CN219957598U - Two-way immunochromatography test strip for rapid six-way detection of carbapenemase - Google Patents
Two-way immunochromatography test strip for rapid six-way detection of carbapenemase Download PDFInfo
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- CN219957598U CN219957598U CN202321652827.1U CN202321652827U CN219957598U CN 219957598 U CN219957598 U CN 219957598U CN 202321652827 U CN202321652827 U CN 202321652827U CN 219957598 U CN219957598 U CN 219957598U
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model discloses a carbapenemase rapid six-joint detection two-way immunochromatography test strip, which comprises a bottom plate; NC films symmetrically fixed on the bottom plate; gold mark/milk mark pads and absorption pads symmetrically fixed at two ends of the NC film; a sample pad lapped on the gold/milk label pad; three detection lines and a quality control line are respectively arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, and goat anti-mouse monoclonal antibodies are coated on the quality control line; the gold mark/emulsion mark pad is sprayed with each monoclonal antibody marked by colloidal gold/emulsion microsphere. The test strip can detect the carbapenemase (A/B/D) common in clinic at present, has higher sensitivity and specificity, can obtain a visual result within 15min, is suitable for being detected immediately beside a bed, and can be widely applied to the rapid identification of carbapenemase-producing bacteria in clinical specimens.
Description
Technical Field
The utility model relates to the field of biological detection, in particular to a carbapenemase rapid six-joint detection two-way immunochromatography test strip.
Background
At present, the global bacteria resistant situation is more severe, wherein the bacteria (CRE) of the order Enterobacteriaceae are most serious, and the drug resistant mechanism of the bacteria of the order Enterobacteriaceae to carbapenems is mainly based on carbapenemases, and can be classified into class A (KPC), class B (NDM/VIM/IMP) and class D (OXA-23/OXA-48-like) enzymes according to the Ambler classification method. Because of different antibacterial activities of different medicines on the outside of the strain producing different carbapenemases, for example, the activities of class A and class D enzymes can be inhibited by a new generation of beta lactamase inhibitor, namely, avibactam, and clinical medication recommends ceftazidime-avibactam. However, class B metalloenzymes cannot be inhibited by β -lactamase inhibitors, and need to be replaced with therapeutic agents such as tigecycline or polymyxin, which would not only prevent bacterial clearance but increase mortality if abamectin is used continuously. Therefore, the rapid detection and identification typing of the carbapenemase are beneficial to the clinical doctors to develop targeted treatment in time, thereby reducing the disability rate of patients.
As a relatively popular non-invasive detection means in recent years, the immunochromatography technology (immunochromatography assay, ICA) has the advantages of simple operation, high sensitivity and specificity, low cost and the like, and is suitable for on-site instant detection (Point of care testing, POCT). The current guidelines recommend a NG-Test Carba 5 carbapenemase detection kit, which can rapidly detect KPC, VIM, IMP, NDM, OXA-48 five common enzyme types within 15min, and covers about 93% of drug-resistant strains clinically. However, the above method cannot detect infection with the carbapenem-resistant acinetobacter baumannii (carbapenem-resistant Acinetobacter baumannii, CRAB) because OXA-23 is the main carbapenemase mediating CRAB resistance, and is not in the detection range, and is prone to missed diagnosis and misdiagnosis.
Therefore, the specific monoclonal antibody aiming at the potential target antigen OXA-23 of Acinetobacter baumannii is screened and prepared in the earlier stage of research, a six-joint detection bidirectional immunochromatography test strip is developed, the space and the cost are further saved, the main detection enzymes comprise KPC, VIM, IMP, NDM, OXA-23 and OXA-48, and one-card six detection is realized. The detection range can cover various bacteria including enterobacteriales bacteria, acinetobacter baumannii, pseudomonas aeruginosa and the like, and well overcomes the defects of the prior detection technology.
