TH17937A3 - One-step method for detecting the mutant bases of isoniazid-resistant tuberculosis on the INSHA gene (inhA) with a single color band. - Google Patents

Abstract

Summary of the invention which will appear on the advertisement page ReadFile:------16/12/2020------(OCR) Method for detecting base mutations in isonia-resistant TB. Ioniazid on the inhA gene with a single-step color band was initiated by designing a sequence-specific five-line primer for isoniazid-resistant tuberculosis. isoniazid with a base mutated at the 15th promoter, where the base C transforms into a base (C-15-T) of the gene inhA (inhA), which yields primer 1. One line was labeled with biotin or digoxigenin and one primer was labeled with fluorescent agent (FITC) to monitor the reactions occurring on the dip. In this system, the target DNA is quantified under 61°C for 1 hour in the heatingblock and then read on a dipstick. A pink test line appears at the test band (T) and control band (C), indicating that in the sample, a mutated base was found at the 15th promoter position, where the base C converted to a base (C-15-T) of gene INHA (inhA), but if negative, the pink test line appears only in the control band (C). This is equivalent to real-time PCR monitoring and does not require PCR and electrolysis to monitor the reaction's effects. -----------Page 1 of 1 page Summary of Invention of a method for detecting the mutant base of isoniazid-resistant tuberculosis (isoniazid) on the INHA gene. The first step was to design a five-line primer specific to isoniazid-resistant tuberculosis with mutant bases at the promo site. The 15th promoter in which base C is converted to base T (C-15-T) requests the gene inha (inhA), which is labeled with biotin or biotin. Digoxigenin and another primer labeled with fluorescence (FITC) to monitor the moist reaction on the dipstick. The dose was added under 61°C for 1 h in the heating block and then read on the dipstick. When the result was positive, a pink test line appeared on the test strip (T) and The control bar (C) indicates that the mutant base in the sample was found at the promoter location. In this method, the 15th promoter C base is converted to base T (C-15-T) of the INHA gene (inhA), but if negative, the pink test line appears only in the control band (C). It is equivalent to real-time PCR monitoring and does not require PCR and electrolysis to monitor the reaction results.

Claims (5)

