ORIGINAL RESEARCH article
Front. Genet.
Sec. Genetics of Common and Rare Diseases
Volume 16 - 2025 | doi: 10.3389/fgene.2025.1564565
Development and Validation of mPCR-CEFA for Detecting Multiple Deletion and Non-Deletion Thalassemia Genotypes
Provisionally accepted
- 1School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, Guangdong Province, China
- 2Yaneng BIOscience (Shenzhen) Co., Ltd, Guangdong, China
- 3Photonics Research Centre, Shenzhen University, shenzhen, China
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Background: Thalassemia is a common hereditary blood disorder caused by genetic variants in globin genes, leading to abnormal hemoglobin production. Rapid and accurate genotyping is essential for molecular screening and prenatal genetic diagnosis to prevent the birth of individuals with severe forms of the disease.Methods: We developed a multiplex PCR-capillary electrophoresis fragment analysis (mPCR-CEFA) method to detect 16 α-thalassemia and 24 β-thalassemia genotypes simultaneously. Genomic DNA extracted from clinical blood samples underwent a two-tube multiplex PCR amplification. The amplification products were analyzed using capillary electrophoresis to detect mutation peaks in different fluorescent channels and to calculate α1/α2 and Y1/Y2 ratios for genotype determination. The performance of mPCR-CEFA was validated against conventional methods, including Gap-PCR and PCR-RDB.Results: The α1/α2 and Y1/Y2 peak ratios exhibited stable and reproducible values, allowing for precise genotyping of thalassemia events involving homologous recombination, such as -α3.7, -α4.2, αααanti3.7, αααanti4.2 and HKαα. Mutation peaks in different fluorescent channels also facilitated the differentiation of various genotypes, including deletion and non-deletion types. The method demonstrated a high accuracy rate of 99.5%. It successfully detected complex compound genotypes like αCD 74α/-α4.2, βCD 17/βN and αWSα/--SEA, βCD 37/βN (or αWSα/--SEA, βCD 37/βCD 37), which were challenging for traditional approaches. Conclusion: The mPCR-CEFA method is a reliable, efficient, and scalable tool for genetic diagnosis of thalassemia. Its ability to detect multiple genotypes simultaneously and resolve complex cases makes it particularly valuable for large-scale screening and clinical applications. This approach holds significant potential for improving thalassemia prevention strategies and supporting public health efforts in high-prevalence regions.
Keywords: thalassemia1, Multiplex PCR2, capillary electrophoresis3, genotyping4, genetic screening5, prenatal diagnosis6
Received: 21 Jan 2025; Accepted: 27 Jun 2025.
Copyright: © 2025 Xu, Ren, Fang, Lin, Xing and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Jingping Xu, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, Guangdong Province, China
Jingting Lin, Photonics Research Centre, Shenzhen University, shenzhen, China
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