Bacterial infections seriously threaten human health and also bring huge financial burden. It is critical to construct multifunctional platforms for effectively inactivating bacteria right after point-of-care testing (POCT). Chemiluminescence (CL) bioassays are considered as powerful candidates for POCT as they are free from using an excitation light source, while the flash-type emission limits their further application. Herein, a CL system with long, persistent, and intensive intensity was constructed based on the peroxidase-like property of 4-mercaptophenylboronic acid (MPBA)-functionalized CuSe nanoprobes (CuSeNPs@MPBA), which improved the detection accuracy and sensitivity. By further integrating a smartphone as an analyzer, quantitative POCT of bacteria was realized with high sensitivity. The limit of detection was as low as 1.25 and 1.01 cfu mL-1 for Staphylococcus aureus and Escherichia coli detection, respectively. Specifically, bacteria can be eliminated with high efficiency due to excellent photothermal property of CuSeNPs@MPBA. The developed multifunctional platform also has advantages of simple operation with low cost, suggesting its high potential for use in food safety, environment monitoring, and clinical applications.

Long-Lasting Chemiluminescence-Based POCT for Portable and Visual Pathogenic Detection and In Situ Inactivation.

The SARS-CoV-2 virus has evolved into new strains that increase viral transmissibility and reduce vaccine protection. The rapid circulation of these more harmful strains across the globe has created a pressing need for alternative public health screening tools. REVEALR (RNA-encoded viral nucleic acid analytic reporter), a rapid and highly sensitive DNAzyme-based detection system, functions with perfect accuracy against patient-derived clinical samples. Here, we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR identified the correct variant [Wuhan-Hu-1, alpha (B.1.1.7), gamma (P.1), epsilon (B.1.427/9), delta (B.1.617.2), and omicron (B.1.1.529)] with 100% accuracy. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.

REVEALR-Based Genotyping of SARS-CoV-2 Variants of Concern in Clinical Samples

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is responsible for the global epidemic of Coronavirus Disease 2019 (COVID-19), with a significant impact on the global economy and human safety. Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard for detecting SARS-CoV-2, but because the virus’s genome is prone to mutations, the effectiveness of vaccines and the sensitivity of detection methods are declining. Variants of concern (VOCs) include Alpha, Beta, Gamma, Delta, and Omicron, which are able to evade recognition by host immune mechanisms leading to increased transmissibility, morbidity, and mortality of COVID-19. A range of research has been reported on detection techniques for VOCs, which is beneficial to prevent the rapid spread of the epidemic, improve the effectiveness of public health and social measures, and reduce the harm to human health and safety. However, a meaningful translation of this that reduces the burden of disease, and delivers a clear and cohesive message to guide daily clinical practice, remains preliminary. Herein, we summarize the capabilities of various nucleic acid and protein-based detection methods developed for VOCs in identifying and differentiating current VOCs and compare the advantages and disadvantages of each method, providing a basis for the rapid detection of VOCs strains and their future variants and the adoption of corresponding preventive and control measures.

/pdf/an-update-on-detection-technologies-for-sars-cov-2-variants-2wq2irs1.pdf

An Update on Detection Technologies for SARS-CoV-2 Variants of Concern

https://www.mdpi.com/2079-6374/12/11/978/pdf?version=1667977901

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection, such as multiplexed detection for viral variant surveillance, may limit their widespread adoption. Herein, we developed a robust multiplexed CRISPR-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose SARS-CoV-2 infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool, CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance which can be locally manufactured, may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

/pdf/a-multiplexed-cas13-based-assay-with-point-of-care-3h1qor0q.pdf

A multiplexed Cas13-based assay with point-of-care attributes for simultaneous COVID-19 diagnosis and variant surveillance

