A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Methods: Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. Results: We found that the freeze-dried PCR mixes with~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18~25°C) for 28 days, and for 14 and 10 days when stored at 37°C and 56°C, respectively. Conclusion: The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.
In view of the complex procedure of nucleic acid extraction, there exists a huge challenge for the widespread use of point-of-care diagnostics for nucleic acid testing. To achieve point-of-care applications in a more rapid and cost-efficient manner, we designed a snake pipe-shaped microfluidic chip so as to accomplish reagents-prestored, time-saving, operation-simple nucleic acid extraction. All reagents needed for this process, including lysis buffer, wash buffer, elution buffer, and so on, were preloaded in the snake pipe and securely isolated by membrane valves, without the need for using any specialized equipment. By an integrated chip and a powerful ultrasonic, this device could complete virus nucleic acid extraction from sophisticated serum samples in less than 1 min. We used hepatitis B virus (HBV) and human immunodeficiency virus (HIV) mixed with different sources of serum as samples to be extracted. The coefficient of variation of HBV and HIV extraction on-chip was 1.32% and 2.74%, respectively, and there were no significant differences between on-chip and commercial instrument extraction (P > 0.05, α = 0.05) in different dilution ratios, which showed that the extraction device we established had excellent stability and sensitivity.
As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18–25 °C) for 1–2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q 10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.
19A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) : bioRxiv preprint 23 19, the need to transport and store these mixes at low temperatures presents challenges 24 to already overburdened logistics networks. Here, we present an optimized freeze-25 drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at 26 ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were 27 freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by 28 Karl-Fischer titration. We found that freeze-dried PCR mixes with ~1.2% residual 29 moisture are optimal for storage, transport, and reconstitution. The sensitivity, 30 specificity, and repeatability of the freeze-dried reagents were similar to those of freshly 31 prepared, wet reagents. The freeze-dried mixes retained activity at room temperature 32 (18~25℃) for 28 days, and for 14 and 10 days when stored at 37℃ and 56℃, 33 respectively. The uptake of this approach will ease logistical challenges faced by 34 transport networks and make more cold storage space available at diagnosis and 35 hospital laboratories. This method can also be applied to the generation of freeze-dried 36 PCR mixes for the detection of other pathogens. 37 38
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