ABSTRACT
COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 µL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.
Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections , Pandemics , Pneumonia, Viral , Betacoronavirus/genetics , Biological Assay , COVID-19 , COVID-19 Testing , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Humans , Nucleic Acid Amplification Techniques , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
The corrected legend is given below.
ABSTRACT
Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.
Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , RNA, Viral/metabolism , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing , Humans , Limit of Detection , SARS-CoV-2/isolation & purificationABSTRACT
INTRODUCTION: COVID-19 is a newly emerging life-threatening respiratory disease caused by a newly identified coronavirus SARS-CoV-2. METHODOLOGY: We included 28 COVID-19 patients admitted to Nantong Third Hospital from January 23 to February 26, 2020. SARS-CoV-2 infection was confirmed using real-time RT-PCR. The demographic, epidemiological, clinical, laboratory parameters were obtained from each patient. RESULTS: The vast majority (71.4%) of confirmed COVID-19 patients were brought in from outside of the city, and all others had contact history with these confirmed cases. The median age of patients was 50 years old and half had underlying diseases. The most common symptoms at the onset of illness were fever (96.4%), cough (67.9%), and chilly (28.6%), and 75.0% patients had two or more symptoms. Increased erythrocyte sedimentation rate, serum ferritin and C-reactive protein levels, and reduced absolute counts of total lymphocytes and T lymphocyte subsets were observed among the patients. The vast majority (85.7%) of patients showed bilateral or unilateral pneumonia, and three symptomatic patients and one asymptomatic case did not show abnormalities in their CT image. Among the 28 admitted patients, 24 were discharged as of February 26, 2020, with an average hospital stay of 14.96 (±4.27) days, which was not significantly associated with the interval between the onset of symptoms and admission. CONCLUSIONS: In the absence of specific antiviral drugs or a vaccine, quarantine or isolation is the most effective intervention strategy for preventing the spread of the virus. Adequate supportive medical care is crucial for good prognosis of COVID-19 patients.