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PCR fluorescence detection kit of clostridium difficile toxin B and application of kit

A Clostridium difficile toxin and fluorescence detection technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of unbalanced sensitivity and specificity

Inactive Publication Date: 2020-05-19
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The innovations of the present invention are: 1) the kit can accurately detect different genotypes of Clostridium difficile producing toxin B, and will not be affected by other non-Clostridium difficile sequences, achieving high sensitivity and high specificity, solving 2) The "cormorant" biological big data mining system adopted in the present invention, according to key technical parameters such as copy number, primer dimer and sequence space structure, Find the best target sequence to greatly improve the detection efficiency and accuracy

Method used

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  • PCR fluorescence detection kit of clostridium difficile toxin B and application of kit
  • PCR fluorescence detection kit of clostridium difficile toxin B and application of kit
  • PCR fluorescence detection kit of clostridium difficile toxin B and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Screening of Primers for PCR Fluorescent Detection Kit of Clostridium difficile Toxin B

[0082] 1. Sample Collection

[0083] Take diarrhea feces and send them for inspection in time. 16 samples were collected, all clinical samples were from Shenzhen Children's Hospital.

[0084] 2. Sample extraction

[0085] (1) Take internal reference fragments (2×10 4 Copes / mL) 10 μl was added to 500 μl sample treatment solution (10 mM Tris-HCl, pH=8.0; 6M guanidine hydrochloride; 25 mM NaOH; 1% TritonX-100; 1% NP-40; 1 mM EDTA);

[0086] (2) Take 100-200 mg of feces sample. If the sample is liquid, draw 0.15-0.20 ml of sample, add 500 μl of sample treatment solution, shake and mix, and treat at 100°C for 10 minutes;

[0087] (3) Centrifuge at 12,000g for 5 minutes;

[0088] (4) Take 400 μl supernatant, add 200 μl absolute ethanol, and mix well;

[0089] (5) Transfer the suspension to a spin column, centrifuge at 12,000g for 1 minute, and install the spin column into a new col...

Embodiment 2

[0130] Specificity and Sensitivity Evaluation of PCR Fluorescence Detection Kit for Clostridium difficile Toxin B

[0131] 1. Sample preparation:

[0132] (1) Positive sample: Cultivate and clone the engineering bacteria of Clostridium difficile toxin B gene and extract the plasmid, measure the concentration with Qubit, then calculate its concentration, and dilute the plasmid with AE buffer to 1.0×10 5 copies / mL~1.0×10 2 copies / mL four concentration gradients.

[0133] (2) Negative samples: physiological saline and nucleic acids of Escherichia coli, Salmonella, Enterococcus faecium, and Enterococcus faecalis (confirmed by mass spectrometry and Sanger sequencing for bacteria obtained from feces culture).

[0134] 2. Sample adding system:

[0135] The PCR reaction solution includes PCR reaction solution A and PCR reaction solution B. Wherein the reaction solution A includes 12.5mM Tris-HCl, pH9.0, 50mM KCl, 0.125% X-100, 3.125mM MgCl 2 , 0.44625mM dATP, 0.44625mMdGTP, 0.4...

Embodiment 3

[0153] The PCR fluorescence detection kit of Clostridium difficile toxin B of the present invention and Comparison experiment of Clostridium difficile glutamate dehydrogenase antigen and toxin detection kit (enzyme-linked immunochromatography)

[0154] 1. Sample Collection

[0155] Take diarrhea feces and send them for inspection in time. A total of 97 samples were collected, all clinical samples were from Shenzhen Children's Hospital.

[0156] 2. Clostridium difficile glutamate dehydrogenase antigen and toxin detection kit (ELISA) (National Food and Drug Administration (Jin) Zi 2014 No. 3403929) operation process:

[0157] (1) Before use, place all reagents and corresponding number of reaction plates at room temperature.

[0158] (2) Prepare a small test tube for each sample and mark it. If necessary, it can be used as an external quality control.

[0159] (3) Take the diluent (bottle with a black dropper cap), and add 750 μL (the second scale from the tip) of the dilu...

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Abstract

The invention discloses a PCR fluorescence detection kit of clostridium difficile toxin B and an application of the kit, and belongs to the technical field of biology. The kit mainly comprises a specific primer, a probe and a PCR reaction reagent, wherein the specific primer and the probe consist of specific primers and probes of clostridium difficile toxin B and internal reference fragments. Thekit can accurately detect clostridium difficile with different genotypes producing toxin B, and cannot be influenced by sequences of other non-clostridium difficile, so that high sensitivity and highspecificity are realized, and the difficult problem that sensitivity and specificity cannot be both excellent in the present design of primers is solved. Meanwhile, the kit adopts a ''cormorant'' biological large data mining system, and an optimal target sequence is searched according to key technical parameters such as copy number, primer dimer, sequence space structure and the like, so that detection efficiency and accuracy are greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a PCR fluorescence detection kit for Clostridium difficile toxin B and an application thereof. Background technique [0002] Clostridium difficile, also known as Clostridium difficile, Clostridium difficile or Clostridium difficile, is an anaerobic bacterium that generally lives in the human intestinal tract. Anaerobic bacteria are those that grow better in an oxygen-free environment than in an oxygen-free environment, and the human gut is a relatively oxygen-free environment. If certain antibiotics are taken excessively, the intestinal flora will be unbalanced, which will lead to the rapid growth of Clostridium difficile, causing inflammation and diarrhea. Rapidly growing C. difficile produces large amounts of exotoxins A and B. Exotoxin A is an intestinal toxin that can invade mucosal cells and cause bleeding. Exotoxin B is a kind of cytotoxin. In the body, exotoxin B cannot inv...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/101C12Q2545/113
Inventor 马东礼刘孝荣邢志浩姜含芳
Owner SHENZHEN CHILDRENS HOSPITAL