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. 2020 Apr 5:13:303-311.
doi: 10.2147/RMHP.S241399. eCollection 2020.

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes

Wei-Hua Wang #  1 , Min Lin #  2 , Hai-Liang Li  3 , Jun-Yun Huang  1 , Jiang-Tao Chen  4   5 , Xian-Song Fang  6 , Dong-Mei Huang  1 , Xu-Xiang Xi  1 , Qing-Fei Zhao  1 , Fang-Li Song  7 , Shao Huang  7 , Tian-Yu Zhong  1
Affiliations

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes

Wei-Hua Wang et al. Risk Manag Healthc Policy. .

Erratum in

Abstract

Purpose: Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three α-thalassemia genes including -Southeast Asian (SEA), -α3.7, and -α4.2 and five β-thalassemia genes including 654M, 41/42M, -28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP).

Methods: Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light.

Results: We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20-60 pg/μL and 20-60 ng/μL. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation.

Conclusion: The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resource-limited areas, especially those with primary hospitals and rural areas.

Keywords: diagnosis; fast; loop-mediated isothermal amplification; low cost; thalassemia genes.

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Conflict of interest statement

Fang-li Song and Shao Huang were employed by company Jiangxi Shiningmed Medical Technology Ltd. All other authors declare no competing interests in this work.

Figures

Figure 1
Figure 1
LAMP assays for the positive and negative samples of thalassemia. (A–H) PCR reactions were performed using positive samples and negative samples of thalassemia as templates under these same real-time LAMP reaction conditions. Abbreviations: SEA, Southeast Asian; LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction.
Figure 2
Figure 2
The LAMP reaction tube with a reaction time of 60 minutes was exposed to ultraviolet light for observing color. The positive sample tube showed strong green fluorescence while the negative tube showed light green fluorescence. (A) –SEA positive sample and negative sample; (B) 654M positive sample and negative sample; (C) 27/28 positive sample and negative sample. Abbreviations: SEA, Southeast Asian; LAMP, loop-mediated isothermal amplification.
See this image and copyright information in PMC

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