Carbapenemase immunochromatographic test strip, its preparation method, kit and application

Abstract

The present invention provides a carbapenemase immunochromatographic test strip and its preparation method, kit and application, which relate to the field of biotechnology. The carbapenemase immunochromatographic test strip includes a labeled antibody and a detection antibody, and the labeled antibody is labeled with a labeling agent; the labeled antibody includes an antibody against at least one carbapenemase among KPC, OXA-48, VIM, IMP and NDM; the detection antibody is composed of a paired antibody of the carbapenemase antibody in the labeled antibody; the immunochromatographic test strip is sequentially provided with a conjugate release area, a detection area and a quality control area along the flowing direction of the sample to be tested; the conjugate release area is coated with the labeled antibody; the detection area is coated with the detection antibody. The carbapenemase immunochromatographic test strip alleviates the problem in the prior art of lacking a simple and rapid product for detecting carbapenemase.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a carbapenemase immunochromatographic test strip, a preparation method thereof, a kit and an application thereof. Background Art

[0002] Currently, bacterial resistance has become a major challenge in the field of global public health, especially the infection situation caused by carbapenem-resistant Enterobacterales (CRE) is the most severe. Carbapenem antibiotics include imipenem, meropenem, ertapenem, etc., and are one of the most effective antibiotics for treating infections caused by multi-drug resistant Gram-negative bacilli. With the wide clinical application of such drugs, the resistance rate of Enterobacterales bacteria to carbapenem antibiotics has shown a rapid upward trend year by year.

[0003] Carbapenem-resistant Enterobacteriaceae (CRE) cause hospital infections and are opportunistic pathogens. The monitoring results of the CHINET China Bacterial Resistance Surveillance Network over the years show that the resistance rate of clinically isolated Klebsiella pneumoniae to carbapenem antibiotics in China has rapidly climbed from 3% in 2005 to over 25% in 2019, with an increase of up to 8 times. The data of the National Bacterial Resistance Surveillance Network (CARSS) in 2018 showed that the average resistance rate of clinically isolated Klebsiella pneumoniae from 1429 hospitals across the country to carbapenem antibiotics was 10.1%, and in some provinces and cities it even exceeded 20%. Since CRE strains usually also carry genes resistant to other antibiotics and show characteristics of extensive resistance or even pan-resistance to antibiotics, it makes the clinical anti-infection treatment face the dilemma of having no available drugs. When the body's immune function declines, bacteria can cause infections in many parts such as the lungs and urinary tract. As an important drug for clinical treatment of infections caused by Enterobacteriaceae bacteria, in recent years, with the wide use of these drugs, carbapenem-resistant Klebsiella pneumoniae (CPKP) has appeared all over the world, and the detection volume has increased year by year, attracting extensive attention in the medical field. In hospital infections caused by CRE in China, the most common is the hospital infection caused by carbapenem-resistant Klebsiella pneumoniae.

[0004] Carbapenemase production is the most important mechanism of Enterobacterales bacteria's resistance to carbapenems. The types of carbapenemases produced vary among different regions, hospitals, populations, and bacteria. The carbapenemases produced by CRE strains isolated clinically in China are mainly of the KPC and NDM types, and a small number of strains produce OXA-48, IMP, and VIM types of carbapenemases. Since different types of antibacterial drugs have different in vitro antibacterial activities against strains producing different carbapenemases, accurate and rapid detection of carbapenemases produced by CRE is of great value for precise medication in clinical anti-infection treatment and prevention and control of hospital infections.

[0005] At present, the methods for laboratory detection of carbapenemases are divided into phenotypic detection and genotypic detection. Phenotypic detection methods include the Carba NP test, modified carbapenem inactivation tests (including mCIM and eCIM), carbapenemase inhibitor enhancement tests, and time-of-flight mass spectrometry technology, etc.; genotypic detection methods include immunochromatography technology and gene detection technology. Except for the Carba NP test, several other methods are basically culture-based, taking as long as 3 - 7 days, and different enzyme detection methods are different, with complex operations, requiring technical personnel with professional backgrounds, and being powerless for difficult-to-culture or slow-growing bacteria, making it difficult to meet clinical needs. The Carba NP test requires special reagents, some of which need to be self-made, have a short shelf life, and low sensitivity. Gene detection technology has higher sensitivity compared to other diagnostic methods, but is costly, requires 4 - 6 hours for the experiment, has high operation requirements, and its application is restricted. Therefore, a product that can simply and rapidly detect carbapenemases is needed in the current market.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The first object of the present invention is to provide a carbapenemase immunochromatographic test strip, which alleviates the problem in the prior art of lacking a simple and rapid product for detecting carbapenemases.

[0008] The second object of the present invention is to provide a preparation method of the above-mentioned carbapenemase immunochromatographic test strip.

[0009] The third object of the present invention is to provide a kit containing the above-mentioned carbapenemase immunochromatographic test strip and their applications.

[0010] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0011] According to one aspect of the present invention, the present invention provides a carbapenemase immunochromatographic test strip, which includes a labeled antibody and a detection antibody; the labeled antibody includes an antibody against at least one carbapenemase among KPC, OXA-48, VIM, IMP, and NDM; the detection antibody is composed of a paired antibody of the carbapenemase antibody in the labeled antibody.

[0012] The immunochromatographic test strip is sequentially provided with a conjugate release area, a detection area, and a quality control area along the flowing direction of the sample to be tested; the conjugate release area is coated with the labeled antibody; the detection area is coated with the detection antibody.

[0013] The labeled antibody is labeled with a labeling agent.

[0014] According to another aspect of the present invention, the present invention further provides a carbapenemase detection kit, which contains the above-mentioned carbapenemase immunochromatographic test strip.

[0015] According to another aspect of the present invention, the present invention further provides a preparation method of the above-mentioned carbapenemase immunochromatographic test strip, which includes coating the labeled antibody on the conjugate release area and coating the detection antibody on the detection area.

