TH17934U - A one-step staining method for detecting Mycobacterium tuberculosis. - Google Patents

Abstract

Page 1 of 1. Summary of the Invention: A one-step staining method for detecting Mycobacterium tuberculosis. The process begins with the design of five primers specific to the base sequence of the tuberculosis bacteria on the IS6110 (/56110) gene. One primer is labeled with biotin or digoxigenin, and another with a fluorescent substance (FITC) to track the reaction on a dipstick. In this system, the target DNA is amplified at 61°C for 45 minutes in a heating block, and the result is read on a dipstick. A positive result shows a pink test line in the test band (T) and control band (C), indicating the presence of tuberculosis in the sample. A negative result shows no pink test line. Only the control band (C) is used. This method is equivalent to real-time PCR testing and does not require a PCR machine or electrophoresis device to monitor the reaction results.

Claims (5)

------04/12/2020------(OCR) 1. A single-step diagnostic method for Mycobacterium tuberculosis using a dipstick strip, comprising a LAMP reaction and the application of a dipstick strip. The LAMP reaction solution (25 μL) contains 50 picomoles each of primers 3 and 4, 5 picomoles each of primers 1, 2, and 5, 0.8 millimolar dNTP mixed with 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSO4), 8 U of BST DNA polymerase, and buffer solution. The LAMP reaction is performed at 61 °C for 45 minutes. 5 μL of the solution is then drawn into a tube containing 100 μL of buffer solution at room temperature. A dipstick is then immersed in the solution for 5 minutes, and the result is read. A positive result shows a pink test line in the test band (T) and the control band (0), indicating the presence of tuberculosis bacteria in the sample. A negative result shows a pink test line only in the control band (C). This method is used to determine the base sequence of tuberculosis bacteria on the IS6110 (/56110) gene in patients suspected of having tuberculosis infection for diagnosis and prevention of disease outbreaks without the need for a PCR thermal cycler and electrochemical separator. This procedure designs primers for the LAMP (Low-Acting Amplification) technique used for tuberculosis detection. The LAMP primer consists of five primers specific to the base sequence of the tuberculosis bacterium on the IS6110 gene. Primer 4 is labeled with either biotin or digoxigenin, and primer 5 is labeled with a fluorescent substance (FITC) to track the reaction on a dipstick as follows: Primer 1 (5'-3'): CgATggCgAACTCAAggAg; Primer 2 (5'-3'): TggCCggATCAgCgATC; Primer 3 (5'-3'): AgCCAC ACTTT gCgggCACTTTCAC AT c AgCCgCgT cc; Primer 4 (5'-3'): biotin/digoxigenin. (Digoxigenin) gAggTggCCAgATgCACCgTCTTTT ggTCCTgCgggCTTTg 5-base primer (5'-3') FITC-ACggCTgATgAC CAAACTCg ------------ Revised 04/12/2018 Page 1 of 1 page Claim 1. A single-step staining method for detecting Mycobacterium tuberculosis, consisting of a LAMP reaction combined with the application of... A 25 µl dipstick test strip contains 50 picomoles each of primers 3 and 4, 5 picomoles each of primers 1, 2, and 5, 0.8 millimolar of dNTP mixed with 0.6 molar of betaine, 4 millimolar of magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, and buffer solution. The LAMP reaction is performed at 61°C for 45 minutes. 5 µl of this solution is then transferred to a tube containing 100 µl of buffer solution at room temperature. The dipstick is immersed in the solution for 5 minutes, and the result is read. A positive result will show a pink test line. The test band (T) and control band (C) indicate the presence of tuberculosis bacteria in the sample. If the result is negative, a pink test line will appear only on the control band (C). This method is used to determine the base sequence of the tuberculosis bacteria on the IS 6110 (/S6110) gene in patients suspected of having tuberculosis infection, for diagnosis and disease prevention, without the need for a PCR (thermal cycler) or electrophoresis machine. The process involves designing primers for the LAMP (Low-Acting Amplification) technique used in tuberculosis detection. The LAMP primer consists of five primers specific to the base sequence of the tuberculosis bacteria on the IS 6110 (/S6110) gene, with primer 4 labeled with biotin or digoxigenin. (Digoxigenin) and use primer 5 labeled with a fluorescent substance (FITC) to track the reaction occurring on a dipstick as follows: Primer 1, base sequence (5'-3'): CgATggCgAACTCAAggAg; Primer 2, base sequence (5'-3'): TggCCggATCAgCgATC; Primer 3, base sequence (5'-3'): AgCC ACACTTT gCgggCACTTT c ACAT c AgCCgCgTcc; Primer 4, base sequence (5'-3'): biotin/Dig-gAggT ggCCAgAT gCACCgT CTTTT ggT CCT gCgggCTTT g; Primer 5, base sequence (5'-3'): FITC-ACggCTgATgAC CAAACTCg Dig = Digoxigenin ---------------------------------------------------------------------------------------------------- Page 1 of 2 pages. Claim. 1. A one-step staining method for detecting Mycobacterium tuberculosis using a LAMP reaction combined with the application of a dipstick. The 25 µl LAMP solution contains 50 picomoles each of primers 3 and 4, 5 picomoles each of primers 1, 2, and 5, 0.8 millimolar dNTP mixed with 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, and a buffer solution for sequencing the tuberculosis bacteria. Tuberculosis on the IS 6110 (/56110) gene: The design of primers for the LAMP (Low-Low-Acting Amplification) technique used for tuberculosis detection in the LAMP reaction consists of 5 primers specific to the base sequence of the tuberculosis bacteria on the IS 6110 (/56110) gene. Primer 4 is labeled with biotin or digoxigenin. (Digoxigenin) and use a fluorescently labeled primer 5 (FITC) to track the reaction on a dipstick as follows: Primer 1 (5'-3') CgATggCgAACTCAAggAg; Primer 2 (5'-3') TggCCggATCAgCgATC; Primer 3 (5'-3') AgCC ACACTTT gCgggCACTTT c ACAT c AgCCgCgTcc; Primer 4 (5'-3') biotin/Dig-gAggT ggCCAgAT gCACCgT CTTTT ggT CCT gCgggCTTT g; Primer 5 (5'-3') FITC-ACggCTgATgAC CAAACTCg Dig = Digoxigenin 2. A single-step staining procedure for detecting Mycobacterium tuberculosis using a dye strip, as per Patent 1, where the optimal conditions for detecting the LAMP product on a dipstick are specific: the LAMP reaction is performed at 61°C for 45 minutes, 5 µL of the solution is aspirated into a tube containing 100 µL of buffer solution at room temperature, the dipstick is immersed in the solution for 5 minutes, and the result is read. 3. A single-step colorimetric detection method for Mycobacterium tuberculosis, as per Patent 1, in which the LAMP reaction involves amplifying the genetic material of the tuberculosis bacteria for 45 minutes at 61°C. Primer 4 is labeled with biotin or digoxigenin, and primer 5 is labeled with a fluorescent substance (FITC). The reaction is monitored by reading the results on a dipstick. A positive result shows a pink test line on the test (T) and control (C) bands, indicating the presence of tuberculosis in the sample. A negative result shows a pink test line only on the control (C) band. 4. A single-step colorimetric detection method for Mycobacterium tuberculosis, as per Patent 1, which is characterized by the absence of a PCR thermal cycler and electrophoretic separator. (Page 2 of 2) 5. A single-step colorimetric detection method for Mycobacterium tuberculosis, as per Patent 1, which is a single-step colorimetric detection technique for tuberculosis in patients suspected of having the infection, for the purpose of diagnosis and prevention of disease outbreaks.