Nasal-anthracnose-like disease marker, kit and application
By using phage peptidoglycan binding protein (PPEP), the problem of early diagnosis of melanoids is solved, and high accuracy and sensitivity detection is achieved, suitable for rapid diagnosis in resource-poor areas.
Patent Information
- Application Number
- CN202311855034.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-07-22
AI Technical Summary
The detection of mesys is difficult to perform early, quickly and accurately. The existing culture methods are time-consuming and have low sensitivity. The bacteria are easily mistaken for Pseudomonas, lacking specific monoclonal antibodies, and IgG has limited host protection.
Using phage peptidoglycan binding protein (PPEP) as a biomarker, ELISA kits and immunochromatography products were developed to detect specific IgM antibodies in in vitro samples to achieve early diagnosis of Burkholderella myeloids.
It has achieved early, rapid and accurate diagnosis of mesysoids, with a detection accuracy of 95.54% and a sensitivity of 90.91%. It can distinguish Bp from other Gram-negative bacteria and is suitable for simple and low-cost testing in resource-poor areas.
Smart Images
Figure CN120352618A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of melioidosis, and specifically to melioidosis markers, kits and applications. Background Art
[0002] Melioidosis is a highly pathogenic tropical zoonotic infectious disease caused by Burkholderia pseudomallei (B.pseudomallei, Bp), with a high mortality rate (19 - 36%). Detection or diagnosis of melioidosis is difficult because the bacteria are usually not easily isolated from clinical specimens, and even if isolated, they may not be correctly identified. Bp is often misidentified as Pseudomonas because of similar colony morphology in blood agar, Gram staining (Gram-negative and safety pin appearance), and biochemical tests such as the oxidase test (oxidase-positive). In addition, the culture method is time-consuming and has low sensitivity (60%) because it requires culturing with selective or enrichment media for non-sterile samples. Therefore, early, rapid, and accurate diagnosis of melioidosis is extremely important for improving the cure rate and survival rate. Summary of the Invention
[0003] Vesicles secreted by Gram-negative bacteria at all stages of growth and development contain outer membrane proteins (OMPs), lipids, and some soluble substances. These proteins are involved in the formation and encapsulation of the cell wall / membrane and the transmembrane transport of ions or small molecules. It involves the interaction between the host and the bacteria mainly through direct or indirect contact with the host's immune cells or immune molecules. Bacteria secrete signal small molecules through vesicles to complete signal transduction with the host. For example, bacteria can fuse phage protein genes and express them on the bacterial outer membrane. This protein can bind to the same type of phage to transmit the false signal that the bacteria are locked by the phage, which is a way for bacteria to evade phages.
[0004] The inventors of this application isolated proteins from the extracellular vesicles of cells infected with Bp. Through mass spectrometry analysis, it was found that the extracellular vesicles of cells infected with Bp carried proteins of bacterial origin, and some proteins were highly expressed. For example, the expression of phage peptidoglycan-binding protein (PPEP) increased by 10.65 times in the infected group, while other similar proteins only appeared in different strains of Bp or their phages. Through the analysis of serum samples of patients, volunteers, and other individuals infected with bacteria, it was found that PPEP can specifically recognize Burkholderia pseudomallei.
[0005] Based on this, in a first aspect, the embodiment provides the use of phage peptidoglycan-binding protein contained in an in vitro sample as a biomarker for any one of A1) to A5): A1) preparing a product for detecting Burkholderia pseudomallei; A2) preparing a product for evaluating Burkholderia pseudomallei; A3) preparing a product for preventing Burkholderia pseudomallei; A4) preparing a product for inhibiting or killing Burkholderia pseudomallei; A5) preparing a product for differentiating Burkholderia pseudomallei infection. The said use is for non-disease detection, evaluation, prevention, inhibition or killing.
[0006] In the embodiment, the in vitro sample is selected from at least one of a blood sample, a saliva sample, a sweat sample, a urine sample, a throat swab sample, a milk sample, a semen sample, a skin swab sample, a fecal sample, and a sputum sample.
