A rapid inflammation detection kit

A kit and a specific technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of expensive testing, long time-consuming, long cycle, etc., and achieve the benefits of treatment, accuracy improvement, and early diagnosis Effect

Active Publication Date: 2021-12-14
FOSHAN DIAN MEDICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Cytotoxicity tests are not suitable for routine clinical laboratories due to complicated operations, high prices and long time-consuming
The toxin-producing culture is to amplify the toxin gene by polymerase chain reaction (PCR) after the Clostridium difficile culture is first cultivated to further confirm the typing. The advantage is that it has high specificity and sensitivity, but it is difficult. The cultivation of Clostridium is difficult, takes a long time and requires the assistance of sophisticated instruments
Glutamate dehydrogenase (GDH) is an antigenic protein expressed on the surface of Clostridium difficile, which has high stability and sensitivity, but it has no ability to distinguish whether the strain is toxic, so it needs to be combined with other methods application
[0005] GDH has high sensitivity, but there are false positives and defects that cannot determine whether C. difficile carries toxin genes
Nucleic acid detection can accurately detect toxin genotyping, but the cost of gene detection for a large number of samples is huge, the cycle is long, and it needs the assistance of high-precision instruments, and the detection cost is expensive
Therefore, the clinical sensitivity of existing methods is not very ideal

Method used

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Examples

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Effect test

Embodiment 1

[0037] Example 1. Preparation of Clostridium difficile GDH antigen

[0038] After the Clostridium difficile C.difficile-630 strain was amplified and cultured, the DNA was extracted using a DNA extraction kit (Tiangen). Using the whole genome of the bacterial liquid as a template, the GDH gene was amplified by PCR and connected to the prokaryotic expression vector pET-30a. This expression vector was named "pET-30a-GDH". Transform the competent cells with the recombinant expression plasmid pET-30a-GDH, and after culturing for 12-14 hours, pick the positive single bacteria, put them in 2 mL / tube of LB (kana+) liquid medium, and shake the bacteria overnight at 180r at 37°C. The next day, mix the bacterial solution and LB freezing solution 1:1, use it as seed bacteria, and store it at -80°C for later use. Take 500 μl of seed bacteria, and expand the culture after recovery. After shaking the bacteria for about 4 hours, wait until it enters the logarithmic growth phase, and the OD60...

Embodiment 2

[0039] Example 2. Preparation of biotinylated Clostridium difficile GDH antigen

[0040] According to the instructions, the GDH antigen was biotinylated using biotinylated ligase B0101A (GnenCopoia). The biotinylated GDH antigen was named Biotin-GDH. Add BufferA / B and BirA ligase to the GDH antigen, and incubate at 30°C for 2h. The biotinylation efficiency was detected by ELISA method. The Biotin-GDH with an initial concentration of 500ng / ml was diluted 1:2 times, and coated with ELISA plates, followed by incubation with SA-HRP. Using the biotinylation standard as a control, the biotinylation labeling efficiency of Biotin-GDH was finally determined to be 75%.

Embodiment 3

[0041] Example 3. Preparation of anti-clostridium difficile GDH antibody

[0042] The recombinant protein GDH purified in Example 1 was used as an antigen to immunize Balb / c female mice. The recombinant protein was mixed with Freund's adjuvant in equal volume (1mL: 1mL), fully emulsified by syringe method, and immunized mice by abdominal multi-point injection method, and the immunization dose was 60μg / mouse. For the second and third immunizations, the mice were immunized with the same volume of Freund's incomplete adjuvant and recombinant protein GDH, and the mice were immunized by multi-point injection in the abdomen. The immunization dose was 30 μg / mouse. Seven days after the third booster immunization, the mouse serum was taken to detect the titer, and the mouse with the highest titer was immunized by tail vein injection, and the antigen was mixed with normal saline, and the dose was 50 μg / mouse. The splenocytes of the immunized Balb / c mice were taken and fused with the my...

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Abstract

The invention discloses a rapid inflammation detection kit. The present invention screens and obtains monoclonal antibodies that specifically recognize the Clostridium difficile GDH antigen, and prepares antibody fluorescent quantum dot rapid detection test paper by marking the monoclonal antibodies with quantum dots and other antibody-labeled nitrocellulose membranes, and at the same time targeting Clostridium difficile toxins Analyze the DNA sequence of B, select the conserved region to design RPA primers and probes, and prepare them into corresponding test strips; the combination of the two detection methods can further improve the accuracy of detection, so that early diagnosis is beneficial to patients Treatment as soon as possible is suitable for large-scale promotion and use.

Description

technical field [0001] The invention relates to the field of biological detection, and more particularly to a rapid inflammation detection kit. Background technique [0002] Clostridium difficile (CD), also known as Clostridium difficile or Clostridium difficile, is a spore-forming, anaerobic, Gram-positive bacillus that exists in the environment, in the intestines of animals and humans. in the way. In 1935, Holl et al. isolated the bacterium from the stool of healthy newborns for the first time, and named it Bacillus difficilis (Bacillus difficilis) to reflect the difficulty of its cultivation. In 1978, Larson et al. and Bartlett et al. identified Clostridium difficile as the causative bacterium of pseudomembranous colitis, and the presence of toxins was associated with pathogenicity. Currently, Clostridium difficile is the main pathogenic bacteria of nosocomial diarrhea in developed countries. After Clostridium difficile invades the human body, the body's immune system ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12Q1/689C12Q1/6844G01N33/569G01N33/577G01N33/58
CPCC07K16/1282C12Q1/689C12Q1/6844G01N33/56911G01N33/588G01N33/577C07K2317/56C07K2317/565G01N2333/33C12Q2521/507C12Q2522/101
Inventor 马红妙张玲
Owner FOSHAN DIAN MEDICAL LAB
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