
Compared with the prior art, the pathogenic candida detection and identification method provided by the invention has the following beneficial effects:
(1) the invention realizes the separation and identification of pathogenic candida for the first time, and adopts the common antibodies of candida albicans, candida tropicalis, candida parapsilosis and candida glabrata enolase as markers, so that 4 bacteria can be detected on the same detection and identification card, the trouble of determining which strain is infected by single blind detection in sequence is saved, and the method is convenient and economic.
(2) The invention overcomes the defect that the original enolase polyclonal antibody can not be typed, adopts the specific monoclonal antibodies of candida albicans, candida tropicalis, candida parapsilosis and candida glabrata enolase as the capture coating antibody, can specifically detect each bacterium by the double antibody sandwich method, and has higher accuracy compared with the competition method used in the patent document CN 105004865A.
(3) The invention adopts the method of coating the premixed pigment, so that the color change of 4 detection lines and 1 quality control line is clear and easy to see and is not easy to be confused, and the strip can be observed only when the color is developed by the traditional colloidal gold technology, and misdiagnosis is easy to occur due to the position offset of the strip.
(4) The invention indirectly detects bacteria through bacterial secretion and is assisted by the high sensitivity of an immunodiagnosis technology, and can detect the viable bacteria infection under the condition of extremely low bacterial quantity, which can not be realized by the traditional separation culture method.
(5) According to the invention, the non-woven fabric is covered and bonded on the sample adding hole, so that particle impurities contained in the sample can be filtered, the detection of samples such as bacterial culture solution, pustular and the like is facilitated, the chromatographic effect of the samples polluted by particles is not easily influenced, the pH of the sample is buffered and adjusted by the sample pad, the influence of pH abnormal samples such as vaginal secretion and the like on the detection result is avoided, the good detection effect on samples such as serum, plasma, vaginal secretion, urine, sputum, cerebrospinal fluid, skin test sample, throat test sample, hospital environment monitoring sample or bacterial culture solution is achieved, and the detection accuracy is improved.
Drawings
FIG. 1 is a schematic view of the structure of the pathogenic Candida detection and identification card of the present invention, wherein 1-sample addition zone, 2-PVC base plate, 3-sample pad, 4-marker pad, 5-nitrocellulose membrane, 6-detection line A, 7-detection line B, 8-detection line C, 9-detection line D, 10-quality control line, and 11-absorbent paper.
Fig. 2 is a photograph of the front of the upper cover of the card case.
Fig. 3 is a photograph of the reverse side of the upper cover of the card case.
Fig. 4 is a photograph of the lower cover of the card case.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following detailed description is given with reference to the accompanying drawings in conjunction with the preferred embodiments of the present invention.
Example 1
A method for detecting and identifying pathogenic candida comprises the following steps: specific enolase secreted by pathogenic candida is used as a detection target molecule, a monoclonal antibody, a marker and a coating carrier of the candida specific enolase are produced by adopting genetic engineering and cell engineering technologies, and the pathogenic candida is detected and identified by a double-antibody sandwich method.
Further, the pathogenic candida includes candida albicans, candida tropicalis, candida parapsilosis, and candida glabrata.
Furthermore, in the candida specific enolase monoclonal antibodies, the specificity enolase secreted by candida albicans corresponds to the specificity monoclonal antibody A, the specificity enolase secreted by candida tropicalis corresponds to the specificity monoclonal antibody B, the specificity enolase secreted by candida parapsilosis corresponds to the specificity monoclonal antibody C, the specificity enolase secreted by candida glabrata corresponds to the specificity monoclonal antibody D, and the specific monoclonal antibody has no cross reaction with other enolases; the specificity enolase secreted by the candida albicans, candida tropicalis, candida parapsilosis and candida glabrata corresponds to the specificity monoclonal antibody E.
A pathogenic candida detection and identification card comprises a card shell and a test strip, wherein the card shell is divided into an upper piece and a lower piece and used for fixing the test strip, the upper piece is provided with a hollowed observation window and a sample adding hole, and the inner side of the sample adding hole is covered and adhered by non-woven fabric; the test strip comprises a sample treatment pad, a marker pad, a nitrocellulose membrane and absorbent paper.
Furthermore, the test strip sample pad is made of glass fiber paper, and is obtained by soaking the test strip sample pad in PBST-EDTA buffer solution and then drying the test strip sample pad.
