Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea.
Department of Colorectal Cancer, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea.
Biosensors (Basel). 2022 Aug 4;12(8):597. doi: 10.3390/bios12080597.
Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize mutations in the DNA of colorectal cancer tissues (HSSP-G12D () and HSSP-G13D ()) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold () values between HSSP-G12D and HSSP-G13D. The limit of detection to detect mutations ( and ) was 5-10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for and 92% for ) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.
检测癌基因的突变对早期诊断、定制治疗、治疗进展和耐药性监测具有重要意义。在这里，我们介绍了一种基于热点特异性探针（HSSP）的快速、敏感、特异的突变检测方法，与传统技术相比，具有更好的临床应用价值。我们设计 HSSP 与实时 PCR 相结合，识别结直肠癌组织中的突变（HSSP-G12D()和 HSSP-G13D()）。在 PCR 分析过程中，HSSP 与目标突变序列结合，干扰扩增。然后，我们通过计算 HSSP-G12D 和 HSSP-G13D 之间的循环阈值（）值差异来确定突变检测效率。检测 突变（和）的检测限为野生型人群中突变等位基因的 5-10%。这优于传统方法（≥30%的突变等位基因）。此外，该技术耗时短（不到 1.5 小时），每个样本的成本低至 2 美元。我们使用 69 份结直肠癌患者的组织样本验证了其临床实用性。与直接测序法相比，HSSP 法的临床灵敏度和特异性更高（84%和 92%）。因此，HSSP 与实时 PCR 相结合，为检测癌症相关突变提供了一种快速、高度敏感、特异且成本低廉的方法。与 NGS 等金标准方法相比，该技术显示了快速突变检测现场应用的可能性，可能在定制治疗和癌症监测等多种应用中具有价值。