[0007] 90% of acute melioidosis patients produce specific IgG antibodies at 4 - 5 weeks after onset, which can be maintained for at least 1 year. However, the protective effect of IgG on the host is limited and it has little significance for the early diagnosis of melioidosis. Immunoglobulin M (IgM) is the first antibody to react with an antigen and is also the earliest reactive circulating antibody, which is usually used as an indicator of acute infection. Due to the current lack of specific monoclonal antibodies against Bp protein and the important diagnostic value of IgM-type antibodies in acute bacterial infections.
[0008] In the embodiment, PPEP is used as an antigen to coat a solid surface to construct an indirect ELISA kit and detection method for detecting whether specific IgM antibodies against HP are contained in an in vitro sample. The results show that the detection accuracy of PPEP is 95.54%, and the sensitivity is 90.91%, which is better than ompA (93.75% and 86.36%) and BLF1 (91.96% and 88.64%).
[0009] For this reason, in a second aspect, the embodiment discloses an ELISA kit. The ELISA kit includes a liquid preparation containing phage peptidoglycan-binding protein and a liquid preparation of an enzyme-labeled antibody. In some embodiments, the enzyme-labeled antibody is an antibody labeled with horseradish peroxidase.
[0010] In a third aspect, the embodiment discloses an immunochromatographic detection product. The immunochromatographic product includes a carrier and phage peptidoglycan-binding protein coated on the carrier. The immunochromatographic product loads phage peptidoglycan-binding protein on the carrier, and a drop or immersion of an in vitro sample is used to promote its immunological binding with the phage peptidoglycan-binding protein on the carrier to form a complex, and the complex undergoes a chromatographic effect after passing through the labeled area (such as the control line), thereby realizing detection (such as causing the control line to change color).
[0011] In an embodiment, the carrier is selected from a microporous filter membrane or an NC membrane.
[0012] In an embodiment, the product is selected from at least one of an immunogold test strip, an immunogold microwell plate, an immunogold stick, an immunofluorescent test strip, an immunofluorescent microwell plate, and an immunofluorescent stick for detecting anti-burkholderia pseudomallei antibodies in an in vitro sample.
[0013] In a fourth aspect, an embodiment discloses a method for detecting anti-burkholderia pseudomallei antibodies in an in vitro sample. The method includes: obtaining a liquid reagent containing phage peptidoglycan-binding protein; mixing and incubating the liquid reagent with the in vitro sample; and correlating the signal generated by the complex of the phage peptidoglycan-binding protein and the anti-burkholderia pseudomallei antibody with the presence and / or concentration of the anti-burkholderia pseudomallei antibody.
[0014] In an embodiment, the signal is selected from at least one of an optical signal or an electrical signal. For example, the antibody in the in vitro sample undergoes immunological binding with the phage peptidoglycan-binding protein to form a complex, and then an optical signal is achieved through an antibody labeled with a fluorescent group and / or a chromogenic agent.
[0015] In a fifth aspect, an embodiment discloses a method for detecting anti-burkholderia pseudomallei antibodies in an in vitro sample. The method includes: coating at least one small hole of at least one microwell plate with a coating solution containing phage peptidoglycan-binding protein; adding a clinical serum sample to the coated small hole; adding a secondary antibody solution to the small hole; and performing a color reaction on the small hole to which the secondary antibody is added. After the color reaction ends, a termination solution is added to terminate the color reaction, and the OD450 value is read.
[0016] In a sixth aspect, an embodiment discloses the application of phage peptidoglycan-binding protein. The application is selected from: B1) preparing a product for detecting Burkholderia pseudomallei; B2) preparing a product for evaluating Burkholderia pseudomallei; B3) preparing a product for preventing Burkholderia pseudomallei; B4) preparing a product for inhibiting or killing Burkholderia pseudomallei; B5) preparing a product for differentiating Burkholderia pseudomallei infection.
[0017] In some embodiments, the selected phage peptidoglycan-binding protein is used to evaluate the detection of serum samples or sputum samples that are positive for other Gram-negative bacteria. The serum samples of melioidosis patients have significantly higher absorbance at OD450. This indicates that the phage peptidoglycan-binding protein has excellent performance in differentiating Bp from other bacteria and shows good species specificity.
