A Novel Technique to Detect EGFR Mutations in Lung Cancer

Abstract

Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples.

Recombinase polymerase amplification: Basics, applications and recent advances

“…In solution-phase, due to the unimpeded diffusion of primers and reaction reagents, amplification kinetics are favoured and the achieved limit of detection is subsequently usually better and amplification is achieved in a faster time than solid-phase. Nevertheless, solidphase and bridge amplification present some advantages, such as the potential for spatially resolved multiplexed amplification or the possibility to couple the amplification with diverse detection techniques including ring resonators [27e29], electrochemical [30e36] and colorimetric detection [5,6,30,33,37,38]. Several methods have been developed with solid phase amplification with performances usually inferior to that achieved with solution phase amplification [5,27e30,39] as primer accessibility is more restricted impeding amplification efficiency, and future work will need to focus on strategies to decrease amplification time.…”

“…Background amplification was reduced via the use of peptide nucleic acids, as PNA-DNA interactions are stronger than DNA-DNA, and one single mismatch is more destabilising than a normal DNA-DNA mismatch, thus improving specificity. However, an extra step is required to allow genomic DNA e PNA hybridization, heating to 99 C and then cooling down to 66 C, moving away from the attractive isothermal nature of RPA [38]. An alternative approach exploiting the use of shorter primers (19e21mer) to decrease the stability between primers and targets and increase specificity towards SNPs has also been reported, where a mismatch in the 3 0 -of the primer was included to increase the specificity.…”

Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene

“…A similar product formation was obtained when the RPA reaction was produced within the 37-42 ºC range, showing high tolerance to temperature fluctuations. Therefore, the selected conditions were similar to previous studies for human cancer tissues [26].…”

A Probe Directed Recombinase Amplification Assay for Detection of MTHFR A1298C Polymorphism Associated with Congenital Heart Disease

“…Among them, loop-mediated isothermal amplification (LAMP) was most commonly used in SNP detection [1113]; however, the complex LAMP primer design andthe requirement for high temperature (65C) have limited its application. A few studies have reported that the recombinase polymerase amplification (RPA) was able to detect point mutations of atumor gene by the use of intercalating dye, internal control and peptide nucleic acid [14,15], but have shown limited specificity and sensitivity. Recombinase-aided amplification (RAA) is a novel isothermal amplification and detection assay, utilizing specific enzymes and protein for rapid detection of nucleic acids at 39C in less than 30min.The RAA assay depends on three major proteins: single-strand DNA-binding protein (SSB), recombinase UvsX extracted from Escherichia coli and DNA polymerase.…”