Background: Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. Methods: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-ofconcept testing. Swabs were spiked in serial dilutions from 1.2 × 10 6 to 1.2 × 10 1 copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 μL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. Results: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 10 3 viral copies/mL with 96.30% sensitivity. Conclusion: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.
Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.
Mechanical lysis of bacteria is a common alternative to enzymatic, detergent, or tonicity‐based methods. Mechanical lysis is needed in areas of research where introducing chemicals can compromise either the assay or the analyte and is achieved in a variety of ways, one of which is bead beating. Bead beating utilizes small beads of various sizes and composition to contact the sample with high energy to facilitate lysis. This type of lysis can be achieved using many different types of equipment from laboratory vortexers to specialized bead mills. Maximizing the amount of lysis is important for both consistency of analyte extractions and increasing yield when sample size is limited.Herein, we evaluate two bead media compositions (borosilicate glass and yttria‐stabilized zirconium oxide) and two sizes of each of those compositions (0.5 mm and 0.1 mm) to analyze what factors are most important to achieve the maximum amount of lysis using cultures of Escherichia coli, Staphylococcus epidermidis, and Nocardia brasiliensis. Speed, time, number of beads, and mass were all controlled to identify the ideal size, composition, force and duration to maximize cell lysis while maintaining nucleic acid integrity. In order to evaluate lysis efficiency cells were disrupted then plated to enable colony counting. After each processing step, genomic DNA was purified, quantified, and analyzed by gel electrophoresis to evaluate genomic DNA integrity.In all cases, it was determined that bead diameter played a greater role in lysis efficiency when compared to bead material even though kinetic energy of the zirconium oxide beads is 2X greater than the glass at a given tube velocity. E. coli was lysed at greater than 90% total cell lysis in three minutes at a tube velocity of 4.2 m/s (Figure 1). No DNA degradation was observed after homogenization for 1 minute. Increasing amounts of DNA shearing was observed as processing was increased (Figure 2).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Context Rapid influenza diagnostic tests (RIDTs) are becoming increasingly accurate, available, and reliable as the first line of testing when suspecting influenza infections, although the global burden of influenza infections remains high. Rapid diagnosis of influenza infections has been shown to reduce improper or delayed treatment and to increase access to diagnostic measures in public health, primary care, and hospital-based settings. Objectives As the use of RIDTs continues to expand in all healthcare settings, there is a multitude of molecular techniques being employed by these various testing platforms. With this in mind, we compare the sensitivity, specificity, and time to diagnosis for nine highly utilized commercial RIDTs. Methods Nine commercially available RIDTs were identified from the US Centers for Disease Control and Prevention (CDC) website, which were also referenced on PubMed by name within the title or abstract of peer-reviewed publications examining the sensitivity and specificity of each test against a minimum of three influenza A virus (IAV) strains as well as seasonal influenza B virus (IBV). Data from the peer-reviewed publications and manufacturers’ websites were combined to discuss the sensitivity, specify, and time to diagnosis associated with each RIDT. Results The sensitivity and specificity across the examined RIDTs were greater than 85.0% for both IAV and IBV across all platforms, with the reverse transcriptase–polymerase chain reaction (RT-PCR) assays maintaining sensitivity and specificity greater than 95.0% for all viruses tested. However, the RT-PCR platforms were the longest in time to diagnosis when compared to the other molecular methods utilized in the examined RIDTs. Conclusions Herein, we discussed the benefits and limitations of nine commercially available RIDTs and the molecular techniques upon which they are based, showing the relative accuracy and speed of each test for IAV and IBV detection as reported by the peer-reviewed literature and commercial manufacturers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.