[0018] Therefore, the embodiments of the present application also disclose the use of phage peptidoglycan-binding protein in the preparation of products for differentiating Burkholderia pseudomallei infection. In some embodiments, the product is an ELISA kit. By coating or loading the phage peptidoglycan-binding protein and adding an in vitro sample, the antibodies in the in vitro sample can immunologically bind to the phage peptidoglycan-binding protein to form a complex, and then a so-called optical signal can be achieved through an antibody labeled with a fluorescent group and / or a chromogenic agent.
[0019] In some embodiments, a cell model of bacterial infection was constructed using normal human lung epithelial cells (BEAS-2B). This model can accurately reflect the state of cells carrying bacteria and has good representativeness. Therefore, when the cells are infected with Bp, the bacterial proteins carried by the cell-derived extracellular vesicles can be detected through the corresponding antibodies in the body fluid, providing technical support for the early detection of the disease.
[0020] The ELISA kit, immunochromatographic detection product, method and application provided by the present application can accurately and sensitively detect the corresponding antibodies against vesicle proteins and toxins in serum, and also have strong specificity. This method is simple to operate and low in cost, providing strong technical support for the early and accurate detection of melioidosis in remote areas. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Micrographs of vesicles of bacteria (Bp, a) / cells (BEAS-2B, b) and infection models (Bp / BEAS-2B, c) provided for the examples.
[0022] Figure 2 Schematic diagram of the construction of the expression vector provided for the examples.
[0023] Figure 3 WB test result graphs of the purified proteins of BLF1 and ompA (A) and PPEP protein (B) provided for the examples.
[0024] Figure 4 ROC curve of PPEP provided for the examples.
[0025] Figure 5 ROC curve of BLF1 provided for the examples.
[0026] Figure 6 ROC curve of ompA provided for the examples.
[0027] Figure 7 Dot plots of OD450 of ELISA detection of three proteins, namely PPEP (A), BLF1 (B) and ompA (C), in serum samples of the control group and the patient group provided for the examples.
[0028] Figure 8 ELISA detection of three proteins, PPEP (a), BLF1 (b), and ompA (c), provided for the examples, showing the O450 dot plot of serum samples from single pathogen-infected patients.
[0029] Figure 9 ELISA detection of three proteins, PPEP (A), BLF1 (B), and ompA (C), provided for the examples, showing the O450 dot plot of serum samples from different populations. Detailed implementation manners
[0030] In order to make the objectives, technical solutions, and advantages of the present application more clear and understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. Reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.
[0031] Vesicle protein extraction
[0032] Since pulmonary infection is the most obvious and main symptom of patients seeking medical treatment after Bp infection, the corresponding strains are often isolated from sputum and blood cultures during clinical diagnosis. Therefore, in this step, human normal lung epithelial cells BEAS-2B are used to simulate the changes in Bp vesicle proteins in humans.
[0033] (1) Establishment of the infection model
[0034] Passage culture BEAS-2B (No.: B164023, brand: Mingzhoubio) until the cells cover the bottom of the culture dish. Subsequently, discard the culture medium and wash twice with PBS, then add trypsin for digestion and centrifuge at 800 rpm for 3 min. Discard the supernatant, resuspend the cells with sterile PBS, and dilute 20 times. Use a neubauer hemocytometer counting plate to count the cells according to the white blood cell counting method. Take 1×10 5 cells, shake well in a 12-well plate, and culture overnight (the cells grow completely adherently).
[0035] Bp group: Culture Bp alone in LB liquid medium overnight until the OD600 reaches 1.0, and take the supernatant of the bacterial culture (solution 1).
[0036] Bp / BEAS-2B group: The diluted Bp bacterial suspension (Burkholderia pseudomallei, Bp, TS225421, Testobio) was added to the culture medium of BEAS-2B obtained above at a multiplicity of infection of 5 (MOI = 5), and continuously infected for 6 h. Then, the culture supernatant was discarded, and the cells were washed 2-3 times with PBS. Subsequently, DMEM containing 750 ng / mL antibiotics kanamycin and amikacin was added to the culture dish to kill all extracellular bacteria for 4 h. Then, the bacteria-infected cells were continuously cultured in a new non-resistant medium for 18-24 h. The above cleaning process was repeated. And the cell morphology was continuously observed, and the cell culture supernatant (solution 2) was retained.