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a new coronavirus causing Coronavirus Disease 2019 (COVID-19), is a major topic of global human health concern. The Delta and Omicron variants have caused alarming responses worldwide due to their high transmission rates and a number of mutations. During a one-year follow-up (from June 2020 to June 2021), we included 114 patients with SARS-CoV-2 infection to study the long-term dynamics and the correlative factors of neutralizing antibodies (NAbs) in convalescent patients. The blood samples were collected at two detection time points (at 6 and 12 months after discharge). We evaluated the NAbs response of discharged patients by performing a micro-neutralization assay using a SARS-CoV-2 wild type. In addition, a total of 62 serum samples from discharged COVID-19 patients with Alpha, Beta, Delta, and Omicron variants of infection were enrolled to perform cross-neutralization tests using the original SARS-CoV-2 strain and VOCs variants (including Alpha, Beta, Gamma, Delta, and Omicron variants) and to assess the ability of NAbs against the SARS-CoV-2 variants. NAbs seroconversion occurred in 91.46% of patients (n = 82) in the first timepoint and in 89.29% of patients (n = 84) in the second detection point, and three kinds of NAbs kinetics curves were perceived. The NAbs levels in young patients had higher values than those in elder patients. The kinetics of disease duration was accompanied by an opposite trend in NAbs levels. Despite a declining NAbs response, NAbs activity was still detectable in a substantial proportion of recovered patients one year after discharge. Compared to the wild strain, the Omicron strain could lead to a 23.44-, 3.42-, 8.03-, and 2.57-fold reduction in neutralization capacity in “SAlpha”, “SBeta”, “SDelta”, and “SOmicron”, respectively, and the NAbs levels against the Omicron strain were significantly lower than those of the Beta and Delta variants. Remarkably, the NAbs activity of convalescent serum with Omicron strain infection was most obviously detectable against six SARS-CoV-2 strains in our study. The role of the vaccination history in NAbs levels further confirmed the previous study that reported vaccine-induced NAbs as the convincing protection mechanism against SARS-CoV-2. In conclusion, our findings highlighted the dynamics of the long-term immune responses after the disappearance of symptoms and revealed that NAbs levels varied among all types of convalescent patients with COVID-19 and that NAbs remained detectable for one year, which is reassuring in terms of protection against reinfection. Moreover, a moderate correlation between the duration of disease and Nabs titers was observed, whereas age was negatively correlated with Nabs titers. On the other hand, compared with other VOCs, the Omicron variant was able to escape the defenses of the immune system more significantly, and the convalescent serum infected with the Omicron variant played a critical part in protection against different SARS-CoV-2 variants. Recovery serum from individuals vaccinated with inactivated vaccine preceding infection with the Omicron strain had a high efficacy against the original strain and the VOCs variants, whereas the convalescent serum of persons vaccinated by inactivated vaccine prior to infection with the Delta variant was only potent against the wild-type strain.

/pdf/progress-of-the-covid-19-persistence-effectiveness-and-2liyccbs.pdf

Progress of the COVID-19: Persistence, Effectiveness, and Immune Escape of the Neutralizing Antibody in Convalescent Serum

Histamine is an important monoamine neurotransmitter that regulates multiple physiological activities in both vertebrates and invertebrates. Clearance and recycling of histamine are critical for sustaining histaminergic transmission. However, unlike other monoamine neurotransmitters, a histamine-specific transporter capable of clearing histamine from the synaptic cleft has not been identified. Here, through an in vitro histamine uptake screening, we identified an epithelial glia–expressing transporter, HisT (Histamine Transporter), that specifically transports histamine into cells. HisT misexpression in both pre- and postsynaptic neurons revealed a critical in vivo role for HisT in histamine transport and synaptic transmission. Last, we generated null hist alleles and demonstrated key physiological roles of HisT in maintaining histamine pools and sustaining visual transmission when the de novo synthesis of histamine synthesis was reduced. Our work identifies the first transporter that specifically recycles histamine and further indicates that the histamine clearance pathway may involve both the uptake-1 and uptake-2 transport systems.

https://www.science.org/doi/pdf/10.1126/sciadv.abq1780?download=true

Drosophila HisT is a specific histamine transporter that contributes to histamine recycling in glia

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-COV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of $10^{-17}$ M (approximately 6 copies/$\mu$L). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of $10^{-16}$ M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

Yufeng Liang

Papers

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

Progress of the COVID-19: Persistence, Effectiveness, and Immune Escape of the Neutralizing Antibody in Convalescent Serum

Drosophila HisT is a specific histamine transporter that contributes to histamine recycling in glia

Rapid and Accurate Detection of SARS-CoV-2 Mutations using a Cas12a-based Sensing Platform