用于简单快速检测α mRNA的逆转录环介导等温扩增技术的开发
Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of α mRNA.
作者信息
Hashimoto Yuki, Hatayama Yuki, Kojima Nao, Morishita Shota, Matsumoto Satoko, Hosoda Yuzuru, Hara Ayako, Motokura Toru
机构信息
Division of Clinical Laboratory, Tottori University Hospital, Yonago 683-8504, Japan.
†Department of Hematology, Tottori University Hospital, Yonago 683-8504, Japan; ‡Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan.
出版信息
Yonago Acta Med. 2016 Dec 26;59(4):262-269. eCollection 2016 Dec.
BACKGROUND
Acute promyelocytic leukemia (APL) is a disease characterized by expression of α (α) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP).
METHODS
An RT-LAMP primer set was designed to detect three types of α mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 α sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays.
RESULTS
Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods.
CONCLUSION
We developed an RT-LAMP assay for simple and rapid α mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis.
背景
急性早幼粒细胞白血病（APL）是一种以α（α）嵌合mRNA表达为特征的疾病。尽管APL是可治愈的，但出血导致的早期死亡是一个主要问题。在此，我们报告了一种基于逆转录环介导等温扩增（RT-LAMP）的简单快速的APL诊断方法的开发。
方法
设计了一套RT-LAMP引物，用于在单一反应中检测三种类型的α mRNA。使用含有bcr1、bcr2或bcr3 α序列的质粒DNA系列稀释液以及从6例APL患者骨髓穿刺物中提取的RNA，比较RT-LAMP和巢式PCR检测结果。
结果
质粒DNA通过RT-LAMP进行了扩增，其反应时间比巢式RT-PCR短>4小时，检测下限更高。7个样本中有6个通过两种方法检测均为阳性。
结论
我们开发了一种用于简单快速检测α mRNA的RT-LAMP检测方法，这可能在临床即时检测和APL诊断中具有实用价值。
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