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CN-121995051-A - Joint inspection kit for diagnosis of meliosis and application thereof

CN121995051ACN 121995051 ACN121995051 ACN 121995051ACN-121995051-A

Abstract

The invention provides a joint inspection kit for diagnosis of melioidosis and application thereof, wherein the joint inspection kit is used for firstly verifying that Hcp1 is a melioidosis specific virulence factor by constructing a melioidosis Hcp1 gene knockout strain and a anaplerosis strain, then verifying that E-selectin is regulated and controlled by the Hcp1 through a cell experiment and is related to current disease infection, and combining clinical serum sample detection to confirm that the E-selectin can be used as a serological index of current disease infection. Based on the above verification, a joint inspection kit containing a first reagent component, a second reagent component and a general reagent was prepared and detected by ELISA method. The kit can accurately diagnose the infection of the melioidosis bacteria, effectively identify the current disease and the past infection, has the advantages of quick detection, simple and convenient operation, high specificity and sensitivity, is suitable for clinical detection of medical institutions at all levels, has important clinical value for early screening, accurate diagnosis, prevention and control of the melioidosis, and overcomes the defects of the existing single detection method.

Inventors

  • HAN CHAOHUI
  • HAN LEMING
  • WU QINGYAN

Assignees

  • 海口市第三人民医院

Dates

Publication Date
20260508
Application Date
20260226

Claims (10)

  1. 1. The antibody joint detection kit for the meliotic bacteria diagnosis is characterized by comprising a first reagent component for detecting an anti-meliotic bacteria Hcp1 protein antibody in serum and a second reagent component for detecting soluble endothelial cell selectin E-selectin protein in serum, wherein the first reagent component comprises a solid-phase carrier coated with the meliotic bacteria recombinant Hcp1 protein, an anti-Hcp 1 monoclonal antibody and an enzyme-labeled secondary antibody, and the second reagent component comprises a solid-phase carrier coated with an anti-human soluble E-selectin protein antibody, a biotin-labeled anti-human soluble E-selectin protein antibody and an enzyme-labeled streptavidin.
  2. 2. The joint inspection kit according to claim 1, wherein the recombinant Hcp1 protein of melioidosis is coated on a solid carrier at a concentration of 4-6 μg/mL at a temperature of 3-5 ℃ overnight.
  3. 3. The joint inspection kit of claim 1, wherein the anti-Hcp 1 monoclonal antibody is used at a dilution of 1:1000 to 1:1024000.
  4. 4. The joint test kit of claim 1, wherein the second reagent component further comprises an E-selectin protein standard configured with a concentration gradient of 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312.5pg/mL, 156.25pg/mL, 78.13pg/mL, 0pg/mL.
  5. 5. The joint inspection kit according to claim 1, further comprising an auxiliary reagent comprising at least one or more of a coating liquid, a blocking liquid, a washing liquid, a light-shielding color-developing agent, a stop liquid, and a sample dilution liquid.
  6. 6. The joint inspection kit according to claim 5, wherein the blocking solution is 1.5% -2.5% BSA solution, the blocking condition is 35-38 ℃ incubation for 1.5-2.5 h, the stop solution is sulfuric acid stop solution, and the enzyme-labeled secondary antibody is murine enzyme-labeled secondary antibody.
  7. 7. The joint inspection kit according to claim 1, wherein the solid phase carrier is an enzyme-labeled plate and the enzyme-labeled streptavidin is HRP-labeled streptavidin.
  8. 8. The joint detection kit according to any one of claims 1 to 7, wherein the kit is used for detecting serum samples by an enzyme-linked immunosorbent ELISA method, the absorbance detection wavelength of the detection being 450nm.
  9. 9. Use of a joint inspection kit according to any one of claims 1-7 for the preparation of a detection reagent for the diagnosis of infection with melioidosis.
  10. 10. Use of a joint inspection kit according to any one of claims 1-7 for the preparation of a detection reagent for the identification of an infection with a present disease of melioidosis and a past infection.

