Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

Rapid molecular detection of CMY-2, and CTX-M group 1 and 9 variants via recombinase polymerase amplification.

Background: Due to their prevalence worldwide, the ß-lactamases CTX-M and plasmid-mediated CMY-2 are important antimicrobial resistance enzymes in a clinical setting. While culture- and PCR-based detection methods exist for these targets, they are time consuming and require specialist equipment and trained personnel to carry out. Methods: In this study, three rapid diagnostic single-plex and a prototype triplex assay were developed, using recombinase polymerase amplification with lateral flow detection (RPA-LF), and tested for their sensitivity and specificity using two isolate DNA panels (n = 90 and n = 120 isolates). In addition, the RPA-LF assays were also tested with a small number of faecal extract samples (n = 18). Results: The RPA-LF assays were able to detect bla CXT-M-group-1, bla CTX-M-group-9 and bla CMY-2-type variants with high sensitivity (82.1%-100%) and specificity (100%) within a short turnaround time (15-20 min for amplification and detection). Conclusions: RPA-LF assays developed in this study have the potential to be used at or close to the point of care, as well as in low-resource settings, producing rapid results to support healthcare professionals in their treatment decisions.

Molecular effects of a variable environment on Sydney rock oysters, Saccostrea glomerata: Thermal and low salinity stress, and their synergistic effect.

Bivalves are frequently exposed to salinity and temperature fluctuations in the estuary. This study explored the molecular effect of these fluctuations by exposing Sydney rock oysters, (Saccostrea glomerata), native to Australia, to either low salinity, elevated temperature or a combined salinity and temperature stress. Following the exposures, RNA-Seq was carried out on the collected oyster tissues. Differential transcript analysis resulted in a total of 1473, 1232 and 2571 transcripts, which were differentially expressed in S. glomerata exposed to low salinity (10 ppt), elevated temperature (30 °C) or the combined stressor (15 ppt and 30 °C), respectively, when compared to control oysters. All stress treatments had some effect on molecular processes such as innate immune response or respiration, with overall the strongest effects seen in S. glomerata exposed to the combined stressor. Additionally, most transporters putatively involved in osmoregulation were found to be suppressed in response to the combined stressor and the low salinity exposure. This study provides insight into the oyster's responses to both, single and dual stressors commonly found in an estuarine environment.

Combined exposure to pyrene and fluoranthene and their molecular effects on the Sydney rock oyster, Saccostrea glomerata.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously detected in the water column, associated with particulate matter or in the tissue of marine organisms such as molluscs. PAH exposure and their resultant bioaccumulation in molluscs can cause a range of serious physiological effects in the affected animals. To examine the molecular response of these xenobiotics in bivalves, Sydney rock oysters (Saccostrea glomerata) were exposed to pyrene and fluoranthene for seven days. Chemical analysis of the soft-tissue of PAH stressed S. glomerata confirmed that pyrene and fluoranthene could be bioaccumulated by these oysters. RNA-Seq analysis of PAH-exposed S. glomerata showed a total of 765 transcripts differentially expressed between control and PAH-stressed oysters. Closer examination of the transcripts revealed a range genes encoding enzymes involved in PAH detoxification (e.g. cytochrome P450), innate immune responses (e.g. pathogen recognition, phagocytosis) and protein synthesis. Overall, pyrene and fluoranthene exposure appears to have resulted in a suppression of pathogen recognition and some protein synthesis processes, whereas transcripts of genes encoding proteins involved in clearance of cell debris and some transcripts of genes involved in PAH detoxification were induced in response to the stressors. Pyrene and fluoranthene exposure thus invoked a complex molecular response in S. glomerata, with results suggesting that oysters focus on removing the stressors from their system and dealing with the downstream effects of PAH exposure, potentially at the exclusion of other, less immediate concerns (e.g. protection from infection).

Transcriptome Analysis of the Sydney Rock Oyster, Saccostrea glomerata: Insights into Molluscan Immunity.

BACKGROUND: Oysters have important ecological functions in their natural environment, acting as global carbon sinks and improving water quality by removing excess nutrients from the water column. During their life-time oysters are exposed to a variety of pathogens that can cause severe mortality in a range of oyster species. Environmental stressors encountered in their habitat can increase the susceptibility of oysters to these pathogens and in general have been shown to impact on oyster immunity, making immune parameters expressed in these marine animals an important research topic. RESULTS: Paired-end Illumina high throughput sequencing of six S. glomerata tissues exposed to different environmental stressors resulted in a total of 484,121,702 paired-end reads. When reads and assembled transcripts were compared to the C. gigas genome, an overall low level of similarity at the nucleotide level, but a relatively high similarity at the protein level was observed. Examination of the tissue expression pattern showed that some transcripts coding for cathepsins, heat shock proteins and antioxidant proteins were exclusively expressed in the haemolymph of S. glomerata, suggesting a role in innate immunity. Furthermore, analysis of the S. glomerata ORFs showed a wide range of genes potentially involved in innate immunity, from pattern recognition receptors, components of the Toll-like signalling and apoptosis pathways to a complex antioxidant defence mechanism. CONCLUSIONS: This is the first large scale RNA-Seq study carried out in S. glomerata, showing the complex network of innate immune components that exist in this species. The results confirmed that many of the innate immune system components observed in mammals are also conserved in oysters; however, some, such as the TLR adaptors MAL, TRIF and TRAM are either missing or have been modified significantly. The components identified in this study could help explain the oysters' natural resilience against pathogenic microorganisms encountered in their natural environment.

Molecular characterisation of colour formation in the prawn Fenneropenaeus merguiensis.

INTRODUCTION: Body colouration in animals can have a range of functions, with predator protection an important aspect of colour in crustaceans. Colour determination is associated with the carotenoid astaxanthin, which is taken up through the diet and stabilised in the tissues by the protein crustacyanin. As a variety of genes are found to play a role in colour formation in other systems, a holistic approach was employed in this study to determine the factors involved in Fenneropenaeus merguiensis colouration. RESULTS: Full length F. merguiensis crustacyanin subunit A and C sequences were isolated. Crustacyanin subunit A and C were found in the F. merguiensis transcriptomes of the muscle/cuticle tissue, hepatopancreas, eye stalk and nervous system, using 454 next generation sequencing technology. Custom microarray analysis of albino, light and dark F. merguiensis cuticle tissue showed genes encoding actin, sarcoplasmic calcium-binding protein and arginine kinase to be 4-fold or greater differentially expressed (p<0.05) and down-regulated in albinos when compared to light and dark samples. QPCR expression analysis of crustacyanin and total astaxanthin pigment extraction revealed significantly (p<0.05) lower crustacyanin subunit A and C gene transcript copy numbers and total astaxanthin levels in albinos than in the light and dark samples. Additionally, crustacyanin subunit A and C expression levels correlated positively with each other. CONCLUSIONS: This study identified gene products putatively involved in crustacean colouration, such as crustacyanin, sarcoplasmic calcium-binding protein and forms of actin, and investigated differences in gene expression and astaxanthin levels between albino, light and dark coloured prawns. These genes open a path to enhance our understanding of the biology and regulation of colour formation.