Claims (all) which will not appear on advertisement pages:------16/12/2020------(OCR) 1. Method for detecting base mutation of resistant tuberculosis. isoniazid on the gene inhA (inhA) with a single-step color band consisting of LAMP reaction (LAMP) in combination with the application of a dipstick. The 25 µL dipstick consists of primer 3 and primer 4 each, 50 picomoles, primer 1, primer 2 and primer. 5 each 5 picomoles, DNTP (dNTP) 0.8 mmol, mixed with betaine (betaine) 0.6 molar, magnesium sulfate (MgSO4) 4 mmol, enzyme BST DNA polymerase (BstDNApolymerase) 8U and LAMP buffer solution (LAMP) were made at 61°C for 1 h, after which 5 μL of the solution was sucked into tubes. with 100 µL buffered solution at room temperature, then dip the dipstick in the solution for 5 min and read the results on the dipstick. When positive, a pink test line appears on the test strip ( T) and the control band (C) showed that the base mutated in the sample was found at the 15th promoter at which the base changed. This method is used to detect the mutant base of the germline. Isoniazid-resistant tuberculosis in the inha (inhA) gene with a single-step colored band in patients with suspected infection for the diagnosis and prevention of outbreaks of drug-resistant tuberculosis. isoniazid without PCR (PCRthermalcycler) and electrolysis of this genome separator has been designed as a primer for the LAMP technique in the detection of tuberculosis. The base primer used in the lamp reaction (LAMP) consists of five primers specific to the mutant sequence at the 15th promoter position. Converted to bases (C-15-T) for the TB gene's inhA (inhA) by having primer 4 labeled with biotin or digoxigenin. where the last base of the 3' tip in primer 4 corresponds to the mutated base position at the 15th promoter, where the base C transforms into the base C-15-T of the gene. INHA (inhA) and primer 5 labeled with fluorescent agent (FITC) was used to trace the reaction. The reactions occurring on the dipstick are as follows: primer 1 base sequence (5'-3')gCTgAgTCACACCgACAAAC primer 2 sequence (5'-3')CCggTTTCCTCCggTAACC primer. 3bases (5'-3')CCACgAgCgTAACgTggCTgCTTTTgTCACgAgCgTAACCCCAprimer4seeds (5'-3')biotin(biotin)/digoxigenin(Digoxigenin)ATCgCAgCCACgTTACgCTTCTTTTgTCACCCCgACAACCTATprimer (5'-3')FITC-TggACATACCgATTTCggCCC------------Edit 04/12/2018 Page 1 of the number 2 claims page 1. A method for detecting the mutant bases of isoniazid-resistant tuberculosis on the INHA gene with a single-step color band consisting of a lamp reaction. (LAMP) in combination with the application of a dipstick in the lamp reaction (LAMP) 25 µl contains primer 3 and primer 4 each, 50 picomoles, primer. Primer 1, primer 2 and primer 5 each 5 picomoles, 0.8 mmolar DNTP (dNTP) mixed with 0.6 molar betaine, magnesium compounds. Sulfate (MgSO4) 4 mmolar, Bst DNA Polymerase 8U enzyme (BstDNApolymerase) and lab buffer solution (LAMP) were made at 61 ° C for 1 h. After that, 5 μL of the solution was aspirated into a tube containing 100 μL buffered solution at room temperature, then immersed in a dipstick for 5 minutes and read on the dipstick. A positive test line shows a pink test line on the test band (T) and control band (C), indicating that in the sample, a mutated base was found at the 15th peomoter where the base C converted to a base (C-15). -T) of the gene inhA (inhA), but if negative, a pink test line will appear only on the C bar. This method was used to determine the base mutation of isoniazid-resistant tuberculosis (isoniazid) in the INSHA gene (inhA) with a single-step color band. In patients with suspected infection for the diagnosis and prevention of isoniazid-resistant tuberculosis without the use of a PCR (PCRthermalcycler) and an electrolysis separator. A primer has been designed for the LAMP technique (LAMP) to detect TB with only one mutated base. Specific to the mutant gene at the 15th promoter position, where the base C transforms into the baset (C-15-T) of the tuberculosis gene inhA (inhA) by primer. 3 Label with biotin or digoxigenin, assuming that the final base of the 3' tip in primer 4 corresponds to the mutated base at the promoter position ( The 15th promoter in which the base C was converted to the baset (C-15-T) of the INHA gene iinhA) and primer 5 was labeled with fluorescence (FITC) to monitor the reaction. on the dipstick as follows: Primer 1 base sequence (5'-3')gCTgAgTCACACCgACAAAC 2 base sequence primers (5'-3')CCggTTTCCTCCggTAACC Primer 3 bases (5'-3') CCACgAgCgTAACgTggCTgCTTTTTTCACgAgCgTAACCCCA Primer 4-base (5'-3') Biotin/Dig-ATCgCAgCCACgTTACgCTTCTTtgTCACCCCgACAACCTATTT Primer 5 base sequence (5'-3')FITC-TggACATACCgATTTCggCCC Dig=Digoxigenin --------------------------------------------------------- ---------------------------------------------------- --- Page 1 of 2 pages Claims 1. Method for detecting the mutant bases of isoniazid-resistant tuberculosis on the inha (inhA) gene with a single-step color band consisting of a lamp reaction. (LAMP) in combination with the application of a dipstick within the lamp reaction (LAMP) 25 µl contains primer 3 and primer 4 each, 50 picomoles, primer. 1 primer 2 and primer 5 each 5 picomoles, 0.8 mmolar DNTP (dNTP) mixed with 0.6 molar betaine, magnesium sulfate. The 4 mmolar phase (MgS04), the enzyme BST DNA polymerase (BstDNApolymerase)8, and the buffer solution were used in the mutation sequencing at the 15th promoter site. Basec transformed into baset (C-15-T) of tuberculosis inhA (inhA) gene, primer design for LAMP technique (LAMP) in the assay of tuberculosis with mutant bases. Only one base primer used in a lamp reaction (LAMP) consists of five primers specific to the mutant sequence at the 15th promoter, which the bases convert to bases. C-15-T request for the TB gene inhA (inhA) by having primer 3 labeled with biotin or digoxigenin. It determined that the last base of the 3' tip in primer 4 corresponded to the mutated base at the 15th promoter, where the base C transformed into the base T (C-15-T) of the I gene. NHA) and primer 5 was labeled with fluorescence (FITC) to monitor the reactions occurring on the dipstick as follows: Primer 1 base sequence (5'-3')gCTgAgTCACACCgACAAAC 2 base sequence primers (5'-3')CCggTTTCCTCCggTAACC Primer 3 base sequence (5'-3') CCACgAgCgTAACgTggCTgCTTTTTTCACgAgCgTAACCCCA Primer 4-base (5'-3') Biotin/Dig-ATCgCAgCCACgTTACgCTTCTTtgTCACCCCgACAACCTATTT Primer 5 base sequence (5'-3')FITC-TggACATACCgATTTCggCCC Dig=Digoxigenin 2. A single-step, color-banded, single-step assay for isoniazid-resistant tuberculosis of tuberculosis resistant to isoniazid, where conditions at It is suitable to test the output from the lamp (LAMP) on the dipstick. The characteristic is that the lamp reaction (LAMP) at 61 degrees Celsius for 1 hour and then absorb 5 microliters of such solution. Place in a tube containing 100 µL buffered solution at room temperature, then dip the dipstick in the solution for 5 minutes and read the results. 3. The method for assaying the mutant bases of isoniazid-resistant tuberculosis on the inha (inhA) gene with a single-step color band according to claim 1, where the reaction and LAMP was carried out for 1 h at 61°C in which the primer 4 was labeled with biotide. Biotin or Digoxigenin and primer 5 was labeled with fluorescence (FITC) to monitor the reaction by reading the results on a dipstick. When positive, a pink test line appears at the test band (T) and the control band (C), indicating that the mutated base in the sample was found at the 15th promoter position, where the base C shifted the base (C- 15-T) of the gene inhA (inhA), but if negative, the pink test line appears only for the control band (C). Page 2 of the number 2 pages 4. The method for detecting the mutant base of isoniazid-resistant tuberculosis (isoniazid) on the INHA gene with a single-step color band according to claim 1, which is characterized by The only thing is that it does not require a PCR (PCRthermalcycler) and an electrolysis separator. 5. The method for detecting the mutant bases of isoniazid-resistant tuberculosis (isoniazid) in NHA (inhA) with single-step color bands according to claim 1 used for Detection of the mutant base of isoniazid-resistant tuberculosis (isoniazid) on the inha (inhA) gene with altered bands in patients with suspected infection for diagnosis and Prevent the outbreak of isoniazid-resistant tuberculosis (isoniazid).