Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

Citations Over TimeTop 13% of 2017 papers

Abstract

In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

Related Papers

• → Viral diagnostics in the era of digital polymerase chain reaction(2012)137 cited
• → A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis(2009)13 cited
• → Validating the Sensitivity of High‐Resolution Melting Analysis for JAK2 V617F Mutation in the Clinical Setting(2016)6 cited
• Detection of the JAK2 V617F mutation in chronic myeloproliferative disorders by high resolution melting curve analysis(2011)
• → [High resolution melting analysis for detecting of JAK2V617F mutation in patients with myeloproliferative neoplasms].(2014)