Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform

Abstract

Background: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans.

Methods: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses).

Results: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%).

Conclusions: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Keywords: Avian influenza; Colorimetric visualization; Multiplex detection; RT-LAMP; Seasonal influenza.

Fig. 1

Highly conserved regions of HA (Influenza A H1, H3, H5, H7) and NA (Influenza B viruses) genes used to design RT-LAMP primers. Nucleotide sequences from conserved regions within HA gene of Influenza A viruses (H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) and NA gene of Influenza B viruses were obtained using CLC Main workbench 7 (version 7.6.4.). a Primer mapping (primer conversation average). These primers were designed to roughly 200 bp of this conserved sequence. b Sequence homology among target regions. The primer region sequence distance of Influenza virus B was calculated by comparing with NA subtype gene. H1, H3, H5, and H7 of Flu A were calculated for each primer region sequence distance of HA. For more information on Influenza virus RT-LAMP primer sequences, see Table 1

Fig. 2

Specificity of influenza RT-LAMP. To evaluate specificity of each RT-LAMP primer set, RT-LAMP was performed to assess cross-reactivity using (a) individual and (b) mixed influenza virus samples. RT-LAMP reactions were conducted by incubation at 65 °C for 1 h. Positive RT-LAMP reactions resulted in a color change from pink to yellow. For confirmation of colorimetric RT-LAMP, see image of agarose gel electrophoresis in Additional file 1: Figure S1. B-Vic: B/Brisbane/60/2008 (Victoria lineage); B-Yam: B/Phuket/3073/2013 (Yamagata lineage); hH1N1: A/California/04/2009; H3N2: A/Perth/16/2009; aH5N1: A/Em/Korea/w149/2006; hH5N6 vac: A/Sichuan/26221/2014; aH5N8: A/Em/Korea/w468/2014; aH5N8 vac: A/gyrfalcon/Washington/41088–6/2014; hH7N9: A/Anhui/1/2013; N.C.: Negative control (D.W.)

Fig. 3

Specificity of Influenza RT-LAMP compared to other subtypes of influenza viruses. RT-LAMP reactions were performed using RNA from H2N3, H4N4, H6N2, H8N6, H9N2, H10N7, H11N9, and H12N5 and each primer set (B, A/H1, A/H3, A/H5, A/H7) to evaluate whether this RT-LAMP assay could cross-react with other influenza virus subtypes. RNAs from other influenza virus subtypes were confirmed using 1-step RT-PCR. Please see Additional file 1: Table S1 for additional details of one-step RT-PCR primer sequences