TH19326Y - A one-step staining method for detecting kanamycin-resistant tuberculosis base mutations. - Google Patents

Abstract

------January 22, 2021------(OCR) A one-step method for detecting kanamycin-resistant tuberculosis mutations using colorimetric strips. The process begins with designing five primers specific to the kanamycin-resistant tuberculosis sequence with a mutation at position 1401, where the A base changes to G in the RRS gene. A forward inner primer (FIP) is labeled with biotin or digoxigenin, and a probe primer (FITC) is labeled with a fluorescent substance to monitor the reaction on a dipstick. In this system, the target DNA is amplified at 61°C for one hour in a heating block, and the result is read on the dipstick. A positive result appears as a pink test line. The test band (T) and control band (C) indicate that a base mutation is present in the sample at position 1401, where base A changes to base G in the RRS gene. However, if the result is negative, a pink test line will appear only on the control band (C). This method is equivalent to real-time PCR testing and does not require a PCR machine or electrophoresis to monitor the reaction results. ------13/11/2019------(OCR) Page 1 of 1 page. Summary of the Invention: A single-step colorimetric method for detecting base mutations in kanamycin-resistant tuberculosis. The process begins by designing five primers specific to the base sequence of kanamycin-resistant tuberculosis bacteria containing a mutation at position 1401, where the base A changes to base G (A --> G) in the RRS gene. Forward inner primers (FIPs) are labeled with biotin or digoxigenin, and probe primers are labeled with a fluorescent substance (FITC) to monitor the reaction on a dipstick. In this system, the target DNA is amplified at 61°C for one hour in a heating block, and the result is read on the dipstick. A positive result shows a pink test line in the test band (T) and control band (C), indicating the presence of the mutation at position 1401 (A --> G) in the RRS gene. 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 to monitor the reaction. ------------ Page 1 of 1 page. Summary of the Invention. A single-step method for detecting base mutations in kanamycin-resistant tuberculosis using color bands. It begins with the design of 5 primers specific to the base sequence of kanamycin-resistant tuberculosis with a base mutation at position 1401, where the A base changes to G (A --> G) in the RRS gene. One primer is labeled with biotin or digoxigenin, and another primer is labeled with a fluorescent substance (FITC) to monitor the reaction occurring on a dipstick. In this system, DNA... The goal is to amplify the sample at 61°C for one hour in a heating block, and then read the results on a dipstick. A positive result will show a pink test line in the test (T) and control (C) bands, indicating the presence of a base mutation at position 1401, where base A changes to base G in the RRS gene. A negative result will show a pink test line only in the control (C) band. This method is equivalent to real-time PCR testing and does not require a PCR machine or electrophoresis to monitor the reaction results.

Claims (1)