Further, the test strip marker pad is obtained by uniformly spraying the colloidal gold-labeled monoclonal antibody E on a nitrocellulose membrane and drying;
further, the test strip cellulose nitrate membrane is respectively coated with a monoclonal antibody A, B, C, D and a goat anti-mouse quality control antibody; the pigment with the color different from that of the marked antibody is added into the antibody to be coated on the coating carrier, such as the nitrocellulose membrane, so that the lines on the coated nitrocellulose membrane are clear and easy to see.
A preparation method of the pathogenic candida detection and identification card comprises the following specific steps:
step one, diluting the colloidal gold-labeled monoclonal antibody E with PBS buffer solution containing 30% BSA to 0.1mg/mL respectively to prepare a gold-labeled solution, spraying the gold-labeled solution with the gold spraying amount of 5 muL/cm onto glass fiber paper to serve as a marker pad;
step two, respectively diluting monoclonal antibodies A, B, C and D with PBS buffer solution containing 30% BSA to 0.1mg/mL to prepare coating solutions, respectively adding 0.1mg/mL sunset yellow, and sequentially coating the coating solutions on a nitrocellulose membrane from left to right by using a membrane scribing instrument to serve as 4 detection lines; diluting goat anti-mouse antibody to 1mg/mL with the same PBS buffer solution, adding 0.1mg/mL sunset yellow, coating on the nitrocellulose membrane on the right side of the D line by using a membrane scribing instrument to serve as a quality control line, and drying to obtain 5 light yellow strips on the nitrocellulose membrane;
step three, soaking the glass fiber paper in 0.01mol/mL PBST-EDTA buffer solution (pH 7.2), and drying the soaked glass fiber paper by using a constant-temperature air-blast drying oven to obtain a sample pad;
step four, assembling the test strip, namely assembling the sample pad, the marker pad, the nitrocellulose membrane and the absorbent paper on a PVC (polyvinyl chloride) base plate in sequence according to the attached drawing 1 and then cutting the test strip;
step five, adhering a layer of 100-mesh non-woven fabric filter cloth on the inner side of the sample adding hole by using a waterproof quick-drying adhesive on the upper cover of the clamping shell, wherein the non-woven fabric can completely cover the sample adding hole;
and sixthly, placing the test strip into the card shell, aligning the sample adding hole with the sample pad, aligning the observation window with the nitrocellulose membrane on the test strip, and pressing the upper card shell and the lower card shell tightly.
Example 2
A method for detecting and identifying pathogenic candida comprises the following steps: specific enolase secreted by pathogenic candida is used as a detection target molecule, a monoclonal antibody, a marker and a coating carrier of the candida specific enolase are produced by adopting genetic engineering and cell engineering technologies, and the pathogenic candida is detected and identified by a double-antibody sandwich method.
Further, the pathogenic candida includes candida albicans, candida tropicalis, candida parapsilosis, and candida glabrata.
Furthermore, in the candida specific enolase monoclonal antibodies, the specificity enolase secreted by candida albicans corresponds to the specificity monoclonal antibody A, the specificity enolase secreted by candida tropicalis corresponds to the specificity monoclonal antibody B, the specificity enolase secreted by candida parapsilosis corresponds to the specificity monoclonal antibody C, the specificity enolase secreted by candida glabrata corresponds to the specificity monoclonal antibody D, and the specific monoclonal antibody has no cross reaction with other enolases; the specificity enolase secreted by the candida albicans, candida tropicalis, candida parapsilosis and candida glabrata corresponds to the specificity monoclonal antibody E.
A pathogenic candida detection and identification card comprises a card shell and a test strip, wherein the card shell is divided into an upper piece and a lower piece and used for fixing the test strip, the upper piece is provided with a hollowed observation window and a sample adding hole, and the inner side of the sample adding hole is covered and adhered by non-woven fabric; the test strip comprises a sample treatment pad, a marker pad, a nitrocellulose membrane and absorbent paper.
Furthermore, the test strip sample pad is made of glass fiber paper, and is obtained by soaking the test strip sample pad in PBST-EDTA buffer solution and then drying the test strip sample pad.
Further, the test paper strip marker pad is obtained by uniformly spraying a marker-labeled monoclonal antibody E on a nitrocellulose membrane and drying; the label may be, but is not limited to, peroxidase, acridinium ester, alkaline phosphatase, latex microspheres, or a fluorescent substance.