[0037] (2) Extraction of vesicles
[0038] Vesicles were extracted from solutions 1 and 2 using ultracentrifugation. Solutions 1 and 2 were centrifuged at 4000 rpm for 15 min at 4 °C to remove cells / bacteria (pellet). The supernatant was transferred to a new labeled centrifuge tube at 4 °C and centrifuged at 12000 rpm for 20 min to remove cell / bacterial debris (pellet). The supernatant obtained in step 2 was further filtered through a filter with a pore size of 0.22 μm to remove cell / bacterial debris, and the filtrate was collected; the supernatant collected from 3 was transferred to a high-speed centrifuge tube at 4 °C and centrifuged at 150000 g for 2 h, and the supernatant was discarded. After resuspending the pellet with sterile PBS, it was centrifuged at 150000 g for 1.5 h at 4 °C and the supernatant was discarded to produce the corresponding extracellular vesicles.
[0039] As Figure 1 shown, vesicles from single bacteria (Bp, a) / cells (BEAS-2B, b) and the infection model (Bp / BEAS-2B, c) all have a vesicular structure with a diameter of 50-150 nm.
[0040] (3) Vesicle protein analysis
[0041] The extracted vesicles were lysed and proteins were extracted, and the types of proteins were analyzed by mass spectrometry. Bioinformatics techniques were used to predict the functional structures and clustering analysis of these proteins.
[0042] There were 154 differentially expressed proteins between the Bp group and the Bp / BEAS-2B group. Among them, in the infected group, the expression levels of 111 proteins decreased, while those of 43 proteins increased. Among the 43 highly expressed proteins, 5 proteins were related to post-transcriptional modification; four proteins were related to the biogenesis of cell wall / membrane / envelope; three proteins were related to intracellular transport, secretion function, and vesicle transport; six proteins were involved in energy production and conversion; 14 proteins were involved in the transport and metabolism of amino acids, coenzymes, nucleic acids, and carbohydrates. According to the subcellular localization of the proteins, 7 proteins were located in the outer cell membrane, and 13 proteins were located in the cell envelope.
[0043] The expression of PPEP increased 10.65-fold in the infected vesicles. It specifically binds to peptidoglycan and is considered one of the pattern recognition proteins in the host innate immunity. It has 100% similarity to the peptidoglycan-binding protein on Burkholderia phage PK23 (Table 1). Therefore, the phage-encoded peptidoglycan-binding protein (PPEP) was selected as the biomarker to be verified.
[0044] Table 1 Similarity of PPEP
[0045]
[0046]
[0047] It has been reported that the structural protein ompA of Bp is a candidate immunogenic vaccine for Bp infection and has been confirmed as a potential biomarker for Bp infection in Thailand. Bacterial lethal factor is a toxic substance secreted by Gram-negative bacteria during their growth and reproduction, which is harmful to the body. It can trigger an immune response in the body and be used to manufacture antitoxins and toxoids for the treatment and prevention of diseases. Therefore, ompA and the exotoxin BLF1 were selected as the markers for the control group.
[0048] Preparation of markers
[0049] The embodiment of the present application also discloses a preparation method of PPEP, which specifically includes the following steps: construction of expression vector, construction of expression host, recombinant expression, and purification.
[0050] 1. Construction of expression vector and expression host
[0051] As shown in Table 2, the corresponding primers were synthesized according to the gene CDS sequences of PPEP (BTHAI0001_RS26005), ompA (Gene ID: 56596437), and BLF1 (Gene ID: 56529274), and inserted into the protein expression vector with His tags at both the N-terminal and C-terminal through digestion and ligation. Figure 2) and transferred into Escherichia coli BL21(DE3) cells (Novagen) for expression.