Disclosure of Invention
Aiming at the defects of the current clinical carbapenem-resistant bacteria detection method, the utility model provides a two-way immunochromatographic test strip for quick six-joint detection of carbapenemase, which realizes qualitative and semi-quantitative quick synchronous detection of common clinical carbapenem-resistant gram-negative bacteria by using an immunochromatographic technology, and can realize synchronous quick detection of KPC, NDM, VIM, IMP, OXA-23 and OXA-48 six carbapenemases; 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 two-way immunochromatography test strip, which comprises:
a bottom plate;
NC films symmetrically fixed on the bottom plate;
gold mark/milk mark pads and absorption pads symmetrically fixed at two ends of the NC film;
a sample pad lapped on the gold/milk label pad;
three detection lines and a quality control line are respectively arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, and goat anti-mouse monoclonal antibodies are coated on the quality control line;
the gold mark/emulsion mark pad is sprayed with each monoclonal antibody marked by colloidal gold/emulsion microsphere.
Further preferably, the detection lines and the quality control lines on each 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/latex microsphere has a particle 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 of the test strip is set according to practical conditions, for example, the test strip can be 5mm.
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 in the United states, the sample pad is Ahlstrom 8964 glass cellulose film in Finland, the gold/milk mark pad is Ahlstrom 6613 polyester fiber film in Finland, and the absorbent pad is cellulose filter paper.
Advantageous effects
The rapid six-joint detection two-way immunochromatography 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 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 makes up 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 immunochromatographic test strip detection;
FIG. 2 is a schematic diagram of the structural hierarchy of the novel immunochromatographic test strip;
FIG. 3 is a schematic diagram of a product;
FIG. 4 is a diagram of result interpretation;
1-sample pad; 2-gold/milk label pad AB; 3-an absorbent pad; 4-NC film AB; a 5-PVC bottom plate; 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. 2 and 3, a carbapenemase rapid six-joint detection immunochromatography test strip comprises a PVC base plate 5 and NC membranes 4 symmetrically fixed on the base plate; the gold mark/milk mark pad 2 and the absorption pad 3 are symmetrically fixed at two ends of the NC film 4; the NC film 4 is respectively provided with three detection lines 7 and a quality control line 6, the six detection lines 7 are respectively coated with monoclonal antibodies of each carbapenemase, the quality control line 6 is coated with goat anti-mouse monoclonal antibodies, and the gold mark/milk mark pad is sprayed with each antibody marked by colloidal gold/latex microspheres;
wherein, the NC film 4 is provided with a detection line 71, a detection line 72, a detection line 73 and one quality control line 6, and the other NC film is provided with a detection line 74, a detection line 75, a detection line 76 and the other quality control line 6, wherein the detection lines and the quality control lines on each NC film are arranged at equal intervals, the widths are 5mm, and the intervals are 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/latex microsphere marking particle size is 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 adhesive, the NC film is Millipore135, the sample pad is a glass cellulose film of Ahlstrom 8964, finland, the gold/milk label pad is a polyester 6613, finland, and the absorbent pad is cellulose filter paper.
Example 2
Based on the embodiment 1, the utility model prepares a carbapenemase rapid six-joint detection two-way immunochromatography test strip. The preparation process comprises the following steps:
(1) The preparation method of the immunochromatography 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
Putting the polyester fiber film into 10% BSA blocking solution, soaking for a period of time, taking out, drying in a 37 ℃ oven for 5 hours, spraying the prepared six gold-labeled antibodies on the dried polyester fiber film by using a BIO DOT spot film tester, and preserving at 4 ℃ for later use;
(C) Preparation of sample pad
Soaking a glass cellulose membrane in a 10% BSA blocking solution for a period of time, 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 5, the second layer is NC membrane 4, six detection line antibodies (T1, T2, T3, T4, T5, T6) and quality control antibody (C) are coated, the third layer is a gold mark pad, the fourth layer is a sample pad 1, the sample pad 1 is used as a starting end, the absorption pad 3 is stuck to the end point end of each NC membrane 4, the above components are connected end to end, the components are sequentially stacked and assembled, and note that the sample pad 1 is lapped on two gold mark pads. 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.
