TH19325U - A one-step, staining method for detecting genes of Chlamydia trachomatis, the pathogen causing non-gonococcal urethritis. - Google Patents

Abstract

------January 22, 2021------(OCR) A one-step genetic detection method for Chlamydia trachomatis, the pathogen causing chlamydia, using colorimetric banding. The process begins with designing five primers specific to the base sequence of Chlamydia trachomatis on a plasmid gene. One primer is labeled with biotin or digoxigenin, and another with a fluorescent agent (FITC) to monitor 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 appears as a pink test line. The test band (T) and control band (C) indicate the presence of Chlamydia trachomatis in the sample. If the result is negative, only the control band (C) will show a pink test line. 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. Summary of the Invention: A one-step, colorimetric method for detecting the genes of Chlamydia trachomatis, the pathogen causing chlamydia. It begins with the design of five primers specific to the base sequence of Chlamydia trachomatis on a plasmid gene. The forward inner primer (FIP) is labeled with biotin or digoxigenin. (Digoxigenin) and a fluorescently labeled probe (FITC) is used to monitor 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 line on the test (T) and control (C) bands, indicating the presence of Chlamydia trachomatis in the sample. A negative result shows a pink line only on 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. ------------ Page 1 of 1 page Summary of the Invention A one-step genetic detection method for Chlamydia trachomatis using color banding begins with designing five primers specific to the Chlamydia trachomatis base sequence on a plasmid 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 the dipstick. A positive result shows a pink test line in the test band (T) and control band (C), indicating the presence of Chlamydia trachomatis in the sample. A negative result will not show a pink test line. Only the control band (C) is used. This method is equivalent to real-time PCR monitoring and does not require a PCR machine or electrophoresis device to monitor the reaction results.

Claims (1)

------January 22, 2021------(OCR) 1. A one-step genetic detection method for Chlamydia trachomatis, a pathogen causing non-gonococcal urethritis, using a staining strip consisting of a LAMP reaction combined with the application of a dipstick. 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 (MgSO4), and the enzyme BS DNA polymerase (Bst DNA). Chlamydia trachomatis was detected using a dipstick in a tube containing 8 U of Chlamydia polymerase and buffer solution. The dipstick was then subjected to a LAMP reaction at 61°C for 45 minutes. 5 µL of this solution was then transferred to a new tube containing 100 µL of buffer solution. A dipstick was immersed in the solution, and after 5 minutes, the result was read. A positive result showed a pink line on the test band (T) and control band (C), indicating the presence of Chlamydia trachomatis. A negative result showed a pink line only on the control band (C). This dipstick test is used to detect Chlamydia trachomatis in suspected patients for diagnosis and prevention of disease outbreaks. This procedure designs primers for the LAMP technique used to detect Chlamydia trachomatis. The LAMP reaction consists of five primers specific to the Chlamydia trachomatis sequence on a plasmid gene. The forward inner primer (FIP) is labeled with biotin or digoxigenin, and the probe primer is labeled with a fluorescent substance (FITC) to track the reaction on a dipstick as follows: Forward outer primer (F3) sequence (5'-3') gAAAAggCgATTTAAAAACCAA; Backward outer primer (B3) sequence (5'-3') ggAACACATgATgCgAAgT. Backward inner primer (BIP) base sequence (5'-3') gTTAgAgAAACgATAgAT AAgTCT gTTTTTAATT ggCgATT CTTcr CT g AA Forward inner primer (FIP) base sequence (5'-3') biotin / digoxigenin (Digoxigenin; Dig) ATCCAACACCCTTATCgCCgTTTTggTCg AT gT gATAgggAAAg Probe primer (probe) Base sequence (5'-3') FITC- Tg AgTTCg AC ATTcc AC ATA ------------ ------13/11/2019------(OCR) Page 1 of 1 page Claims 1. A one-step genetic detection method for Chlamydia trachomatis, the pathogen causing non-gonococcal urethritis, using a staining strip consisting of LAMP reaction combined with dipstick application. 25 μL of LAMP reaction containing 50 picomoles each of forward inner primer (FIP) and backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3), and probe primer. A sample of Chlamydia trachomatis was prepared using a probe containing 5 picomoles each, 0.8 millimolar dNTP mixed with 0.6 molar betaine, 4 millimolar magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, and buffer solution. The sample was then subjected to a LAMP reaction at 61°C for 45 minutes. 5 µL of this solution was then transferred to a new tube containing 100 µL of buffer solution. A dipstick was immersed in the solution, and after 5 minutes, the result was read on the dipstick. A positive result showed a pink test line on the test band (T) and control band (C), indicating the presence of Chlamydia trachomatis. A negative result showed no pink test line. Only the control band (C) is used for dipstick detection of Chlamydia trachomatis in suspected patients for diagnosis and prevention of disease outbreaks. This procedure designs primers for the LAMP technique used to detect Chlamydia trachomatis in the LAMP reaction. It consists of 5 primers specific to the Chlamydia trachomatis base sequence on the plasmid gene. The forward inner primer (FIP) is labeled with biotin or digoxigenin, and the probe primer is labeled with a fluorescent substance (FITC) to track the reaction occurring on the dipstick as follows: Forward outer primer (F3), base sequence (5-3') gAAAAggCgATTTAAAAACCAA Backward outer primer (B3) base sequence (5'-3') ggAACACATgATgCgAAgT Backward inner primer (BIP) base sequence (5'-3') gTTAgAgAAACgATAgATAAgTCTgTTTTTAATTggCgATTCTTCTCTgAA Forward inner primer (FIP) sequence (5'-3'): biotin/Dig- ATCCAACACCCTTATCgCCgTTTTggTCgATgTgATAgggAAAg Probe primer sequence (5'-3'): FITC- TgAgTTCgACATTCCACATA Dig = Digoxigenin ------------ Page 1 of 1 page. Claim 1. A one-step genetic detection method for Chlamydia trachomatis using a staining strip consisting of a LAMP reaction combined with the application of a dipstick. The LAMP reaction uses 25 μL of primers 3 and 4 (5 picomoles each), primers 1, 2, and 5 (5 picomoles each), and dNTP. A 0.8 mmol solution is mixed with 0.6 mmol betaine, 4 mmolol magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, and buffer solution. The solution is then subjected to a LAMP reaction at 61°C for 45 minutes. 5 µl of this solution is then transferred to a new tube containing 100 µl of buffer solution at room temperature. A dipstick is immersed in the solution for 5 minutes, and the result is read from the dipstick. A positive result shows a pink test line on the test (T) and control (C) bands, indicating the presence of Chlamydia trachomatis. A negative result shows a pink test line only on the control (C) band. This method does not require a PCR thermal cycler or electrochemical chromatography (PCR) machine. This method involves the detection of Chlamydia trachomatis using a dipstick in patients suspected of infection, for diagnosis and prevention of disease outbreaks. The process designs primers for the LAMP (Large-Acting Amplification) technique used to detect Chlamydia trachomatis in the LAMP reaction. It consists of five primers specific to the base sequence of Chlamydia trachomatis on a plasmid gene, with four primers 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') gAAAAggCgATTTAAAAACCAA; Primer 2 (5'-3') ggAACACATgATgCgAAgT; Primer 3 (5'-3') gTTAgAg AAACg ATAg ATAAgT CT gTTTTTAATT ggCg ATT CTT CTCT g AA; Primer 4 (5'-3') bioti n/Dig- ATCC AACACCCTTATCgCCgTTTT ggTCgAT gT gATAgggAAAg; Primer 5 (5'-3') FITC- T g AgTTc g AC ATT cc AC ATA Dig = Digoxigenin