BACKGROUND We have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called "Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)", generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. METHODS Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays. RESULTS Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at - 20 °C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. CONCLUSIONS We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

中文翻译：

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一种用于检测膀胱癌患者尿液中FGFR3突变的新型超灵敏方法-Urodiag®PCR试剂盒的设计，用于监测非肌肉浸润性膀胱癌（NMIBC）患者。

背景技术我们最近开发了一种名为Urodiag®的高度精确的基于尿液的测试，该测试将FGFR3突变和DNA甲基化分析相关联，用于低，中和高风险NMIBC患者的复发监测。以前，检测四个FGFR3突变（G372C，R248C，S249C和Y375C）需要扩增步骤，并通过毛细管电泳（等位基因特异性PCR，AS-PCR）分析PCR产物，这既昂贵又费时。在这里，我们提出了一种新型的超灵敏多重PCR检测方法，称为“突变等位基因特异性寡核苷酸PCR（MASO-PCR）”，可产生一种经济有效，简单，快速且可用于临床的检测FGFR3突变的检测方法排尿。方法对263份尿液DNA样本（87种FGFR3突变和176种FGFR3野生型）进行MASO-PCR和AS-PCR技术的比较临床表现。在开发Urodiag®PCR Kit时，我们研究了用于MASO-PCR和QM-MSPCR（定量甲基化特异性）的每种多合一PCR主混合物（包括所有必需的PCR组分的单一反应混合物）的稳定性和可重复性。 PCR以共同扩增SEPTIN9，HS3ST2和SLIT2甲基化基因）测定。结果MASO-PCR和AS-PCR结果完全一致（100％）。每种PCR预混液在-20°C下储存12个月后均显示出出色的重现性和稳定性，测定内标准偏差低于0.3 Ct，变异系数（CV）低于1％。在95％的野生型背景中，MASO-PCR的检出限（LoD）为5％突变检测。QM-MSPCR的定量限（LoQ）为10 pg亚硫酸氢盐转化的DNA。结论我们开发并临床验证了MASO-PCR测定法，产生了成本有效，简单，快速且临床上可用于尿液中FGFR3突变检测的测定法。我们还设计了Urodiag®PCR试剂盒，其中包括MASO-PCR和QM-MSPCR分析。该套件适用于常规临床实验室（简单，准确），将为泌尿科医师对低，中和高风险NMIBC的患者进行复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。QM-MSPCR的定量限（LoQ）为10 pg亚硫酸氢盐转化的DNA。结论我们开发并临床验证了MASO-PCR测定法，产生了成本有效，简单，快速且临床上可用于尿液中FGFR3突变检测的测定法。我们还设计了Urodiag®PCR试剂盒，其中包括MASO-PCR和QM-MSPCR分析。该套件适用于常规临床实验室（简单，准确），将为泌尿科医师对低，中和高风险NMIBC患者的复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。QM-MSPCR的定量限（LoQ）为10 pg亚硫酸氢盐转化的DNA。结论我们开发并临床验证了MASO-PCR测定法，产生了成本有效，简单，快速且临床上可用于尿液中FGFR3突变检测的测定法。我们还设计了Urodiag®PCR试剂盒，其中包括MASO-PCR和QM-MSPCR分析。该套件适用于常规临床实验室（简单，准确），将为泌尿科医师对低，中和高风险NMIBC的患者进行复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。生成经济有效，简单，快速且可在临床上应用的检测尿中FGFR3突变的检测方法。我们还设计了Urodiag®PCR试剂盒，其中包括MASO-PCR和QM-MSPCR分析。该套件适用于常规临床实验室（简单，准确），将为泌尿科医师对低，中和高风险NMIBC的患者进行复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。生成经济有效，简单，快速且可在临床上应用的检测尿中FGFR3突变的检测方法。我们还设计了Urodiag®PCR试剂盒，其中包括MASO-PCR和QM-MSPCR分析。该套件适用于常规临床实验室（简单，准确），将为泌尿科医师对低，中和高风险NMIBC患者的复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。该套件将为泌尿科医师对低，中和高风险NMIBC患者的复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。该套件将为泌尿科医师对低，中和高风险NMIBC患者的复发监测提供很大帮助。减少不必要的膀胱镜检查的次数，它将对患者（无痛）和医疗系统（低成本）产生极其有益的影响。