To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and control the spread of coronavirus disease (COVID‐19), there is an urgent need for highly sensitive on‐site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas)‐based molecular diagnostic method was developed for this purpose. Here, a CRISPR system‐mediated lateral flow assay (LFA) for SARS‐CoV‐2 was established based on multienzyme isothermal rapid amplification, CRISPR‐Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/μl in both fluorescence‐ and immunochromatography‐based assays. To enhance the quality control of the CRISPR‐based LFA method, glyceraldehyde‐3‐phosphate dehydrogenase was detected as a reference using a triple‐line strip design in a lateral flow strip. In total, 52 COVID‐19‐positive and 101 COVID‐19‐negative clinical samples examined by reverse transcription polymerase chain reaction (RT‐PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT‐PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR‐mediated LFA and provides a crucial tool for on‐site detection of SARS‐CoV‐2.
Human adenoviruses (HAdVs) can cause acute respiratory diseases (ARDs) worldwide, and HAdV-55 is a reemergent pathogen in recent years. In the study, we investigated an outbreak of ARD at a school due to HAdV-55 in Beijing, China, during the early outbreak of coronavirus disease 2019 . The epidemic prevention team was dispatched to the school to collect epidemiologic data and nasopharyngeal samples. Then, real-time reverse transcription polymerase chain reaction (PCR) and multiplex PCR assays were used to detect severe acute respiratory syndrome coronavirus 2 and other respiratory pathogens, respectively.One representative HAdV-55 isolate was selected and submitted for whole-genome sequencing using a MiSeq system and the whole-genome phylogenetic tree was conducted based on the maximum likelihood method. The outbreak lasted from January 27 to February 6, 2020, and 108 students developed fever, among whom 60 (55.56%) cases were diagnosed with HAdV-55 infection in the laboratory using realtime PCR and 56 cases were hospitalized. All the confirmed cases had a fever and 11 cases (18.33%) presented with a fever above 39°C. Other main clinical symptoms included sore throat (43.33%) and headache (43.33%). We obtained and assembled the full genome of one isolate, BJ-446, with 34 761 nucleotides in length. HAdV-55 isolate BJ-446 was 99.85% identical to strain QS-DLL, which was the first HAdV-55 strain in China isolated from an ARD outbreak in Shanxi in 2006. One and four amino acid mutations were observed in the hexon gene and the coding region of L2 pV 40.1 kDa protein, respectively. We identified the first HAdV-55 infection associated with the ARD outbreak in Beijing since the emergence of COVID-19. The study suggests that improved surveillance of HAdV is needed, although COVID-19 is still prevalent in the world.
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