Feb 18, 2025
Development of LAMP-colorimetric assay for detecting hemoglobin Constant Spring and hemoglobin Pakse'
- Wittaya Jomoui1
- 1Srinakharinwirot university
Protocol Citation: Wittaya Jomoui 2025. Development of LAMP-colorimetric assay for detecting hemoglobin Constant Spring and hemoglobin Pakse' . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr9ykpvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2025
Last Modified: February 18, 2025
Protocol Integer ID: 120610
Keywords: developed lamp colorimetric phenol red assay, colorimetric assay, detecting hemoglobin constant spring, hemoglobin constant spring, novel molecular detection method, colorimetric phenol, red assay, colorimetric lamp with phenol, colorimetric lamp, using primer explorer v5 software, lamp primer set, primer explorer v5 software, specific with hb c, probe in this study, hb ps for the first time
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Abstract
A novel molecular detection method based on a colorimetric LAMP with phenol red assay was developed to detect Hb CS and Hb PS for the first time. All LAMP primer sets and probe in this study were designed using Primer Explorer V5 software (http://primerexplorer.jp /lampv5e/index.html). The common primer set including the outer primers (F3 and B3) and the inner primers (FIP and BIP) was shown in the Table. Furthermore, two loop probe (LP) used in this study were designed for specific with Hb CS (LP-CS) and Hb PS (LP-PS) as also shown in Table . These primers set and probes were protected according to petty patent submission number 2203001286 (Thailand). The developed LAMP colorimetric phenol red assay was performed with reaction mixture and temperature as detailed. The LAMP mixture was incubated at an isothermal temperature of 65 °C for 35 minutes. The positive result was observed as yellow color by the naked eye compared with negative with pink color.
Troubleshooting
LAMP-colorimetric assay for detecting hemoglobin Constant Spring
All LAMP primer sets and probe in this study were designed using Primer Explorer V5
software (http://primerexplorer.jp /lampv5e/index.html).
Primers for Hb Constant spring
| A | B | |
| Primers & Probes | Primer’s sequence (5’> 3’) | |
| F3 | TCCCTGGACAAGTTC | |
| B3 | GTTGGCACATTCCGGGATA | |
| FIP | CGGGCAGGAGGAACGGCTACGAGCACCGTGCTGACCT | |
| BIP | CCTTGCACCGGCCCTTCCTGAGAACCCAGGCACACAC | |
| LP-CS | CAGCTTGACGGT |
Primers for Hb Pakse'
| A | B | |
| Primers & Probes | Primers's sequence (5'>3') | |
| F3 | TCCCTGGACAAGTTC | |
| B3 | GTTGGCACATTCCGGGATA | |
| FIP | CGGGCAGGAGGAACGGCTACGAGCACCGTGCTGACCT | |
| BIP | CCTTGCACCGGCCCTTCCTGAGAACCCAGGCACACAC | |
| LP-PS | CCAGCATAACGGTA |
Reaction mixture: Hb Constant Spring or Hb Pakse'
A total of 12.5 μL contained 1.5 μL of (20-50) ng/μL genomic DNA, 6.25 μL of
WarmStart‱ Colorimetric LAMP, 1.0 μL of 5M Betaine, 0.2 μL of 10 μM each primer
of F3 and B3, 0.4 μL of 40 μM each primer of FIP and BIP, 1.5 μL of 10 μM of LP-CS,
and the remainder distilled water.
Isothermal temperature: 65 0C, 35 minutes
The positive result was observed as yellow color by the naked eye compared with negative with
pink color.