[0016] According to another aspect of the present invention, the present invention further provides the application of the above-mentioned carbapenemase immunochromatographic test strip or the above-mentioned carbapenemase detection kit in detecting carbapenemase for non-diagnostic and therapeutic purposes, or detecting carbapenem-resistant bacteria.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The carbapenemase immunochromatographic test strip provided by the present invention includes a labeled antibody and a detection antibody; the labeled antibody includes an antibody against at least one carbapenemase among KPC, OXA-48, VIM, IMP, and NDM; the detection antibody is composed of a paired antibody of the carbapenemase antibody in the labeled antibody. Its detection principle is the double antibody sandwich method. If the sample to be tested contains carbapenemase antigen and the antigen concentration is higher than the minimum detection limit, the carbapenemase in it binds to the labeled antibody to form a complex, and under the action of chromatography, the complex moves forward along the test strip and binds to the pre-coated detection antibody when passing through the detection area. If a labeled agent-labeled antibody-antigen-detection antibody immune complex is formed, a detectable signal appears in the detection area; on the contrary, if the sample to be tested does not contain carbapenemase antigen or the antigen concentration is lower than the minimum detection limit, no immune complex can be formed, and no detectable signal appears in the detection area, and the result is negative at this time.

[0019] The carbapenemase immunochromatographic test strip provided by the present invention is characterized by being rapid and simple. The entire detection process only takes 15 minutes, without the need for any other instrument and equipment, without the need for professional personnel, is convenient to carry, and can be tested at any time. Moreover, the specimens can be tested in batches or individually. Each single test can detect at most whether one or several of five carbapenemases (KPC, NDM, OXA-48, IMP, VIM) exist in the samples to be tested. This alleviates the problem in the prior art of lacking a simple and rapid product for detecting carbapenemases.

[0020] Based on the inventive concept of the above-mentioned carbapenemase immunochromatographic test strip, the kit containing the above-mentioned carbapenemase immunochromatographic test strip and their applications provided by the present invention can accurately and rapidly detect carbapenemases or carbapenemase-resistant bacteria, and have important value for the precise medication of clinical anti-infection treatment and the prevention and control of hospital infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the SDS-PAGE electrophoresis diagram of antibodies KPC-1 and KPC-2;

[0023] Figure 2 It is the SDS-PAGE electrophoresis diagram of antibodies OXA-48-1 and OXA-48-2;

[0024] Figure 3 It is the SDS-PAGE electrophoresis diagram of antibodies VIM-1 and VIM-2;

[0025] Figure 4 It is the SDS-PAGE electrophoresis diagram of antibodies IMP-1 and IMP-2;

[0026] Figure 5 It is the SDS-PAGE electrophoresis diagram of antibodies NDM-1 and NDM-2;

[0027] Figure 6 It is the structural schematic diagram of the colloidal gold immunochromatographic test strip in Example 2;

[0028] Figure 7 It is the top view structural schematic diagram of the colloidal gold immunochromatographic test strip in Example 2;

[0029] Figure 8Detection results of five carbapenemase antigens by the colloidal gold immunochromatographic test strip in Example 2. Detailed implementation mode

[0030] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0031] Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0032] It should be noted that:

[0033] In the present invention, if there is no special description, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution; all the technical features and preferred features mentioned herein can be combined with each other to form a new technical solution; the components or their preferred components involved can be combined with each other to form a new technical solution.

[0034] In the present invention, unless otherwise stated, the numerical range "a~b" represents the abbreviated representation of any real number combination between a and b, where a and b are both real numbers.

[0035] The "range" disclosed in the present invention can be in the form of one or more lower limits and one or more upper limits, respectively.

[0036] Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0037] According to one aspect of the present invention, the present invention provides a carbapenemase immunochromatographic test strip for detecting at least one of the following types of carbapenemases: KPC, OXA-48, VIM, IMP, and NDM.

[0038] The carbapenemase immunochromatographic test strip comprises a labeled antibody and a detection antibody; the labeled antibody comprises an antibody against at least one carbapenemase among KPC, OXA-48, VIM, IMP and NDM; the detection antibody is composed of the paired antibodies of the carbapenemase antibodies in the labeled antibody, and the labeled antibody is labeled with a labeling agent. The paired antibodies refer to two antibodies that can bind to an antigen molecule simultaneously and are used to detect the antigen in a sample by the sandwich immunoassay method.

[0039] In some optional embodiments, when the labeled antibody is selected from the antibodies against one of the carbapenemases among KPC, OXA-48, VIM, IMP and NDM, the detection antibody is selected from its corresponding paired antibody; specific examples include: the labeled antibody is a KPC antibody, and the detection antibody is another paired antibody of the KPC antibody; the labeled antibody is an OXA-48 antibody, and the detection antibody is another paired antibody of the OXA-48 antibody; the labeled antibody is a VIM antibody, and the detection antibody is another paired antibody of the VIM antibody; the labeled antibody is a VIM antibody, and the detection antibody is another paired antibody of the VIM antibody; or, the labeled antibody is an NDM antibody, and the detection antibody is another paired antibody of the NDM antibody.

[0040] In some other optional embodiments, when the labeled antibody is selected from the antibodies against at least two carbapenemases among KPC, OXA-48, VIM, IMP and NDM, the detection antibody contains the corresponding paired antibodies of each type of carbapenemase antibody in the labeled antibody; specific examples include but are not limited to: the labeled antibody comprises a KPC antibody and an OXA-48 antibody, and the detection antibody comprises another paired antibody of the KPC antibody and another paired antibody of the OXA-48 antibody; the labeled antibody comprises a VIM antibody, an IMP antibody and an NDM antibody, and the detection antibody comprises another paired antibody of the VIM antibody, another paired antibody of the IMP antibody and another paired antibody of the NDM antibody; the labeled antibody comprises a KPC antibody, an OXA-48 antibody, a VIM antibody, an IMP antibody and an NDM antibody; the detection antibody comprises another paired antibody of the KPC antibody, another paired antibody of the OXA-48 antibody, another paired antibody of the VIM antibody, another paired antibody of the IMP antibody and another paired antibody of the NDM antibody.