Further, the test strip cellulose nitrate membrane is respectively coated with a monoclonal antibody A, B, C, D and a goat anti-mouse quality control antibody; the pigment with the color different from that of the marked antibody is added into the antibody to be coated on the coating carrier such as the nitrocellulose membrane, so that lines on the coated nitrocellulose membrane are clear and easy to see.
A preparation method of the pathogenic candida detection and identification card comprises the following specific steps:
step one, diluting the colloidal gold-labeled monoclonal antibody E with PBS (phosphate buffer solution) containing 30% BSA (bovine serum albumin) to 0.5mg/mL respectively to prepare a gold-labeled solution, spraying the gold-labeled solution with the gold spraying amount of 5 muL/cm onto glass fiber paper to serve as a marker pad;
step two, respectively diluting monoclonal antibodies A, B, C and D with PBS buffer solution containing 30% BSA to 0.5mg/mL to prepare coating solutions, respectively adding 0.1mg/mL sunset yellow, and sequentially coating the coating solutions on a nitrocellulose membrane from left to right by using a membrane scribing instrument to serve as 4 detection lines; diluting goat anti-mouse antibody to 1mg/mL with the same PBS buffer solution, adding 0.1mg/mL sunset yellow, coating on the nitrocellulose membrane on the right side of the D line by using a membrane scribing instrument to serve as a quality control line, and drying to obtain 5 light yellow strips on the nitrocellulose membrane;
step three, soaking the glass fiber paper with 0.05mol/mL PBST-EDTA buffer solution (pH 7.2), and drying the soaked glass fiber paper by using a constant-temperature air-blast drying oven to obtain a sample pad;
step four, assembling the test strip, namely assembling the sample pad, the marker pad, the nitrocellulose membrane and the absorbent paper on a PVC (polyvinyl chloride) base plate in sequence according to the attached drawing 1 and then cutting the test strip;
step five, covering the upper cover of the clamping shell, and adhering a layer of 200-mesh non-woven fabric filter cloth on the inner side of the sample adding hole by using waterproof quick-drying adhesive, wherein the non-woven fabric can completely cover the sample adding hole;
and sixthly, putting the test paper strip into the card shell, aligning the sample adding hole with the sample pad, aligning the observation window with the nitrocellulose membrane on the test paper strip, and pressing the upper card shell and the lower card shell tightly.
Example 3
A method for detecting and identifying pathogenic candida comprises the following steps: specific enolase secreted by pathogenic candida is used as a detection target molecule, a monoclonal antibody, a marker and a coating carrier of the candida specific enolase are produced by adopting genetic engineering and cell engineering technologies, and the pathogenic candida is detected and identified by a double-antibody sandwich method.
Further, the pathogenic candida include candida albicans, candida tropicalis, candida parapsilosis, and candida glabrata.
Furthermore, in the candida specific enolase monoclonal antibodies, the specificity enolase secreted by candida albicans corresponds to the specificity monoclonal antibody A, the specificity enolase secreted by candida tropicalis corresponds to the specificity monoclonal antibody B, the specificity enolase secreted by candida parapsilosis corresponds to the specificity monoclonal antibody C, the specificity enolase secreted by candida glabrata corresponds to the specificity monoclonal antibody D, and the specific monoclonal antibody has no cross reaction with other enolases; the specific enolase secreted by Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata all correspond to the specific monoclonal antibody E.
A pathogenic candida detection and identification card comprises a card shell and a test strip, wherein the card shell is divided into an upper piece and a lower piece and used for fixing the test strip, the upper piece is provided with a hollowed observation window and a sample adding hole, and the inner side of the sample adding hole is covered and adhered by non-woven fabric; the test strip comprises a sample treatment pad, a marker pad, a nitrocellulose membrane and absorbent paper.
Furthermore, the test strip sample pad is made of glass fiber paper, and is obtained by soaking the test strip sample pad in PBST-EDTA buffer solution and then drying the test strip sample pad.
Further, the test paper strip marker pad is obtained by uniformly spraying a marker-labeled monoclonal antibody E on a nitrocellulose membrane and drying; the label may be, but is not limited to, peroxidase, acridinium ester, alkaline phosphatase, latex microspheres, or a fluorescent substance.
