CN219348880U - Colloidal gold immunochromatography test strip for rapid six-joint detection of carbapenemase - Google Patents

Colloidal gold immunochromatography test strip for rapid six-joint detection of carbapenemase Download PDF

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CN219348880U
CN219348880U CN202320719661.4U CN202320719661U CN219348880U CN 219348880 U CN219348880 U CN 219348880U CN 202320719661 U CN202320719661 U CN 202320719661U CN 219348880 U CN219348880 U CN 219348880U
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pad
colloidal gold
rapid
test strip
carbapenemase
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任建安
吴秀文
李家扬
吴文琦
周志涛
王家杰
吴美琳
田赛
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Eastern Theater General Hospital of PLA
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Eastern Theater General Hospital of PLA
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract

The utility model discloses a colloidal gold immunochromatographic test strip for rapid six-way detection of carbapenemase, which comprises a PVC (polyvinyl chloride) bottom plate, an NC (numerical control) film, a sample pad, a gold mark pad and an absorption pad, wherein the NC film is fixed on the PVC bottom plate, the gold mark pad and the absorption pad are respectively fixed at two ends of the NC film, and the sample pad is fixed at the outer side end of the gold mark pad; six detection lines and a quality control line are arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, goat anti-mouse IgG antibodies are coated on the quality control line, and 6 monoclonal antibodies marked by colloidal gold are sprayed on the gold mark pad. The test strip can detect the gram-negative bacillus resistant to carbapenems common clinically at present, has higher sensitivity and specificity, can obtain a visual result within 15min, is suitable for bedside instant detection, and can be widely applied to quick identification of carbapenemase-producing bacteria in clinical specimens.

