Visual detection of human infection with influenza A (H7N9) virus by subtype-specific reverse transcription loop-mediated isothermal amplification with.

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Visual detection of human infection with influenza A (H7N9) virus by subtype-specific reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye K. Nie, X. Zhao, X. Ding, X.D. Li, S.M. Zou, J.F. Guo, D.Y. Wang, R.B. Gao, X.Y. Li, W.J. Huang, Y.L. Shu, X.J. Ma Clinical Microbiology and Infection Volume 19, Issue 8, Pages e372-e375 (August 2013) DOI: 10.1111/1469-0691.12263 Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1 Specificity and sensitivity analyses of subtype-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of human-infected influenza A H7N9 virus. (a,b) Colorimetric and turbidity analyses of the specificities of H7 (a) and N9 (b) subtype-specific RT-LAMP assays for the detection of influenza A H7N9 virus. RT-LAMP reaction mixture contained 1 μL Bst 2.0 DNA polymerase (8 U/μL, New England Biolabs, Ipswitch, MA, USA), 1 μL avian myeloblastosis virus reverse transcriptase (10 U/μL; Promega, Madison, WI, USA), 10 μL reaction mix (Deaou Biotechnology Co., Ltd., Guang Zhou, China), 1 μL hydroxynaphthol blue (3 mM, Lemongreen, Shanghai, China) and 0.8 μL of each primer (F3 and B3: 5 pmol/μL; BIP and FIP: 40 pmol/μL; Loop-F and Loop-B: 20 pmol/μL). The RT-LAMP reaction was incubated in a conventional water bath at 63°C for 60 min. Tube 1: No template control (NTC); 2: human pandemic influenza A H1N1 virus (H1N1); 3: human seasonal influenza A H3N2 virus; 4: human influenza A H5N1 virus; 5: human influenza H9N2 (G1) virus; 6: human influenza H9N2 (G9) virus; 7: human influenza B virus (HB); 8: human influenza A H7N9 virus. (c,d) Colorimetric and turbidity analyses of the sensitivities of H7 (c) and N9 (d) subtype-specific RT-LAMP assays for the detection of influenza A H7N9 virus and the comparison with qRT-PCR. Tubes 1–8: a serial ten-fold dilution (101–108) of extracted reference virus. The qRT-PCR results were defined as positive for Ct not higher than 38 for both haemagglutinin and neuraminidase genes and the corresponding Ct values are shown in parentheses. Clinical Microbiology and Infection 2013 19, e372-e375DOI: (10.1111/1469-0691.12263) Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions