Ultrasensitive detection of serum miRNA biomarkers related to papillary thyroid cancer using ligation-initiated phosphorothioated primer-based loop-mediated isothermal amplification

Accurately distinguishing of papillary thyroid cancer (PTC) from benign nodules, at the same time, avoiding overdiagnosis is of great significance. In consideration of the role of miRNA in pathogenesis and progression of tumors, miRNA might also be useful as diagnostic marker for cancer. In this study, a ligation-initiated phosphorothioated primer-based loop-mediated isothermal amplification (PS-LAMP) strategy was established to quantify miRNAs in serum sample, thereby achieving noninvasive diagnosis of PTC. Two linker probes designed from dumbbell-shaped amplicons of LAMP are ligated to obtain an active amplicon only when they are perfectly hybridized with target miRNA, exhibiting high specificity. The introduction of PS modification into inner primers (BIP/FIP) facilitated extra efficient hairpin formation and extension for LAMP, which significantly improved sensitivity. Then, the proposed method was successfully applied in the early diagnosis of PTC, in which circulating serum level of miR-146b-5p and miR-199b-5p could draw a clear distinction between patients and controls. This ligation-initiated PS-LAMP assay opens new avenues for highly sensitive quantification of miRNA biomarkers and thus exhibits a considerable potential utility in clinical diagnosis and prognosis.