Hot-Spot-Specific Probe (HSSP) for Rapid and Accurate Detection of KRAS Mutations in Colorectal Cancer

Hyo Joo Lee, Bonhan Koo, Yoon Ok Jang, Huifang Liu, Thuy Nguyen Thi Dao, Seok Byung Lim, Yong Shin

Research output: Contribution to journalArticlepeer-review


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 KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGT→GAT) and HSSP-G13D (GGC→GAC)) 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 (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) 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 G12D and 92% for G13D) 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.

Original languageEnglish
Article number597
Issue number8
Publication statusPublished - 2022 Aug

Bibliographical note

Funding Information:
This study was supported by the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation of Korea (NRF) (2020R1A2C2007148); supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI22C0306), Republic of Korea; and also supported by the Yonsei University Research Fund of 2022-22-0113, Republic of Korea.

Publisher Copyright:
© 2022 by the authors.

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biotechnology
  • Biomedical Engineering
  • Instrumentation
  • Engineering (miscellaneous)
  • Clinical Biochemistry


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