Method and kit for quickly detecting mycobacterium tuberculosis

A Mycobacterium tuberculosis and kit technology, applied in the fields of immunology, protein detection, and molecular biology, can solve the problems that the rapid phage amplification method is not fast enough, and the sensitivity and specificity of serological diagnosis methods are not high, so as to meet the requirements of rapid diagnosis of pulmonary tuberculosis. needs, improved sensitivity and specificity, and the effect of strong specificity

Active Publication Date: 2010-02-17
ABBOTT DIAGNOSTICS (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The technical problem to be solved in the present invention is to provide a method for rapid detection of Mycobacterium tuberculosis, which overcomes the shortcomings of the existing bacteriophage rapid amplification method that is not fast enough, and the sensitivity a

Method used

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  • Method and kit for quickly detecting mycobacterium tuberculosis
  • Method and kit for quickly detecting mycobacterium tuberculosis
  • Method and kit for quickly detecting mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Preparation and identification of monoclonal antibodies

[0033] 1. Plasmid construction: construct the eukaryotic expression vector and prokaryotic expression vector of protein gp17 (corresponding gene number: NC_046832) and gp23 (corresponding gene number: NC_046839). The eukaryotic expression vector selects the plasmid pcDNA3.1 / myc-His(-)A, the prokaryotic expression vector selects the plasmid pET22b(+), after PCR, gel recovery, enzyme digestion, purification, ligation, transformation, colony PCR, the selection is positive Cloning, extracting the plasmid and sequencing to obtain the correct clones gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A).

[0034] 2. Recombinant protein expression and purification

[0035] The plasmids gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) were transformed into E. coli BL21, and the expression was induced according to routine experiments, the induction temperature was 30 degrees Celsius, and the IPTG concentration was 0.8 mM. After obtaining the...

Embodiment 2

[0122] Except for the following steps, other steps 1, 2, 5, and 6 are the same as in Example 1.

[0123] 3. Preparation of other components of D29 phage rapid detection kit:

[0124] 1. Sample treatment solution (2% sodium hydroxide solution)

[0125] Weigh an appropriate amount of sodium hydroxide, add purified water to prepare a 2% sodium hydroxide solution, filter it with a 0.22um sterile filter, and place it in a 50ml plastic bottle (25ml / bottle).

[0126] 2. Enriching liquid

[0127] Mie's 7H9 medium 5g, calcium chloride 1g, ampicillin 25g, amphotericin B25g, bovine serum albumin 100g, glucose 10g, oleic acid 1g and catalase 0.1g, all prepared by dissolving in 1000g pure water After filtering with a 0.22um sterile filter, it is packed into 50ml plastic bottles (40ml / bottle) in a local 100-level ultra-clean workbench.

[0128] 3. Mycobacterium D29 phage

[0129] After the amplified mycobacterial D29 phage was calibrated by the plaque counting method, it was diluted to 10 with Mie 7...

Embodiment 3

[0143] Except for the following steps, other steps 1, 2, 5, and 6 are the same as in Example 1.

[0144] 3. Preparation of other components of D29 phage rapid detection kit:

[0145] 1. Sample treatment solution (3% sodium hydroxide solution)

[0146] Weigh an appropriate amount of sodium hydroxide, add purified water to prepare a 3% sodium hydroxide solution, filter it with a 0.22um sterile filter, and dispense it into 50ml plastic bottles (25ml / bottle).

[0147] 2. Enriching liquid

[0148] Michaelis 7H9 medium 6g, calcium chloride 1.5g, ampicillin 37.5g, amphotericin B37.5g, bovine serum albumin 150g, glucose 12.5g, oleic acid 1.5g and catalase 0.15g, all dissolved in Prepared from 1000g of pure water; after filtering with a 0.22um sterilizing filter, it is divided into 50ml plastic bottles (40ml / bottle) in a local 100-level ultra-clean workbench.

[0149] 3. Mycobacterium D29 phage

[0150] After the amplified mycobacterial D29 phage was calibrated by the plaque counting method, it...

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Abstract

The invention discloses a method for quickly detecting mycobacterium tuberculosis, which comprises the following steps: 1, infecting phage with the mycobacterium tuberculosis; 2, killing the phage notinfecting outside the mycobacterium tuberculosis; 3, amplifying the infecting phage; and 4, after labeling gold with antiphage antibody, adopting quick immunochromatography to detect whether the phage exists or not so as to judge whether the mycobacterium tuberculosis exists in a sample. In addition, the invention also discloses a corresponding kit, which comprises sample treating fluid, proliferous liquid, a killing agent, mycobacterium smegmatis, mycobacteriophages and a phage colloidal gold method immunity-chromatography test pen. The method and the kit can quickly and accurately detect the mycobacterium tuberculosis so as to meet the requirement of quickly diagnosing pulmonary tuberculosis and timely medicate the patient.

Description

Technical field [0001] The invention relates to the technical fields of molecular biology, immunology and protein detection. More specifically, it relates to a method and kit for rapid detection of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis (Tuberculosis) is a zoonotic chronic infectious disease caused by Tubercle Bacillus. It is currently the main cause of death by a single infectious bacterium. In recent years, with the epidemic of AIDS, the increase of patients with multiple malignancies, and the emergence of multi-drug resistant strains, the difficulties in the prevention and treatment of tuberculosis infection have increased. Statistics show that there are 2 billion people infected with tuberculosis (one-third of the total population) in the world, infecting one person every second, and 8 million new tuberculosis bacteria infect each year. If anti-proliferation measures are not taken immediately, tuberculosis will be in the future. Over 30 milli...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/577
Inventor 胡志能王亚新吴伟清
Owner ABBOTT DIAGNOSTICS (SHANGHAI) CO LTD
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