(2) Principle of detection
And (3) adding the sample into a sample adding hole, dissolving the colloidal gold/latex microsphere labeled antibody on the gold-labeled pad by the 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, the complex is trapped by forming a gold-labeled antibody-antigen-capture antibody sandwich complex with the solid-phase monoclonal antibody at the detection line due to the upward chromatography, and the colloidal gold/latex microsphere 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.
(3) Specific detection method
(1) 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 an immunochromatographic test strip, and the result is observed after 10 minutes;
(2) Colony specimen treatment: after a single separated colony is picked by a 1uL sampling rod and evenly mixed in 200uL sample treatment liquid, 200uL liquid is sucked and slowly added into an immunochromatographic test strip sample adding hole, and the result is observed after 10 min;
(3) 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 utility model 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" in the present utility model means that each exists alone or both exist.
"connected" as used herein means either a direct connection between components or an indirect connection between components via other components.
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 (8)
1. A carbapenemase rapid six-joint detection two-way immunochromatography test strip is characterized in that,
a bottom plate;
NC films symmetrically fixed on the bottom plate;
gold mark/milk mark pads and absorption pads symmetrically fixed at two ends of the NC film;
a sample pad lapped on the gold/milk label pad;
three detection lines and a quality control line are respectively arranged on the NC film, monoclonal antibodies of the carbapenemases are respectively coated on the six detection lines, and goat anti-mouse monoclonal antibodies are coated on the quality control line;
the gold mark/emulsion mark pad is sprayed with each monoclonal antibody marked by colloidal gold/emulsion microsphere.
2. The rapid six-way detection bi-directional 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 carbapenemase rapid six-joint detection two-way immunochromatographic test strip of claim 1, wherein the detection lines and the quality control lines on each NC membrane are arranged at equal intervals, the width is 5mm, and the interval is 5mm.
4. The carbapenemase rapid six-joint detection two-way immunochromatographic test strip of claim 1, wherein the colloidal gold/latex microsphere label particle size is 20-40 nm.
5. The carbapenemase rapid six-joint detection two-way immunochromatographic test strip of claim 1, wherein the test strip is sleeved with a plastic cartridge and is provided with a sample adding hole and a result observation window.
6. The carbapenemase rapid six-joint detection two-way immunochromatographic strip of claim 5, wherein the result observation window has a width of 5mm.
7. The rapid six-way carbapenemase test strip of claim 6, wherein the sample is isolated as a single colony or as a positive blood sample.
8. The rapid six-joint detection two-way immunochromatographic test strip for carbapenemase according to claim 1, wherein the bottom plate is a PVC bottom plate with an adhesive, the NC film is Millipore135 in America, the sample pad is a glass cellulose film of Ahlstrom 8964 in Finland, the gold/milk mark pad is a polyester fiber film of Ahlstrom 6613 in Finland, and the absorption pad is cellulose filter paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202321652827.1U CN219957598U (en) | 2023-06-28 | 2023-06-28 | Two-way immunochromatography test strip for rapid six-way detection of carbapenemase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202321652827.1U CN219957598U (en) | 2023-06-28 | 2023-06-28 | Two-way immunochromatography test strip for rapid six-way detection of carbapenemase |
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| Publication Number | Publication Date |
|---|---|
| CN219957598U true CN219957598U (en) | 2023-11-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202321652827.1U Active CN219957598U (en) | 2023-06-28 | 2023-06-28 | Two-way immunochromatography test strip for rapid six-way detection of carbapenemase |
Country Status (1)
| Country | Link |
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| CN (1) | CN219957598U (en) |
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- 2023-06-28 CN CN202321652827.1U patent/CN219957598U/en active Active
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