RECEIVED DATE (to be automatically inserted after your manuscript is accepted if requiredaccording to the journal that you are submitting your paper to) *colston1@llnl.gov ABSTRACT. The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10 6 smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of ~18, twenty cycles earlier than 2 commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 μL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.

The ongoing severe acute respiratory syndrome-coronavirus (SARS-CoV-2) has triggered the coronavirus pandemic (COVID-19) that has claimed hundreds of thousands of lives worldwide. This virus spreads predominantly by human-to-human transmission via respiratory droplets. However, the presence of this virus in the fecal and anal swabs of infected patients has triggered the need for research into its waterborne transmission. The various environmental factors that impact the persistence of coronavirus in different water matrices include temperature, UV exposure, organic matter, disinfectants as well as adversarial microorganisms. This review summarizes the most recent research data on the effect of various factors on coronavirus in aqueous environments. The available data suggest that: (i) increasing temperature decreases the overall persistence of the virus; (ii) the presence of organic matter can increase the survivability of coronavirus; (iii) chlorine is the most effective and economic disinfectant; (iv) membrane bioreactors in wastewater treatment plants are hosts of competitive microorganisms that can inactivate coronaviruses; (v) ultraviolet irradiation is another effective option for virus inactivation. However, the inactivation disinfection kinetics of coronaviruses are yet to be fully understood. Thus, further research is needed to understand its fate and transport with respect to the water cycle so that effective strategies can be adopted to curb its effects. These strategies may vary based on geographic, climatic, technical, and social conditions around the globe. This paper explores possible approaches and especially the conditions that local communities and authorities should consider to find optimal solutions that can limit the spread of this virus.

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