Description

Colloidal gold immunochromatography test strip for rapid six-joint detection of carbapenemase
Technical Field
The utility model relates to the field of biological detection, in particular to a colloidal gold immunochromatographic test strip for rapid six-joint detection of carbapenemase.
Background
Carbapenem antibiotics are the last means antibiotics for the current clinical treatment of bacterial infections of the order enterobacteriales, due to their broad-spectrum antibacterial activity, stability to various beta-lactamases and low toxicity. However, recent years of non-standardized use of antibiotics have resulted in an increase in the isolation rate of carbapenem-resistant enterobacteriales (carbapenem-resistant Enterobacteriaceae, CRE) year by year. Carbapenemases are still the most prominent drug resistant mechanism for carbapenem-resistant gram-negative bacilli. Common carbapenemases include KPC (Klebsiella pneumoniae carbapenemases), NDM (new delhi metallo-. Beta. -lactase), IMP (imipenemase metallo-. Beta. -lactase), VIM (verona integral-encoded meta-o-. Beta. -lactase) and OXA (oxacillinase carbapenemases) in class D. Because the therapeutic drugs for producing different carbapenemase strains are different, the quick detection and identification of carbapenemase are classified, which is helpful for clinicians to make accurate individual therapeutic schemes in time, thereby improving prognosis of patients and reducing mortality.
The colloidal gold immunochromatography (gold immunochromatography assay, GICA) is a popular bacterial detection method in recent years, is widely applied to the field of biomedical detection, has high sensitivity and specificity, is simple and quick to operate, does not need professional training for detection personnel and does not need any special instrument, and is suitable for point-of-care detection. The NG-Test Carba 5 carbapenemase detection kit which is marketed at present can complete the visual detection of five enzyme types KPC, VIM, IMP, NDM, OXA-48 within 15min, and covers about 93% of drug-resistant strains clinically. However, this method is not able to detect carbapenem-resistant acinetobacter baumannii (carbapenem-resistant Acinetobacter baumannii, CRAB) infection, because OXA-23, the most widely transmitted carbapenemase as CRAB, is not included in the detection range and is prone to false negative results.
Therefore, a specific monoclonal antibody aiming at a potential target antigen OXA-23 of Acinetobacter baumannii is screened and prepared in the earlier stage of research, and a six-joint detection colloidal gold immune test strip is developed on the basis, wherein the main detection enzymes comprise KPC, VIM, IMP, NDM, OXA-23 and OXA-48. The detection range can cover various bacteria including enterobacteriales bacteria, acinetobacter baumannii, pseudomonas aeruginosa and the like, and the detection efficiency is greatly improved.
Disclosure of Invention
Aiming at the defects of the current clinical carbapenem-resistant bacteria detection method, the utility model provides a colloidal gold immunochromatography test strip for rapid six-joint detection of carbapenemases, and the qualitative and semi-quantitative rapid synchronous detection of the clinical common carbapenem-resistant gram-negative bacteria can be realized by utilizing the colloidal gold immunochromatography technology, and the KPC, NDM, VIM, IMP, OXA-23 and OXA-48 six carbapenemases can be synchronously and rapidly detected; the test strip is simple to operate, convenient to carry and short in result interpretation time, and can be used under various conditions.
The technical scheme adopted for solving the technical problems is as follows:
the utility model firstly protects a carbapenemase rapid six-joint detection colloidal gold immunochromatographic test strip, which comprises a bottom plate, an NC film, a sample pad, a gold mark pad and an absorption pad, wherein the NC film is fixed on the bottom plate, the gold mark pad and the absorption pad are respectively fixed at two ends of the NC film, and the sample pad is fixed at the outer side end of the gold mark pad; six detection lines and a quality control line are arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, goat anti-mouse IgG antibodies are coated on the quality control line, and 6 monoclonal antibodies marked by colloidal gold are sprayed on the gold mark pad.
As a further preferred aspect of the present utility model, the monoclonal antibodies are KPC protein monoclonal antibody, NDM protein monoclonal antibody, VIM protein monoclonal antibody, IMP protein monoclonal antibody, OXA-23 protein monoclonal antibody and OXA-48 protein monoclonal antibody, respectively.
Further preferably, the detection lines and the quality control lines on the NC film are arranged at equal intervals, for example, the width and the interval may be set according to actual needs, for example, the width is set to 5mm, and the interval is set to 5mm.
As a further preferred aspect of the present utility model, the monoclonal antibodies are KPC protein monoclonal antibody, NDM protein monoclonal antibody, VIM protein monoclonal antibody, IMP protein monoclonal antibody, OXA-23 protein monoclonal antibody and OXA-48 protein monoclonal antibody, respectively.
As a further preferred aspect of the present utility model, the colloidal gold-labeled particles have a size of 20 to 40nm, such as 20nm,25nm,30nm,35nm,40nm, etc.
As a further preferable mode of the utility model, the test strip is sleeved with the plastic clamping shell, a sample adding hole and a result observation window are arranged, and the width is set according to practical conditions, such as 4mm, 5mm, 6mm, 7mm, 8mm and the like.
Preferably, the test strip can be further filled into an aluminum foil packaging bag to form a test strip product.
In use, the sample to be tested may be a single isolated colony or a positive blood sample.
As a further preferred aspect of the utility model, the substrate is a PVC substrate, the NC film is Millipore135 of America, the sample pad is Ahlstrom 8964 glass cellulose film of Finland, the gold mark pad is Ahlstrom 6613 polyester fiber film of Finland, and the absorption pad is cellulose filter paper.
Advantageous effects
The rapid six-joint detection colloidal gold immunochromatographic test strip for carbapenemases can realize synchronous and rapid detection of KPC, NDM, VIM, IMP, OXA-23 and OXA-48 carbapenemases on one test strip, is simple to operate, does not need large-scale equipment and instruments, has low professional requirements on surrounding environment and detection personnel, has high detection speed, can obtain a visual result within 15min, can provide information such as gram-negative bacillus carbapenemases for clinicians and researchers in time, greatly overcomes the defect of clinical examination work at the present stage, and guides clinical work to timely and pertinently use antibiotics.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold immunochromatographic test strip;
FIG. 2 is a schematic diagram of the structural hierarchy of a novel colloidal gold immunochromatographic test strip;
FIG. 3 is a schematic diagram of a product;
FIG. 4 is a diagram of result interpretation;
1-gold mark pad; 2-sample pad; 3-PVC bottom plate; 4-NC film; 5-an absorbent pad; 6-a quality control line; 7-detecting lines; 71-detection line 1; 72-detecting line 2; 73-detection line 3; 74-detecting line 4; 75-detecting line 5; 76-detection line 6.
Detailed Description
In order that the present utility model may be more readily understood, a more particular description of the utility model will be rendered by reference to specific embodiments that are illustrated in the appended drawings. The utility model will now be described in further detail with reference to the accompanying drawings. The drawings are simplified schematic representations which merely illustrate the basic structure of the utility model and therefore show only the structures which are relevant to the utility model.