[0052] Table 2 Primers
[0053]
[0054] 2. Recombinant expression and purification
[0055] The screened positive clone cells were incubated in LB broth supplemented with 100 mg / L ampicillin at 37 °C for 4 h. When OD600 reached 0.6, 1 mM IPTG was added to induce protein expression.
[0056] Before protein purification, Escherichia coli cells were lysed on ice using non-denaturing lysis buffer. The ultrasonic crusher was operated for 10 s every 25 s for 15 min until the cell suspension became transparent. The lysate was clarified by centrifugation at 6000×g for 20 min. The clarified lysate was loaded onto a nickel-NTA (nitrilotriacetic acid) agarose column and eluted with 50 mM imidazole buffer (pH 8). Elution with 250 nM imidazole was carried out for 2 column volumes. His-tag Protein Purification Kit (Beyotime, P2229S-His-tag Protein Purification Kit (Denaturation-resistant Form)) was used for protein purification.
[0057] WB detection: The purified recombinant protein was denatured at 100 °C for 5 min and subjected to SDS-PAGE with a 12% separating gel. Electrophoresis was carried out at 110 V for 30 min first, and then the voltage was adjusted to 120 V for 90 min. The gel after electrophoresis was placed on the transfer membrane plate and a PVDF membrane soaked in methanol was covered on top of the gel. Transfer was carried out at 90 V in the transfer buffer for 80 min. The PVDF membrane was taken out and blocked in 2% BSA at room temperature for 2 h. The PVDF membrane was then placed in a solution containing mouse anti-His-tag primary antibody (Product No.: AH367, Beyotime Biotechnology Co., Ltd., diluted 1:5000) and incubated at 4 °C with 180 rpm for 16 h. The PVDF membrane was washed three times with PBST washing solution for 10 min each time. Subsequently, rabbit anti-mouse secondary antibody (Rabbit Anti-Mouse IgG H&L(HRP), Catalog No.: 701051, ZEN-BIOSCIENCE Co., Ltd., Chengdu, diluted 1:5000) was added and reacted at room temperature for 2 h. The PVDF membrane was washed three times with PBST washing solution for 10 min each time. Finally, chemiluminescent reagent was added to the PVDF membrane for detection.
[0058] As Figure 3 A and Figure 3As shown in Figure B, the recombinant vectors expressed recombinant proteins of the expected sizes, namely 29 kDa (PPEP), 23 kDa (BLF1), and 27 kDa (ompA), which were consistent with the expected sizes. Only a single protein band was observed in the results for the molecular weight of the purified proteins, indicating a high protein purity. The band showed good homogeneity, demonstrating the successful expression and purification of the recombinant proteins.
[0059] ELISA kit for detecting serum antibodies
[0060] Using the PPEP prepared in the above embodiments, the embodiments of the present application also constructed a kit and method for detecting anti-burkholderia pseudomallei antibodies in in vitro samples.
[0061] In some embodiments, the kit includes a liquid reagent containing a phage peptidoglycan-binding protein and an enzyme-labeled antibody. In some embodiments, the enzyme-labeled antibody is a goat anti-human enzyme-linked antibody, and the enzyme is horseradish peroxidase.
[0062] In some embodiments, the method includes obtaining a liquid reagent containing a phage peptidoglycan-binding protein; mixing and incubating the liquid reagent with the in vitro sample; the signal generated by the complex of the phage peptidoglycan-binding protein and the anti-burkholderia pseudomallei antibody is associated with the presence and / or concentration of the anti-burkholderia pseudomallei antibody. In some embodiments, the signal is selected from at least one of an optical signal or an electrical signal.
[0063] In some embodiments, the method includes the following steps 1) to 4):
[0064] 1) Coating: Coating at least one small hole of at least one well plate with a coating solution of PBS at pH 7.4 containing the recombinant protein (PPEP).