Further, the test strip cellulose nitrate membrane is respectively coated with a monoclonal antibody A, B, C, D and a goat anti-mouse quality control antibody; the pigment with the color different from that of the marked antibody is added into the antibody to be coated on the coating carrier, such as the nitrocellulose membrane, so that the lines on the coated nitrocellulose membrane are clear and easy to see.
A preparation method of the pathogenic candida detection and identification card comprises the following specific steps:
step one, diluting the colloidal gold-labeled monoclonal antibody E with PBS buffer solution containing 30% BSA to the concentration of 1mg/mL respectively to prepare a gold-labeled solution, spraying the gold with the amount of 5 muL/cm, and spraying the gold-labeled solution on glass fiber paper to serve as a marker pad;
step two, respectively diluting the monoclonal antibodies A, B, C and D with PBS buffer solution containing 30% BSA to the concentration of 1mg/mL to prepare coating solutions, respectively adding 0.1mg/mL sunset yellow, and sequentially coating the coating solutions on a nitrocellulose membrane from left to right by using a membrane scribing instrument to serve as 4 detection lines; diluting goat anti-mouse antibody to 1mg/mL with the same PBS buffer solution, adding 0.1mg/mL sunset yellow, coating on the nitrocellulose membrane on the right side of the D line by using a membrane scribing instrument to serve as a quality control line, and drying to obtain 5 light yellow strips on the nitrocellulose membrane;
step three, soaking the glass fiber paper with 0.1mol/mL PBST-EDTA buffer solution (pH is 7.2), and drying the soaked glass fiber paper by using a constant-temperature air blast drying oven to obtain a sample pad;
step four, assembling the test strip, namely assembling the sample pad, the marker pad, the nitrocellulose membrane and the absorbent paper on a PVC (polyvinyl chloride) base plate in sequence according to the attached drawing 1 and then cutting the test strip;
step five, covering the upper cover of the clamping shell, and adhering a layer of 300-mesh non-woven fabric filter cloth on the inner side of the sample adding hole by using waterproof quick-drying adhesive, wherein the non-woven fabric can completely cover the sample adding hole;
and sixthly, placing the test strip into the card shell, aligning the sample adding hole with the sample pad, aligning the observation window with the nitrocellulose membrane on the test strip, and pressing the upper card shell and the lower card shell tightly.
Sensitivity and specificity test
Respectively carrying out pure culture on candida albicans, candida tropicalis, candida parapsilosis and candida glabrata at 36 ℃ by using a liquid culture medium, grinding and crushing bacterial suspensions after 48 hours of culture, filtering, carrying out gradient centrifugation to remove impurities, precipitating enolase by using an ammonium sulfate method, purifying by using a glucan molecular sieve purification column, concentrating into an enolase solution, and determining the corresponding concentration of the enolase solution.
Diluting four candida enolase solutions to three concentrations of 0.1ng/ml, 100ng/ml and 25ng/ml by using normal saline, and detecting on a test strip, wherein when the test strip of 0.1ng/ml, 0.15ng/ml and 0.2ng/ml candida albicans enolase is added, only the first strip and the fifth strip are changed into red, and the rest strips are not changed in color; when the test strips of 0.1ng/ml, 0.15ng/ml and 0.2ng/ml candida tropicalis enolase are added, only the second and fifth strips turn red, and the rest do not change color; when the test strips of 0.1ng/ml, 0.15ng/ml and 0.2ng/ml candida parapsilosis enolase are added, only the third and fifth strips become red, and the others do not change color; only the fourth and fifth bands turned red, and none of the others turned off, when the test strips of Candida glabrata enolase at 0.1ng/ml, 0.15ng/ml, and 0.2ng/ml were added.
Detecting the culture solution of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida glabrata for 48 hours by using a test strip, wherein the corresponding detection strip and quality control strip are red; the yeast culture solution, the escherichia coli culture solution, the staphylococcus aureus culture solution and the pseudomonas aeruginosa culture solution are detected by a test strip, and the strips are not changed in color except for the fact that the quality control strips are changed into red.