Example 1
Referring to fig. 1 and 2, a colloidal gold immunochromatographic test strip for rapid six-joint detection of carbapenemase comprises a PVC bottom plate 3, an NC membrane 4, a sample pad 2, a gold-labeled pad 1, and an absorbent pad 5, wherein the NC membrane 4 is fixed on the PVC bottom plate 3, the gold-labeled pad 1 and the absorbent pad 5 are respectively fixed at two ends of the NC membrane 4, and the sample pad 2 is fixed at the outer end of the gold-labeled pad 1; six detection lines 7 and a quality control line 6 are arranged on the NC film 4, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines 7, goat anti-mouse IgG antibodies are coated on the quality control line 6, and 6 monoclonal antibodies marked by colloidal gold are sprayed on a gold mark pad;
the inspection lines 7 (inspection lines 71, 72, 73, 74, 75, and 76) and the quality control line 6 on the NC film 4 are arranged at equal intervals, each having a width of 5mm and a spacing of 5mm.
Wherein, the monoclonal antibodies of the carbapenemases are KPC protein monoclonal antibody, NDM protein monoclonal antibody, VIM protein monoclonal antibody, IMP protein monoclonal antibody, OXA-23 protein monoclonal antibody and OXA-48 protein monoclonal antibody respectively.
In further embodiments, the colloidal gold-labeled particles have a size of 20 to 40nm.
In a further embodiment, the test strip is sleeved with a plastic cartridge, provided with a sample application hole and a result observation window, and has a width of 5mm.
In further embodiments, the sample to be tested may be an isolated single colony or a positive blood sample.
In other embodiments, the substrate is a PVC substrate with an adhesive, the NC film is Millipore135, the sample pad is a glass cellulose film of Ahlstrom 8964, finland, the gold mark pad is a polyester fiber film of Ahlstrom 6613, finland, and the absorbent pad is cellulose filter paper.
Example 2
Based on example 1, a rapid six-joint detection colloidal gold immunochromatography test strip for carbapenemase is prepared. The preparation process comprises the following steps:
(1) Preparation of colloidal gold particles
By trisodium citrate reduction, 100ml of the mixture was first prepared0.01% HAuCl 4 Adding the solution into a flask, and performing 300 r.min -1 Mechanically stirring, heating to boil, rapidly adding 1ml 1% trisodium citrate aqueous solution, boiling, stopping heating when bright red appears, stirring for 15min, naturally cooling, packaging in glass bottles, and adding a small amount of antiseptic for preservation at 4deg.C for several months.
(2) Preparation of colloidal gold-labeled antibody
Taking 50ml of the colloidal gold solution prepared in the step (1), and using 0.1mol K 2 CO 3 The pH of the solution is regulated to 8.0, and the commercial KPC monoclonal antibody 10E1 is slowly added according to the optimal proportion, and is stirred and mixed evenly by magnetic force. 10% BSA was added to a final concentration of 0.5% and magnetically stirred for 30min. 10% PEG20000 was added to a final concentration of 0.2% and magnetically stirred for 30min. Centrifuge at 12000rpm,4℃for 30min, discard supernatant, leave colloidal gold precipitate. One tenth of the original volume of colloidal Jin Chong suspension (0.025g PEG20000,2.5g sucrose, 0.5g HSA and 50uL Tween-20 in 50ml10mM Tris-HCl buffer pH 8.0) was added.
The other five monoclonal antibodies are marked by colloidal gold in turn by the same method: NDM mab 17D2, VIM mab 2F7, IMP mab 7C11, OXA-23 mab 1F10, OXA-48 mab 8H11.
(3) The preparation method of the colloidal gold immunochromatographic test strip for rapid six-up detection of carbapenemase comprises the following steps:
(A) NC membrane antibody coating
Coating six commercial monoclonal antibodies on six detection lines of an NC film by using a BIO DOT spot film instrument, coating a goat anti-mouse monoclonal antibody on a quality control line, and closing the rest blank sites;
(B) Preparation of gold mark pad
Soaking a polyester fiber membrane in 10% BSA blocking solution for a period of time, taking out, drying in a 37 ℃ oven for 5 hours, spraying the six gold-labeled antibodies prepared in the step (2) on the dried polyester fiber membrane by using a BIO DOT spot-film instrument, and preserving at 4 ℃ for later use;
(C) Preparation of sample pad
Soaking a glass cellulose membrane in a 10% BSA blocking solution, taking out, drying in a 37 ℃ oven for 5 hours, and preserving in a room temperature drying environment for later use;
(D) Assembly of test strips
The first layer is PVC bottom plate 3, and the second layer is NC membrane 4, and the coating has six detection line antibodies (T1, T2, T3, T4, T5, T6) and quality control antibody (C), and the third layer is gold mark pad 1, and the fourth layer is sample pad 2, regard sample pad as the starting end, and absorption pad 5 pastes at the terminal end, and above each component links up end to end, stacks in proper order and assembles. The strips were cut into individual strips 5mm wide by a slitter and placed into special plastic card shells with sample addition holes and result viewing windows as shown in FIG. 3. Sealing, drying and preserving.
(4) Principle of detection
And (3) adding the sample into a sample adding hole, dissolving a colloidal gold-labeled antibody on a gold-labeled pad by a sample solution, wherein if the sample contains bacteria to be detected, bacterial antigens can be specifically combined with the gold-labeled monoclonal antibody to form a complex, and the complex is retained by forming a gold-labeled antibody-antigen-capture antibody sandwich complex with the solid-phase monoclonal antibody at a detection line due to the upward chromatography, so that the colloidal gold is gathered in a large amount to form red color, and the color depth is related to the amount of the detected antigen. And the excessive gold-labeled antibody continues to ascend, and is combined with goat anti-mouse IgG at a quality control line to aggregate, precipitate and develop color, so that the chromatography is smooth, and the detection result is effective.
(5) Specific detection method
(A) Treatment of positive blood samples: after the positive blood sample is cracked and washed, the positive blood sample is centrifuged at 13000rpm for 5min, 100uL of supernatant is sucked and mixed with 100uL of sample treatment liquid uniformly, and then the positive blood sample is kept stand at room temperature for 10min. 200uL of liquid is sucked and slowly added into a sample adding hole of the colloidal gold immunochromatographic test strip, and the result is observed after 10 minutes;
(B) Colony specimen treatment: after a single separated colony is picked up by a 1uL sampling rod and uniformly mixed in 200uL sample treatment liquid, 200uL liquid is sucked and slowly added into a sample adding hole of a colloidal gold immunochromatography test strip, and the result is observed after 10 min;
(C) Interpretation of the results: and developing by a quality control line, wherein the color development of the corresponding detection line represents a positive result, and the non-color development of the detection line represents a negative result. The quality control line does not develop color, and whether the detection line develops color or not, the test strip is invalid, and the result is invalid and needs to be detected again, as shown in fig. 4.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The meaning of "and/or" as referred to in this application means that each exists alone or both.
As used herein, "connected" means either a direct connection between elements or an indirect connection between elements via other elements.
The protection of the present utility model is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the utility model without departing from the spirit and scope of the inventive concept, and the scope of the utility model is defined by the appended claims.

