Rapid and Visual Molecular Detection of High Hb F Determinants; HPFH6, Asian Indian inv-del (Aγδβ)0-Thalassemia, and Thai del-inv-ins (Aγδβ)0-Thalassemia Using LAMP Colorimetric Phenol Red Assays.

Abstract

Hemoglobin (Hb) F, or fetal hemoglobin, is the predominant Hb in fetuses and is converted to adult hemoglobin (Hb A) at the age of 2 years. However, high Hb F levels in adults are typically present in conditions such as β-thalassemia disease and high Hb F determinants including large deletional β-globin gene clusters, and hereditary persistence of fetal hemoglobin (HPFH). The accurate detection of these conditions is crucial for effective disease management and genetic counseling. Several molecular techniques have been used to identify high Hb F determinants but require advanced instrumentation, highly skilled personnel, high cost, long time duration, and post-PCR processing. This study aimed to develop a rapid and cost-effective molecular assay for detecting common high Hb F determinants using colorimetric loop-mediated isothermal amplification (LAMP) with phenol red assays. We focused on the detection of HPFH6, Asian Indian inv-del (^Aγδβ)⁰-thalassemia, and Thai del-inv-ins (^Aγδβ)⁰-thalassemia. A total of 331 DNA samples encompassing 21 genotypes were screened using the developed LAMP assays, which were optimized to detect these determinants within 60-70 min. The assays showed high sensitivity (100%) and specificity (99.6-100%) in each mutation with detection limits of 2.5 ng/reaction. Validation by comparison with conventional methods confirmed the efficacy of the LAMP assays, which is simple, inexpensive, and suitable for use in low-resource settings. Rapid performance, visual detection, and accurate diagnosis may be useful for genetic counseling, particularly in Thailand and Southeast Asia. This innovation is suitable for application in thalassemia screening programs, especially in remote areas.