Nested Phosphorothioated Hybrid Primer-Mediated Isothermal Amplification for Specific and Dye-Based Subattomolar Nucleic Acid Detection at Low Temperatures

Developing dye-based isothermal nucleic acid amplification (INAA) at low temperatures such as 37 °C remains a technical challenge. Here, we describe a nested phosphorothioated (PS) hybrid primer-mediated isothermal amplification (NPSA) assay which only utilizes EvaGreen (a DNA-binding dye) to achieve specific and dye-based subattomolar nucleic acid detection at 37 °C. The success of low-temperature NPSA essentially depends on employing Bacillus smithii DNA polymerase, a strand-displacing DNA polymerase with wide range of activation temperature. However, the NPSA's high efficiency entails nested PS-modified hybrid primers and the additives of urea and T4 Gene 32 Protein. To address the inhibition of urea on reverse transcription (RT), one-tube two-stage recombinase-aided RT-NPSA (rRT-NPSA) is established. By targeting human Kirsten rat sarcoma viral (KRAS) oncogene, NPSA (rRT-NPSA) stably detects 0.2 aM of KRAS gene (mRNA) within 90 (60) min. In addition, rRT-NPSA possesses subattomolar sensitivity to detect human ribosomal protein L13 mRNA. The NPSA/rRT-NPSA assays are also validated to obtain consistent results with PCR/RT-PCR methods on qualitatively detecting DNA/mRNA targets extracted from cultured cells and clinical samples. As a dye-based, low-temperature INAA method, NPSA inherently facilitates the development of miniaturized diagnostic biosensors.