Ultrasensitive Isothermal Detection of SARS-CoV‑2 Based on Self-Priming Hairpin-Utilized Amplification of the G‑Rich Sequence

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints

Comparison of out-of-hospital cardiac arrests during the COVID-19 pandemic with those before the pandemic: an updated systematic review and meta-analysis

The coronavirus disease of 2019 (COVID-19) pandemic, directly and indirectly, affected the emergency medical care system and resulted in worse out-of-hospital cardiac arrest (OHCA) outcomes and epidemiological features compared with those before the pandemic. This review compares the regional and temporal features of OHCA prognosis and epidemiological characteristics. Various databases were searched to compare the OHCA outcomes and epidemiological characteristics during the COVID-19 pandemic with before the pandemic. During the COVID-19 pandemic, survival and favorable neurological outcome rates were significantly lower than before. Survival to hospitalization, return of spontaneous circulation, endotracheal intubation, and use of an automated external defibrillator (AED) decreased significantly, whereas the use of a supraglottic airway device, the incidence of cardiac arrest at home, and response time of emergency medical service (EMS) increased significantly. Bystander CPR, unwitnessed cardiac arrest, EMS transfer time, use of mechanical CPR, and in-hospital target temperature management did not differ significantly. A subgroup analysis of the studies that included only the first wave with those that included the subsequent waves revealed the overall outcomes in which the epidemiological features of OHCA exhibited similar patterns. No significant regional differences between the OHCA survival rates in Asia before and during the pandemic were observed, although other variables varied by region. The COVID-19 pandemic altered the epidemiologic characteristics, survival rates, and neurological prognosis of OHCA patients.Review registration: PROSPERO (CRD42022339435)

Rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA)

We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/μL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19

Multifunctional self-priming hairpin probe-based isothermal nucleic acid amplification and its applications for COVID-19 diagnosis

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings

Impact of scannable healing abutment type on implant impression accuracy

Aim or Purpose: The purpose of this in vitro study was to evaluate the impact of scannable healing abutment shape and height on the accuracy of implant impression. Materials and Methods: IS III active (Neobiotech Co., Seoul, Korea), TS III (Osstem Implant Co., Seoul, Korea), and Bright tissue level (Dentium Co., Seoul, Korea) were installed in the mandibular right second premolar and first molar areas of each 3d-printed partially edentulous mandibular model. With scanbodies and scannable healing abutments connected, reference STL files were generated by the model scanner (inEos X5, Dentsply Sirona, Bensheim, Germany) and 10 STL files of each model were generated with the intraoral scanner (PRIMESCAN®, Dentsply Sirona, Bensheim, Germany). By using exocad DentalCAD (exocad GmbH, Darmstadt, Germany), STL files were aligned on the coordinate system and digital laboratory analogues were matched to each STL file. The deviation of the angle between the long axes of fixtures and the reference plane and the 3d-distance deviation of the fixture tops' centers were measured using the Geomagic® Control X (3D Systems Inc., Rock Hill, SC, USA). Kruskal-Wallis test, Mann-Whitney test and Bonferroni correction were used for statistical analysis. Results: Scannable healing abutments of the three companies showed comparable 3d-distance and angular deviation, although there were some differences depending on the location. Adding a plastic cap on the scannable healing abutment of Bright implant significantly increased the angular deviation, while the 3d-distance deviation was significantly decreased. Conclusions: In the limitation of this study, scannable healing abutment shape did not affect the implant impression accuracy while adding a cap increased the angular deviation