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

PLoS One. 2013 Aug 1;8(8):e69941. doi: 10.1371/journal.pone.0069941. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dogs
  • Influenza A Virus, H1N1 Subtype / genetics
  • Influenza A Virus, H1N1 Subtype / isolation & purification
  • Influenza A Virus, H3N2 Subtype / genetics
  • Influenza A Virus, H3N2 Subtype / isolation & purification
  • Influenza A Virus, H7N9 Subtype / genetics*
  • Influenza A Virus, H7N9 Subtype / isolation & purification*
  • Laboratories
  • Madin Darby Canine Kidney Cells
  • Reverse Transcriptase Polymerase Chain Reaction / instrumentation*
  • Temperature
  • Time Factors

Grants and funding

This study was supported in part by the National Major Science & Technology Projects for Infectious Disease Control and Prevention (2013ZX10004103-006), the Jiangsu Province Health Development Project with Science and Education (ZX201109), the Jiangsu Province Key Medical Talent Foundation (RC2011191, RC2011084, JKRC2011001), and the Science and Technology Pillar program of Jiangsu Province (BE2011796). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.