Description

Joint inspection kit for diagnosis of meliosis and application thereof Technical Field The invention relates to the technical field of biomedical detection, in particular to a joint inspection kit for diagnosis of melioidosis and application thereof. Background The meliosis is a zoonosis infectious disease caused by meliosis Bokkaido (Burkholderia pseudomallei, B.pseudomullei, abbreviated as meliosis bacteria) infection, and is popular in tropical and subtropical areas. The clinical manifestations of the disease are complex and various, diagnosis is difficult, and standardized diagnostic kits are lacking. The current clinical biochemical identification system is easy to be misjudged, so that missed diagnosis or misdiagnosis is caused, and the natural drug resistance spectrum of the strain is wide, so that timely and accurate laboratory diagnosis is particularly critical. The type VI secretory system of the melioidosis bacteria (Type VI Secretion System, T6 SS) is an important virulence factor. Among them, the hemolysin co-regulatory protein 1 is a key structural protein and virulence factor of T6 SS-5. In T6SS-5, the hemolysin co-regulatory protein 1 (Hemolysin coregulated protein, hcp1) is an important structural protein and virulence factor, and during the assembly process of T6SS-5, the Hcp1 can be polymerized into hexamers and stacked into a secretion tube of T6SS-5, and researches show that serum of patients in acute stage of melioidosis can react with Hcp1 strongly with antigen-antibody, and ELISA or immunochromatography kits based on recombinant Hcp1 protein can be used for auxiliary diagnosis of melioidosis. However, in the epidemic region, high levels of anti-Hcp 1 antibodies are also detected in the serum of past infected persons, and therefore, this method makes it difficult to distinguish between the present infection of melioidosis and the past infection. Endothelial cell selectin (E-selectin) is a key molecule that mediates adhesion and fusion of leukocytes to vascular Endothelial cells. Studies show that infection with melioidosis bacteria can up-regulate the expression of host cell E-selectin, and that higher levels of E-selectin protein can be detected in serum of patients with acute infection with melioidosis. Serum E-selectin levels are an important indicator for the assessment of sepsis, reflecting the status of symptomatic infection, but with insufficient specificity. Based on this, there are limitations in either single Hcp1 antibody detection or E-selectin protein detection. The invention aims to provide a kit and a method for jointly detecting the two, so as to improve the accuracy of the diagnosis of the melioidosis and realize the identification of the current infection and the past infection. Disclosure of Invention In view of the above, the invention provides a joint inspection kit for the diagnosis of melioidosis and application thereof. The kit can more accurately and comprehensively diagnose and identify the melioidosis infection by jointly detecting the anti-melioidosis Hcp1 protein antibody and the soluble endothelial cell selectin protein in serum. The technical scheme of the invention is realized as follows: A joint inspection kit for the diagnosis of meliosis The kit comprises a first reagent component for detecting an anti-melioidosis bacteria Hcp1 protein antibody in serum, a second reagent component for detecting a soluble endothelial cell selectin E-selectin protein in serum, and a general reagent component for assisting detection; The first reagent component comprises a solid phase carrier coated with the recombinant Hcp1 protein of the melioidosis bacteria, an anti-Hcp 1 monoclonal antibody with the concentration of 7-9 mg/mL and a murine enzyme-labeled secondary antibody, wherein the coating concentration of the recombinant Hcp1 protein of the melioidosis bacteria on the solid phase carrier is 4-6 mug/mL, the coating condition is 3-5 ℃ overnight, and the use dilution of the anti-Hcp 1 monoclonal antibody is 1:1000-1:1024000; A second reagent component comprising a solid phase carrier coated with an anti-human soluble E-selectin protein antibody, a biotin-labeled anti-human soluble E-selectin protein antibody, HRP-labeled streptavidin, and an E-selectin protein standard, wherein the concentration gradient of the E-selectin protein standard is 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312.5pg/mL, 156.25pg/mL, 78.13pg/mL, and 0pg/mL; The universal reagent comprises at least one or more of coating liquid, 1.5% -2.5% BSA blocking liquid, washing liquid, light-proof color developing agent, sulfuric acid stopping liquid and sample diluting liquid; the solid phase carriers are ELISA plates, and the reaction holes of the ELISA plates are designed detachably, so that single sample or multiple sample detection is facilitated. (II) detection method of the joint inspection kit The ELISA method is adopted to detect the serum sample, the detection wavelength of absorbance is 450