First report on disinfection performance for SARS-CoV-2 in medical wastewater • Incomplete removal of SARS-CoV-2 RNA under WHO guideline • High level of DBPs when SARS-CoV-2 is completed removed.

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNAtriggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.

Fewm ethods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in as ingle test, whichi sakey measure to improve detection accuracy,a sa dopted by the gold standardR T-qPCR method. Developed here is aCRISPR/Cas9-mediated triple-line lateral flowa ssay(TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in as ingle strip test. This assayi sc haracterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNAstandards,showing asensitivity of 100 RNA copies per reaction (25 mL). Furthermore,d ual-gene analysis of 64 nasopharyngeal swab samples showed 100 %n egative predictive agreement and 97.14 %p ositive predictive agreement. This platform will provide am ore accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.

The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription− polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.

Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multisignal readout lateral flow immunoassay for Salmonella typhimurium (S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multifunctional target separation and enrichment, multisignal readout, and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labeled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88 × 104 CFU/mL S. typhi, and 3.75 × 103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2–4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88 × 104 to 1.88 × 107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum, and whole blood) indicated its potential application in real samples.

Rapid detection of highly contagious pathogens is the key to increasing the probability of survival and reducing infection rates. We developed a sensitive and quantitative lateral flow assay for detection of Ebola virus (EBOV) glycoprotein with a novel multifunctional nanosphere (RNs@Au) as a reporter. Each RNs@Au contains hundreds of quantum dots and dozens of Au nanoparticles and can achieve enhanced dual-signal readout (fluorescence signal for quantitative detection and colorimetric signal for visual detection). Antibody (Ab) and streptavidin (SA) were simultaneously modified onto the RNs@Au to label the target and act as signal enhancer. After the target was labeled by the Ab-RNs@Au-SA and captured on the test line, biotin-modified RNs@Au was used to amplify the dual signal by the reaction of SA with biotin. The assay enables naked-eye detection of 2 ng/mL glycoprotein within 20 min, and the quantitative detection limit is 0.18 ng/mL. Additionally, the assay has been successfully tested in field work for detecting EBOV in spiked urine, plasma, and tap water samples and is thus a promising candidate for early diagnosis of suspect infections in EBOV-stricken areas.

Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/ reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.

Background The longitudinal antigen-specific immunity in COVID-19 convalescents is crucial for long-term protection upon individual re-exposure to SARS-CoV-2, and even more pivotal for ultimately achieving population-level immunity. To better understand the features of immune memory in individuals with different disease severities at one year post-disease onset we conducted this cohort study. Methods We conducted a systematic antigen-specific immune evaluation in 101 COVID-19 convalescents, who had asymptomatic, mild, moderate, or severe disease, through two visits at months 6 and 12 post-disease onset. The SARS-CoV-2-specific antibodies, comprising NAb, IgG, and IgM, were assessed by mutually corroborated assays, i.e. neutralization, enzyme-linked immunosorbent assay (ELISA), and microparticle chemiluminescence immunoassay (MCLIA). Meanwhile, the T-cell memory against SARS-CoV-2 spike, membrane and nucleocapsid proteins was tested through enzyme-linked immunospot assay (ELISpot), intracellular cytokine staining (ICS), and tetramer staining-based flow cytometry, respectively. Results SARS-CoV-2-specific IgG antibodies, and also NAb can persist among over 95% COVID-19 convalescents from 6 months to 12 months after disease onset. At least 19/71 (26%) of COVID-19 convalescents (double positive in ELISA and MCLIA) had detectable circulating IgM antibody against SARS-CoV-2 at 12m post-disease onset. Notably, the percentages of convalescents with positive SARS-CoV-2-specific T-cell responses (at least one of the SARS-CoV-2 antigen S1, S2, M and N protein) were 71/76 (93%) and 67/73 (92%) at 6m and 12m, respectively. Furthermore, both antibody and T-cell memory levels of the convalescents were positively associated with their disease severity. Conclusions SARS-CoV-2-specific cellular and humoral immunities are durable at least until one year after disease onset.