[0065] In one embodiment, the concentration of the recombinant protein (PPEP) in the coating solution is 0.1 to 100 μg / μL, such as 0.1 μg / μL, 0.5 μg / μL, 1 μg / μL, 5 μg / μL, 10 μg / μL, 20 μg / μL, 30 μg / μL, 40 μg / μL, 50 μg / μL, 60 μg / μL, 70 μg / μL, 80 μg / μL, 90 μg / μL, 100 μg / μL. In one embodiment, the coating solution is a pH 7.4 PBS solution containing PPEP. In one embodiment, the orifice of the coated microplate is sealed with a sealing film, and incubated overnight at 4 °C and 100 rpm. In one embodiment, the microplate is washed with a PBS buffer containing 0.02% Tween-20. In one embodiment, the washed microplate is patted dry on absorbent paper and stored at 4 °C for later use to complete the coating. In one embodiment, the microplate is selected from an ELISA plate, a reaction plate, and a cell culture plate. The number of orifices of the microplate is selected from 24, 48, 96, or 384. The material of the microplate is selected from polystyrene, polypropylene, polycarbonate, glass, ceramic, or metal.
[0066] 2) Sample addition: Add the diluted clinical serum sample to the orifice coated in step 1).
[0067] In one embodiment, the clinical serum sample is diluted 50-fold with pH 7.4 PBS. In one embodiment, the volume of the clinical serum sample is the same as the volume of the coating solution in step 1). In one embodiment, after sample addition, the reaction is carried out at 37 °C for 1 h.
[0068] 3) Secondary antibody addition: Add the secondary antibody solution to the orifice in step 2).
[0069] In one embodiment, the secondary antibody solution is a dilution of goat anti-human enzyme-linked secondary antibody. In one embodiment, the dilution ratio of the secondary antibody solution is 1:5000. In one embodiment, the secondary antibody is from Chengdu ZEN-BIOSCIENCE Co., Ltd., product number: 550115. In one embodiment, after adding the secondary antibody, the reaction is carried out at 37 °C for 30 min, and the microplate is washed with a PBS buffer containing 0.02% Tween-20. In one embodiment, the volume of the dilution of the secondary antibody is the same as the volume of the coating solution in step 1).
[0070] 4) Color development: Perform a color development reaction on the orifice to which the secondary antibody is added. Immediately after the color development ends, add the termination solution to terminate the color development reaction, and read the OD450 value. In one embodiment, TMB is used for color development.
[0071] In some embodiments, the method further includes determining a critical value (Cutoff value) according to steps 1) to 4), and determining whether Burkholderia pseudomallei is contained in the sample to be tested according to the critical value. In some embodiments, an ROC curve is made based on the OD450 obtained from steps 1) to 4) and the positive rate of the clinical sample actually containing Burkholderia pseudomallei, and the critical value (Cutoff value) is determined according to the ROC curve.
[0072] Test example
[0073] 1. Sample to be tested
[0074] 44 clinical serum samples infected with Burkholderia pseudomallei, 23 serum samples infected with other pathogenic bacteria, and 47 serum samples from volunteers in non-Burkholderia pseudomallei epidemic prevention areas. The samples are all from the Second Affiliated Hospital of Hainan Medical College.
[0075] 2. Detection of serum samples
[0076] Using steps 1) to 4) provided in the above embodiments, OD450 is detected.
[0077] 3. ROC curve and scatter plot
[0078] Figures 4-6 The ROC curve made from OD450 obtained by the ELISA kits of PPEP, BLF1 and ompA and the detection method of in vitro samples and the clinical true positive samples is shown. It can be calculated according to the ROC curve that the critical value corresponding to PPEP is OD450 = 0.1095, the critical value corresponding to BLF1 is OD450 = 0.125, and the critical value corresponding to ompA is OD450 = 0.108. Values greater than the critical value are judged as positive, and values lower than the critical value are judged as negative.
[0079] According to the obtained critical values, 44 serum specimens of patients diagnosed with Burkholderia pseudomallei by blood culture are used as the patient group (patients), and 47 serum samples from volunteers in non-Burkholderia pseudomallei epidemic prevention areas are used as the control group (control) for the detection of the sample to be tested. Figure 7 The scatter plot drawn from the ELISA test results of the serum samples of these control groups and patient groups is shown. The dotted line in the figure represents the critical value of each index, and those lower than this line are judged as negative.
