title: Ultrafast, sensitive and large-volume on-chip real-time PCR for the molecular diagnosis of bacterial and viral infections.
abstract: To control future infectious disease outbreaks, like the 2014 Ebola epidemic, it is necessary to develop ultrafast molecular assays enabling rapid and sensitive diagnoses. To that end, several ultrafast real-time PCR systems have been previously developed, but they present issues that hinder their wide adoption, notably regarding their sensitivity and detection volume. An ultrafast, sensitive and large-volume real-time PCR system based on microfluidic thermalization is presented herein. The method is based on the circulation of pre-heated liquids in a microfluidic chip that thermalize the PCR chamber by diffusion and ultrafast flow switches. The system can achieve up to 30 real-time PCR cycles in around 2 minutes, which makes it the fastest PCR thermalization system for regular sample volume to the best of our knowledge. After biochemical optimization, anthrax and Ebola simulating agents could be respectively detected by a real-time PCR in 7 minutes and a reverse transcription real-time PCR in 7.5 minutes. These detections are respectively 6.4 and 7.2 times faster than with an off-the-shelf apparatus, while conserving real-time PCR sample volume, efficiency, selectivity and sensitivity. The high-speed thermalization also enabled us to perform sharp melting curve analyses in only 20 s and to discriminate amplicons of different lengths by rapid real-time PCR. This real-time PCR microfluidic thermalization system is cost-effective, versatile and can be then further developed for point-of-care, multiplexed, ultrafast and highly sensitive molecular diagnoses of bacterial and viral diseases.
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com and École Normale Supérieure-PSL Research University, 24 rue Lhomond, 75005, Paris, France. yong.chen@ens.fr.
authors.forename: Timothée
authors.initials: T
authors.lastname: Houssin
authors.affiliation: École Normale Supérieure-PSL Research University, 24 rue Lhomond, 75005, Paris, France. yong.chen@ens.fr and Cherry Biotech, Université de Rennes 1, Campus Santé, Bât8, Et2, 2 Av. du Pr. Léon Bernard, CS 34317, 35043 Rennes Cedex, France. jeremy.cramer@cherrybiotech.com.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Cramer
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Rébecca
authors.initials: R
authors.lastname: Grojsman
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Lyes
authors.initials: L
authors.lastname: Bellahsene
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Colas
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Hélène
authors.initials: H
authors.lastname: Moulet
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Walter
authors.initials: W
authors.lastname: Minnella
authors.affiliation: Episteme Research and Consulting, 1 rue Rabelais, 92170, Vanves, France. christophe.pannetier@epistemerc.eu.
authors.forename: Christophe
authors.initials: C
authors.lastname: Pannetier
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Maël
authors.initials: M
authors.lastname: Leberre
authors.affiliation: Elvesys - Innovation Center, 83 avenue Philippe Auguste, 75011, Paris, France. houssintimothee@gmail.com mael.leberre@elvesys.com adrien.plecis@elvesys.com.
authors.forename: Adrien
authors.initials: A
authors.lastname: Plecis
authors.affiliation: École Normale Supérieure-PSL Research University, 24 rue Lhomond, 75005, Paris, France. yong.chen@ens.fr.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000881:Anthrax / Q000175:diagnosis*; D019142:Hemorrhagic Fever, Ebola / Q000175:diagnosis*; D056656:Lab-On-A-Chip Devices; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014777:Virus Diseases / Q000175:diagnosis*