All-in-one approaches for rapid and highly specific quantifcation of single nucleotide polymorphisms based on ligase detection reaction using molecular beacons as turn-on probes

Abstract

Rapid, simple, specific and sensitive approaches for single nucleotide polymorphisms (SNPs) detection are essential for clinical diagnosis. In this study, all-in-one approaches, consisting of the whole detection process including ligase detection reaction (LDR) and real time quantitative polymerase chain reaction performed in one PCR tube by a one-step operation on a real-time PCR system using molecular beacon (MB) as turn-on probe, were developed for rapid, simple, specific and sensitive quantifcation of SNPs. High specificity of the all-in-one approach was achieved by using the LDR, which employs a thermostable and single-base discerning Hifi Taq DNA ligase to ligate adjacently hybridized LDR-specific probes. In addition, a highly specific probe, MB, was used to detect the products of all-in-one approach, which doubly enhances the specificity of the all-in-one approach. The linear dynamic range and high sensitivity of mutant DNA (MutDNA) and wild-type DNA (WtDNA) all-in-one approaches for the detection of MutDNA and WtDNA were studied in vitro, with a broad linear dynamic range of 0.1 fM to 1 pM and detection limits of 65.3 aM and 31.2 aM, respectively. In addition, the MutDNA and WtDNA all-in-one approaches were able to accurately detect allele frequency changes as low as 0.1%. In particular, the epidermal growth factor receptor T790M MutDNA frequency in the tissue of five patients with non-small cell lung cancer detected by all-in-one approaches were in agreement with clinical detection results, indicating the excellent practicability of the developed approaches for the quantification of SNPs in real samples. In summary, the developed all-in-one approaches exhibited promising potential for further applications in clinical diagnosis.