[0080] 4. Sensitivity, specificity and accuracy evaluation
[0081] Table 1
[0082] Number of clinically positive samples Number of clinically negative samples Number of ELISA-detected positive samples A B Number of ELISA-detected negative samples C D
[0083] Calculate the sensitivity, specificity, and accuracy of detecting Burkholderia pseudomallei in vitro serum samples by ELISA after coating with PPEP, BLF1, and ompA respectively according to the A, B, C, and D values in Table 1. Among them, sensitivity = 100% × A / (A + C), specificity = 100% × B / (B + D), and accuracy = 100% × (A + D) / (A + B + C + D).
[0084] Table 2
[0085] Marker PPEP BLF1 ompA Number of clinically serum samples with detected melioidosis infection 40 39 38 Number of clinically serum samples with detected other pathogenic bacteria infections 1 2 1 Number of serum samples of volunteers in non-melioidosis epidemic prevention areas detected 0 2 0 Sensitivity (%) 90.91 88.64 86.36 Specificity (%) 98.53 94.12 98.53 Accuracy (%) 95.54 91.96 93.75
[0086] Table 2 shows the number of clinical serum samples infected with Burkholderia pseudomallei, serum samples infected with other pathogenic bacteria, and serum samples of volunteers in non-Burkholderia pseudomallei epidemic prevention areas, as well as the number of positive samples obtained by using ELISA kits of PPEP, BLF1, and ompA and the detection methods of in vitro samples, and the corresponding sensitivity, specificity, and accuracy.
[0087] It can be seen that the sensitivity, specificity, and accuracy of the ELISA kit using PPEP and the detection method of in vitro samples are higher than those of BLF1 and ompA.
[0088] Test example: Specificity evaluation of different pathogenic bacteria
[0089] 1. Test samples
[0090] 6 serum samples infected with a single Klebsiella pneumoniae, 5 serum samples infected with a single Pesu. aeruginosa, 4 serum samples infected with a single Acinetobacter baumannii, 3 serum samples infected with a single Stenotrophomonas maltophilia, 5 serum samples infected with a single Escherichia coli, and 23 serum samples infected with a single Burkholderia pseudomallei. The samples are all from the Second Affiliated Hospital of Hainan Medical College.
[0091] 2. Detection of serum samples
[0092] Using the steps 1) - 4) provided in the above embodiments, OD450 was detected. According to the calculation of the ROC curve, the critical value corresponding to PPEP was OD450 = 0.1095, the critical value corresponding to BLF1 was OD450 = 0.125, and the critical value corresponding to ompA was OD450 = 0.108. Values greater than the critical value were judged as positive, and values lower than the critical value were judged as negative.
[0093] 3. Results
[0094] Figure 8 The OD450 values of different single-infected serum samples are shown. It can be seen from the results that the sera of melioidosis patients had significantly higher absorbance at OD450, and there was a good distinction between the sera of melioidosis patients and those of other bacterial infection patients. Among them, PPEP and ompA each had one positive result, while BLF1 had two positive results. After analysis, it was found that these 4 positive results all came from the sera of a single patient infected with Pseudomonas aeruginosa. Bp belongs to β-proteobacteria and may also be diagnosed as Pseudomonas aeruginosa in clinical diagnosis. The ELISA kit and detection method established with PPEP provided in the embodiments of the present application have high anti-interference ability, can distinguish Bp from other Gram-negative bacterial infections, and have important diagnostic value in the diagnosis of melioidosis.
[0095] Test example: Detection of serum samples of different populations
[0096] 1. Test samples
[0097] 47 serum samples from volunteers in non-melioidosis epidemic prevention areas (Control), 44 clinically diagnosed serum samples (patients), 95 serum samples from physically normal individuals during physical examinations (Examiner), and 5 serum samples from clinically cured patients (Convalescent) were all from the Second Affiliated Hospital of Hainan Medical University.
[0098] 14 serum samples from high-risk individuals participating in melioidosis detection (High risk individuals) were from the Tropical Biomedical Technology Laboratory of Hainan Medical University.
[0099] 11 serum samples from farm workers in farms infected with Bp (Farm labourers) were from Farmlabourers.
