Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

Ying Chi,Bingjie Zou,Lunbiao Cui,Minghao Zhou,Chuan Su,Zhiyang Shi,Bin Liu,Kangchen Zhao,Qian Bian,Xian Qi,Fengcai Zhu,Yiyue Ge

doi:10.1038/srep44924

Abstract

Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings.

Highlights

Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed
We developed a multiplex RT-loop-mediated isothermal amplification (LAMP) assay for simultaneous amplification of three prominent subtypes of influenza A viruses (H5, H7 subtypes of avian influenza A and 2009A/H1N1 viruses), and the multiplex reverse transcriptase LAMP (RT-LAMP) amplicons were further identified by cascade invasive reaction[32,33] and gold nanoparticle hybridization in separate target-specific detection tubes
The results showed that the optimal multiplex RT-LAMP reaction was obtained with primer sequences shown in Table 1 and the primer concentrations described in Materials and Methods

Summary

Introduction

Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. We developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). Molecular diagnostic methods based on nucleic acid amplification tests are more rapid and sensitive than traditional techniques including virus isolation and serological assays. RT-PCR (including real-time RT-PCR) is at present the powerful method for detection of influenza viruses[11,12,13,14,15] It requires bulky and expensive equipments, as well as highly skilled technicians, which make these methods not suitable for use in resource limited regions or for field use. RT-LAMP methods have been developed to detect various RNA pathogens including influenza viruses[19,20,21,22,23]

Methods

Results

Conclusion