基于LAMP技术的地中海贫血快速基因诊断方法的建立和应用研究

Thalassemia is a serious inherited hematologic disease, rapid and accurate diagnosis of which is the key for its prevention and control. The current diagnosis methods have obvious disadvantages. Hematological phenotype screening method has a high misdiagnosis rate while gene diagnostic method is not only expensive but also requires strict technology platform capabilities; none of them is suitable for application in fundamental hospitals and rural areas. Therefore, we aim to develop a simple and rapid gene diagnosis method of thalassemia with high sensitivity and low cost. According to the distribution of thalassemia genotype in southern Jiangxi, we choose 3 α- thalassemia (- SEA,-α3.7,-α4.2) and 5 β-thalassemia (654M, 41/42M -28M, 17M, 27/28M) subtypes as detected objects and establish a gene diagnosis method of thalassemia based on LAMP technology. The clinical value of the method would be judged by comparing the results of LAMP method with other currently used clinical methods (erythrocyte osmotic fragility, hemoglobin electrophoresis analysis, Gap-PCR, PCR -RDB). If the research objective is achieved, this method would significantly reduce the misdiagnosis of thalassemia, and would replace the hematological phenotype analysis for thalassemia screening in fundamental hospitals and rural areas, promising a good future for application.

地中海贫血（地贫）是一组严重威胁人类健康的遗传性血液病。快速、准确的进行地贫检测是其防控的关键。目前血液学表型筛查方法漏诊、误诊率高；基因诊断方法成本昂贵，技术平台要求高，难以在基层医院和农村推广。因此，我们拟建立敏感性强、成本低、操作简单的地贫快速基因诊断方法。根据赣南地区地贫的基因型分布特点，以3种缺失型α-地贫（--SEA、-α3.7、-α4.2）和5种β-地贫（654M、41/42M、-28M、17M、27/28M）为研究对象，建立基于LAMP技术的地贫快速基因诊断方法。通过与目前临床上使用的地贫检测方法（红细胞渗透脆性一管法、血分析、血红蛋白电泳分析、Gap-PCR、PCR-RDB）比较，评判其临床应用价值。如能取得预期效果，本研究将大幅度减少地贫的漏诊误诊，并有可能在基层医院和广大农村替代血液学表型分析用于地贫筛查，具有广阔的应用前景。

地中海贫血（地贫）是一组严重威胁人类健康的遗传性血液病。快速、准确的进行地贫检测是其防控的关键。目前血液学表型筛查方法漏诊、误诊率高；基因诊断方法成本昂贵，技术平台要求高，难以在基层医院和农村推广。本研究基于Chelex-100树脂核酸提取方法、LAMP扩增技术和钙黄绿素荧光法建立了一套用于点突变和片段缺失的DNA检测技术，并进一步利用不同荧光标记的分子信标探针结合LAMP技术建立了一套可同时识别多个待检测片段的LAMP检测技术，增强了LAMP技术的应用范围。在上述技术平台上，根据赣南地区地贫的基因型分布特点，本研究开发了一套用于检测包括3 种缺失型α-地贫（--SEA、-α3.7、-α4.2）、3种突变型α-地贫（HbCS、HbQS、HbWS）及5 种突变型β-地贫（654M、41/42M、-28M、17M、27/28M）的检测试剂。收集了400例临床地贫阳性患者的血液标本进行了对照试验，与临床常用的Gap-PCR、反向膜杂交技术进行临床对比，LAMP准确度达到90%以上，并且具有简单、快速、方便、廉价的特点。本研究进一步利用上述方法，开发了检测镰状细胞贫血点突变相关检测试剂，正在进行临床验证。