Moss, C. | bioRxiv Science

bioRxiv2021

Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.

molecular biology↗

bioRxiv2020

Inflight head stabilization associated with wingbeat cycle and sonar emissions in the Egyptian fruit bat.

Sensory processing of environmental stimuli during locomotion is critical for the successful execution of goal-directed behaviors and navigating around obstacles. The outcome of these sensorimotor processes can be challenged by head movements that perturb the sensory coordinate frames directing behaviors. In the case of visually-guided behaviors, visual gaze stabilization results from the integrated activity of the vestibuloocular reflex and motor efference copy originating within circuits driving locomotor behavior. A recent videographic study showed that echolocating bats exhibit inflight head stabilization during a target identification and landing task, though compensatory timing of the bats sonar signals was not reported. In the present investigation we tested hypotheses that head stabilization is more broadly implemented during epochs of exploratory flight, and is temporally associated with emitted sonar signals, which would optimize acoustic gaze. This was achieved by measuring head and body kinematics with motion sensors secured to the head and body of free-flying Egyptian fruit bats. These devices were integrated with ultrasonic microphones to record the bats sonar emissions and elucidate their temporal association with periods of head stabilization. Head accelerations in the Earth-vertical axis were asymmetric with respect to wing downbeat and upbeat relative to body accelerations. This indicated that inflight head and body accelerations were uncoupled, outcomes consistent with the implementation of head movements that limit vertical acceleration during wing downbeat. Furthermore, sonar emissions during stable flight occurred most often during wing downbeat and head stabilization, supporting the conclusion that head stabilization behavior optimized sonar gaze and environmental interrogation via echolocation. Summary statementDirect measurements of head and body kinematics from affixed motion sensors revealed head stabilization behaviors during exploratory flights in bats. Most sonar emissions were temporally correlated with this behavior, thereby contributing to the optimization of acoustic gaze.

animal behavior and cognition↗