Method and kit for quickly detecting mycobacterium tuberculosis
A technology of Mycobacterium tuberculosis and reagent kits, which is applied in the fields of immunology, protein detection, and molecular biology, can solve the problems of low sensitivity and specificity of serological diagnostic methods, and insufficient speed of phage rapid amplification method, so as to improve sensitivity and Specificity, meet the needs of rapid diagnosis of pulmonary tuberculosis, and facilitate the treatment of pulmonary tuberculosis
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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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