Antibody for detecting Burkholderia cepacia flora, immunodetection method and application

Abstract

The invention discloses an antibody for detecting Burkholderia cepacia flora, an immunodetection method and application. The antibody provided by the invention comprises a BCC-F1 antibody and / or a BCC-F2 antibody; a light chain CDR1, a light chain CDR2 and a light chain CDR3 of the BCC-F1 are sequences as shown in SEQ ID NO.1 to SEQ ID NO.3 in sequence, and a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 are sequences as shown in SEQ ID NO.4 to SEQ ID NO.6 in sequence; a light chain CDR1, a light chain CDR2 and a light chain CDR3 of the BCC-F2 are sequences as shown in SEQ ID NO.7 to SEQ ID NO.9 in sequence, and a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 are sequences as shown in SEQ ID NO.10 to SEQ ID NO.12 in sequence. The Burkholderia cepacia flora is subjected to immunochromatography detection based on the antibody, the operation is simple, the sensitivity is high, the specificity is strong, the rapid detection of the Burkholderia cepacia flora can be realized, and the Burkholderia cepacia flora antibody has important significance for preventing pollution and personnel infection.

Description

Technical Field

[0001] The present application relates to the technical field of detection of cepacia Burkholderia, and in particular to an antibody, a kit and an application thereof for detecting cepacia Burkholderia. Background Art

[0002] Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria belonging to the genus Burkholderia that are ubiquitous in environments with similar phenotypes but different genotypes.

[0003] As a typical unacceptable microorganism, cepacia contamination is the leading cause of drug microbial contamination recalls and one of the main causes of cosmetic microbial contamination recalls. As a conditional pathogen, cepacia Burkholderia can cause a variety of hospital infections, including cystic fibrosis, chronic granuloma, sepsis, endocarditis, pneumonia, wound infections and abscesses. It has a strong ability to spread between patients and has multidrug resistance, making it difficult to treat.

[0004] The detection of onion Burkholderia is mainly carried out through microbiological inspection methods, mass spectrometry identification methods, etc. The currently commonly used microbiological inspection methods and mass spectrometry identification methods require 48-72 hours of bacterial enrichment culture and 48-72 hours of selection and separation culture. The detection is time-consuming and labor-intensive, and requires expensive instruments and equipment and professional personnel to operate. The "Draft Public Notice of the Onion Burkholderia Inspection Method" issued in November 2022 formulated for the first time a method for detecting onion Burkholderia for drugs. This method uses culture and inspection methods. The patent application with application number 202211167257.7 discloses a kit, primers, method and application for detecting onion Burkholderia. This method uses a PCR molecular detection method, which also requires expensive instruments and equipment and professional personnel to operate.

[0005] There is currently no rapid immunoassay for Burkholderia cepacia. It is necessary and important to establish a rapid, real-time, low-cost, highly sensitive and specific rapid immunoassay for Burkholderia cepacia detection and to prevent contamination and infection of personnel. Summary of the invention

[0006] The purpose of the present application is to provide a new antibody, immunoassay method, kit and application for detecting Burkholderia cepacia.

[0007] This application adopts the following technical solutions:

[0008] The first aspect of the present application discloses an antibody for detecting Burkholderia cepacia, the antibody comprising a BCC-F1 antibody and / or a BCC-F2 antibody; the BCC-F1 antibody comprises a heavy chain and a light chain, and the three light chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the light chain are sequences shown in sequence from SEQ ID NO.1 to SEQ ID NO.3, and the three heavy chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are sequences shown in sequence from SEQ ID NO.4 to SEQ ID NO.6; the BCC-F2 antibody comprises a heavy chain and a light chain, and the three light chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the light chain are sequences shown in sequence from SEQ ID NO.7 to SEQ ID NO.9, and the three heavy chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are sequences shown in sequence from SEQ ID NO.10 to SEQ ID NO.12.