[0041] In some preferred embodiments, the labeled antibody comprises antibodies against five carbapenemases, namely KPC, OXA-48, VIM, IMP and NDM. Correspondingly, the detection antibody comprises antibodies that can pair with the antibodies against the five carbapenemases, namely KPC, OXA-48, VIM, IMP and NDM in the detection antibody, so as to achieve the technical effect of simultaneously detecting five carbapenemases.

[0042] The immunochromatographic test strip provided by the present invention is sequentially provided with a conjugate release area, a detection area and a quality control area along the flowing direction of the sample to be tested; the conjugate release area is coated with a labeled antibody; the detection area is coated with a detection antibody. The detection principle of the immunochromatographic test strip is the double antibody sandwich method. If the sample to be tested contains a carbapenemase antigen and the antigen concentration is higher than the minimum detection limit, the carbapenemase in it binds to the labeled antibody labeled with a label to form a complex. Under the action of chromatography, the complex moves forward along the test strip and binds to the pre-coated detection antibody when passing through the detection area. If a labeled antibody-antigen-detection antibody immune complex is formed, a detectable signal appears in the detection area; on the contrary, if the sample to be tested does not contain a carbapenemase antigen or the antigen concentration is lower than the minimum detection limit, an immune complex cannot be formed, and no detectable signal appears in the detection area, and the result is negative at this time.

[0043] In some optional embodiments, the quality control area is coated with an antibody that specifically binds to the antigen or labeled antibody coated in the conjugate release area; optionally, the conjugate release area is further coated with a quality control antigen, and the quality control area is coated with an antibody that specifically binds to the quality control antigen; optionally, the quality control area is directly coated with an antibody that specifically binds to the labeled antibody. A detectable signal should appear in the quality control area when detecting a sample. Whether a detectable signal appears in the quality control area is the standard for determining whether the chromatography process is normal and also serves as an internal control standard for the reagent. The detectable signals in the detection area and the quality control area can be detected by an instrument, such as a fluorescence signal formed by fluorescent microspheres; or directly form a signal observable by the naked eye, such as the detection area or the quality control area showing color due to the enrichment of colored microspheres, colloidal gold, colloidal silver, and colloidal carbon.

[0044] The antibodies used in the carbapenemase immunochromatographic test strip provided by the present invention can be selected from commercially available antibodies. In some preferred embodiments, all or part of the antibodies used in the carbapenemase immunochromatographic test strip are selected from the following paired antibodies:

[0045] The heavy chain variable region of the antibody in the following paired antibodies includes three complementary determining regions CDR-H1, CDR-H2 and CDR-H3; the light chain variable region includes three complementary determining regions CDR-L1, CDR-L2 and CDR-L3.

[0046] The KPC paired antibody includes KPC-1 and KPC-2. The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of KPC-1 are shown as Seq_1, Seq_2 and Seq_3 respectively; the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown as Seq_4, Seq_5 and Seq_6 respectively;

[0047] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of KPC-2 are shown in Seq_11, Seq_12, and Seq_13 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_14, Seq_15, and Seq_16 respectively.

[0048] The OXA-48 paired antibodies include OXA-48-1 and OXA-48-2;

[0049] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of OXA-48-1 are shown in Seq_21, Seq_22, and Seq_23 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_24, Seq_25, and Seq_26 respectively;

[0050] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of OXA-48-2 are shown in Seq_31, Seq_32, and Seq_33 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_34, Seq_35, and Seq_36 respectively.

[0051] The VIM paired antibodies include VIM-1 and VIM-2;

[0052] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of VIM-1 are shown in Seq_41, Seq_42, and Seq_43 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_44, Seq_45, and Seq_46 respectively;

[0053] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of VIM-2 are shown in Seq_51, Seq_52, and Seq_53 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_54, Seq_55, and Seq_56 respectively.

[0054] The IMP paired antibodies include IMP-1 and IMP-2;

[0055] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of IMP-1 are shown in Seq_61, Seq_62, and Seq_63 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_64, Seq_65, and Seq_66 respectively;

[0056] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of IMP-2 are shown as Seq_71, Seq_72, and Seq_73 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_74, Seq_75, and Seq_76 respectively.

[0057] NDM paired antibodies, including NDM-1 and NDM-2;

[0058] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of NDM-1 are shown as Seq_81, Seq_82, and Seq_83 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_84, Seq_85, and Seq_86 respectively;

[0059] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of NDM-2 are shown as Seq_91, Seq_92, and Seq_93 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_94, Seq_95, and Seq_96 respectively.

[0060] It is well known in the art that the binding specificity and affinity of antibodies are mainly determined by the CDR sequences. According to mature and well-known existing technologies, the amino acid sequences of non-CDR regions can be easily changed to obtain variants with similar biological activities. The variants of the antibodies of the present invention having CDR sequences exactly the same as the above CDR sequences have similar biological activities because they have the same CDR sequences as the above antibodies, and can be used for the carbapenemase immunochromatographic test strip provided by the present invention. Therefore, the carbapenemase immunochromatographic test strip of the antibody having the above CDR sequences belongs to the protection scope of the present invention.

[0061] In some preferred embodiments, the amino acid sequence of the heavy chain variable region of KPC-1 is preferably as shown in Seq_7, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_8; the amino acid sequence of the heavy chain variable region of KPC-2 is preferably as shown in Seq_17, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_18; the amino acid sequence of the heavy chain variable region of OXA-48-1 is preferably as shown in Seq_27, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_28; the amino acid sequence of the heavy chain variable region of OXA-48-2 is preferably as shown in Seq_37, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_38; the amino acid sequence of the heavy chain variable region of VIM-1 is preferably as shown in Seq_47, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_48; the amino acid sequence of the heavy chain variable region of VIM-2 is preferably as shown in Seq_57, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_58; the amino acid sequence of the heavy chain variable region of IMP-1 is preferably as shown in Seq_67, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_68; the amino acid sequence of the heavy chain variable region of IMP-2 is preferably as shown in Seq_77, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_78; the amino acid sequence of the heavy chain variable region of NDM-1 is preferably as shown in Seq_87, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_88; the amino acid sequence of the heavy chain variable region of NDM-1 is preferably as shown in Seq_97, and the amino acid sequence of the light chain variable region is preferably as shown in Seq_98.

