Fast and Sensitive Detection of SARS-CoV-2 Nucleic Acid Using a Rapid Detection System Free of RNA Extraction

Abstract

Objectives. To establish and evaluate the analytical and clinical performance of the Flash20 SARS-CoV-2 nucleic acid rapid detection system free of RNA extraction. Methods. The limit of detection (LoD) was determined using a negative nasopharyngeal swab matrix spiked with different concentrations of SARS-CoV-2 virus; a total of 734,337 reference sequences of viral genomes from GenBank were used for the in-silico analysis to assess the inclusivity of the assay. The specificity of the system was evaluated by testing 27 medically relevant organisms. A total of 115 clinical specimens were collected and tested on the Flash20 SARS-CoV-2 detection system and with an FDA-approved comparator test to assess the clinical performance of the system. Results. The LoD of the Flash20 SARS-CoV-2 detection system is 250 copies/mL with a positive rate ≥90% (n = 20); alignments results showed that over 99% identity of the primer and probe of the Flash20 SARS-CoV-2 nucleic acid rapid detection system to the available SARS-CoV-2 sequences; the omicron samples tested 100% positive. None of the 27 organisms showed cross-reactivity with the Flash20 SARS-CoV-2 nucleic acid rapid detection system. Among all the 215 clinical samples, the Flash20 SARS-CoV-2 nucleic acid rapid detection system exhibits a high sensitivity of 99.24% (131/132) and 100% (83/83) specificity. Conclusion. The nucleic acid rapid detection system provides sensitive and accurate detection of SARS-CoV-2 free of RNA extraction. The high sensitivity and short time to results of approximately 35 minutes may impact earlier infection control and disease management.

Point-of-Care Molecular Test for the Detection of 14 High-Risk Genotypes of Human Papillomavirus in a Single Tube

“…Every hrHPV strain was amplified during RPA. Cross-reactivity, specificity, and selectivity to potential contaminating pathogens were determined in silico using BLASTn by comparing the primers and probe to common pathogens and human DNA, similar in approach to prior research. , No significant homology to common STIs or other pathogens commonly present in the vaginal canal for both primers was found (Table S2). The analytical LoD was determined by a serial dilution that was performed to identify the lowest concentration that was reproducibly detected.…”

Advances in Simple, Rapid, and Contamination-Free Instantaneous Nucleic Acid Devices for Pathogen Detection

“…To enhance the purity of nucleic acid extraction, purification methods such as magnetic bead extraction [66] and centrifugal column purification are employed. Additionally, a nucleic-acid-free extraction method has been developed to reduce the overall testing time [67,68]. By using sample release agents, the amount of nucleic acid extraction is reduced, simplifying the nucleic acid testing process, shortening the testing time, improving detection rates, and making the procedure simple and cost-effective.…”

Development and application of a universal extraction-free reagent based on an algal glycolipid

“…19 Baksh et al 20 described an extraction-free real-time reverse-transcriptase PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate, cost-effective, high-capacity solution to COVID-19 testing. Ma et al 21 developed a real-time RT-PCR-based SARS-CoV-2 POCT by using sample release agents, which allows direct nucleic acid amplification on a PCR cycler without RNA extraction, and the entire process from sample to result can be completed in 35 minutes. Although these methods are fast and simple, there are some problems, such as the incomplete release of nucleic acids, low purification efficiency, and interference of cleavage components with subsequent amplification, which will affect the effect of nucleic acid amplification.…”