TH17934A3 - One-step color-coded detection method for Mycobacteriumtuberculosis - Google Patents

Abstract

A summary of the invention which will appear on the advertisement page ReadFile:Page 1 of 1 Page Summary of the invention of the Mycobacteriumtuberculosis detection method with colored stripes in the step-by-step process. A single primer was initially designed with a sequence-specific five-strand tuberculosis primer on the IS6110(/56110) gene, in which one primer was labeled with biotin or di Digoxigenin and another primer labeled with fluorescence (FITC) to monitor the reactions occurring on the dipstick. In this system, target DNA is added. Dosage under 61°C for 45 minutes in the heating block and then read the results on the dipstick. When the result is positive, a pink test line will appear on the test strip (T) and control strip. (C) shows TB in the sample, but if negative, only the control band (C) will show pink test line. This method is equivalent to real-time PCR monitoring. It also does not require PCR and electrolysis to monitor the reaction.

Claims (5)

Claims (all) which will not appear on the classifieds page:------04/12/2020------(OCR) 1. A one-step color-coded detection method for Mycobacteriumtuberculosis that consists of LAMP reaction (LAMP) in combination with the application of colored stripes. 25 µl lamp reaction dipstick (LAMP) consisting of primer 3 and primer 4 each, 50 picomoles, primer 1, primer 2 and primer 4 each. Mer 5 each 5 picomoles, 0.8 mmol DNTP (dNTP) mixed with betaine (betaine) 0.6 molar, magnesium sulfate (MgS04) 4 mmola. The enzyme BST DNA polymerase (BstDNApolymerase) 8U and buffer solution by LAMP reaction (LAMP) at 61 ° C for 45 minutes, then the aqueous solution of 5 μL was added to it. In a tube containing 100 µL buffered solution at room temperature, dip a dipstick in the solution for 5 min and read the results. When positive, pink test lines appear at the test strips (T) and the control V strips (0. ) indicates that TB is found in the sample, but if negative, only the control band (C) is pink in the test line. Suspected infection with this tuberculosis Diagnosis and prevention of disease outbreaks without the use of PCR (PCRthermalcycler) and electrolysis genome separator, this method is designed as a primer for the LAMP technique used in the detection of pathogens. Lamp reaction tuberculosis (LAMP) consists of five primers specific to the TB gene sequence on the IS 6110(IS6110) gene, where primer 4 is labeled with biotin. or digoxigenin and primer 5 was labeled with fluorescence (FITC) to monitor the reactions occurring on the dipstick as follows: primer 1 base sequence. (5'-3')CgATggCgAACTCAAggAg Primer 2 Sequence (5'-3')TggCCggATCAgCgATCPrimer 3 Sequence (5'-3')AgCCACACTTTgCgggCACTTTCACATcAgCCgCgTccPrimer 4 Sequence (5'-3') ')Biotin(biotin)/Digoxigenin(Digoxigenin)gAggTggCCAgATgCACCgTCTTTTggTCCTgCgggCTTTg5 sequence primer(5'-3')FITC-ACggCTgATgACCAAACTCg------------edit 04/12/2018 Page 1 of 1 Claims Page 1. A single-step color-banded detection method for Mycobacteriumtuberculosis that consists of LAMP reaction. ) In conjunction with the application of a dipstick, 25 μl LAMP consisted of 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. 4 mmol phase (MgSO4), BST DNA polymerase (BstDNApolymerase) 8U and buffer solution by lamp reaction (LAMP) at 61°C for 45 minutes from 5 μL of the solution was aspirated into a tube containing 100 μL buffered solution at room temperature, and then immersed in a dipstick for 5 minutes and read the result. T) and control band (C) indicate that TB is found in the sample, but if negative, a pink test line appears only for control band (C). /S6110) in the suspected patient This TB infection for the diagnosis and prevention of outbreaks without the use of PCR (PCRthermalcycler) and electrolysis of the genome is designed as a primer for the LAMP technique. Used in the lamp reaction TB assay (LAMP). It consists of five primers specific to TB genes on the IS6110(/S6110) gene by bi-labeled primer 4. Biotin or Digoxigenin and a primer 5 labeled with fluorescence (FITC) to monitor the reactions occurring on the dipstick are as follows: Primer 1 base sequence (5'-3')CgATggCgAACTCAAggAg Primer 2 base sequence (5'-3')TggCCggATCAgCgATC Primer 3 bases (5'-3') AgCCACACTTTgCgggCACTTTTTcACATcAgCCgCgTcc Primer 4 base sequence (5'-3') biotin/Dig-gAggTggCCAgATgCACCgTCTTTTggTCCTgCgggCTTTg Primer 5 bases (5'-3') FITC-ACggCTgATgACCAAACTCg Dig=Digoxigenin ------------------------------ ---------------------------------------------------- -------------------- Page 1 of 2 Pages Claims 1. Method for the detection of mycobacterium tuberculosis (TB) 25 μl LAMP consists of primer 3. and primer 4 each of 50 picomoles, primer 1, primer 2 and primer 5 each of 5 picomoles, 0.8 mmolar DNTP (dNTP). Formulated with 0.6 molar betaine, 4 mmolar magnesium sulfate (MgS04), 8U of BstDNApolymerase and buffer solution. Tuberculosis sequencing on IS6110(/56110) gene The primer design for the LAMP technique (LAMP) used in the lamp reaction (LAMP) assay consists of a primer. 5 strands specific to tuberculosis sequencing on the IS 6110(/56110) gene by primer 4 labeled with biotin or digoxigenin and Primer 5 should be labeled with fluorescent agent (FITC) to monitor the reactions occurring on the dipstick as follows: Primer 1 base sequence (5'-3')CgATggCgAACTCAAggAg Primer 2 base sequence (5'-3')TggCCggATCAgCgATC Primer 3 bases (5'-3') AgCCACACTTTgCgggCACTTTTTcACATcAgCCgCgTcc Primer 4 base sequence (5'-3') biotin/Dig-gAggTggCCAgATgCACCgTCTTTTggTCCTgCgggCTTTg Primer of 5 bases (5'-3') FITC-ACggCTgATgACCAAACTCg Dig=Digoxigenin 2. Mycobacteriumtuberculosis is a one-step color band detection method according to claim 1, where the optimal conditions for the LAMP assay On a dipstick, it is characterized by a lamp reaction (LAMP) at 61°C for 45 min and 5 μl of such solution is aspirated into a tube containing 100 μL buffered solution at temperature. room, then dip the dipstick in the solution for 5 minutes and read the results. 3. Mycobacteriumtuberculosis is a one-step detection method for Mycobacteriumtuberculosis by Claim 1, where LAMP reaction has an amplification step. Tuberculosis genotype for 45 min at 61°C with primer 4 labeled with biotin or digoxigenin and primer 5 labeled. With fluorescence reagent (FITC) to monitor the moist reaction by reading the results on a dipstick, when a positive result shows a pink test line around the test strip (T) and control strip (C). Shows that tuberculosis is found in the sample, but if negative, a pink test line will appear only on the control band (C). 4. Mycobacteriumtuberculosis is a one-step detection method for Mycobacteriumtuberculosis with a single color band according to claim 1, which is characterized by that it does not require PCR (PCRthermalcycler). ) and an electric separator Page 2 of the number 2 pages 5. Method for the detection of Mycobacterium tuberculosis with single-stage bands according to claim 1, which is a technique for detecting tuberculosis by colored bands in a single-stage step. The only episode in patients with suspected tuberculosis infection for the diagnosis and prevention of outbreaks.