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.
SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.
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
For systematical NVH development of vehicle (especially for mass-production passenger vehicles) electric powertrain, an optimized V-Model is designed and has been implemented in the entire component-vehicle development, which integrates three individual branches: simulation, validation and optimization. Compared to the V-models in the traditional sense, this optimized V-model is not only driven by requirement and task accomplishment but also maximum optimization of NVH system performance. In this case, developing procedures are capable to be efficiently iterated and the NVH engineering can be expanded into 3D with this V-model.
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