key: cord-0799889-xrow2j71 authors: Sakai, Jun; Tarumoto, Norihito; Orihara, Yuta; Kawamura, Rieko; Kodana, Masahiro; Matsuzaki, Nanako; Matsumura, Ryo; Okane, Kana; Kawamura, Toru; Takeuchi, Shinichi; Imai, Kazuo; Murakami, Takashi; Maesaki, Shigefumi; Takuya, Maeda title: Evaluation of a high-speed but low-throughput RT-qPCR system for SARS-CoV-2 detection date: 2020-05-22 journal: J Hosp Infect DOI: 10.1016/j.jhin.2020.05.025 sha: 7612ea1717fe5101d7a4841d8df0b63810b9951d doc_id: 799889 cord_uid: xrow2j71 With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with COVID-19, a high-speed and convenient detection technology should be at the forefront of medical care worldwide. In this study, we evaluated the usefulness of GeneSoC®, a compact, high-speed reciprocal flow RT-qPCR system, for the detection of SARS-CoV-2. Our results support the use of this system for the rapid identification of SARS-CoV-2. This approach can contribute to the strategic selection of initial management strategies for patients with COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19), first detected in Wuhan, China, has become a global pandemic [1, 2] . With the rapid increase in the number of patients, SARS-CoV-2 detection is a key component of comprehensive medical care to reduce nosocomial infections, including transmission to health care workers. Accordingly, there is urgent demand for high-speed and practical diagnostic technologies [3] . Recently, a compact, reciprocal flow PCR system, GeneSoC ® (https://genesoc.jp/en/), has become available for specific gene amplification in a very short time (within 15 min) [4, 5] . The system has one heater for the RT reaction and two heaters for thermal cycling, with two micro-blowers at both flow ends for the high-speed shuttle of the PCR solution. It is also possible to monitor the fluorescence intensity of the cycling solution in real time (Fig. 1) . The aim of our study was to clarify the utility of GeneSoC ® for the rapid diagnosis of COVID-19 and to consider its relative advantages and disadvantages as a point-of-care test. Purified and quantified SARS-CoV-2 RNA isolated from an infected Japanese patient was provided by the National Institute of Infectious Diseases, Japan as a standard specimen for the molecular diagnosis of COVID-19. Analytical sensitivity was determined using ten-fold serially diluted standard RNA ranging from 1.0 × 10 6 to 1.0 × 10 0 copies/μL, stored at −30°C until required. High-speed RT-qPCR amplification using GeneSoC ® (Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan) was performed using SpeedSTAR HS DNA Polymerase (Takara Bio Inc., Shiga, Japan) and real-time detection of the amplification product was simultaneously monitored by the measurement of emission. The detailed composition of reaction reagents, together with the primers and probe targeting the specific sequence of the N gene of SARS-CoV-2 are listed in Table 1 [6]. Finally, 5 μL of the RNA template was added to the amplification mixture, and distilled water was added to a final volume of 20 μL. Amplification conditions were as follows: 42°C for 60 s, followed by 50 cycles of 96°C for 4 s and 58°C for 8 s. Amplification results were considered positive if an exponential amplification curve was generated that could be distinguished from that of the negative control. All reactions were performed in duplicate. Recent viral outbreaks that have spread rapidly across health infrastructures have presented major challenges in hospital settings [7] . RT-qPCR from nasopharyngeal swabs or sputum samples is still a core technology for determining the need for the hospitalization and 7 isolation of patients with COVID-19, but this method generates false negatives and is time consuming [8] . A history of exposure to patients with COVID-19 along with the characteristic CT findings of viral pneumonia strongly suggest COVID-19 and can provide a basis for patient management, even if a sample is negative by RT-qPCR [9, 10] . However, CT images are not specific to COVID-19, emphasizing the importance of RT-qPCR with extremely high diagnostic specificity for comprehensive diagnosis. The GeneSoC ® assay, which requires a short test period and is not labor-intensive, can contribute to the diagnosis and management of patients and thereby to the prevention of hospital-acquired infections. With respect to sensitivity, the limit of detection of the GeneSoC ® assay was 1.0 × 10 1 copies/reaction within 15 min. The main advantages of the platform are its simplicity and portability, its capacity for high-speed and real-time monitoring, and the use of a single disposable tip per analysis. GeneSoC ® can be easily transported and does not require highly equipped laboratories. However, it has some disadvantages. First, the analytical sensitivity was lower than that of the conventional RT-qPCR assay used as a reference. Second, the results could be influenced by the sampling procedure and timing, similar to conventional RT-qPCR. Third, the simplification and refinement of the RNA extraction procedure is required. Our findings support the use of GeneSoC ® for rapid, low-throughput detection for the strategic selection of initial management strategies for patients with COVID-19 and for the prevention of hospital-acquired infections. The authors declare no conflicts of interest. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China of Novel Coronavirus-Infected Pneumonia Exploring the reasons for healthcare workers infected with novel coronavirus disease 2019 (COVID-19) in China Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system The Role of Super-Spreaders in Infectious Disease Research use only 2019-novel coronavirus (2019-nCoV) real-time RT-PCR primer and probe information COVID-19): Role of Chest CT in Diagnosis and Management Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases The authors thank the Advanced Photonics and Biosensing Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology (AIST) for providing technical and scientific guidance on nucleonic amplification with GeneSoC ® .