[0009] It should be noted that antibodies with high sensitivity and good specificity are the key to the successful development and implementation of immunoassay technology. Based on monoclonal antibody technology, the present application obtains specific antibodies against flagellin of Burkholderia cepacia, namely monoclonal antibody BCC-F1 and monoclonal antibody BCC-F2. The use of the specific antibodies of the present application for immunochromatographic detection is simple to operate, highly sensitive, and highly specific, and can achieve rapid detection of Burkholderia cepacia. It can be understood that the BCC-F1 antibody and BCC-F2 antibody of the present application both have the advantages of high sensitivity and high specificity, and can be used alone for immunoassay or in combination. For example, in one implementation of the present application, BCC-F1 antibody and BCC-F2 antibody are used for double antibody sandwich detection.

[0010] The second aspect of the present application discloses a nucleic acid encoding the antibody of the present application.

[0011] The third aspect of the present application discloses a recombinant plasmid containing a nucleic acid encoding the specific antibody against the flagellin of Burkholderia cepacia group of the present application.

[0012] The fourth aspect of the present application discloses a microorganism or cell containing a nucleic acid encoding a specific antibody against Burkholderia cepacia flagellin of the present application or containing a recombinant plasmid of the present application.

[0013] It should be noted that in one implementation of the present application, the specific antibody against the flagellin of the Burkholderia cepacia group of the present application is obtained by screening using hybridoma cell technology; it can be understood that on this basis, the coding sequence of the specific antibody of the present application can be artificially synthesized according to the sequence of the specific antibody of the present application, and then expressed by cloning to obtain the specific antibody of the present application. The specific nucleic acid sequence can be optimized according to the preference of the cloned expression microorganism or cell adopted, and the specific vector sequence and host microorganism or cell can also refer to the existing antibody cloning expression technology, which is not specifically limited here.

[0014] The fifth aspect of the present application discloses a chromatographic test strip for detecting cepacia Burkholderia, wherein the antibody of the present application is coated on the detection pad T line of the chromatographic test strip.

[0015] In one implementation of the present application, one of the BCC-F1 antibody and the BCC-F2 antibody is coated on the T line of the detection pad, and the other antibody is coated on the sample pad of the chromatography test strip, and the antibody coated on the sample pad is labeled with a signal-enhancing nanomaterial. In one implementation of the present application, the C line of the detection pad is coated with an anti-mouse IgG antibody for quality control.

[0016] It should be noted that the key to the chromatography test strip of the present application is the use of the specific antibody of the present application. As for the preparation method of the chromatography test strip, reference can be made to the prior art. In one implementation of the present application, a double antibody sandwich method is specifically adopted. Therefore, one of the BCC-F1 antibody and the BCC-F2 antibody is coated on the T line of the detection pad, and the other antibody is coated on the sample pad of the chromatography test strip, and the antibody coated on the sample pad is labeled with a signal-enhancing nanomaterial. The signal-enhancing nanomaterials that can be used in the present application include but are not limited to colloidal gold, colored latex microspheres, fluorescent microspheres, and quantum dot nanomaterials. For example, in one implementation of the present application, colloidal gold is labeled on the BCC-F2 antibody and coated on the sample pad; the BCC-F1 antibody is coated on the T line of the detection pad.

[0017] The sixth aspect of the present application discloses the use of the specific antibody of the present application, the nucleic acid encoding the specific antibody of the present application, the recombinant plasmid of the present application, the microorganism or cell of the present application, or the chromatography test strip of the present application in the preparation of a kit for detecting cepacia.

[0018] The seventh aspect of the present application discloses a kit for detecting Burkholderia cepacia, which contains the specific antibody of the present application, the nucleic acid encoding the specific antibody of the present application, the recombinant plasmid of the present application, the microorganism or cell of the present application, or the chromatography test strip of the present application.

[0019] It should be noted that the kit for detecting Burkholderia cepacia of the present application may directly contain the chromatographic test strip of the present application. When used, the chromatographic test strip is directly used to detect Burkholderia cepacia. The kit of the present application may also contain the specific antibody of the present application. When used, the specific antibody of the present application is used for immunoassay, or the chromatographic test strip is prepared using the specific antibody of the present application. The kit of the present application may also contain the nucleic acid, recombinant plasmid or cloned microorganism or cell of the present application. At this time, the kit of the present application is mainly used as a raw material to prepare the specific antibody of the present application for the detection of Burkholderia cepacia.

[0020] The eighth aspect of the present application discloses an immunodetection method of cepacia for non-diagnostic and therapeutic purposes, which comprises using the antibody of the present application to perform immunodetection on cepacia.

