Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA) signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 μL PCR reaction includes primer(s) (0.3–0.5 μM each), dNTP mixture (160 μM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 μL), DNA template (250 ng/50 μL), BSA (0.4 μg/μL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.

中文翻译：

---

通过优化的多重 PCR 测定法同时检测肌营养不良蛋白基因的 13 个外显子以筛选杜兴氏/贝克尔肌营养不良症

聚合酶链反应 (PCR) 技术和脱氧核糖核酸 (DNA) 信号和靶标扩增等其他技术的进步已成为分子诊断的关键程序。PCR 能够在体外合成核酸，通过它可以以半保守的方式特异性地复制 DNA 片段，从而进行缺失和突变分析。多重 PCR (M-PCR) 优于标准和长 PCR，因为它可以使用相应的引物组扩增多个位点。与此一致，本研究旨在优化 M-PCR，然后优化其化学和条件，以筛选杜氏肌营养不良症 (DMD) [OMIM #310200] 和贝克尔肌营养不良症 (BMD) [OMIM #300376]。肌营养不良症 (MDs) 是一组广泛的遗传性、进行性和退行性肌肉疾病。X 连锁隐性 D/BMD 是由编码抗肌萎缩蛋白 [UniProt#P11532] 的抗肌萎缩蛋白基因 [OMIM #300377] 突变引起的。由于抗肌萎缩蛋白是具有 79 个外显子的人类元基因，因此突变分析非常具有挑战性。Chamberlain 集合 (10 plex)、Beggs 集合 (9 Plex) 和 Kunkel 集合 (7 Plex) 多年来一直用于诊断这种情况。然而，在这项研究中，Beggs 集定制了 13 个外显子，以在单个反应中筛选 DMD 基因突变。M-PCR 的优化设计有许多物理化学参数。根据文献，经过多次评估，本研究证明了各种化学成分的最充分浓度和最佳循环条件，以优化改良的 Beggs 组 (13 Plex)。50 μL PCR 反应包括引物（每个 0.3–0.5 μM）、dNTP 混合物（每个 160 μM）、Dream Taq 缓冲液 (1X)、Taq DNA 聚合酶 (6U/50 μL)、DNA 模板 (250 ng/50 μL)、BSA (0.4 μg/μL) 和 MgCl2 (1.4 mM)。为获得最有效的结果，循环条件为 94°C 初始变性 10 分钟、62°C 退火温度和 72°C 延伸温度下 35 个 PCR 循环。因此，该研究成功地为 >3000 bp 制定了一种成本较低且简单的方法，用于筛选 D/BMD。最后，开发的 M-PCR 组合具有特异性和灵敏度的独特组合以及极大的灵活性，导致了分子诊断学的真正革命。在 72°C 延伸温度下进行 35 个 PCR 循环。因此，该研究成功地为 >3000 bp 制定了一种成本较低且简单的方法，用于筛选 D/BMD。最后，开发的 M-PCR 组合具有特异性和灵敏度的独特组合以及极大的灵活性，导致了分子诊断学的真正革命。在 72°C 延伸温度下进行 35 个 PCR 循环。因此，该研究成功地为 >3000 bp 制定了一种成本较低且简单的方法，用于筛选 D/BMD。最后，开发的 M-PCR 组合具有特异性和灵敏度的独特组合以及极大的灵活性，导致了分子诊断学的真正革命。