Classification between Viable and Non-Viable Influenza A Virus and Differentiation of H1N1 and H3N2 Subtypes with a Propidium Monoazide xx (PMAxx) Based Loop-Mediated Isothermal Amplification (LAMP) Assay

Abstract

Influenza A virus outbreaks pose a significant burden to global health. However, detecting and subtyping the virus is challenging due to its high mutation rate, and it is difficult to distinguish viable viruses that cause infection from clinical samples. Here is a novel PMAxx-LAMP assay for the classification of viable and non-viable Influenza A virus together with differentiation between H1N1 and H3N2 subtypes. Our loop-mediated isothermal amplification (LAMP) primers displayed limits of detection of 10² copies/μL for both subtypes, high specificity, reproducibility, and good linearity with correlation coefficients of 0.9762 and 0.9901 for H1N1 and H3N2, respectively. PMAxx effectively distinguished between viable and non-viable viruses at an optimal concentration of 10 μM. In the LAMP method, the clinical specificity and sensitivity for the H1N1 subtype were both 100%, while for the H3N2 subtype, they were 94.44 and 92.31%, respectively. With the PMAxx-LAMP assay, the clinical specificity and sensitivity were 68% and 65% for the H1N1 subtype, and 81% and 69% for the H3N2 subtype, respectively. The reduced clinical specificity and sensitivity observed in the PMAxx-LAMP assay can be attributed to the presence of non-viable viruses, which are labeled with PMAxx and subsequently eliminated from positive samples. The PMAxx-LAMP offers potential benefits in determining the stage of infection, enabling early screening of patients, and shortening the window period for virus detection. The simplicity and efficiency of this assay make it suitable for use in resource limited settings and point-of-care (POCT) applications.

Keywords:

• Influenza A virus
• loop-mediated isothermal amplification (LAMP)
• propidium monoazide xx (PMAxx)
• subtype differentiation
• viable virus detection

Author contributions

Yue Su: conceptualization, methodology, validation, formal analysis, investigation, visualization, and writing. Lei Zhang: conceptualization, methodology, investigation, review, and editing. Shuting Tan: methodology, investigation, validation, writing, and editing. Qin Huang: methodology, review, and editing. Huiping Yang: resources and review. Xiangyu Jin: methodology, investigation, and review. Zeyin Mao: methodology, investigation, and review. Anni Deng: methodology, investigation, and review. Ming Pan and Guoliang Huang: conceptualization, investigation, resources, review, and supervision.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was funded by the National Key Research and Development Program of China, Grant Number 2018YFA0704000; the Sichuan Science and Technology Program, Grant Numbers 2021YFQ0060 and 2022YFS0637; the National Natural Science Foundation of China, Grant Numbers 62375148, 61927819, 81827808, and 62105177; Vanke Special Fund for Public Health and Health Discipline Development, Tsinghua University, Grant Number 2022Z82WKJ002; the Tsinghua University Spring Breeze Fund, Grant Number 2020Z99CFG011; the Beijing Lab Foundation, and the Tsinghua Autonomous Research Foundation, Grant Numbers 20194180031, 20201080058, 20201080510; and Tsinghua Laboratory Innovation Fund, Grant Number 100020019.