[0062] In some preferred embodiments, a signal peptide with the amino acid sequence as shown in Seq_121 is further connected to the N-terminus of the heavy chain variable regions of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2.

[0063] In some preferred embodiments, a signal peptide with the amino acid sequence as shown in Seq_122 is further connected to the N-terminus of the light chain variable regions of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2.

[0064] In some preferred embodiments, the antibody types of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2 can be, for example, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. More preferably, the above antibodies contain the sequence of the constant region of any one of rabbit antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. The amino acid sequence of the heavy chain constant region is preferably as shown in Seq_123, and the amino acid sequence of the light chain constant region is preferably as shown in Seq_124.

[0065] In some alternative embodiments, the labeled antibody is selected from at least one of KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1. Correspondingly, the detection antibody is selected from KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2 as the paired antibody paired with the labeled antibody.

[0066] In some other alternative embodiments, the labeled antibody is selected from at least one of KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2. Correspondingly, the detection antibody is selected from KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1 as the paired antibody paired with the labeled antibody.

[0067] In some preferred embodiments, it is preferred to use at least one of KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1 as the labeled antibody.

[0068] In some preferred embodiments, when the detection area of the immunochromatographic test strip is coated with antibodies of at least two types of carbapenemases, the antibodies of various types of carbapenemases are respectively coated on the test lines arranged at intervals in the detection area, and it is determined whether the corresponding type of carbapenemase is contained in the sample to be tested according to the results of the test lines.

[0069] In some alternative embodiments, the conjugate release area of the immunochromatographic test strip is coated with KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1; the detection area is coated with KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2; or, the conjugate release area is coated with KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2; the detection area is coated with KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1.

[0070] In some alternative embodiments, the conjugate release zone of the immunochromatographic test strip is coated with KPC-1, OXA-48-1, VIM-1, IMP-1, and NDM-1; the test zone consists of five test lines, which are respectively coated with KPC-2, OXA-48-2, VIM-2, IMP-2, and NDM-2.

[0071] In some alternative embodiments, the marker includes one or more of latex particles, fluorescent molecule labels, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver, and colloidal carbon.

[0072] In some preferred embodiments, the immunochromatographic test strip is a colloidal gold immunochromatographic test strip, that is, the labeled antibody labels colloidal gold. The particle size of the colloidal gold labeled by the labeled antibody is preferably 5-50 nm, for example, it can be but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm, or a mixture of particle size ranges between any two of the above point values. The particle size of the colloidal gold is preferably 20-40 nm. The colloidal gold is preferably prepared by the chloroauric acid-sodium citrate reduction method.

[0073] In some preferred embodiments, the carbapenemase immunochromatographic test strip further comprises a housing.

[0074] According to another aspect of the present invention, the present invention also provides a carbapenemase detection kit, which contains the above-mentioned carbapenemase immunochromatographic test strip.

[0075] In some preferred embodiments, the kit further includes a sample lysis reagent, and the sample lysis reagent includes a buffer reagent and a surfactant. The buffer system preferably uses PBS as the buffer system, and more preferably uses 0.01 mol / L PBS buffer solution as the buffer system. The surfactant includes but is not limited to one or several of Tween-20, Tween-80, CHAPS, Brij35, Brij23, Triton-X100, SDS, or sodium lauroyl sarcosine.

[0076] According to another aspect of the present invention, the present invention also provides a preparation method of the immunochromatographic test strip in the above embodiments. The preparation method includes coating the labeled antibody on the conjugate release zone and coating the detection antibody on the test zone.

[0077] In some alternative embodiments, the marker is colloidal gold. The colloidal gold is labeled on the labeled antibody by physical adsorption or chemical coupling methods, preferably by physical adsorption. More preferably, the labeled antibody is prepared as follows: adjust the pH of the colloidal gold solution, then add the labeled antibody and stir, then add the blocking solution and continue to stir. After centrifugation, remove the supernatant, and then redissolve with the reconstitution solution; the blocking solution contains casein or BSA, and the reconstitution solution contains sucrose and BSA, and uses Tris-HCl as the buffer system. Among them, the colloidal gold is preferably prepared by the chloroauric acid-sodium citrate reduction method.

[0078] In some alternative embodiments, the antibody coated on the control region and the detection antibody are respectively diluted to a final concentration of 0.5-2 mg / mL with the coating dilution solution. For example, it can be but is not limited to 0.5, 1, 1.5 or 2 mg / mL, and then streaked.

[0079] In some preferred embodiments, each type of carbapenemase antibody in the detection antibody is respectively diluted to a final concentration of 0.5-2 mg / mL with the coating dilution solution. For example, it can be but is not limited to 0.5, 1, 1.5 or 2 mg / mL, and streaked in the detection region respectively to form detection lines capable of detecting different types of carbapenemases, so as to distinguish the antigen types in the sample.

[0080] In some preferred embodiments, the coating dilution solution is streaked according to the ratio of the volume of the coating dilution solution to the length of the detection line or the control line of 0.5-2 μL / cm. For example, it can be but is not limited to 0.5, 1, 1.5 or 2 μL / cm, and preferably 1 μL / cm.

[0081] In some alternative embodiments, the immunochromatographic test strip is installed as follows: sequentially paste the detection pad, the absorbent pad, the labeled pad and the sample pad on the fixed bottom plate. The absorbent pad and the labeled pad each press the detection pad by 1-2 mm, and the sample pad presses the labeled pad by 1-2 mm. After assembly, the test strip is cut into a test strip with a width of 4±0.4 mm and installed in the housing.

[0082] According to another aspect of the present invention, the present invention also provides the above-mentioned carbapenemase immunochromatographic test strip or kit for detecting carbapenemase for non-diagnostic and therapeutic purposes, or for detecting carbapenem-resistant bacteria. Examples of detecting carbapenem-resistant bacteria include but are not limited to detecting carbapenem-resistant Enterobacteriaceae bacteria, such as detecting Klebsiella pneumoniae. The above-mentioned carbapenemase immunochromatographic test strip uses the principle of forming an antibody-antigen-antibody complex to detect carbapenemase or carbapenem-resistant bacteria. It has simple operation and short reaction time, and is suitable for quickly identifying whether one or several of the five carbapenemases exist in the sample.

[0083] In some preferred embodiments, it is preferred to pretreat the sample to be tested with the sample lysis reagent provided in the above embodiments, and then detect the sample. Currently, common methods for bacterial lysis used in the market include: repeated freeze-thaw method, ultrasonic treatment method, organic solvent treatment method, etc. These treatment methods generally require the aid of instrument devices, and the reagents need to be stored at low temperature or have a short shelf life. Pretreating the sample to be tested with the above sample lysis reagent does not require the aid of instruments and is easy to operate.

[0084] The technical solutions and technical effects of the present invention will be further described below in conjunction with preferred embodiments:

[0085] Example 1

[0086] Antibody preparation:

[0087] (1) Through NCBI sequence alignment, the conserved sequence of the KPC enzyme was obtained. Using conventional restriction enzyme digestion and ligation techniques in the field of molecular biology, the expression plasmid pET 28a(+)PM was constructed, transferred into competent cells to obtain positive clones, and the positive clones were transformed into Escherichia coli BL21(DE3) competent cells and then cultured and induced for expression. After purification, the KPC antigen was obtained. The OXA-48, VIM, IMP, and NDM antigens were prepared by the same method.

[0088] (2) The antigen was used to immunize New Zealand white rabbits. The New Zealand white rabbits were immunized by multiple subcutaneous injections on the back; two weeks later, a booster immunization was performed, and then a booster immunization was performed every week thereafter, for a total of six immunizations. And starting from the third immunization, 200 - 500 μL of ear marginal vein blood of the New Zealand white rabbits was taken one week after immunization to measure the titer and affinity; after the last immunization, the spleen was taken for cell fusion to prepare hybridoma cells.

[0089] (3) Logarithmic growth phase SP2 / 0 myeloma cells were fused with splenocytes isolated from rabbit spleens to prepare hybridoma cells. The obtained hybridoma cells were screened by ELISA method. The growth of the cells was observed on the 5th day after fusion, and the titer of the cell culture supernatant was detected by indirect ELISA method on the 10th - 14th day. The positive hybridoma cells with the strongest titer were expanded in culture until the positive cell rate reached 100%, and the hybridoma cell line was established and stored in liquid nitrogen for later use.

[0090] (4) After homogenizing the hybridoma cells screened in step (3), the PCR product obtained by extracting and reverse transcribing RNA for PCR was identified by 1% agarose gel electrophoresis, the target fragment was recovered, sent for sequencing, and the sequencing result was compared with the IMGT database (http: / / www.imgt.org / IMGT_vquest / vquest) to obtain the variable region gene fragment of the antibody.

[0091] (5) Homologous recombination primers were used to add homologous recombination arms and signal peptide sequences to both ends of the antibody heavy chain variable region gene and both ends of the light variable region gene. The amino acid sequence of the signal peptide of the heavy chain variable region is as shown in Seq_121, and the amino acid sequence of the signal peptide of the light chain variable region is as shown in Seq_121. The expression plasmids containing the rabbit antibody heavy chain IgG1 constant region and the expression plasmid containing the rabbit antibody light chain IgG1 constant region were linearized to generate homologous recombination arms; the variable region gene fragments with added homologous recombination arms and the linearized plasmids were ligated by homologous recombination to construct complete light chain expression vectors and heavy chain expression vectors. The recombinant products were transformed into TOP10 Escherichia coli competent cells to amplify the plasmids. The obtained monoclonal antibody heavy and light chain expression plasmids were transfected into 293T cells at a ratio of 1:1. After expression and purification, the purified monoclonal antibody was concentrated using a concentrator centrifugal column. PBS was used as the buffer for antibody storage, and finally, the concentration of the concentrated antibody was determined using the BSA protein concentration detection method. The molecular weight of the monoclonal antibody was identified by SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis pattern is as shown in Figures 1 to 5 shown. KPC-1 and KPC-2 are as shown in Figure 1 shown, OXA-48-1 and OXA-48-2 are as shown in Figure 2 shown, VIM-1 and VIM-2 are as shown in the figure, IMP-1 and IMP-2 are as shown in Figure 4 shown, NDM-1 and NDM-2 are as shown in Figure 5 shown.

[0092] The sequence information of KPC-1 and KPC-2, OXA-48-1 and OXA-48-2, VIM-1 and VIM-2, IMP-1 and IMP-2, and NDM-1 and NDM-2 is shown in Tables 1 to 10. The amino acid sequence of the heavy chain constant region of the following antibody is as shown in Seq_123, and the nucleotide sequence is as Seq_125; the amino acid sequence of the light chain constant region is as shown in Seq_124, and the nucleotide sequence is as Seq_126.

[0093] Table 1 KPC-1

[0094]

[0095] Table 2 KPC-2

[0096]

[0097] Table 3 OXA-48-1

[0098]

[0099] Table 4 OXA-48-2

[0100]

[0101]

[0102] Table 5 VIM-1

[0103]

[0104] Table 6 VIM-2

[0105]

[0106] Table 7 IMP-1

[0107]

[0108] Table 8 IMP-2

[0109]

[0110]

[0111] Table 9 NDM-1

[0112]

[0113] Table 10 NDM-2

[0114]

[0115] The nucleotide sequences of the heavy-chain variable regions of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2 are shown as Seq_101, Seq_103, Seq_105, Seq_107, Seq_109, Seq_111, Seq_113, Seq_115, Seq_117 and Seq_119 respectively; the nucleotide sequences of the light-chain variable regions are shown as Seq_102, Seq_104, Seq_106, Seq_108, Seq_110, Seq_112, Seq_114, Seq_116, Seq_118 and Seq_120 respectively.