Claims (7)

1. The rapid six-joint detection colloidal gold immunochromatographic test strip for carbapenemase is characterized by comprising a bottom plate, an NC film, a sample pad, a gold mark pad and an absorption pad, wherein the NC film is fixed on the bottom plate, the gold mark pad and the absorption pad are respectively fixed at two ends of the NC film, and the sample pad is fixed at the outer side end of the gold mark pad; six detection lines and a quality control line are arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, goat anti-mouse IgG antibodies are coated on the quality control line, and 6 monoclonal antibodies marked by colloidal gold are sprayed on the gold mark pad.
2. The rapid six-joint detection colloidal gold immunochromatographic strip for carbapenemase according to claim 1, wherein the monoclonal antibodies are a KPC protein monoclonal antibody, an NDM protein monoclonal antibody, a VIM protein monoclonal antibody, an IMP protein monoclonal antibody, an OXA-23 protein monoclonal antibody and an OXA-48 protein monoclonal antibody, respectively.
3. The rapid six-joint detection colloidal gold immunochromatographic test strip for carbapenemase according to claim 1, wherein the detection lines and the quality control lines on the NC membrane are arranged at equal intervals, the widths are 5mm, and the intervals are 5mm.
4. The rapid six-joint detection colloidal gold immunochromatographic test strip for carbapenemase according to claim 1, wherein the size of the colloidal gold-labeled particles is 20-40 nm.
5. The rapid six-joint detection colloidal gold immunochromatographic test strip for carbapenemase according to claim 1, wherein the test strip is sleeved with a plastic cartridge, is provided with a sample adding hole and a result observation window, and has a width of 5mm.
6. The rapid six-joint detection colloidal gold immunochromatographic strip for carbapenemase according to claim 1, wherein the sample to be tested is an isolated single colony or a positive blood specimen.
7. The rapid six-joint detection colloidal gold immunochromatographic strip for carbapenemase according to claim 4, wherein the bottom plate is a PVC bottom plate, the NC membrane is Millipore135 in America, the sample pad is a glass cellulose membrane of Ahlstrom 8964 in Finland, the gold label pad is a polyester fiber membrane of Ahlstrom 6613 in Finland, and the absorption pad is cellulose filter paper.
CN202320719661.4U 2023-04-04 2023-04-04 Colloidal gold immunochromatography test strip for rapid six-joint detection of carbapenemase Active CN219348880U (en)

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