The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and other modifications or equivalent substitutions made by the technical solution of the present invention by the ordinary skilled in the art should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (5)
1. A method for detecting and identifying pathogenic candida is characterized in that specific enolase secreted by pathogenic candida is used as a detection target molecule, a gene engineering and cell engineering technology is adopted to produce a monoclonal antibody, a marker and a coating carrier of the candida specific enolase, the pathogenic candida is detected and identified by a double-antibody sandwich method, the pathogenic candida comprises candida albicans, candida tropicalis, candida parapsilosis and candida glabrata, a specific enolase secreted by the candida albicans in the monoclonal antibody of the candida specific enolase corresponds to a specific monoclonal antibody A, a specific enolase secreted by the candida tropicalis corresponds to a specific monoclonal antibody B, a specific enolase secreted by the candida parapsilosis corresponds to a specific monoclonal antibody C, a specific enolase secreted by the candida glabrata corresponds to a specific monoclonal antibody D, the specific monoclonal antibody has no cross reaction with other enolase; the specificity enolase secreted by the candida albicans, candida tropicalis, candida parapsilosis and candida glabrata corresponds to the specificity monoclonal antibody E.
2. The pathogenic candida detection and identification card according to the pathogenic candida detection and identification method of claim 1, comprising a card shell and a test strip, wherein the card shell is divided into an upper piece and a lower piece for fixing the test strip, the upper piece is provided with a hollowed observation window and a sampling hole, and the inner side of the sampling hole is covered and adhered by a non-woven fabric filter cloth; the test strip comprises a sample treatment pad, a marker pad, a nitrocellulose membrane and absorbent paper, wherein the marker pad of the test strip is uniformly sprayed on the nitrocellulose membrane by using a marker to mark a monoclonal antibody E; the label includes, but is not limited to, colloidal gold, peroxidase, acridinium ester, alkaline phosphatase, latex microspheres, or fluorescent substance; the nitrocellulose membrane is respectively coated with a monoclonal antibody A, B, C, D and a goat anti-mouse quality control antibody; the pigment with the color different from that of the marked antibody is added into the antibody to be coated on the nitrocellulose membrane, so that the lines coated on the nitrocellulose membrane are clear and easy to see.
3. The pathogenic candida detection and identification card as set forth in claim 2, wherein the sample treatment pad is a glass fiber paper impregnated with PBST-EDTA buffer and dried.
4. The pathogenic candida detection and identification card as claimed in claim 2, wherein the mesh number of the non-woven fabric filter cloth is 100-300.
5. A method for preparing the pathogenic Candida detection and identification card according to any of claims 2 to 4, wherein the specific steps are as follows:
step one, respectively diluting a monoclonal antibody E marked by a marker with PBS (phosphate buffer solution) containing 30% BSA (bovine serum albumin) to 0.1-1 mg/mL to prepare a marking solution, and spraying the marking solution on glass fiber paper to serve as a marker pad;
step two, respectively diluting monoclonal antibodies A, B, C and D with PBS (phosphate buffer solution) containing 30% BSA (bovine serum albumin) to be 0.1-1 mg/mL to prepare coating solutions, respectively adding 0.1mg/mL sunset yellow, and sequentially coating the coating solutions to a nitrocellulose membrane from left to right by using a membrane scribing instrument to serve as 4 detection lines; diluting goat anti-mouse antibody to 1mg/mL with the same PBS buffer solution, adding 0.1mg/mL sunset yellow, coating on the nitrocellulose membrane on the right side of the D line by using a membrane scribing instrument to serve as a quality control line, and drying to obtain 5 light yellow strips on the nitrocellulose membrane;
soaking the glass fiber paper with 0.01-0.1 mol/mL PBST-EDTA buffer solution with pH =7.2, and drying the soaked glass fiber paper by using a constant-temperature blast drying oven to serve as a sample processing pad;
assembling the test strip, namely assembling the sample treatment pad, the marker pad, the nitrocellulose membrane and the absorbent paper on a PVC (polyvinyl chloride) base plate in sequence and then cutting the PVC base plate;
step five, covering the upper cover of the clamping shell, and adhering a layer of non-woven fabric filter cloth on the inner side of the sample adding hole by using waterproof quick-drying adhesive, wherein the non-woven fabric can completely cover the sample adding hole;
and step six, placing the test strip into the card shell, aligning the sample adding hole with the sample processing pad, aligning the observation window with the nitrocellulose membrane on the test strip, and pressing the upper card shell and the lower card shell tightly.
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| 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 |
| 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 |
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