Highly Sensitive Detection of PTENR130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

An analytical method based on amplification refractory mutation system- real time fluorescencequantitative polymerase chain reaction ( ARMS-qPCR) was developed for detection of gene mutation PTENR130* mutation in thyroid cancer. Allele-specific and reference primers for the PTENR130* mutation were designed for allele-specific and reference amplification of each sample at the same time, and the Delta Ct value was obtained by Ct-allele-specific value minus Ct-reference value to analyze the variant allele frequency ( VAF). The ARMS- qPCR method could detect VAF as low as 0. 01%, with a linear range of 0. 1% to 90% VAF, and could identify PTEN mutations in genomic DNA as low as 4 copies. This method successfully detected 5 cases of mutation with VAF> 0. 1% in 24 thyroid tumor samples. In addition, the ARMS-qPCR method also showed good detection performance for PTENR130* mutant spiked plasma samples under the electrochemical enrichment, indicating clinical practicability and the potential to detect trace PTENR130* mutation in complex samples.