TH20199U - A one-step colorimetric method for detecting APOE gene mutations and Alzheimer's risk. - Google Patents

Abstract

------January 22, 2021------(OCR) A one-step colorimetric method for detecting APOE gene mutations and Alzheimer's risk. The process begins with designing 10 primers specific to the mutated base sequences RS (rs) 7412 and RS (ro) 429358 on the APOE gene, which are associated with Alzheimer's risk. For specificity at each position, one primer is labeled with biotin or digoxigenin, and another with a fluorescent substance (FITC) to monitor the reaction on a dipstick. In this system, DNA is amplified at 61°C for 60 minutes 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 the presence of a mutated base sequence at position RS (rs) 7412 or RS (rs) 429358 on the APOE gene, which is associated with Alzheimer's risk. 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. ------------ Page 1 of 1 page. Summary of the Invention. A one-step method for detecting APOE gene mutations associated with Alzheimer's risk using color bands. This begins with the design of 10 primers specific to the mutated base sequences RS (rs) 7412 and RS (rs) 429358 on the APOE gene, which is associated with Alzheimer's risk. For specificity at each position, one primer is labeled with biotin or digoxigenin. (Digoxigenin) and another primer labeled with a fluorescent substance (FITC) are used to monitor the reaction on a dipstick. In this system, the DNA is amplified at 61 degrees Celsius for 60 minutes in a heating block, and the result is read on the dipstick. A positive result will show a pink test line in the test band (T) and control band (C), indicating the presence of a mutated base at position RS 7412 or RS 429358 on the APOE gene, which is associated with Alzheimer's risk. If the result is negative, a pink test line will only appear in the control band (C). This method is equivalent to real-time PCR testing without the need for a PCR machine and electrophoresis to monitor the reaction results.

Claims (2)

------January 22, 2021------(OCR) 1. A one-step staining method for detecting APOE gene mutations (Alzheimer's risk) using a colorimetric banding system. This method involves a LAMP reaction combined with the application of a dipstick. 25 μL of LAMP at each mutated base site 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 a buffer solution. The LAMP reaction is performed at 61°C for 60 minutes. 5 µl of the solution is then transferred to a new 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 on the dipstick. A positive result shows a pink line on the test band (T) and control band (C), indicating the presence of a mutated base at position RS (rs)7412 or RS (rs)429358 on the APOE gene, which is associated with Alzheimer's risk. A negative result shows a pink line only on the control band (C). This method does not require a PCR thermal cycler or electrophoresis. It is a dipstick-based diagnostic and preventative measure for detecting APOE gene abnormalities in individuals suspected of having Alzheimer's disease. This process designs primers for the LAMP (Landing-Age-Lamping) technique used to detect base mutations at positions RS (rs) 7412 or RS (rs) 429358 on the APOE gene associated with Alzheimer's risk. Each LAMP reaction consists of 5 primers specific to the rs (rs) 7412 or rs (rs) 429358 mutant sequences on the APOE gene. Primer 4 is labeled with biotin or digoxigenin, and primer 5 is labeled with the fluorescent substance FITC. The reaction is tracked on a dipstick as follows: Primers specific to the rs (rs) 429358 mutant sequence are: Primer 1: (5'-3') ggACgAgACCATgAAggAgT; Primer 2: (5'-3') gCTTgCgCAgggTgggA; Primer 3: (5'-3') CgCggACATggAggACgTTT TCTgCACCAggCggCCgTg; Primer 4: [No specific primer number provided]. (5'-3') biotin / digoxigenin -TCCTTggACAgCCgTgCCCTTTTggCCTA CAAATCggAACTgg Primer 5 base sequence (5'-3') FITC- ACCggggT CAgTT gTTCCT Primers specific to the rs (rs) 7412 mutant base are as follows: Primer 1 base sequence (5'-3') gAggTgCAggCCATgCTc Primer 2 base sequence (5'-3') CTCgCggATggCgCTgAg Primer 3 base sequence (5'-3') AT gCCgAT gACCT gCAgAATTTT CCT ggTACACT gCCAggTA Primer 4 base sequence (5'-3') biotin / digoxigenin (digoxigenin) - ggAggAgCCgCTTACgCA TTTTgCCAgAgCACCgAggA Primer 5 base sequence (5'-3')FITC- gCTTgCgCAggTgggAg ------------ Page 1 of 1 page. Claims 1. A one-step method for detecting APOE gene mutations, an Alzheimer's risk factor, using a colorimetric banding system consisting of a LAMP reaction combined with the application of a dipstick. LAMP reaction: 25 μL at each mutated base position 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), and the enzyme BST DNA polymerase. 8 U of Bst DNA polymerase and buffer solution were used in a LAMP reaction at 61°C for 60 minutes. 5 µl of this solution was then transferred to a new 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 from the dipstick. A positive result showed a pink line on the test band (T) and control band (C), indicating the presence of a mutated base at position RS 7412 or RS 429358 on the APOE gene, which is associated with Alzheimer's risk. A negative result showed a pink line only on the control band (C). This method does not require a PCR thermal cycler or electrophoresis machine. This method is a dipstick test to detect abnormalities in the APOE gene in individuals suspected of having Alzheimer's disease. For the diagnosis and prevention of the disease, this process involves designing primers for the LAMP technique to detect the RS (rs) 7412 or RS (rs) 429358 mutation on the APOE gene, which is associated with Alzheimer's risk. Each LAMP set consists of five primers specific to the RS (rs) 7412 or RS (rs) 429358 mutation on the APOE gene. Primer 4 is labeled with biotin or digoxigenin, and primer 5 is labeled with a fluorescent substance (FITC) to track the reaction on a dipstick as follows: 1. Primers specific to the mutated base at position RS (rs) 429358 are as follows: Primer 1 (5'-3'): ggACgAgACCATgAAggAgT; Primer 2 (5'-3'): gCTTgCgCAgggTgggA; Primer 3 (5'-3'): CgCggACATggAggACgTTTTCTgCACCAggCggCCgTg; Primer 4 (5'-3'): biotin/Dig- TCCTTggACAgCCgTgCCCTTTTggCCTACAAATCggAACTgg; Primer 5 (5'-3'): FITC- ACCggggTCAgTTgTTCCT 2. Primers specific to the mutant base at position RS (rs) 7412 are as follows: Primer 1 (5'-3') gAggTgCAggCCATgCTC; Primer 2 (5'-3') CTCgCggATggCgCTgAg; Primer 3 (5'-3') ATgCCgATgACCTgCAgAATTTTCCTggTACACTgCCAggTA; Primer 4 (5'-3') biotin/Dig- ggAggAgCCgCTTACgCATTTTgCCAgAgCACCgAggA; Primer 5 (5'-3') FITC-gCTTgCgCAggTgggAg Dig = Digoxigenin