[0116] Example 2

[0117] This example provides a colloidal gold immunochromatographic test strip:

[0118] This example provides a colloidal gold immunochromatographic test strip, as Figure 6 and Figure 7As shown in the figure (where the icons are represented as: 10: fixed base plate; 20: sample pad; 30: labeling pad; 40: detection pad; 41: detection area; 42: quality control area; 50: absorbent pad), along the flowing direction of the sample to be tested, it includes a sample pad, a labeling pad, a detection pad, and an absorbent pad that are sequentially arranged on a PVC fixed base plate; the labeling pad and the detection pad are made of nitrocellulose membrane; the conjugate release area is arranged on the labeling pad; the detection area and the quality control area are arranged at intervals on the detection pad; the detection area is composed of a KPC detection line, an OXA-48 detection line, a VIM detection line, an IMP detection line, and an NDM detection line.

[0119] The conjugate release area is coated with the antibodies KPC-1, OXA-48-1, VIM-1, IMP-1, and NDM-1 prepared in Example 1; the detection area is respectively coated with KPC-2, OXA-48-2, VIM-2, IMP-2, and NDM-2; the quality control area is coated with goat anti-rabbit antibody. The specific preparation method is as follows:

[0120] (1) Preparation method of colloidal gold and its particle size: Colloidal gold is prepared by the chloroauric acid-sodium citrate reduction method, and the particle size is 20 - 40 nm.

[0121] (2) Process of labeling antibody with colloidal gold: Take the colloidal gold solution, add K2CO3 solution to adjust the pH, add KPC-1, OXA-48-1, VIM-1, IMP-1, and NDM-1 respectively, stir for 2 h, then add the blocking solution (5% casein or BSA) and stir for 30 min. Centrifuge at 10000 g for 30 min, discard the supernatant, and finally redissolve with the redissolution solution (0.05 M Tris-HCl containing 10% sucrose and 1% BSA).

[0122] (3) Preparation of colloidal gold pad: After mixing the colloidal gold solutions labeled with the five antibodies in step (2) evenly, perform gold spraying treatment, and the drying method is drying at 37°C.

[0123] (4) Coating of detection line and quality control line: Use the coating dilution solution (0.01 M PBS solution containing 1% sucrose) for goat anti-rabbit IgG, KPC-2, OXA-48-2, VIM-2, IMP-2, and NDM-2 respectively, with a final concentration of 0.5 - 2 mg / mL. Draw the detection line at 1 μL / cm respectively, and draw the quality control line with goat anti-rabbit IgG, and the drying method is drying at 37°C.

[0124] (5) Assembly of test strip: Paste the detection pad, absorbent pad, labeling pad, and sample pad on the PVC board in sequence. The absorbent pad and the labeling pad each press the detection pad by 1 - 2 mm, and the sample pad presses the labeling pad by 1 - 2 mm. After assembly, the test strip is cut into a width of 4 ± 0.4 mm and installed in the cartridge. The cartridge and the desiccant are put into an aluminum foil bag for plastic packaging. Stick the label and pack it in a box to obtain the finished test strip.

[0125] Example 3

[0126] This example provides a carbapenemase detection kit, including the colloidal gold immunochromatographic test strip of Example 2 and a sample lysis reagent; the sample lysis reagent uses 0.01 mol / L PBS as the buffer system and also contains 0.2% v / v Tween-20 and 1% w / v SDS. Taking the preparation of 1 L as an example, weigh 0.20 g of KCl, 0.24 g of KH2PO4, 3.58 g of Na2HPO4·12H2O, 8.00 g of NaCl, 2 mL of Tween-20, and 10 g of SDS, dissolve them with ultrapure water and make up the volume to 1 L.

[0127] Example 4

[0128] This example provides a method for detecting carbapenem-resistant bacteria. Using the kit provided in Example 4 for detection, the detection steps are as follows:

[0129] Use a 1 μL inoculation loop to pick up a loop of bacterial sample and place it in an EP tube containing 100 μL of lysis solution, vortex and mix well, and let it stand at room temperature for 10 minutes. Add 60 - 80 μL of the sample to the sample addition hole of the test card; observe the result after standing for 15 - 30 minutes.

[0130] Effect Example 1

[0131] The ELISA method was used to detect the titer of the antibody prepared in Example 1. The results are shown in Table 11.

[0132] Table 11 Antibody titer prepared in Example 1

[0133]

[0134] Effect Example 2

[0135] (1) Minimum detection limit: Prepare carbapenemase antigen solutions using five carbapenemase antigens and the sample lysis reagent in Example 3, namely 0.5 ng / mL KPC antigen solution, 1 ng / mL NDM antigen solution, 0.5 ng / mL OXA-48 antigen solution, 0.25 ng / mL IMP antigen solution, and 2 ng / mL VIM antigen solution. The colloidal gold immunochromatographic test strips prepared in Example 2 can detect all the above antigens. The detection results are shown in Figure 8 .

[0136] (2) Specificity analysis: The following strains were detected using the detection method of Example 4. Bacteria that do not produce carbapenemase and produce extended-spectrum β-lactamases (ESBLs) (including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended-spectrum β-lactamase-producing Klebsiella pneumoniae, multidrug-resistant Pseudomonas aeruginosa), intestinal infection bacteria that do not produce carbapenemase (including Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella oxytoca, Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Proteus mirabilis), respiratory infection bacteria that do not produce carbapenemase (including Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Staphylococcus epidermidis, Corynebacterium diphtheriae, Corynebacterium, Bordetella pertussis), digestive tract infection bacteria that do not produce carbapenemase (including Escherichia coli, Shigella, Salmonella typhi, Vibrio cholerae, Helicobacter pylori, Vibrio parahaemolyticus), common bacteria in the air (including Staphylococcus aureus, Lactobacillus, Bacillus subtilis, Clostridium, Bifidobacterium), and other microorganisms (including Saccharomyces cerevisiae, Candida albicans, Aspergillus fumigatus, Cryptococcus). The test results were all negative, indicating that there was no cross-reaction between the above strains and the colloidal gold immunochromatographic test strip of Example 2.

