key: cord-0751364-uk4bjr0y authors: Wang, Ji; Cai, Kun; He, Xiaozhou; Shen, Xinxin; Wang, Jinrong; Liu, Jun; Xu, Junqiang; Qiu, Feng; Lei, Wenwen; Cui, Lunbiao; Ge, Yiyue; Wu, Tao; Zhang, Yanjun; Yan, Hao; Chen, Yin; Yu, Jiezhong; Ma, Xiaojun; Shi, Hua; Zhang, Ruiqing; Li, Xinna; Gao, Yuan; Niu, Peihua; Tan, Wenjie; Wu, Guizhen; Jiang, Yongzhong; Xu, Wenbo; Ma, Xuejun title: A multiple center clinical evaluation of an ultra-fast single-tube assay for SARS-CoV-2 RNA date: 2020-05-15 journal: Clin Microbiol Infect DOI: 10.1016/j.cmi.2020.05.007 sha: ffc6d7874122f92a88f58780c22da3b5e87a01ff doc_id: 751364 cord_uid: uk4bjr0y OBJECTIVES: To evaluate the performance of an ultra-fast single-tube nucleic acid isothermal amplification detection assay for SARS-CoV-2 RNA using clinical samples from multiple centers. METHODS: A reverse transcription recombinase-aided amplification (RT-RAA) assay for SARS-CoV-2 was conducted within 15minutesat39°C with portable instruments after addition of extracted RNA. The clinical performance of RT-RAA assay was evaluated using 947 clinical samples from five institutions in four regions of China, and the approved commercial real-time fluorescent RT-PCR (qRT-PCR) kits were used for parallel detection. The sensitivity and specificity of RT-RAA were compared and analyzed. RESULTS: The RT-RAA test results of 926 samples were consistent with those of qRT-PCR (330 were positive, 596 were negative) and 21 were inconsistent. The sensitivity and specificity of RT-RAA was 97.63% [330/338, 95% confidence interval (CI): 95.21 to 98.90] and 97.87% (596/609, 95% CI: 96.28 to 98.81), respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 96.21% (330/343, 95% CI: 93.45 to 97.88), and 98.68% (596/604, 95% CI: 97.30 to 99.38), respectively. The total coincidence rate was 97.78% (926/947, 95% CI: 96.80 to 98.70) and the Kappa was 0.952 (P <0.05). CONCLUSION: With comparable sensitivity and specificity to the commercial qRT-PCR kits, RT-RAA assay for SARS-CoV-2 exhibited distinctive advantages of simplicity and rapidity in terms of operation and turn-around time. Hubei CDC. The specimens that can be processed within 24 hours were 127 stored at 4 , and those that cannot were stored at ℃ -70 or below. All ℃ 128 aspects of the study were performed in accordance with national ethics 129 regulations and approved by the Institutional Review Boards of local 130 CDCs and hospital mentioned above. We downloaded the whole SARS-CoV-2genome sequences available 134 from the Global Initiative on Sharing All Influenza Data (GISAID). We the target gene to screen the best set of primers and probes, the 142 recombinant plasmid was diluted to 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 2 and 1 143 copies/test, and non-ribozyme water was used as a negative control. We 144 then used the specificity evaluation panel to further explore the 145 specificity of RT-RAA kit for SARS-CoV-2, and finally two primers and 146 one probe with the highest amplification efficiency were chosen. 147 According to the instructions recommended by the manufacturer, the 149 total RNA was extracted from 200 µL of sample preservation solution 150 using Tian Long automatic extraction kit (Tian Long, Soochow, China). 151 The nucleic acid was eluted in 50 µL of nuclease free water and stored at 152 -80 °C until use. We freeze-dried the selected primers and probe to the reaction unit 155 tube, and made a single-tube SARS-CoV-2 nucleic acid isothermal 156 amplification rapid detection kit (RT-RAA kit). A panel of diluted recombinant plasmids (10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 2, 204 and 1 copies / test) were tested to ascertain the endpoint dilution. As 205 shown in Fig. 1 , the sensitivity of RT-RAA kit was 2 copies per reaction. Totally, 947 samples were detected by RT-RAA and qRT-PCR (Table 2 ) 221 (Supplementary Table S2 100 (4/4) 100 (4/4) stool 0 (0/2) 0 (0/2) whole blood 0 (0/1) 0 (0/1) Table 3 Positive rates of RT-RAA and qRT-PCR in different sample types. cross-contamination while has lower cost than that of qRT-PCR kits. 295 The proposed RT-RAA kit had a sensitivity of 2 copies per reaction 296 using DNA template, which might not reflect the true sensitivity using 297 In conclusion, our results strongly demonstrate RT-RAA kit for 319 SARS-CoV-2 shows comparable sensitivity and specificity to the 320 commercial qRT-PCR kits, and exhibits distinctive advantages of 321 simplicity and rapidity over qRT-PCR kits in terms of operation and 322 turn-around time. This RT-RAA kit is therefore a promising tool to be 323 potentially used in the resource-limited scenes or remote areas such as 324 community fever clinics and mobile laboratories. 325 A novel coronavirus 345 outbreak of global health concern Analyzing the 348 epidemiological outbreak of COVID-19: A visual exploratory data 349 analysis approach Severe acute respiratory syndrome-related coronavirus: The 352 species and its viruses-a statement of the Coronavirus Study Group Development and evaluation of recombinase-aided amplification assays 356 incorporating competitive internal controls for detection of human 357 adenovirus serotypes 3 and 7 A rapid and 359 sensitive recombinase aided amplification assay incorporating 360 competitive internal control to detect Bordetella pertussis using the DNA 361 obtained by boiling Development 364 of a duplex reverse transcription recombinase-aided amplification assay 365 for respiratory syncytial virus incorporating an internal control Development 368 of a reverse transcription recombinase-aided amplification assay for the 369 detection of coxsackievirus A10 and coxsackievirus A6 RNA Applicability of duplex real time and lateral flow strip 373 reverse-transcription recombinase aided amplification assays for the 374 detection of Enterovirus 71 and Coxsackievirus A16 A rapid and 377 sensitive recombinase aided amplification assay to detect hepatitis B 378 virus without DNA extraction Technical 385 Guide for Laboratory Detection of pneumonia infected by novel 386 coronavirus (fourth Edition)/pneumonia Prevention and Control 387 Program for novel coronavirus infection COVID-19): Paving the Road for Rapid Detection and Point-of-Care 393 Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an 396 Outbreak of Pneumonia Detection of 2019 novel coronavirus (2019-nCoV) by real-time 399 RT-PCR Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2 Clinical features 404 of patients infected with 2019 novel coronavirus in Wuhan, China. 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