TH20107U - A one-step colorimetric method for detecting APP gene mutations and Alzheimer's risk. - Google Patents

Abstract

------January 22, 2021------(OCR) A one-step method for detecting APP gene mutations for Alzheimer's risk using color bands. The process begins with designing five primers specific to the RS (rs) 63750264 mutation sequence of the APP gene associated with Alzheimer's risk. 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, DNA is amplified at 61°C for 60 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 the RS (rs) 63750264 mutation in the sample. The method for detecting APP gene mutations associated with Alzheimer's risk using a single-step color banding technique begins with the design of five primers specific to the mutated base sequence position RS (rs) 63750264 of the APP gene associated with Alzheimer's risk. One primer is labeled with either biotin or digoxiganin. (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°C for 60 minutes in a heating block, and the result is read on the dipstick. A positive result shows a pink test line in the test (T) and control (C) bands, indicating the presence of a mutated base at position RS 63750264 of the APP gene, which is associated with Alzheimer's risk. If the result is negative, a pink test line will only appear in the control (C) band. This method is equivalent to real-time PCR testing without the need for a PCR machine and electrogenetic separator to monitor the reaction results.

Claims (1)

------January 22, 2021------(OCR) 1. A one-step staining method for detecting APP gene mutations for Alzheimer's risk, consisting of a LAMP reaction combined with the application of a dipstick. The LAMP reaction solution (25 μL) contained 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 BstDNA polymerase enzyme, and 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 63750264 in the APP 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 APP gene abnormalities in individuals suspected of having Alzheimer's disease. This procedure designs primers for the LAMP (Landing, Amplification, and Applications) technique used to detect the rs 63750264 mutation in the APP gene, which is associated with Alzheimer's risk. The LAMP reaction consists of five primers specific to the rs 63750264 mutation sequence of the APP gene. Primer 4 is labeled with either biotin or digoxigenin. (Digoxigenin) and use fluorescently labeled primer 5 (FITC) to track the reaction on a dipstick as follows: Primers specific to the rs (63750264) mutant base: Primer 1, base sequence (5'-3'): ACggAggAgATCTCTgAAg; Primer 2, base sequence (5'-3'): gAATggATgTgTACTgTTTCT; Primer 3, base sequence (5'-3'): AgATgTgggTTCAAACAAAggTTTTTAgCATCACCAAggTgATgCT; Primer 4, base sequence (5'-3'): Biotin/Digoxigenin. (Digoxigenin; Dig) -TgCAAAgAACACCAATTTTTgATgATTTTT gAAgAT ggAT gCAgAATT cc Primer 5 base sequence (5'-3') FITC- T g AACTTCATAT CCT g AgT CAT gTc ------------ Page 1 of 1 page. Claims 1. A one-step method for detecting APP (Applied Polymorphopropyl Dialysis) mutations in Alzheimer's risk using a colorimetric banding system consisting of a LAMP reaction combined with the application of a dipstick. The LAMP reaction used 25 μL of primers 3 and 4 (5 picomoles each), primers 1, 2, and 5 (5 picomoles each), and 0.8 millimolar dNTP mixed with 0.6 molar betaine. A solution of 4 millimolar magnesium sulfate (MgSO4), 8 U of BST DNA polymerase enzyme, 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 results were 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 63750264 of the APP gene, which carries an 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 involves dipstick testing to detect abnormalities in the APP gene in individuals suspected of having Alzheimer's disease for diagnosis and prevention. The process uses primers designed for the LAMP (Landing-Assisted Amplification) technique to detect the RS 63750264 mutation in the APP gene associated with Alzheimer's risk. The LAMP reaction consists of five primers specific to the RS 63750264 mutation sequence of the APP gene. Primer 4 is labeled with either biotin or digoxigenin. (Digoxigenin) and use primer 5 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 (63750264) as follows: Primer 1, base sequence (5'-3'): ACggAggAgATCTCTgAAg; Primer 2, base sequence (5'-3'): gAATgg ATgTgTACTgTTTCT; Primer 3, base sequence (5'-3'): AgATgTgggTTCAAACAAAggTTTTTAgCATCACCAAggTgATgCT; Primer 4, base sequence (5'-3'): biotin/Dig- TgCAAAgAACACCAATTTTTgATgATTTTTgAAgATggATgCAgAATTCC; Primer 5, base sequence (5'-3'): FITC- TgAACTTCATATCCTgAgTCATgTC Dig = Digoxigenin