A primer set and probe for detecting the hemoglobin constant spring (Hb Constant Spring) gene using loop-mediated isothermal amplification (LAMP) with phenol red color change.

Abstract

------08 / 09 / 2568------(OCR) OCR 10KL primers (primers set) and probes specifically labeled for the detection of the hemoglobin constant spring (Hb Constant Spring) gene using loop-mediated isothermal amplification (LAMP) with phenol red color change. The reaction is performed at a single temperature throughout. Testing showed that this method offers a reliability level of 2.0 in terms of detectable DNA.The test uses 5 nanograms per reaction and is specific only to the hemoglobin constant-spring gene, according to the test kit. Furthermore, the test procedure is simple and rapid, taking only 45 minutes, and does not require expensive equipment or complex principles. It can be performed in general laboratories or used for field testing. The resulting LAMP product can be easily identified by visually observing the color change of the phenol red indicator. Given all these advantages, LAMP testing for hemoglobin constant-spring gene is highly beneficial in controlling and preventing thalassemia, enabling rapid treatment planning and genetic counseling for doctors and nurses, and can also be used to determine the prevalence of the hemoglobin constant-spring gene, providing baseline genetic information for the Thai population.

Claims

------08 / 09 / 2568------(OCR) OCR 10KL1. A primer set and probe for detecting the hemoglobin constant spring (Hb Constant Spring) gene using the phenol red LAMP reaction, consisting of 4 primers and 1 probe as follows: 1.1 Forward outer primer (F3(CS-PS))5'-TCCCTGGACAAGTTC -3' 1.2 Backward outer primer (B3 / CS-PS))5'-GTTGGCACATTCCGGGATA -3' 1.3 Forward inner primer (FIP(CS-PS))5'-CGGGCAGGAGGAACGGCTACGAGCACCGTGCTGACCT -3' 1.4 Backward inner primer (FIP(CS-PS))5'-CGGGCAGGAGGAACGGCTACGAGCACCGTGCTGACCT -3' BIP(CS-PS))5'-CCTTGCACCGGGCCCTTCCTGAGAACCCAGGCACACAC -3'1.5 Hemoglobin constant-spring probe (probe; LP-CS)5'- CAGCTTGACGGT -3'2. A procedure for detecting the hemoglobin constant-spring gene using a phenol red LAMP reaction with the primer and probe set as per Patent 1, which involves the following steps: a.The reaction was performed under loop-mediated isothermal amplification (LAMP) conditions at 65°C for 45 minutes. The LAMP product was visually inspected by observing the color change of the phenol red indicator in the reaction tube from purple to yellow. A positive result indicates the presence of yellow in the reaction tube, while a negative result indicates the presence of purple.

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