著者

Kazuya Shirato Akinori Azumano Tatsuko Nakao Daisuke Hagihara Manabu Ishida Kanji Tamai Kouji Yamazaki Miyuki Kawase Yoshiharu Okamoto Shigehisa Kawakami Naonori Okada Kazuko Fukushima Kensuke Nakajima Shutoku Matsuyama

出版者

国立感染症研究所 Japanese Journal of Infectious Diseases 編集委員会

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

vol.68, no.3, pp.256-258, 2015 (Released:2015-05-20)

参考文献数

18

被引用文献数

3 14

著者

Kenichi Komabayashi Yohei Matoba Junji Seto Yoko Ikeda Waka Tanaka Yoko Aoki Tatsuya Ikeda Yoko Matsuzaki Tsutomu Itagaki Kazuya Shirato Katsumi Mizuta

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

pp.JJID.2020.776, (Released:2020-11-30)

参考文献数

31

被引用文献数

7

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Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1×104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.

著者

Kazuya Shirato Naganori Nao Harutaka Katano Ikuyo Takayama Shinji Saito Fumihiro Kato Hiroshi Katoh Masafumi Sakata Yuichiro Nakatsu Yoshio Mori Tsutomu Kageyama Shutoku Matsuyama Makoto Takeda

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

pp.JJID.2020.061, (Released:2020-02-18)

参考文献数

7

被引用文献数

339

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At the end of 2019, pneumonia caused by novel coronavirus 2019 (nCoV) emerged in Wuhan city, China. Many airline travelers moved between Wuhan and Japan at that time, suggesting that Japan is at high risk of invasion by the virus. Diagnostic systems for 2019-nCoV were developed with urgency. Two nested RT–PCR assays and two real-time RT–PCR assays were adapted to local Japanese conditions. As of 8 February 2020, the assays developed have successfully detected 25 positive cases of infection in Japan.

著者

Yuki Higashimoto Masaru Ihira Yoshiki Kawamura Masato Inaba Kazuya Shirato Tadaki Suzuki Hideki Hasegawa Tsutomu Kageyama Yohei Doi Tadayoshi Hata Tetsushi Yoshikawa

出版者

Fujita Medical Society

雑誌

Fujita Medical Journal (ISSN:21897247)

巻号頁・発行日

pp.2022-003, (Released:2022-07-22)

参考文献数

38

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Objectives: To establish a point-of-care test for coronavirus disease 2019 (COVID-19), we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA.Methods: We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture, except for the primers, is dried and immobilized inside the tube lid.Results: To determine the specificity of the kit, 22 viruses associated with respiratory infections, including SARS-CoV-2, were tested. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. No LAMP product was detected in reactions performed with RNA from any pathogens other than SARS-CoV-2. After completing an initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Of the 24 samples, 19 (79.2%) were determined by real-time RT-PCR analysis as being positive for SARS-CoV-2 RNA. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive values of the Loopamp 2019-CoV-2 detection reagent kit were 78.9%, 100%, 100%, and 55.6%, respectively.Conclusions: The dry LAMP method for detecting SARS-CoV-2 RNA is fast and easy to use, and its reagents can be stored at 4°C, solving the cold chain problem; thus, it represents a promising tool for COVID-19 diagnosis in developing countries.

著者

Kazuya Shirato Naganori Nao Shutoku Matsuyama Tsutomu Kageyama

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

vol.73, no.3, pp.181-186, 2020-05-29 (Released:2020-05-22)

参考文献数

21

被引用文献数

13 26

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Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.

著者

Kiyoko Okamaoto Kazuya Shirato NagaNaganori Nao Shinji Saito Tsutomu Kageyama Hideki Hasegawa Tadaki Suzuki Shutoku Matsuyama Makoto Takeda

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

pp.JJID.2020.108, (Released:2020-04-30)

参考文献数

11

被引用文献数

13 33

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The COVID-19 outbreak caused by SARS-CoV-2 in Wuhan (China) in December 2019 is currently spreading rapidly and globally. We recently reported a laboratory diagnostic protocol for SARS-CoV-2 based on real-time RT-PCR assays using two primer sets, N and N2. On 30–31 January 2020, the protocol and reagents for these assays were distributed to local public health institutes and quarantine depots in Japan, and nationwide, SARS-CoV-2 diagnostic testing was started. For further validation, the assays were compared with the commercially available kits using SARS CoV-2 viral RNA and the clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LN S&W-E) assay was highly sensitive for SARS-CoV-2, as was the N2 set, and both assays had perfectly consistent results with the clinical specimens. While the LM S&W-E set targets the highly conserved region of the E gene in SARS-CoV and SARS-CoV-2, the N2 set was designed to target the unique region in the SARS-CoV-2 N gene. Thus, the N2 set has high specificity and sensitivity for SARS-CoV-2 detection. These indicate that the protocol using the N and N2 sets is comparable to commercially available kits and is reliable for the laboratory diagnosis of COVID-19.

著者

Kazuya Shirato Naganori Nao Shutoku Matsuyama Makoto Takeda Tsutomu Kageyama

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

pp.JJID.2020.324, (Released:2020-06-30)

参考文献数

15

被引用文献数

14

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The disease caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in December 2019 is currently spreading rapidly worldwide. SARS-CoV-2 is usually detected via real-time RT-PCR. However, as institutions/hospitals deal with increasing numbers of specimens, a simpler detection system is required. Here, we present an ultra-rapid, real-time RT-PCR assay for SARS-CoV-2 using the PCR1100 device. Although this tests only one specimen at any one time, the amplification period is <20 min, with maintenance of the sensitivity and specificity of conventional real-time RT-PCR performed using large instruments. The method will be very helpful if SARS-CoV-2 testing is required a few times daily, for example to confirm virus-free status prior to discharge.

著者

Kazuya Shirato Naganori Nao Shutoku Matsuyama Tsutomu Kageyama

出版者

National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

雑誌

Japanese Journal of Infectious Diseases (ISSN:13446304)

巻号頁・発行日

pp.JJID.2019.400, (Released:2019-12-25)

参考文献数

21

被引用文献数

26

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Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used and are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These are disadvantages for rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established: a multiplex assay for upE and ORF1a running on the mobile PCR1100 device. As few as five copies of MERS-CoV RNA can be detected within 20 min using the WHO standard assays with similar sensitivity and specificity to those of a conventional real-time PCR instrument such as the LightCyler, enabling timely intervention to control MERS-CoV infection.