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Original research
One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve
  1. Jiachun Qin1,
  2. Jun He2,
  3. Yang Li1,
  4. Nansong Liu3,
  5. Fangchao Tao1,
  6. Pengyi Zhang4,
  7. Weilin Guo3,
  8. Qiongzhen Qin1,
  9. Wanjun Zhou1,5
  1. 1Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China
  2. 2Department of Genetics, Changsha Hospital for Maternal and Child Health Care, Changsha, China
  3. 3Research and Development Center, Yaneng BIOscience (Shenzhen) Co Ltd, Shenzhen, Guangdong, China
  4. 4Neonatal Screening Center, Maternal and Child Health Hospital, Fushan, China
  5. 5Department of Laboratory Medicine, Southern Medical University Nanfang Hospital, Guangzhou, Guangdong, China
  1. Correspondence to Dr Wanjun Zhou, Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China; zhouwanjun72{at}163.com

Abstract

Aims Alpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in α-globin gene cluster, deletional and non-deletional mutations were usually separately detected with different methods. The aim of this study was to develop a novel one-step method for α-thalassaemia genotyping.

Methods A multiplex symmetric PCR melting curve strategy was designed for one-step α-thalassaemia genotyping. Based on this strategy, a novel method was developed to simultaneously detect four common deletional (-α3.7, -α4.2, _ _SEA, --THAI) and five common non-deletional (αCD30(-GAG)α, αCD31(G>A)α, αWSα, αQSα, αCSα) α-thalassaemia mutations in a closed-tube reaction. This method was also evaluated by double-blind detection of 235 genotype-known samples and 1630 clinical samples.

Results All nine α-thalassaemia mutations could be accurately identified by this novel method within 3 hours. The evaluation results also showed a 100% concordance with comparison methods.

Conclusions This method is rapid, accurate, low-cost and easy to operate, which can be used for molecular screening and genetic diagnosis of α-thalassaemia in clinical practice. The multiplex symmetric PCR melting curve strategy designed in this study can also provide an effective approach to the method development for high GC content templates and multiple mutations.

  • Thalassemia
  • Methods
  • GENETICS

Data availability statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

https://doi.org/10.1136/jclinpath-2022-208363

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    Data availability statement

    Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

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    Footnotes

    • Handling editor Runjan Chetty.

    • JQ and JH contributed equally.

    • Contributors WZ is the guarantor of this study. WZ and JH supervised this study and revised the manuscript. JQ conducted the experiments and wrote the original draft. NL and QQ performed statistical analyses of the results. YL and FT drafted the manuscript. PZ and WG collected samples. All authors approved the manuscript.

    • Funding This study was supported by the National Natural Science Foundation of China (81972008), Basic and Applied Basic ResearchFoundation of Guangdong Province (2021A1515012585).

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.