SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.
SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.
复制标题
SMA 诊断：使用新鲜血液 DNA 通过实时 mCOP-PCR 系统检测 SMN1 缺失。
DOI：
--
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
M. Shinohara
中科院分区：
文献类型：
--
作者：
Emma Tabe Eko Niba;Mawaddah Ar Rochmah;N. Harahap;H. Awano;I. Morioka;K. Iijima;Toshio Saito;Kayoko Saito;A. Takeuchi;P. Lai;Y. Bouike;H. Nishio;M. Shinohara
背景
脊髓性肌萎缩症（SMA）是最常见的常染色体隐性遗传病之一。这些症状是由脊髓中的下运动神经元缺陷引起的。超过95%的SMA患者为运动神经元存活基因1（SMN 1）缺失纯合子。我们以前开发了一个筛选系统SMN 1缺失的基础上，改进的竞争性寡核苷酸引物PCR（mCOP-PCR）技术，使用滤纸上的干血斑（DBS）。该系统便于大规模人群筛查和/或偏远地区患者的一级诊断方法。然而，该系统仍然是耗时和费力的，因为它需要预扩增程序，以避免非特异性扩增和凝胶电泳检测SMN 1缺失的存在或不存在。用新鲜血标本代替DBS或用实时荧光PCR代替凝胶电泳，可能是一种更简便、快速的SMA诊断方法。
目的
目的建立一种简便、快速的SMN 1基因缺失诊断方法。
方法
从新鲜血液中提取DNA样品，并在4 ℃下储存1个月。使用实时mCOP-PCR系统测定样品，无需预扩增程序。已通过PCR-限制性片段长度多态性（PCR-RFLP）对DNA样本进行基因分型，显示是否存在SMN 1外显子7。DNA样品直接进行mCOP-PCR步骤。在实时PCR装置中监测mCOP-PCR的扩增。
结果
用新鲜血液DNA建立的实时荧光定量PCR系统的基因分型结果与PCR-RFLP的结果完全一致。在这种使用新鲜血液DNA的实时mCOP-PCR系统中，从DNA提取到检测SMN 1缺失的存在与否仅需4小时，而PCR-RFLP则需要12小时以上。
结论
本研究建立的实时荧光定量mCOP-PCR方法快速、准确，可作为SMA的一级诊断方法。
BACKGROUND
Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA.
AIM
To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA.
METHODS
DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus.
RESULTS
The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP.
CONCLUSION
Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.
影响因子：
34.7
作者：
Burghes, Arthur H. M.;Beattie, Christine E.
通讯作者：
Beattie, Christine E.
影响因子：
--
作者：
Kolb, Stephen J.;Kissel, John T.
通讯作者：
Kissel, John T.