Use of high-resolution fluorescence in situ hybridization for fast and robust detection of SARS-CoV-2 RNAs

• Published in: Scientific reports Vol. 14; no. 1; pp. 20906 - 13
• Main Authors: Hu, Jiapei, Hu, Jiayi, Jin, Li, Hu, Dakang, Nicholls, Philip K., Wang, Tao, Ren, Yonglin, Hu, Dailun, Ma, Bin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08-09-2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647; 631/208; 631/326; 692/308; 692/4017; Cell culture; Coronavirus Envelope Proteins - genetics; Coronavirus Nucleocapsid Proteins - genetics; COVID-19; COVID-19 - diagnosis; COVID-19 - genetics; COVID-19 - virology; Deoxyribonucleic acid; DNA; DNA probes; DNA viruses; Drinking water; Fluorescence; Fluorescence in situ hybridization; HEK293 Cells; Humanities and Social Sciences; Humans; Hybridization; In Situ Hybridization, Fluorescence - methods; Infection; Lymphocytes T; mRNA; multidisciplinary; Phosphoproteins - genetics; Plasmids; Public health; RNA virus; RNA viruses; RNA, Messenger - genetics; RNA, Viral - genetics; Saliva; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Science; Science (multidisciplinary); Severe acute respiratory syndrome coronavirus 2; Sputum; Transfection; COVID-19; Infection; SARS-CoV-2; mRNA; RNA virus; Fluorescence in situ hybridization
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-70980-9

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (−) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.