Wang Wei-Hua, Lin Min, Li Hai-Liang, Huang Jun-Yun, Chen Jiang-Tao, Fang Xian-Song, Huang Dong-Mei, Xi Xu-Xiang, Zhao Qing-Fei, Song Fang-Li, Huang Shao, Zhong Tian-Yu
Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi Province, People's Republic of China.
School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, Guangdong Province, People's Republic of China.
Risk Manag Healthc Policy. 2020 Apr 5;13:303-311. doi: 10.2147/RMHP.S241399. eCollection 2020.
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).
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.
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.
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.
目前，地中海贫血通常采用缺口聚合酶链反应（PCR）和脱氧核糖核酸（DNA）反向斑点杂交进行检测，这对空间、仪器和人员有较高要求。因此，有必要开发一种灵敏度高、成本低、操作简单快速的地中海贫血检测新方法。在本研究中，我们旨在设计并评估一种基于环介导等温扩增（LAMP）检测三种α地中海贫血基因（包括东南亚型（SEA）、-α3.7和-α4.2）和五种β地中海贫血基因（包括654M、41/42M、-28M、17M和27/28M）的新方法。
使用Primer Explorer V4软件设计引物序列。从所有参与者采集5 mL乙二胺四乙酸（EDTA）抗凝血样。使用Chelex 100提取DNA并进行LAMP反应。通过在紫外线下显色检测LAMP产物。
我们发现，地中海贫血阳性样本的LAMP检测在60分钟前达到平台期，而阴性对照样本在70分钟后进入平台期或无扩增。阳性反应的浓度范围为20 - 60 pg/μL和20 - 60 ng/μL。此外，8种地贫亚型之间无交叉反应。对于临床样本，阳性样本管显示强烈的绿色荧光，而阴性管显示浅绿色荧光。根据这些结果，LAMP方法检测地中海贫血具有高灵敏度（252/254）。然而，LAMP检测获得了43例假阳性结果。LAMP检测成本也较低，操作简单快速。
新型LAMP检测使用加热块或水浴可在60分钟内完成，结果可根据颜色变化直观读取以检测地中海贫血。LAMP检测满足现场应用和资源有限地区的要求，尤其是基层医院和农村地区。