[0021] It should be noted that the key to the detection method of the present application is to use the specific antibodies of the present application to perform immune detection on Burkholderia cepacia. It is understood that the detection method of the present application mainly refers to the detection of Burkholderia cepacia in food, medicine, cosmetics, medical materials, medical devices or the environment to ensure the safety of the detection object and avoid the spread of Burkholderia cepacia, and is not used for the diagnosis of related diseases.

[0022] In one implementation of the present application, the immunoassay is an immunochromatographic assay; the immunochromatographic assay includes using a chromatographic test strip to detect the cepacia group using a double antibody sandwich method; wherein the double antibody sandwich method includes simultaneously using BCC-F1 antibody and BCC-F2 antibody to prepare a chromatographic test strip, labeling one of the BCC-F1 antibody and the BCC-F2 antibody with a signal-enhancing nanomaterial and coating it on a sample pad of the chromatographic test strip, and coating the other antibody on the detection pad T line of the chromatographic test strip.

[0023] In one implementation of the present application, the signal enhancing nanomaterial used in the chromatographic test strip is at least one of colloidal gold, colored latex microspheres, fluorescent microspheres and quantum dot nanomaterials.

[0024] The beneficial effects of this application are:

[0025] The antibodies for detecting cepacia bacteria of the present application have high sensitivity and good specificity. Immunochromatographic detection of cepacia bacteria based on the antibodies of the present application is simple to operate, highly sensitive, and highly specific, and can achieve rapid and real-time detection of cepacia bacteria, which is of great significance for preventing cepacia bacteria contamination and human infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1These are the titer test results of the BCC-F1 antibody and the BCC-F2 antibody in the examples of this application. DETAILED DESCRIPTION

[0027] The present application uses Burkholderia cepacia flagellin to immunize animals and prepare monoclonal antibodies to Burkholderia cepacia flagellin, which have the advantages of high binding force and strong specificity for Burkholderia cepacia. The established Burkholderia cepacia rapid immunoassay method is simple to operate, has higher sensitivity and better specificity when detecting Burkholderia cepacia, and can realize rapid real-time detection and monitoring of Burkholderia cepacia.

[0028] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Balb / c mice: Guangdong Provincial Laboratory Animal Center, 5-6 weeks old.

[0030] Example 1 Preparation of monoclonal antibodies against Burkholderia cepacia

[0031] 1. Animal immunization and cell fusion screening

[0032] The recombinant flagellin (FliC protein) of Burkholderia cepacia was diluted with physiological saline and mixed with MontanideGel 01PR adjuvant at a ratio of 9:1 to prepare an injection solution with a FliC protein concentration of 400 μg / mL. Then, three Balb / c mice were immunized by calf muscle injection at an injection volume of 20 μg / mouse. On the 14th day, the immunization was boosted in the same way. On the 21st day, blood was collected from the tail of the mice, and the serum was separated and tested for serum titer by indirect ELISA. The titer reached the fusion requirement, that is, reached 10 6 Three days before cell fusion, spleens were boosted with 100 μg of FliC protein.

[0033] On the third day after the spleen booster immunization, the mice were killed according to the biosafety method. In this case, the carbon dioxide asphyxiation method was used. After disinfection with alcohol, the spleen cells were taken and fused with NS1 myeloma cells in the logarithmic growth phase at a ratio of 1:5 under the action of 50% PEG1500. After the fusion, the cells were cultured in HAT semi-solid screening medium for 8-10 days. After the cells were picked and cloned in HT medium for screening and culture for 2 days, the antibody specificity in the cell culture supernatant was detected by indirect ELISA. The positive cells were cloned once more with HT semi-solid medium to obtain a monoclonal cell line that can stably secrete antibodies. After expanded culture, they were frozen with cell freezing solution containing 10% DMSO, and the cell density was 10 6 / mL. Ascites was prepared by in vivo induction method, and the ascites antibody was purified by Protein A affinity chromatography to obtain purified antibody. After dialyzing in 0.01mol / L phosphate buffer, the protein concentration was determined by Nanodrop. Multiple monoclonal cell lines were obtained. After identifying the antibody titer, two cell lines with the best specificity were screened from the monoclonal cell lines and named hybridoma cell lines BCC-F1 and BCC-F2.

