An efficient isothermal PCR method for on-site detection of nucleic acid

Abstract

Convective PCR (CPCR) is an isothermal nucleic acid amplification technology; however, natural convection exhibits a chaotic and multiplex flow state, resulting in low amplification efficiency and specificity. We placed a polycarbonate strip (p-strip) inside reaction tubes to induce circumfluence by blocking the inner ring that originally allowed fluid to flow at suboptimal temperatures. Moreover, we constructed a dual-temperature instrument to provide appropriate denaturing and annealing zones for CPCR. Tubes containing p-strips exhibited significantly improved efficiency, sensitivity and specificity. For real-time detection, the variation coefficients of three replicates having the same concentrations were less than 2% in more than half of the cases, indicating improved CPCR amplification and potential as a commercial on-site nucleic acid diagnosis tool.