[0137] When adding 1% whole blood sample, 1% saliva sample, 1% sputum sample, 1% bronchoalveolar lavage fluid sample, and 1% urine sample, the test results were the same as those without addition, indicating that the above samples had no interference on the test results. When adding throat swab sample and nasal swab sample, the test results were the same as those without addition, indicating that the above samples had no interference on the test results. When adding 10 mg / mL beef extract, peptone, soy peptone, tryptone, casein peptone, yeast extract, beef liver extract powder, beef heart extract powder, beef brain extract powder, and brain heart infusion, the test results were the same as those without addition, indicating that the above culture medium components had no interference on the test results. When adding 10 mg / mL LB broth, beef extract peptone broth, eosin methylene blue agar, nutrient agar, peptone water medium, Columbia broth, chocolate agar base medium, enterobacteria enrichment broth, MacConkey agar medium, and blood agar base, the test results were the same as those without addition, indicating that the above culture medium products had no interference on the test results.

[0138] (3) Performance detection of three batches of reagents: The colloidal gold immunochromatographic test strips of three batches were respectively taken to detect the quality control strains, and Klebsiella pneumoniae expressing different types of carbapenemase was detected strictly according to the above detection method. The results are shown in Table 12.

[0139] Table 12 Detection results of three batches of reagents

[0140]

[0141] As can be seen from the above table, the detection results of the three batches of kits for the corresponding carbapenemases of the five carbapenemase quality control strains were all positive, the detection results of other types of carbapenemases were all negative, and the detection results of the control negative quality control strains were all negative. Therefore, the coincidence rates of positive and negative samples were both 100%.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A carbapenemase immunochromatographic test strip, characterized in that, It includes a labeled antibody and a detection antibody; the labeled antibody includes antibodies against KPC, OXA-48, VIM, IMP, and NDM carbapenemases; the detection antibody consists of the paired antibodies of the carbapenemase antibodies in the labeled antibody; The immunochromatographic test strip is sequentially provided with a conjugate release area, a detection area, and a quality control area along the flow direction of the sample to be tested; the conjugate release area is coated with the labeled antibody; the detection area is coated with the detection antibody; The labeled antibody is labeled with a label; The labeled antibody and the detection antibody are selected from the paired antibodies of KPC, the paired antibodies of NDM, the paired antibodies of OXA-48, the paired antibodies of IMP, and the paired antibodies of VIM; The paired antibodies of KPC include KPC-1 and KPC-2; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of KPC-1 are shown as Seq_1, Seq_2, and Seq_3 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_4, Seq_5, and Seq_6 respectively; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of KPC-2 are shown as Seq_11, Seq_12, and Seq_13 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_14, Seq_15, and Seq_16 respectively; The paired antibodies of OXA-48 include OXA-48-1 and OXA-48-2; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of OXA-48-1 are shown as Seq_21, Seq_22, and Seq_23 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_24, Seq_25, and Seq_26 respectively; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of OXA-48-2 are shown as Seq_31, Seq_32, and Seq_33 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_34, Seq_35, and Seq_36 respectively; The paired antibodies of VIM include VIM-1 and VIM-2; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of VIM-1 are shown as Seq_41, Seq_42, and Seq_43 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_44, Seq_45, and Seq_46 respectively; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of VIM-2 are shown as Seq_51, Seq_52, and Seq_53 respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown as Seq_54, Seq_55, and Seq_56 respectively; The paired antibodies of IMP include IMP-1 and IMP-2; The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of IMP-1 are shown as Seq_61, Seq_62 and Seq_63 respectively; the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown as Seq_64, Seq_65 and Seq_66 respectively; The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of IMP-2 are shown as Seq_71, Seq_72 and Seq_73 respectively; the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown as Seq_74, Seq_75 and Seq_76 respectively; The paired antibodies of NDM include NDM-1 and NDM-2; The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of NDM-1 are shown as Seq_81, Seq_82 and Seq_83 respectively; the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown as Seq_84, Seq_85 and Seq_86 respectively; The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of NDM-2 are shown as Seq_91, Seq_92 and Seq_93 respectively; the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown as Seq_94, Seq_95 and Seq_96 respectively.

2. The carbapenemase immunochromatographic test strip according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of KPC-1 is shown as Seq_7, and the amino acid sequence of the light chain variable region is shown as Seq_8.

3. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of KPC-2 is shown as Seq_17, and the amino acid sequence of the light chain variable region is shown as Seq_18.

4. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of OXA-48-1 is shown as Seq_27, and the amino acid sequence of the light chain variable region is shown as Seq_28.

5. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of OXA-48-2 is shown as Seq_37, and the amino acid sequence of the light chain variable region is shown as Seq_38.

6. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of VIM-1 is shown as Seq_47, and the amino acid sequence of the light chain variable region is shown as Seq_48.

7. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of VIM-2 is shown as Seq_57, and the amino acid sequence of the light chain variable region is shown as Seq_58.

8. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of IMP-1 is shown as Seq_67, and the amino acid sequence of the light chain variable region is shown as Seq_68.

9. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of IMP-2 is shown as Seq_77, and the amino acid sequence of the light chain variable region is shown as Seq_78.

10. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain variable region of NDM-1 is shown as Seq_87, and the amino acid sequence of the light chain variable region is shown as Seq_88.

11. The carbapenemase immunochromatographic test strip according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of NDM-1 is shown as Seq_97, and the amino acid sequence of the light chain variable region is shown as Seq_98.

12. The carbapenemase immunochromatographic test strip according to claim 1, wherein The N-terminus of the heavy chain variable region of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2 is also connected to a signal peptide with an amino acid sequence as shown in Seq_121.

