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Development of a high-sensitivity and short-duration fluorescence in situ hybridization method for viral mRNA detection in HEK 293T cells.
Hu, Dailun; Wang, Tao; Uddin, Jasim; Greene, Wayne K; Hu, Dakang; Ma, Bin.
  • Hu D; Clinical College, Hebei Medical University, Shijiazhuang, China.
  • Wang T; Telethon Kids Institute, Perth Children's Hospital, Nedlands, WA, Australia.
  • Uddin J; Medical School, University of Western Australia, Nedlands, WA, Australia.
  • Greene WK; School of Veterinary Medicine, Murdoch University, Murdoch, WA, Australia.
  • Hu D; Medical, Molecular and Forensic Sciences, Murdoch University, Murdoch, WA, Australia.
  • Ma B; Department of Laboratory Medicine, Taizhou Municipal Hospital, Taizhou, China.
Front Cell Infect Microbiol ; 12: 960938, 2022.
Статья в английский | MEDLINE | ID: covidwho-2154694
ABSTRACT
Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.
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Полный текст: Имеется в наличии Коллекция: Международные базы данных база данных: MEDLINE Основная тема: SARS-CoV-2 / COVID-19 Тип исследования: Диагностическое исследование / Прогностическое исследование Пределы темы: Люди Язык: английский Журнал: Front Cell Infect Microbiol Год: 2022 Тип: Статья Аффилированная страна: Fcimb.2022.960938

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Полный текст: Имеется в наличии Коллекция: Международные базы данных база данных: MEDLINE Основная тема: SARS-CoV-2 / COVID-19 Тип исследования: Диагностическое исследование / Прогностическое исследование Пределы темы: Люди Язык: английский Журнал: Front Cell Infect Microbiol Год: 2022 Тип: Статья Аффилированная страна: Fcimb.2022.960938