Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device

Yiyue Ge,Zhiyang Shi,Kangchen Zhao,Hua Wang,Lunbiao Cui,Yuhua Qi,Yefei Zhu,Xiling Guo,Minghao Zhou,Xian Qi,Bin Wu

doi:10.1371/journal.pone.0069941

Abstract

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

Highlights

Since February, 2013, a previously unrecognized novel avianorigin influenza A (H7N9) virus associated with human deaths has emerged in China [1]
The lateral flow device (LFD) method which can provide a result within 5–10 min after completion of the RT-Loop-mediated isothermal amplification (LAMP) reaction was as sensitive as real-time turbidity detection (Figure 2)
We describe a sensitive RT-LAMP-LFD method for the specific detection of H7N9 virus for the first time

Summary

Introduction

Since February, 2013, a previously unrecognized novel avianorigin influenza A (H7N9) virus associated with human deaths has emerged in China [1]. Different strains of the novel H7N9 virus have been isolated from poultries, birds and the environmental specimens (Data from Global Initiative on Sharing All Influenza Data, GISAID), the source of infection in most of the human cases still remains to be determined. There has been no direct evidence of human-to-human transmission of this virus, the presence of mutations in the polymerase basic protein 2 (PB2) gene associated with improved replication of avian influenza viruses in mammals might indicate a certain propensity of the H7N9 virus to further adapt to humans [1,3]. The potential for the novel influenza A (H7N9) virus to spread among the human population is still exist

Methods

Results

Conclusion