Method and kit for quickly detecting mycobacterium tuberculosis

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 not infecting 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, and is currently the main factor of death caused by a single infectious bacterium. In recent years, with the prevalence of AIDS, the increase of patients with various malignant tumors and the emergence of multi-drug resistant strains, the difficulty in the prevention and treatment of tuberculosis infection has increased. Statistics show that there are 2 billion tuberculosis infections in the world (accounting for 1 / 3 of the total population), and one person is infected every second. The global tuberculosis bacteria infects 8 million people every year. If non-proliferation measures are not taken immediately, tuberculosis will s...

Examples

Embodiment 1

[0032] 1. Preparation and identification of monoclonal antibodies

[0033] 1. Plasmid construction: construct eukaryotic expression vectors and prokaryotic expression vectors for proteins gp17 (corresponding gene number: NC_046832) and gp23 (corresponding gene number: NC_046839). Select plasmid pcDNA3.1 / myc-His(-)A for eukaryotic expression vector, select plasmid pET22b(+) for prokaryotic expression vector, after PCR, gel recovery, digestion, purification, connection, transformation, after colony PCR, select positive After cloning, extracting the plasmid and sequencing, the sequenced correct clones gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) were obtained.

[0034] 2. Expression and purification of recombinant protein

[0035] The plasmids gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) were transformed into Escherichia coli BL21, and the expression was induced according to conventional experiments, the induction temperature was 30 degrees Celsius, and the IPTG concentration was 0....

Embodiment 2

[0122] Except following steps, other steps one, two, five, six are the same as embodiment 1.

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

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

[0125] Take by weighing an appropriate amount of sodium hydroxide, add purified water, be mixed with 2% sodium hydroxide solution, after filtering with a 0.22um sterilizing filter, pack in 50ml plastic bottles (25ml / bottle).

[0126] 2. Enrichment solution

[0127] Michaelis 7H9 medium 5g, calcium chloride 1g, ampicillin 25g, amphotericin B 25g, bovine serum albumin 100g, glucose 10g, oleic acid 1g and catalase 0.1g, all dissolved in 1000g of pure water to prepare Form; After filtering with a 0.22um sterilizing filter, it is subpackaged in 50ml plastic bottles (40ml / bottle) in a local 100-level ultra-clean workbench.

[0128] 3. Mycobacteriophage D29

[0129] After the amplified mycobacterial D29 phage was calibrated by plaque co...

Embodiment 3

[0143] Except following steps, other steps one, two, five, six are the same as embodiment 1.

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

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

[0146] Take by weighing an appropriate amount of sodium hydroxide, add purified water, be mixed with 3% sodium hydroxide solution, after filtering with a 0.22um sterilizing filter, pack in 50ml plastic bottles (25ml / bottle).

[0147] 2. Enrichment solution

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

[0149] 3. Mycobacteriophage D29

[0150] After the amplified mycobacterial D29 phage was calibrated ...

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, and is currently the main factor of death caused by a single infectious bacterium. In recent years, with the prevalence of AIDS, the increase of patients with various malignant tumors and the emergence of multi-drug resistant strains, the difficulty in the prevention and treatment of tuberculosis infection has increased. Statistics show that there are 2 billion tuberculosis infections in the world (accounting for 1 / 3 of the total population), and one person is infected every second. The global tuberculosis bacteria infects 8 million people every year. If non-proliferation measures are not taken immediately, tuberculosis will s...

Examples

Embodiment 1

[0032] 1. Preparation and identification of monoclonal antibodies

[0033] 1. Plasmid construction: construct eukaryotic expression vectors and prokaryotic expression vectors for proteins gp17 (corresponding gene number: NC_046832) and gp23 (corresponding gene number: NC_046839). Select plasmid pcDNA3.1 / myc-His(-)A for eukaryotic expression vector, select plasmid pET22b(+) for prokaryotic expression vector, after PCR, gel recovery, digestion, purification, connection, transformation, after colony PCR, select positive After cloning, extracting the plasmid and sequencing, the sequenced correct clones gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) were obtained.

[0034] 2. Expression and purification of recombinant protein

[0035] The plasmids gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) were transformed into Escherichia coli BL21, and the expression was induced according to conventional experiments, the induction temperature was 30 degrees Celsius, and the IPTG concentration was 0....

Embodiment 2

[0122] Except following steps, other steps one, two, five, six are the same as embodiment 1.

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

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

[0125] Take by weighing an appropriate amount of sodium hydroxide, add purified water, be mixed with 2% sodium hydroxide solution, after filtering with a 0.22um sterilizing filter, pack in 50ml plastic bottles (25ml / bottle).

[0126] 2. Enrichment solution

[0127] Michaelis 7H9 medium 5g, calcium chloride 1g, ampicillin 25g, amphotericin B 25g, bovine serum albumin 100g, glucose 10g, oleic acid 1g and catalase 0.1g, all dissolved in 1000g of pure water to prepare Form; After filtering with a 0.22um sterilizing filter, it is subpackaged in 50ml plastic bottles (40ml / bottle) in a local 100-level ultra-clean workbench.

[0128] 3. Mycobacteriophage D29

[0129] After the amplified mycobacterial D29 phage was calibrated by plaque co...

Embodiment 3

[0143] Except following steps, other steps one, two, five, six are the same as embodiment 1.

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

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

[0146] Take by weighing an appropriate amount of sodium hydroxide, add purified water, be mixed with 3% sodium hydroxide solution, after filtering with a 0.22um sterilizing filter, pack in 50ml plastic bottles (25ml / bottle).

[0147] 2. Enrichment solution

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

[0149] 3. Mycobacteriophage D29

[0150] After the amplified mycobacterial D29 phage was calibrated ...

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