ABSTRACT
Tracking genomic sequences as microbial biomarkers in wastewater has been used to determine community prevalence of infectious diseases, contributing to public health surveillance programs. Here we report upon a low-cost, rapid, and user-friendly paper microfluidic platform for SARS-CoV-2 and influenza detection, using a loop-mediated isothermal amplification (LAMP), with the signal read simply using a mobile phone camera. Sample-to-answer results were collected in < 1.5 hours providing rapid detection of SARS-CoV-2 and influenza viruses in wastewater, with a detection limit of < 20 copies µL-1. The device was subsequently used for on-site testing of SARS-CoV-2 in wastewater samples from four quarantine hotels at London Heathrow Airport, showing comparable results to those obtained using a gold-standard polymerase chain reaction assay, as reference. Our sensing platform, which enables rapid and localized wastewater surveillance and does not require the sample to be sent to a centralized laboratory, is potentially an important public health tool for a wide variety of future applications, in community settings.
Subject(s)
Communicable DiseasesABSTRACT
In type II CRISPR systems, the guide RNA (gRNA) consists of a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA) which interacts directly with Cas9 and is essential to its guided DNA targeting function. Though tracrRNAs are diverse in sequences and structures across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for particular Cas9 has not been studied adequately. Here, we revealed the high programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9. By reprogramming the crRNA-tracrRNA hybridized sequence, reprogrammed tracrRNAs can repurpose various RNAs as crRNAs to trigger CRISPR function. We showed that the engineered crRNA-tracrRNA pairs enable design of orthogonal cellular computing devices and hijacking of endogenous RNAs as crRNAs. We next designed novel RNA sensors that can monitor the transcriptional activity of specific genes on the host genome and detect SARS-CoV-2 RNA in vitro . The engineering potential of crRNA-tracrRNA interaction has therefore redefined the capabilities of CRISPR/Cas9 system.
ABSTRACT
Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has reached 28 million cases worldwide in eight months. The serological detection of antibodies against the virus will play a pivotal role in complementing molecular tests to improve diagnostic accuracy, contact tracing, vaccine efficacy testing and seroprevalence surveillance. Here, we aimed first to evaluate a lateral flow assays ability to identify specific IgM and IgG antibodies against SARS-CoV-2 and second, to report the seroprevalence of these antibodies among health care workers and healthy volunteer blood donors in Panama. We recruited study participants between April 30th and July 7th, 2020. For the test validation and performance evaluation, we analyzed serum samples from participants with clinical symptoms and confirmed positive RT-PCR for SARS-CoV-2, participants with other confirmed infectious diseases, and a set of pre-pandemic serum samples. We used two by two table analysis to determine the test sensitivity and specificity as well as the kappa agreement value with a 95% confidence interval. Then, we used the lateral flow assay to determine seroprevalence among serum samples from COVID-19 patients, potentially exposed health care workers, and healthy volunteer donors. Our results show this assay reached a positive percent agreement of 97.2% (95% CI 84.2-100.0%) for detecting both IgM and IgG. The assay showed a kappa of 0.898 (95%CI 0.811-0.985) and 0.918 (95% CI 0.839-0.997) for IgM and IgG, respectively. The evaluation of serum samples from hospitalized COVID-19 patients indicates a correlation between test sensitivity and the number of days since symptom onset; the highest positive percent agreement (87% (95% CI 67.0-96.3%)) was observed at [≥]15 days post-symptom onset. We found an overall antibody seroprevalence of 11.6% (95% CI 8.5-15.8%) among both health care workers and healthy blood donors. Our findings suggest this lateral flow assay could contribute significantly to implementing seroprevalence testing in locations with active community transmission of SARS-CoV-2.