Multiplexed detection of single-point mutations in unprocessed human serum with SERS-integrated PCR

Early detection and monitoring of tumor mutations is an important area of research in diagnostics. Polymerase chain reaction (PCR) is considered the gold standard for monitoring tumor mutations. Recently, integration of PCR with SERS has led to enhanced sensitivity and multiplexity of methods for monitoring pathogens and mutations. However, limitations on increasing the number of targets that can be detected using a single set of reaction still exist. The number of mutations that can be analyzed in a single reaction is influenced by the interaction among different fluorophores. For simultaneous detection of more than three mutations with SERS, complicated extraction, and subsequent statistical analysis is required. Here, we present a SERS Hairpin-blocked Integrated PCR (SHIP) system, which integrates PCR with SERS using hairpin-shaped blocking DNA to prevent false positive enhancement of SERS intensity. SHIP enables simultaneously detection of up to four different KRAS single-point mutations in unprocessed serum. The SHIP system holds a great potential for multiplexed detection of mutations and pathogens for diagnostic purposes.