ABSTRACT
The COVID-19 pandemic has challenged testing capacity worldwide. The mass testing needed to stop the spread of the virus requires new molecular diagnostic tests that are faster and with reduced equipment requirement, but as sensitive as the current gold standard protocols based on polymerase chain reaction. We developed a fast (25-35 minutes) molecular test using reverse transcription recombinase polymerase amplification for simultaneous detection of two conserved regions of the virus, targeting the E and RdRP genes. The diagnostic platform offers two complementary detection methods: real-time fluorescence or visual dipstick. The analytical sensitivity of the test by real-time fluorescence was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick readout was 130 (95% CI: 82-500) RNA copies per reaction. The assay showed high specificity with both detection methods when tested against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing, with simple or equipment-free incubation methods and a user-friendly prototype smartphone application was proposed with data capture and connectivity. This ultrasensitive molecular test offers valuable advantages with a swift time-to-result and it requires minimal laboratory equipment compared to current gold standard assays. These features render this diagnostic platform more suitable for decentralised molecular testing.
Subject(s)
COVID-19ABSTRACT
Introduction The Coronavirus (COVID-19) Pandemic has caused significant global mortality and impacted lives around the world. Virus Watch aims to provide evidence on which public health approaches are most likely to be effective in reducing transmission and impact of the virus, and will investigate community incidence, symptom profiles, and transmission of COVID-19 in relation to population movement and behaviours. Methods and analysis Virus Watch is a household community cohort study of acute respiratory infections in England & Wales and will run from June 2020 to August 2021. The study aims to recruit 50,000 people, including 12,500 from minority ethnic backgrounds, for an online survey cohort and monthly antibody testing using home finger prick kits. Nested within this larger study will be a sub-cohort of 10,000 individuals, including 3,000 people from minority ethnic backgrounds. This cohort of 10,000 people will have full blood serology taken between October 2020 and January 2021 and repeat serology between May 2021 and August 2021. Participants will also post self-administered nasal swabs for PCR assays of SARS-CoV-2 and will follow one of three different PCR testing schedules based upon symptoms. Ethics and dissemination This study has been approved by the Hampstead NHS Health Research Authority Ethics Committee. Ethics approval number – 20/HRA/2320. We are monitoring participant queries and using these to refine methodology where necessary, and are providing summaries and policy briefings of our preliminary findings to inform public health action by working through our partnerships with our study advisory group, Public Health England, NHS and Government Scientific Advisory panels. Strengths and limitations of this study Virus Watch is a large national household community cohort study of the occurrence and risk factors for COVID-19 infection that aims to recruit 50,000 people, including 12,500 from minority ethnic backgrounds. Virus Watch is designed to estimate incidence of PCR confirmed COVID-19 in those with respiratory and non-respiratory presentations and the incidence of hospitalisation among PCR confirmed COVID-19 cases. Virus Watch will measure effectiveness and impact of recommended COVID-19 control measures including testing, isolation, social distancing, respiratory and hand hygiene measures on risk of respiratory infection. Only households with a lead householder able to speak English were able to take part in the study up until March 2021. Only households of up to six people were eligible for inclusion and they were also required to have access to an internet connection. These restrictions will limit the generalisability to large or multigenerational households, and those without access to the internet.