Introduction of Multilayered Dual-Signal Nanotags into a Colorimetric-Fluorescent Coenhanced Immunochromatographic Assay for Ultrasensitive and Flexible Monitoring of SARS-CoV-2.

Timely, accurate, and rapid diagnosis of SARS-CoV-2 is a key factor in controlling the spread of the epidemic and guiding treatments. Herein, a flexible and ultrasensitive immunochromatographic assay (ICA) was proposed based on a colorimetric/fluorescent dual-signal enhancement strategy. We first fabricated a highly stable dual-signal nanocomposite (SADQD) by continuously coating one layer of 20 nm AuNPs and two layers of quantum dots onto a 200 nm SiO2 nanosphere to provide strong colorimetric signals and enhanced fluorescence signals. Two kinds of SADQD with red and green fluorescence were conjugated with spike (S) antibody and nucleocapsid (N) antibody, respectively, and used as dual-fluorescence/colorimetric tags for the simultaneous detection of S and N proteins on one test line of ICA strip, which can not only greatly reduce the background interference and improve the detection accuracy but also achieve a higher colorimetric sensitivity. The detection limits of the method for target antigens via colorimetric and fluorescence modes were as low as 50 and 2.2 pg/mL, respectively, which were 5 and 113 times more sensitive than those from the standard AuNP-ICA strips, respectively. This biosensor will provide a more accurate and convenient way to diagnose COVID-19 in different application scenarios.

CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/µL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

Application of ultrasonography in neonatal lung disease: An updated review.

Lung disease is often life-threatening for both preterm and term newborns. Therefore, an accurate and rapid diagnosis of lung diseases in newborns is crucial, as management strategies differ with different etiologies. To reduce the risk of radiation exposure derived from the conventionally used chest x-ray as well as computed tomography scans, lung ultrasonography (LUS) has been introduced in clinical practice to identify and differentiate neonatal lung diseases because of its radiation-free characteristic, convenience, high accuracy, and low cost. In recent years, it has been proved that LUS exhibits high sensitivity and specificity for identifying various neonatal lung diseases. Here, we offer an updated review of the applications of LUS in neonatal lung diseases based on the reports published in recent years (2017 to present).

An integrated fluorescent lateral flow assay for multiplex point-of-care detection of four respiratory viruses.

This work established a highly sensitive and specific quantum dot nanobeads-based lateral flow assay for multiplex detection of four respiratory virus markers at point of care. The respiratory virus antigens were detected by fluorescent lateral flow strips within 20 min. The limits of detection for SARS-CoV-2 antigen, IAV antigen, IBV antigen, and ADV antigen were 0.01 ng/mL, 0.05 ng/mL, 0.31 ng/mL, and 0.40 ng/mL, respectively, which were superior to that of conventional AuNPs-based colorimetric lateral flow assay. The coefficients of variation of the test strip were 6.09%, 2.24%, 7.92%, and 12.43% for these four antigens, which indicated that the proposed method had good repeatability. The specificity of the detection system was verified by different combinations of these four respiratory viruses and several other respiratory pathogens. These results indicated that this method could simultaneously detect SARS-CoV-2, IAV, IBV and ADV in a short assay time, showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

Tetra-primer ARMS-PCR combined with dual-color fluorescent lateral flow assay for the discrimination of SARS-CoV-2 and its mutations with a handheld wireless reader.

Several virulent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged along with the spread of this virus throughout the population. Some variants can exhibit increased transmissibility and reduced immune neutralization reactivity. These changes are deeply concerning issues that may hinder the ongoing effort of epidemic control measures, especially mass vaccination campaigns. The accurate discrimination of SARS-CoV-2 and its emerging variants is essential to contain the coronavirus disease 2019 pandemic. Herein, we report a low-cost, facile, and highly sensitive diagnostic platform that can simultaneously distinguish wild-type (WT) SARS-CoV-2 and its two mutations, namely, D614G and N501Y, within 2 h. WT or mutant (M) nucleic acid fragments at each allelic locus were selectively amplified by the tetra-primer amplification refractory mutation system (ARMS)-PCR assay. Allele-specific amplicons were simultaneously detected by two test lines on a quantum dot nanobead (QB)-based dual-color fluorescent test strip, which could be interpreted by the naked eye or by a home-made fluorescent strip readout device that was wirelessly connected to a smartphone for quantitative data analysis and result presentation. The WT and M viruses were indicated and were strictly discriminated by the presence of a green or red band on test line 1 for the D614G site and test line 2 for the N501Y site. The limits of detection (LODs) for the WT and M D614G were estimated as 78.91 and 33.53 copies per µL, respectively. This assay was also modified for the simultaneous detection of the N and ORF1ab genes of SARS-CoV-2 with LODs of 1.90 and 6.07 copies per µL, respectively. The proposed platform can provide a simple, accurate, and affordable diagnostic approach for the screening of SARS-CoV-2 and its variants of concern even in resource-limited settings.

A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra07756b

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per μL and 14.23 copies per μL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings. Rapid and highly sensitive multiplex detection of SARS-CoV-2 and influenza B virus using water bath PCR-combined fluorescent lateral flow assay.

A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus.

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per µL and 14.23 copies per µL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.