Colorimetric PCR Based on Pyrophosphate Displacement

Abstract

Quantitative PCR (qPCR) is the gold standard for molecular diagnostics. Colorimetric readout can greatly simplify PCR-based diagnostics and make them appealing for decentralized testing. Current colorimetric PCR schemes mostly target the dsDNA amplicons. It is expected that pyrophosphate (Py), the inherent product of PCR (in fact, all nucleic acid amplification schemes), stands as a simpler colorimetric target, due to its versatile complexing with various metal ions/indicators. However, Py-based colorimetric PCR has not been reported yet, partially because it will receive serious competitive complexation from Mg2+ in PCR. Herein, we introduced the Sc3+-hydroxynaphthol blue (Sc-HNB) complex to facilitate Py-based colorimetric PCRs. Because of the higher stability of Sc-HNB over Mg-HNB and Sc-Py over Mg-Py, Py generated during PCR cycles can directly displace HNB from the Sc-HNB complex in a Mg2+-independent manner, yielding a distinct color change. The proposed strategy features simplicity and low cost and can be coupled with PCR in both real-time and end-point manner, with sensitivity for target gene detection comparable to conventional qPCR. Because of the robustness of the Py-displacement reactions, the proposed colorimetric PCR could work with even water bath heating and was compatible with the existing PCR kits. To further validate its feasibility and accuracy, human papillomavirus (HPV) detection in vaginal swab samples was carried out, with results fully consistent with those obtained by qPCR. All of these results indicate that the Py-displacement-based colorimetric PCR can be a promising tool for decentralized nucleic acid testing.