Combination of multiplex reverse-transcription loop-mediated isothermal amplification with an immunochromatographic strip for subtyping influenza A virus

• Multiplex RT-LAMP for HA and M genes was developed for subtyping influenza A virus.
• Multiplex RT-LAMP amplicons were simply analyzed by the colorimetric ICS detection.
• Multiplex RT-LAMP (40 min) and ICS detection (15 min) could be complete in 55 min.
• Detection sensitivity for the multiplex RT-LAMP and ICS was 10 copies of viral RNA.
• Our methodology provides simple, rapid genetic analysis platform for viral detection.

Considering the fatal human victims and economic loss by the annual epidemic influenza virus, the development of a rapid and convenient genetic analysis methodology is demanding for timely on-site pathogen detection. In this study, we utilized reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for multiplex target gene amplification, and the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus. Through the optimized primer design, reaction temperature and time, and concentration of enzymes (Bst DNA polymerase and AMV reverse transcriptase) and dNTP, the HA (H1, H3, and H5 gene) and conserved M gene were amplified. The ICS contains two test lines in addition to a control line in order to detect the presence of the HA and M gene, thereby informing us of influenza virus A type as well as its subtype (H1N1, H3N2, and H5N1). The combination of the multiplex RT-LAMP with the ICS could be complete in 40 min and the pathotyping and subtyping of influenza A virus were performed even with 10 copies of viral RNA templates. Moreover, the subtyping of clinical samples, which were obtained from patients infected by influenza A virus was successfully confirmed using the multiplex RT-LAMP and ICS techniques, showing great feasibility of our methodology for real sample analysis with high speed, simplicity and sensitivity.

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