[0034] In the above method, the indirect ELISA method for detecting serum titer and identifying antibody titer is the same as "II. Titer detection". The preparation method of Burkholderia cepacia recombinant flagellin is as follows:

[0035] The complete gene synthesis sequence of Burkholderia cepacia recombinant flagellin (FliC protein) (GenBank: QCY01824.1, ATCC: 25416) was used to construct the pET-32a (+) vector, with cloning sites: XbaI-XhoI, AMP resistance, N-terminal fusion trxA tag, and C-terminal 6*His tag. Escherichia coli (BL21) competent cells were transfected into TSB liquid medium containing AMP resistance and cultured at 37°C overnight until OD 600 = 0.8, and IPTG was added at a final concentration of 0.5 mM for induction culture at 16 ° C for 12 hours. The cells were collected after centrifugation, and the cell pellet was added to the lysis buffer for resuspending and ultrasonic lysis for 40 minutes. Centrifugation, the lysis supernatant was filtered with a 0.45 μm filter membrane, and the purified FliC protein was obtained by cobalt affinity chromatography.

[0036] 2. Potency detection (indirect ELISA method)

[0037] 1. Antigen coating: Take the recombinant flagellar protein of Burkholderia cepacia, dilute it with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2 μg / mL, add 100 μL / well of the ELISA plate, and coat it at 4°C overnight.

[0038] 2. After completing step 1, take the ELISA plate, wash it 4 times with PBST solution, then add blocking solution (200 μL / well), and incubate at 37°C for 2 hours. Blocking solution: PBST solution containing 1g / 100mL BSA.

[0039] 3. After completing step 2, take the ELISA plate, wash it with PBST solution 4 times, then add antibody diluent (100 μL / well), and incubate at 37°C for 1 hour. Antibody dilution: Take monoclonal antibodies BCC-F1 and BCC-F2, dilute them with pH 7.4, 0.02M PBS buffer to a protein concentration of 10 μg / mL, and then perform 10-fold gradient dilution.

[0040] 4. After completing step 3, take the ELISA plate, wash it with PBST solution 4 times, then add HRP-labeled secondary antibody working solution and incubate at 37°C for 1 hour. HRP-labeled secondary antibody working solution: 1:4000 dilution of HRP-labeled goat anti-mouse secondary antibody.

[0041] 5. After completing step 4, take the ELISA plate, wash it with PBST solution 4 times, then add TMB substrate reaction solution to color for 10 minutes, then add 2M sulfuric acid solution to stop the color development, and then use an ELISA reader to measure the absorbance at 450nm. The results are shown in Figure 1 . Figure 1 The horizontal axis is the exponent of 10, -2 is 10 -2 ng / mL, that is, 0.01ng / mL, and so on.

[0042] Figure 1 The results showed that the EC50 of monoclonal antibody BCC-F1 was lower than 2.08 ng / mL; the EC50 of monoclonal antibody BCC-F2 was lower than 0.49 ng / mL.

[0043] 3. Antibody subtype identification

[0044] The commercial antibody Ig class / subclass / subtype identification kit (Luoyang Bio-Tong Experimental Materials Center, catalog number C060101-L) was used to identify the antibody subtype. The specific method is shown in the kit manual. The results are shown in Table 1.

[0045] Table 1 Antibody subtype test results

[0046] IgG1 IgG2a IgG2b IgG3 IgM IgA LAMDA KAPPA BCC-F1 2.1393 0.0474 0.0497 0.0576 0.055 0.0494 0.0643 1.8757 BCC-F2 2.3623 0.057 0.0479 0.0465 0.0468 0.0466 0.0664 1.8595

[0047] The first row in Table 1 is the subtype, and the first case is the antibody. The results in Table 1 show that both antibody BCC-F1 and antibody BCC-F2 are of IgG1 subtype, and both antibody light chains are Kappa chains.

[0048] Example 2 Monoclonal Antibody Sequence Determination

[0049] The amino acid sequences of the three CDRs of the light chain and heavy chain of antibody BCC-F1 and antibody BCC-F2 were obtained by sequencing.

[0050] According to the sequencing results, the amino acid sequences of the three CDRs of the light chain and heavy chain of antibody BCC-F1 and antibody BCC-F2 were determined with reference to the Kabat and IMGT numbering methods. The specific amino acid sequences are shown in Table 2.

[0051] Table 2 Amino acid sequences of three CDRs in each of the antibody light chain and heavy chain

[0052]

[0053]

[0054] Example 3 Preparation of colloidal gold rapid test paper

[0055] 1. Preparation of colloidal gold labeled antibodies

[0056] 1. Preparation of colloidal gold by trisodium citrate reduction method

[0057] Use a measuring cylinder to measure 495mL of deionized water, pour it into a glass two-necked bottle, add 5mL of 1g / 100mL chloroauric acid aqueous solution; put a magnetic stirrer in the glass two-necked bottle, connect a spherical condenser, and heat to boiling with a controllable temperature electromagnetic heating stirrer; add 5mL of 1g / 100mL trisodium citrate aqueous solution while stirring, the golden yellow chloroauric acid solution turns purple-red within 2 minutes, continue to boil for 5 minutes, and then cool naturally to obtain a colloidal gold solution.

[0058] 2. Preparation of colloidal gold labeled antibodies

[0059] Take 20 volumes of colloidal gold solution, adjust the pH to 7.4 with 0.1M potassium carbonate aqueous solution, then add monoclonal antibody BCC-F2 (6 micrograms of antibody per milliliter of colloidal gold solution), stir at room temperature for 1 hour, then add BSA and make its concentration 1g / 100mL and react for 1 hour; after the reaction is completed, place it in a centrifuge at 4°C and 12000rpm for 20 minutes, discard the supernatant, and resuspend the remaining red precipitate with 1 volume of PBS buffer (pH7.4, 0.02M) containing 1g / 100mL BSA to obtain a colloidal gold-labeled BCC-F2 antibody solution, which is stored at 4°C.

[0060] 2. Preparation of colloidal gold test strips

[0061] 1. Prepare the test pad

[0062] Take the nitrocellulose membrane, use BioDot's XYZ3050 spray system to spray the C solution to the quality control line (C line), spray the T solution to the test line (T line), and then dry for 6 hours to obtain the test pad. Use pH7.4, 0.02M PBS buffer to prepare the goat anti-mouse IgG antibody into a solution with a concentration of 0.5mg / ml, which is the C solution. Use pH7.4, 0.02M PBS buffer to prepare the monoclonal antibody BCC-F1 into a solution with a concentration of 1.5mg / ml (in terms of protein concentration), which is the T solution.

[0063] 2. Preparation of colloidal gold pad

[0064] (1) Take glass fiber paper, soak it in PBS buffer (pH 7.4, 0.02 M) containing 0.1 g / 100 mL Tween-20, 1 g / 100 mL BSA, and 5 g / 100 mL sucrose at 37° C. for half an hour, and then dehumidify it in a drying room with a relative humidity of less than 10% for 4 hours.

[0065] (2) After completing step (1), take the glass fiber paper, use BioDot's XYZ3050 spray system to spray the gold-labeled antibody solution, and then dry it for 6 hours to obtain a colloidal gold pad. Gold-labeled antibody solution: Take the colloidal gold-labeled BCC-F2 antibody solution prepared in step "2. Preparation of colloidal gold-labeled antibodies" and dilute it with a pH 7.4, 0.02M PBS buffer containing 0.1 g / 100 mL Tween-20, 1 g / 100 mL BSA, and 5 g / 100 mL sucrose to make the concentration of the colloidal gold-labeled BCC-F2 antibody 2 μg / ml (based on protein concentration).

[0066] 3. Assembly of test strips

[0067] The test strip includes a base plate (also called a backing), a colloidal gold pad (also called a sample pad), a detection pad and a water absorbent pad. Along the sample flow direction, the colloidal gold pad, the detection pad and the water absorbent pad are fixed on the base plate in sequence (adjacent pads slightly overlap).