------January 22, 2021------(OCR) 1. A single-step staining method for detecting kanamycin-resistant tuberculosis mutagenic bases, consisting of a LAMP reaction combined with dipstick application. The LAMP reaction solution (25 μL) contained 50 picomoles each of forward inner primer (FIP) and backward inner primer (BIP), 5 picomoles each of forward outer primer (F3), backward outer primer (B3), and probe primer, 0.8 millimolar dNTP mixed with 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSOa), 8 U of Bst DNApolymerase enzyme, and buffer solution. The LAMP reaction is performed at 61°C for 1 hour. 5 µL of the solution is then drawn into a tube containing 100 µL of buffer solution. A dipstick is immersed in the solution and left for 5 minutes. The result is read on the dipstick. A positive result shows a pink line in the test band (T) and control band (C), indicating the presence of a base mutation at position 1401, where base A changes to base G in the RRS gene. A negative result shows a pink line only in the control band (C). This method is used to detect kanamycin-resistant tuberculosis (TB) using a single-step colorimetric testing process in suspected patients, enabling diagnosis and prevention of disease outbreaks without the need for a PCR thermal cycler or electrophoresis machine. A primer specifically designed for the LAMP technique is used to detect TB bacteria with only one base mutation. The primers used in the LAMP reaction consisted of five primers specific to the A-to-G mutation sequence of the tuberculosis RRS gene at position 1401. A forward inner primer (FIP) labeled with biotin or digoxigenin and a fluorescently labeled probe (FITC) were used to track the reaction on a dipstick as follows: Forward outer primer (F3) sequence (5'-3'): ACAATggCCggTACAAAgg; Backward outer primer (B3) sequence (5'-3'): ACCTTCgACAgCTCCCTC; Backward inner primer (BIP) sequence (5'-3'): TT gCAgACCCCgAT CCgAACTTTT gAT gCCgCgAggTTAAgC. Forward inner primer (FIP) base sequence (5'-30 biotin/dioxygenin) (Digoxigenin; Dig) TgAAgTCggAgTCgCTAgTATTTTTTgTTAC Cg ACTTTCAT g ACTc Probe primer (probe) Base sequence (5'-3') FTTC-gTTCCCgggCCTTgTACAC AC ------------ ------13/11/2019------(OCR) Page 1 of 1 page Claims 1. A single-step staining method for detecting base mutations in kanamycin-resistant tuberculosis using a LAMP reaction combined with dipstick application. The LAMP reaction consists of 25 μL of forward inner primer (FIP) and backward inner primer (BIP) at 50 picomoles each, forward outer primer (F3), backward outer primer (B3), and probe primer at 50 picomoles each. A mixture of 0.8 millimolar picomole and 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, and buffer solution was prepared using the LAMP reaction at 61°C for 1 hour. 5 µl of this solution was then transferred to a tube containing 100 µl of buffer solution. A dipstick was immersed in the solution, and after 5 minutes, the result was read from the dipstick. A positive result showed a pink test line in the test band (T) and control band (C), indicating the presence of a base mutation at position 1401, where base A changed to base G in the rrs gene. A negative result showed no pink test line. Control band (C) only. This method uses a single-step staining process to detect kanamycin-resistant tuberculosis in suspected patients for diagnosis and outbreak prevention, without the need for a PCR thermal cycler or electrophoresis machine. The process involves the design of primers for the LAMP technique to detect tuberculosis bacteria with only one mutated base. The primers used in the LAMP reaction consisted of five primers specific to the A-to-G mutation sequence of the tuberculosis RRS gene at position 1401. A forward inner primer (FIP) labeled with biotin or digoxigenin and a fluorescently labeled probe (FITC) were used to track the reaction on a dipstick as follows: Forward outer primer (F3) sequence (5'-3'): ACAATggCCggTACAAAgg; Backward outer primer (B3) sequence (5'-3'): ACCTTCgACAgCTCCCTC; Backward inner primer (BIP) sequence (5'-3'): TTgCAgACCCCgATCCgAACTTTTgATgCCgCgAggTTAAgC Forward inner primer (FIP) sequence (5'-3'): biotin/Dig-TgAAgTCggAgTCgCTAgTATTTTTgTTACCgACTTTCATgACTC Probe primer sequence (5'-3'): FITC-gTTCCCgggCCTTgTACAC AC Dig = Digoxigenin ------------ Page 1 of 1 Claim 1. A single-step staining method for detecting kanamycin-resistant tuberculosis mutated chromosomal variants using a LAMP reaction combined with dipstick application. LAMP reaction: 25 μL containing 50 picomoles each of primers 3 and 4, and 5 picomoles each of primers 1, 2, and 5. 0.8 millimolar dNTP was mixed with 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSO4), 8 U of IBST DNA polymerase enzyme, and buffer solution. This mixture was then subjected to a LAMP reaction at 61°C for 1 hour. 5 µl of this solution was then transferred to a tube containing 100 µl of buffer solution at room temperature. A dipstick was immersed in the solution for 5 minutes, and the result was read on the dipstick. A positive result showed a pink test line in the test band (T) and control band (C), indicating the presence of a base mutation at position 1401, where base A changed to base G in the RRS gene. A negative result showed a pink test line only in the control band (C). This method is used to detect base mutations in the RRS gene. A one-step staining method can be used to detect kanamycin-resistant tuberculosis in suspected patients, enabling diagnosis and prevention of outbreaks without the need for a PCR thermal cycler or electrophoresis machine. This process utilizes a primer design for the LAMP technique to detect tuberculosis bacteria with only one base mutation. The LAMP primers consist of five strands specific to the 1401 base change sequence (A --> G) of the RRS gene in the tuberculosis bacteria. Primer 4 is labeled with biotin or digoxigenin. (Digoxigenin) and use a fluorescently labeled primer (FITC) to track the wet reaction on a dipstick as follows: Primer 1 (5'-3') ACAATggCCggTACAAAgg; Primer 2 (5'-3') ACCTTCgACAgCTCCCTC; Primer 3 (5'-3') TT gCAgACCCCgAT CCgAACTTTT gAT gCCgCgAggTTAAgC; Primer 4 (5'-3') bioti n/Di g-T g AAgT Cgg AgTC gCTAgTATTTTT gTTACC g ACTTT CAT g ACT c; Primer 5 (5'-3') FITC-gTTCCCgggCCTTgTACACAC Dig = Digoxigenin