A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses.

Lili Xu,Xiwen Jiang,Yun Zhu,Yali Duan,Taosheng Huang,Zhiwen Huang,Baoping Xu,Zhengde Xie,Chunyan Liu

doi:10.3389/fmicb.2018.01405

Lili Xu, Xiwen Jiang + Show 7 more

Open Access

https://doi.org/10.3389/fmicb.2018.01405

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Abstract

The reliable and rapid detection of viral pathogens that cause respiratory infections provide physicians several advantages in treating patients and managing outbreaks. The Luminex respiratory virus panel (RVP) assay has been shown to be comparable to or superior to culture/direct fluorescent-antibody assays (DFAs) and nucleic acid tests that are used to diagnose respiratory viral infections. We developed a multiplex asymmetric reverse transcription (RT)-PCR assay that can simultaneously differentiate all influenza A virus epidemic subtypes. The amplified products were hybridized with an electrochemical DNA sensor, and the results were automatically acquired. The limits of detection (LoDs) of both the Luminex RVP assay and the multiplex RT-PCR-electrochemical DNA sensor were 101 TCID50 for H1N1 virus and 102 TCID50 for H3N2 virus. The specificity assessment of the multiplex RT-PCR-electrochemical DNA sensor showed no cross-reactivity among different influenza A subtypes or with other non-influenza respiratory viruses. In total, 3098 respiratory tract specimens collected from padiatric patients diagnosed with pneumonia were tested. More than half (43, 53.75%) of the specimens positive for influenza A viruses could not be further subtyped using the Luminex RVP assay. Among the remaining 15 specimens that were not subtyped, not degraded, and in sufficient amounts for the multiplex RT-PCR-electrochemical DNA sensor test, all (100%) were H3N2 positive. Therefore, the sensitivity of the Luminex RVP assay for influenza A virus was 46.25%, whereas the sensitivity of the multiplex RT-PCR-electrochemical DNA sensor for the clinical H1N1 and H3N2 specimens was 100%. The sensitivities of the multiplex RT-PCR-electrochemical DNA sensor for the avian H5N1, H5N6, H9N2, and H10N8 viruses were 100%, whereas that for H7N9 virus was 85.19%. We conclude that the multiplex RT-PCR-electrochemical DNA sensor is a reliable method for the rapid and accurate detection of highly variable influenza A viruses in respiratory infections with greater detection sensitivity than that of the Luminex xTAG assay. The high mutation rate of influenza A viruses, particularly H3N2 during the 2014 to 2016 epidemic seasons, has a strong impact on diagnosis. A study involving more positive specimens from all influenza A virus epidemic subtypes is required to fully assess the performance of the assay.

Highlights

Influenza A virus causes morbidity and mortality in humans, and this is overstated during epidemic years, which is a major public health concern (Neuzil et al, 2000; Petrova and Russell, 2018)
No crossreactivity was observed among the subtypes, and no spurious PCR amplification or voltmeter measurements were observed in the specimens positive for RSV, HAdV, human metapneumovirus (HMPV), HPIV, enteroviruses and rhinoviruses (EV/Rh), human coronavirus (HCoV), and human bocavirus (HBoV)
The clinical specimens were used to compare the sensitivity of the multiplex RT-PCR-electrochemical DNA sensor using comparative methods

Summary

Introduction

Influenza A virus causes morbidity and mortality in humans, and this is overstated during epidemic years, which is a major public health concern (Neuzil et al, 2000; Petrova and Russell, 2018). Based on the antigenic specificity of haemagglutinin (HA) and neuraminidase (NA) proteins, influenza A viruses can be classified into subtypes H1–H18 and N1–N11, respectively (Wu et al, 2014; Pinsent et al, 2016). Rapid detection and subtyping are the first steps in the characterization of influenza A viruses, and can facilitate the appropriate treatments to improve patient clinical outcomes and significantly reduce hospital costs (Bonner et al, 2003). The RVP assay (Luminex Molecular Diagnostics Inc., Toronto, Canada) is a CE-marked, commercially available kit based on suspension microarray technology that enables the detection of many viruses in a single reaction (Krunic et al, 2007; Merante et al, 2007). The Luminex RVP assay has been shown to be more sensitive and specific than culture and antigen detection (Wong et al, 2009; Gadsby et al, 2010). The cost of the kit and instrument is a major disadvantage of the Luminex RVP assay

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