[0068] The sample flow direction is taken as the length direction of the test strip, and the direction perpendicular to the length direction is taken as the width direction of the test strip. The width of the test strip is 3.5 mm. The total length of the test strip is 6 cm, of which the length of the colloidal gold pad is 2 cm, the length of the detection pad is 2.5 cm, and the length of the absorbent pad is 2 cm. The C line is close to the absorbent pad and away from the colloidal gold pad. The T line is close to the colloidal gold pad and away from the absorbent pad.

[0069] The material of the absorbent pad is absorbent paper.

[0070] The bottom plate only plays a supporting role and can be made of any conventional material. For example, PVC is used in this test.

[0071] 3. How to use the test strips

[0072] Slowly drop 60 μL of the sample to be tested vertically into the sample end of the colloidal gold test strip (the sample end of the colloidal gold test strip is the part of the colloidal gold pad close to the free end), place it horizontally at room temperature for 10-15 minutes, and then judge the results as follows:

[0073] The C line is colored, indicating that the results are reliable;

[0074] If the T line is colored, it means that the sample to be tested contains Burkholderia cepacia;

[0075] If the T line does not show color, it means that the sample to be tested does not contain cepacia bacteria.

[0076] 4. Working Principle of Test Strips

[0077] The goat anti-mouse IgG antibody on line C captured the colloidal gold-labeled BCC-F2 antibody, resulting in color development.

[0078] If the sample to be tested contains cepacia, the flagellin of cepacia will form a complex with the colloidal gold-labeled BCC-F2 antibody on the colloidal gold pad. When passing through the detection pad, the BCC-F1 antibody on the T line captures the complex through specific binding with the flagellin of cepacia, thereby developing color.

[0079] If the sample to be tested does not contain cepacia, when passing through the test pad, the BCC-F1 antibody on the T line cannot capture the colloidal gold-labeled BCC-F2 antibody, and thus no color is developed.

[0080] Performance of Example 4 Test Strips

[0081] 1. Detection sensitivity

[0082] Take the standard strain of Burkholderia cepacia (ATCC 25416), inoculate it into TSA medium, culture it at 33℃ for 16-18 hours, pick a single colony and inoculate it into TSB medium, culture it for 18-24 hours, and detect OD 670 Adjust the bacterial solution to 10 8 CFU / mL, and 10-fold gradient dilution, the dilution was inoculated into TSA medium at the same time, cultured at 33℃ for 48 hours, and counted. The gradient diluted bacterial solution was heated at 65℃ for 10 minutes to lyse the bacterial flagella as the test sample, and then the test strip prepared in Example 3 was used to detect each test sample. 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 The samples with CFU / mL were tested and the results are shown in Table 3.

[0083] Table 3 Sensitivity test results

[0084] Bacterial concentration (CFU / mL) <![CDATA[10 1 ]]> <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> <![CDATA[10 7 ]]> <![CDATA[10 8 ]]> Test results Negative Negative Negative Negative Positive Positive Positive Positive

[0085] The results in Table 3 show that the detection sensitivity of the colloidal gold rapid test strip is 10 5 CFU / mL.

[0086] In order to verify the rapid detection effect of the present method after the sample single bacterial contamination was enriched and cultured, the concentration of the Burkholderia cepacia standard strain (ATCC 25416) was diluted to 10 3 CFU / mL, 50 μL was inoculated into 50 mL of TSB medium as the initial sample, that is, the concentration of Bcc in the initial sample was 1 CFU / mL, the initial sample was cultured in two groups for easy sampling, 1 mL of the culture was taken at 0 h, 4 h, 8 h, 12 h, 16 h, 18 h, 20 h, 22 h, and 24 h, respectively, heated at 65 ° C for 10 minutes, as the sample to be tested, and then tested with the test strip prepared in Example 3. The experimental results are shown in Table 4.

[0087] Table 4 Bacterial enrichment culture time test results

[0088] Cultivation time 0h 4h 8h 12h 16h 18h 20h 22h 24h Test results Negative Negative Negative Negative Positive Positive Positive Positive Positive

[0089] The results in Table 4 show that when the initial sample contamination Bcc concentration is no more than 1 CFU / mL, the sample can be tested for Bcc contamination by rapid immunoassay after at least 16 hours of bacterial culture, which is much faster than the one week required for traditional culture.