13. The carbapenemase immunochromatographic test strip according to claim 1, characterized in that The N-terminus of the light chain variable region of KPC-1, KPC-2, OXA-48-1, OXA-48-2, VIM-1, VIM-2, IMP-1, IMP-2, NDM-1 and NDM-2 is also connected to a signal peptide with an amino acid sequence as shown in Seq_122.

14. The carbapenemase immunochromatographic test strip according to claim 1, wherein The amino acid sequence of the heavy chain constant region in the KPC paired antibody, NDM paired antibody, OXA-48 paired antibody, IMP paired antibody and VIM paired antibody is as shown in Seq_123, and the amino acid sequence of the light chain constant region is as shown in Seq_124.

15. The carbapenemase immunochromatographic test strip according to claim 1, characterized in that, The labeled antibody is selected from KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1, and the detection antibody is selected from KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2; Or, the labeled antibody is selected from KPC-2, OXA-48-2, VIM-2, IMP-2 and NDM-2, and the detection antibody is selected from KPC-1, OXA-48-1, VIM-1, IMP-1 and NDM-1.

16. The carbapenemase immunochromatographic test strip according to claim 1, wherein The label includes one or more of latex particles, fluorescent molecule labels, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver and colloidal carbon.

17. The carbapenemase immunochromatographic test strip according to claim 16, characterized in that, The label is colloidal gold.

18. The carbapenemase immunochromatographic test strip according to claim 17, wherein, The particle size of the colloidal gold particles is 5 - 50 nm.

19. The carbapenemase immunochromatographic test strip according to claim 18, wherein The particle size of the colloidal gold particles is 20 - 40 nm.

20. The carbapenemase immunochromatographic test strip according to claim 1, wherein The quality control region is coated with an antibody that specifically binds to the labeled antibody coated in the conjugate release region.

21. The carbapenemase immunochromatographic test strip according to any one of claims 1-20, characterized in that, Along the flow direction of the sample to be tested, it includes a sample pad, a labeling pad, a detection pad and a water absorption pad sequentially arranged on a fixed base plate; the conjugate release region is arranged on the labeling pad; the detection region and the quality control region are arranged at intervals on the detection pad.

22. The carbapenemase immunochromatographic test strip according to claim 21, characterized in that, The detection region includes five detection lines arranged at intervals, and different types of carbapenemase antibodies in the detection antibody are respectively coated on different detection lines.

23. The carbapenemase immunochromatographic test strip according to claim 21, wherein The carbapenemase immunochromatographic test strip also includes a housing.

24. Carbapenemase detection kit, characterized in that, It includes the carbapenemase immunochromatographic test strip according to any one of claims 1 - 23.

25. The carbapenemase detection kit according to claim 24, wherein It also includes a sample lysis reagent.

26. The carbapenemase detection kit according to claim 25, wherein The sample lysis reagent includes a buffer system and a surfactant.

27. The carbapenemase detection kit according to claim 26, wherein The sample lysis reagent uses PBS as the buffer system.

28. The carbapenemase detection kit according to claim 26, wherein, The surfactant includes one or several of Tween-20, Tween-80, CHAPS, Brij35, Brij23, Triton-X100, SDS or sodium lauroyl sarcosinate.

29. The preparation method of the carbapenemase immunochromatographic test strip according to any one of claims 1-23, characterized in that, It includes coating the labeled antibody in the conjugate release region and coating the detection antibody in the detection region.

30. The preparation method according to claim 29, characterized in that, The label is colloidal gold, and the colloidal gold is labeled on the labeled antibody by physical adsorption or chemical coupling.

31. The preparation method according to claim 30, characterized in that, The marker is colloidal gold. The colloidal gold is labeled on the labeled antibody by physical adsorption method, including adjusting the pH of the colloidal gold solution, adding the labeled antibody and stirring, then adding the blocking solution and continuing to stir, centrifuging and removing the supernatant, and then redissolving with the reconstitution solution; the blocking solution contains casein or BSA; the reconstitution solution contains sucrose and BSA, and the reconstitution solution uses Tris-HCl as the buffer system.

32. The preparation method according to claim 31, wherein Dilute the detection antibody with the coating diluent to a final concentration of 0.5 - 2 mg / mL, and draw a line in the detection area to form a detection line.

33. The preparation method according to claim 32, wherein, Dilute each type of carbapenemase antibody in the detection antibody with the coating diluent to a final concentration of 0.5 - 2 mg / mL, and draw lines in the detection area respectively to form detection lines.

34. The preparation method according to claim 32, wherein The volume ratio of the coating diluent to the length of the detection line is 0.5 - 2 μL / cm.

35. The preparation method according to claim 34, wherein, The volume ratio of the coating diluent to the length of the detection line is 1 μL / cm.

36. The preparation method according to claim 31, characterized in that, Paste the detection pad, the absorbent pad, the labeled pad and the sample pad on the fixed base plate in sequence. The absorbent pad and the labeled pad each press the detection pad by 1 - 2 mm, and the sample pad presses the labeled pad by 1 - 2 mm. After assembly, the test strip is cut into a test strip with a width of 4 ± 0.4 mm and installed in the housing. Use of the carbapenemase immunochromatographic test strip according to any one of claims 1 - 23, or the carbapenemase detection kit according to any one of claims 24 - 28 for detecting carbapenemase for non-diagnostic and non-therapeutic purposes, or for detecting carbapenem-resistant bacteria.

38. The application according to claim 37, wherein The carbapenem-resistant bacteria include carbapenem-resistant Enterobacteriaceae.

39. The application according to claim 37, wherein The carbapenem-resistant bacteria include Klebsiella pneumoniae.

40. The application according to claim 37, characterized in that, First, pretreat the sample to be tested with the sample lysis reagent described in any one of claims 25 - 28, and then detect the sample.

41. The application according to claim 40, characterized in that, Place the bacterial sample to be tested in the sample lysis reagent, mix and let stand for 10 - 30 min, take 60 - 80 μL and detect it with the immunochromatographic test strip; observe the result after 15 - 30 minutes of placement.

Images