Diagnostic accuracy of MMCA-PCR in detection of β-thalassemia gene mutation; retrospective sampling validation

Abstract

               Beta-thalassemia is one of the major causes of hereditary anemia with a high prevalence in Thailand, where carrier rates range from 3–9% of the population. The coexistence of hemoglobin E contributes to severe conditions such as homozygous β-thalassemia and β-thalassemia/Hb E. Accurate and rapid detection of β-globin gene mutations is essential for carrier screening and prenatal diagnosis. Conventional methods, including allele-specific PCR (AS-PCR) and reverse dot-blot hybridization (RDB), provide high accuracy but involve complex procedures and carry a risk of contamination.

               This retrospective triple-blind diagnostic accuracy study evaluated multicolor melting curve analysis polymerase chain reaction (MMCA-PCR) for β-thalassemia mutation detection compared with standard methods. A total of 146 residual DNA samples collected from pregnant women and their husbands in Health Region 3 during 2020–2025 were included, consisting of 73 positive and 73 negative samples. The mutations identified covered common β-thalassemia variants, including CD41/42 (-TTCT) in 12 samples, CD17 (A>T) in 5 samples, IVS-I-5 (G>C) in 7 samples, IVS-II-654 (C>T) in 6 samples, CD71/72 (+A) in 10 samples, and CD26 (Hb E) in 10 samples, as well as less common mutations ได้แก่ IVS-I-1 (G>T), -28 (A>G), -31 (A>C), CD43 (GAG>TAG), CD30 (AGG>GGG), initiation codon mutation, and CD14-15 (+G).

All samples were anonymized and independently analyzed using MMCA-PCR and reference methods. Testing was performed on the SLAN-96S real-time PCR platform using the Yaneng® β-thalassemia Genotyping Kit. Diagnostic performance was evaluated using 2×2 contingency table analysis.

               The results demonstrated complete concordance between MMCA-PCR and standard methods, yielding 100% sensitivity, specificity, and overall accuracy without false-positive or false-negative results. In addition, the closed-tube system reduced contamination risk and shortened turnaround time.

               In conclusion, MMCA-PCR showed excellent diagnostic performance comparable to standard techniques and may serve as an effective tool for β-thalassemia screening and prenatal diagnosis in Thailand.