[0090] 2. Detection Specificity

[0091] For the activated representative Bcc strains and non-Bcc strains in Table 5, the bacterial solution was heated at 65° C. for 10 minutes as the test sample, and then tested with the test strip prepared in Example 3. The results are shown in Table 5.

[0092] Table 5 Specificity test results

[0093]

[0094]

[0095] The results in Table 5 show that the detection results of 9 species of cepacia group: cepacia, neocepacia, polyphagic Burkholderia, Vietnamese Burkholderia, stable Burkholderia, bidirectional Burkholderia, mysterious Burkholderia, pyrrolizidine Burkholderia and garden Burkholderia were all positive, and the detection results of the remaining 14 strains of non-cepacia group were all negative, with good specificity.

[0096] 3. Bcc sample test

[0097] 13 samples of Bcc clinical isolates and 22 samples of cosmetic isolates were separated and preserved by Shenzhen Institute of Drug Inspection. The separation method was the "Burkholderia cepacia test method", and then mass spectrometry was used to identify them as Bcc. The isolated bacterial solution was heated at 65°C for 10 minutes and used as the test sample to be tested. The test strip prepared in Example 3 was used for detection, and negative controls and water blank controls were designed. The test results showed that the test results of 35 samples were all positive, and the negative controls and water blank controls were normal and negative, indicating that this test method can specifically detect environmental isolated strains.

[0098] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. An antibody for detecting Burkholderia cepacia, characterized in that: The antibodies include BCC-F1 antibody and / or BCC-F2 antibody; The BCC-F1 antibody comprises a heavy chain and a light chain, wherein the three light chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the light chain are sequentially represented by sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and the three heavy chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are sequentially represented by sequences shown in SEQ ID NO.4 to SEQ ID NO.6; The BCC-F2 antibody comprises a heavy chain and a light chain, wherein the three light chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the light chain are sequences shown in sequence from SEQ ID NO.7 to SEQ ID NO.9, and the three heavy chain variable region complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are sequences shown in sequence from SEQ ID NO.10 to SEQ ID NO.

12.

2. A nucleic acid encoding the antibody according to claim 1.

3. A recombinant plasmid containing the nucleic acid according to claim 2.

4. A microorganism or cell containing the nucleic acid according to claim 2 or the recombinant plasmid according to claim 3.

5. A chromatographic test strip for detecting Burkholderia cepacia, characterized in that: The antibody of claim 1 is coated on the detection pad T line of the chromatography test strip.

6. The chromatography test strip according to claim 5, characterized in that: The detection pad T line is coated with one of the BCC-F1 antibody and the BCC-F2 antibody, the sample pad of the chromatography test strip is coated with another antibody, and the antibody coated on the sample pad is labeled with a signal enhancement nanomaterial; Preferably, the signal enhancing nanomaterial is at least one of colloidal gold, colored latex microspheres, fluorescent microspheres and quantum dot nanomaterials.

7. Use of the antibody according to claim 1, the nucleic acid according to claim 2, the recombinant plasmid according to claim 3, the microorganism or cell according to claim 4, or the chromatography test strip according to claim 5 or 6 in preparing a kit for detecting cepacia.

8. A kit for detecting Burkholderia cepacia, characterized in that: Contains the antibody according to claim 1, the nucleic acid according to claim 2, the recombinant plasmid according to claim 3, the microorganism or cell according to claim 4, or the chromatography test strip according to claim 5 or 6.

9. An immunoassay method for detecting Burkholderia cepacia for non-diagnostic and therapeutic purposes, characterized in that: The method comprises using the antibody described in claim 1 to perform immunodetection on Burkholderia cepacia.

10. The immunoassay method according to claim 9, characterized in that: The immunoassay is an immunochromatographic assay; The immunochromatographic test includes using a chromatographic test strip and a double antibody sandwich method to detect the cepacia group; The double antibody sandwich method includes labeling one of the BCC-F1 antibody and the BCC-F2 antibody with a signal-enhancing nanomaterial and coating it on the sample pad of the chromatographic test strip, and coating the other antibody on the detection pad T line of the chromatographic test strip; Preferably, the signal enhancing nanomaterial is at least one of colloidal gold, colored latex microspheres, fluorescent microspheres and quantum dot nanomaterials.

Images