Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay

Yuki Nakaya,Makoto Fujimaki,Takashi Fukuda,Masato Yasuura,Hiroki Ashiba

doi:10.1186/s12879-020-05317-8

Abstract

BackgroundThe polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation.MethodsIAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays.ResultsA long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3′ termini of each genomic segment and subsequent qPCR of the 5′ regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066).ConclusionsThis study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Highlights

The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity
The results indicated that long-range reverse-transcription quantitative PCR (RT-quantitative PCR (qPCR)) would potentially be applicable to the assessment of influenza A virus (IAV) infectivity by targeting PB2, PB1 and PA
We investigated whether long-range RT-qPCR is applicable to determination of IAV infectivity

Summary

Introduction

The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. With the recent growing demand for the rapid diagnosis of viral infection, novel techniques and devices based on PCR have been developed [13, 14]. These newer methods mostly shorten the assay time and reduce the proportion of false negatives. A maximum sterilizing effect of UV rays on pathogens are shown at 253.7 nm [23, 24]

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