[0100] 2. Detection
[0101] Using the steps 1) - 4) provided in the above - mentioned embodiments, OD450 was detected. According to the calculation of the ROC curve, the critical value corresponding to PPEP is OD450 = 0.1095, the critical value corresponding to BLF1 is OD450 = 0.125, and the critical value corresponding to omp A is OD450 = 0.108. Values greater than the critical value are judged as positive, and values lower than the critical value are judged as negative.
[0102] 3. Results
[0103] Figure 9 The detection results of serum samples from different populations are shown. The results indicate that there are significant differences in the serum detection results between melioidosis patients and other populations.
[0104] By measuring the presence of four IgM proteins in the sera of healthy individuals, high - risk individuals, recovered patients, and farm workers in contact with naturally occurring goats, it can be found that the selected proteins have great practical value in community screening. These data emphasize the usefulness of PPEP as a potential antigen for serological diagnosis of melioidosis by ELISA and can be used to develop other methods such as dot - blot ELISA or immunochromatographic test (ICT) for rapid, simple, cost - effective, and efficient diagnosis in resource - poor areas where melioidosis is endemic.
[0105] As described above, it is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered within the protection scope of the present application.
Claims
1. Use of phage peptidoglycan-binding protein contained in an in vitro sample as a biomarker, for any one of A1) to A5): A1) Preparing a product for detecting Burkholderia pseudomallei; A2) Preparing a product for evaluating Burkholderia pseudomallei; A3) Preparing a product for preventing Burkholderia pseudomallei; A4) Preparing a product for inhibiting or killing Burkholderia pseudomallei; A5) Preparing a product for differentiating Burkholderia pseudomallei infection; The said use is for non-disease detection, evaluation, prevention, inhibition, killing or differentiation.
2. The use according to claim 1, wherein the in vitro sample is selected from at least one of blood sample, saliva sample, sweat sample, urine sample, throat swab sample, milk sample, semen sample, skin swab sample, feces sample, sputum sample.
3. An ELISA kit, comprising: A liquid preparation containing phage peptidoglycan-binding protein and a liquid preparation containing an enzyme-labeled antibody.
4. An immunochromatographic detection product, which comprises a carrier and phage peptidoglycan-binding protein coated on the carrier.
5. The immunochromatographic detection product according to claim 4, wherein the carrier is selected from a microporous filter membrane or an NC membrane.
6. The immunochromatographic detection product according to claim 4, wherein the product is selected from at least one of an immunogold test strip, an immunogold well plate, an immunogold rod, an immunofluorescent test strip, an immunofluorescent well plate, an immunofluorescent rod for detecting anti-Burkholderia pseudomallei antibody in an in vitro sample.
7. A method for detecting anti-Burkholderia pseudomallei antibody in an in vitro sample, which comprises: Obtaining a liquid reagent containing phage peptidoglycan-binding protein; Mixing and incubating the liquid reagent with the in vitro sample; The signal generated by the complex of the phage peptidoglycan-binding protein and the anti-Burkholderia pseudomallei antibody is associated with the presence and / or concentration of the anti-Burkholderia pseudomallei antibody.
8. The method according to claim 7, wherein the signal is selected from at least one of an optical signal or an electrical signal.
9. A method for detecting anti-Burkholderia pseudomallei antibody in an in vitro sample, which comprises: Coating at least one small hole of at least one well plate with a coating solution containing phage peptidoglycan-binding protein; Adding a clinical serum sample into the coated small hole; Adding a secondary antibody solution into the small hole; And Performing a color reaction on the small hole added with the secondary antibody, adding a termination solution to terminate the color reaction after the color reaction ends, and reading the OD450 value.
10. Use of phage peptidoglycan-binding protein, the use is selected from: B1) Preparing a product for detecting Burkholderia pseudomallei; B2) Preparing a product for evaluating Burkholderia pseudomallei; B3) Preparing a product for preventing Burkholderia pseudomallei; B4) Preparing a product for inhibiting or killing Burkholderia pseudomallei; B5) Preparing a product for differentiating Burkholderia pseudomallei infection.