The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

Application of xanthan and locust bean gum mix or sorbitol in the jelly formulation to improved jelly 3D printing using a fused deposition modeling printer.

This study examined the impacts of applying a xanthan and locust bean gum mix or sorbitol to a jelly formulation on the rheological parameters necessary for 3D printing a jelly applying the fused deposition modeling method. A jelly formulation was fortified with a gum mix (xanthan gum:locust bean gum = 0.625:0.375) at 1% (w/w), or added with sorbitol instead of sugar. Both treatments increased the values of storage modulus and yield stress, related to fidelity and shape retention, and adding the gum mix, in particular, increased the gel strength. Applying these treatments to the formulation that lacks the rheological parameters and gel strength required for 3D printing changed those values in a direction fulfilling the material requirements. This research confirmed that the application of xanthan and locust bean gum mix or sorbitol could adjust the properties of materials used in 3D printing for improved printability.

Cold plasma treatment to improve jelly production using a fused deposition modeling 3D printer.

Herein, the effects of cold plasma (CP) on the rheological properties of gelatin-based solutions for 3D jelly printing using a fused deposition modeling method were investigated. The gelatin powder was packaged in a nylon/polyethylene pouch with nitrogen gas and subjected to CP treatment at 15 W for 10 min using atmospheric dielectric barrier discharge plasma treatment. CP treatment reduced the relative proportion of polar functional groups on the surface of the gelatin powder and forged new bonds (e.g., C-C) that reduced the hydrophilicity of the material. Furthermore, it increased the storage modulus and yield stress of the jelly formulation and lowered the phase angle, improving the fidelity and shape retention of the 3D printed jelly. Using CP-treated gelatin, inferior jelly formulations could be tuned to satisfy established printing criteria. CP treatment can control the rheological properties involved in the 3D printing of jelly.

Material requirements for printing cookie dough using a fused deposition modeling 3D printer.

This study examined the requirements for using flour-based formulations in fused deposition modeling (FDM) of cone-shaped cookie dough. By considering the requirements of fidelity, shape retention, and extrudability, the rheological and mechanical parameters, which resulted in high printability (93.88‒96.49%) and dimensional stability (96.36‒97.15%), for formulations containing soft wheat flour, granulated sugar, water, and olive oil were determined to be: storage modulus (G') of 7165‒12,590 Pa, loss modulus (G″) of 4161‒8297 Pa, shear modulus of 6613‒12,804 Pa, yield stress (τ0) of 50.22‒72.80 Pa, phase angle of 30.28‒33.52°, apparent viscosity of 181.25‒230.20 Pa·s, and hardness of 0.65-0.91 N. When olive oil and water were replaced with butter and egg, the formulations demonstrated higher values of G', G″, shear modulus, τ0, and hardness; a smaller phase angle; and a wider range of apparent viscosity. These results provide quantitative information for developing cookie dough formulations suitable for 3D printing by FDM.

Determination of Material Requirements for 3D Gel Food Printing Using a Fused Deposition Modeling 3D Printer.

The material requirements for printing gel food with a fused deposition modeling 3D printer were determined based on fidelity, shape retention, and extrudability, as described by the rheological parameters of storage modulus (G'), yield stress (τ0), and phase angle (δ). The material requirements were determined for printing gel food using three formulations containing gelatin, gelatin and pectin, and gum mixture as the gelling agents. As compared with formulations based on gelatin alone, pectin-containing gelatin-based formulations yielded higher δ and lower G' and τ0 values, while gum mixture-based formulations formed a gel with higher G' and δ values and a wider range of τ0. Overall, this study presents quantitative material requirements for printing gel products containing gelatin, gelatin-pectin, and gum mixtures.