การพัฒนาการตรวจฮีโมโกลบินคอนสแตนท์สปริงและฮีโมโกลบินปากเซ ด้วยเทคนิคมัลติเพล็กซ์เรียลไทม์พีซีอาร์

Worrawalan Lerttham; Nopporn Sawatjui; Prajuab Chaimanee; Patcharee Komvilaisak; Pimjai Ananta; Phatsarawadee Hemwaranon

doi:10.64960/srimedj.v41i3.269930

Authors

Worrawalan Lerttham Clinical Laboratory Section, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002
Nopporn Sawatjui Clinical Laboratory Section, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002
Prajuab Chaimanee Clinical Laboratory Section, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002
Patcharee Komvilaisak Department of Pediatrics, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002
Pimjai Ananta Clinical Laboratory Section, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002
Phatsarawadee Hemwaranon Clinical Laboratory Section, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Thailand 40002

DOI:

https://doi.org/10.64960/srimedj.v41i3.269930

Keywords:

Hemoglobin Constant Spring, Hemoglobin Pakse, hybridization, multiplex real-time PCR

Abstract

Background and objectives: Hemoglobin Constant Spring (Hb CS) and Hemoglobin Pakse (Hb PS) are gene mutations that cause alpha-thalassemia, which are difficult to diagnose in carriers of the disease. Therefore, this study aimed to develop a multiplex real-time PCR method for the detection of both mutations and to evaluate the cost-effectiveness per test kit and the time efficiency of the analytical procedure.

Methods: Three hydrolysis probe-specific primer-probe sets labeled with fluorescent dyes were designed and applied to 67 DNA samples extracted from blood specimens obtained from Srinagarind Hospital. The analysis was performed on a BioRad® CFX96 Touch™ instrument, and results were interpreted using real-time PCR amplification curves in conjunction with allelic discrimination plots. Concordance with the hybridization method was assessed using Cohen's Kappa coefficient.

Results: The multiplex real-time PCR assay developed in this study demonstrated perfect concordance with the hybridization method, yielding a Cohen's Kappa coefficient of 1.0. The assay provided easy result interpretation with no non-specific binding. It reduced the cost by 70.9% and the turnaround time by 73.1% compared to the routine method. Furthermore, Hb CS or Hb PS mutations were detected in samples that had previously been tested negative by hemoglobin typing analysis.

Conclusions: The developed multiplex real-time PCR is suitable for the detection of Hb CS and Hb PS carriers, offering significant cost and time efficiency. Further validation in a larger sample cohort would enhance confidence in implementing this method in a clinical laboratory setting.

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