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. Electronic supplementary material The online version of this article (10.1186/s12879-019-4277-8) contains supplementary material, which is available to authorized users.

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2

“…This intact viral RNA was ten-fold serially diluted to 10 −9 and processed for parallel testing with RT-LAMP assay and qRT-PCR. The serially diluted RNA (2 μL) was mixed with the optimized master mix and subjected to RT-LAMP assay as described previously [32].…”

“…The specificity and sensitivity of this method is generally comparable to those of the conventional PCR and qRT-PCR. The LAMP assays merged with reverse transcription steps have been developed for the detection of RNA viruses, including SARS-CoV, MERS-CoV, influenza, and other respiratory viruses [26][27][28][29][30][31][32].…”

2019 Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics

“…Table 2. Comparison between PCR and loop-mediated isothermal amplification (LAMP) reactions [37,[40][41][42].…”

“…For virus detection, for example, influenza [40] or human norovirus, LAMP assay offers one-step detection [41]. Sample preparation steps are simplified.…”

Establishment and Application of Reverse Transcription Recombinase-aided Amplification as a Rapid Detection Method for SARS-COV-2

“…SARS-CoV-2 is transmitted mainly by air droplets, thus making it easier for the virus to spread through close contact [23]. When a person is infected with SARS-CoV-2 and shows symptoms, the viral load of the nasopharynx increases in a short duration, which makes the carrier as a virus dissemination center [24]. Early detection, early isolation, and treatment are of great signi cance to prevent the transmission of the virus.…”