Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye

Abstract

Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8 min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens.

Introduction

The rise in the number of influenza outbreaks and outpatient visits for influenza-like-illness suggests an increased risk for global human public health (CDC, 2016b). Recently, incidents of human infection by avian and other zoonotic influenza viruses, such as avian influenza (AI) virus subtypes A/H5N1, A/H7N9, and A/H9N2, and swine influenza virus subtypes A/H1N1 and A/H3N2, have been reported (WHO, 2016).

The H7 subtypes of the AI virus are classified into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses based primarily on mortality rates following chicken pathogenicity testing (Belser et al., 2011). Since 2002, H7 subtype viruses have caused more than 100 cases of human infection in Europe and North America, resulting in both ocular and respiratory illnesses (Fouchier et al., 2004, Skowronski et al., 2006, Tweed et al., 2004).

World Health Organization (WHO) recommends rapid influenza testing only in cases of patients with lower respiratory tract illnesses, especially in children and adults with medical conditions that increase their risk for influenza complications (WHO, 2005). Therefore, highly sensitive, rapid, quantitative, low-cost, and reliable tests are urgently needed for diagnosing infectious diseases (Caliendo et al., 2013).

Currently, a major challenge is to develop a highly sensitive point-of-care test (POCT) for the detection of avian influenza virus (Fedorko et al., 2006). In this respect, considerable efforts have been directed toward developing fluorescent probes for immunochromatographic tests in field-deployable biosensors. Fluorescent dyes are increasingly being used for the identification, detection, quantification, and characterization of biological molecules (Mahmoudian et al., 2011). However, few studies targeted the development of fluorescent dye for immunochromatographic tests and most research has been limited to Europium (Juntunen et al., 2012) and quantum dots (Cheng et al., 2014, Di Nardo et al., 2016, Le et al., 2016).

The labeling of antibodies with fluorescent moieties is a key for biological application (Coons and Kaplan, 1950). The presence of multiple primary amines in the active site of an antibody can result in fluorophore conjugation but changes the characteristics of antigen binding and, in extreme cases, completely inactivates the antibody (Werthen and Nygren, 1988). Steric hindrance and the absence of additional reactive sites on the fluorophore are presumed to limit the degree of antibody modification by the conjugation reaction (Vira et al., 2010). Finally, there is a limit to the number of fluorescent molecules that can be attached to an antibody limiting their application as diagnostic agents, as higher photoluminescence is required for better performance. To improve the performance, fluorescent dyes have been examined in terms of photostability and brightness. However, it is largely unknown how changes in the molecular structure of a dye give rise to profound differences in its fluorescence properties (Zhu et al., 2002).

A fluorescent dye is a chemical compound that absorbs light and then remits it at a longer wavelength. There were two main considerations in the selection of the fluorescent dye: a sufficiently large difference between its absorption and emission wavelengths to minimize interference and the ability to easily introduce a linker to the dye which can react with lysine or arginine. We selected a Red dye with a pyridine-styrylpyridine structure, which satisfied both the conditions. Styrylpyridine is one of the most common fluorescence dyes (Ams et al., 2009) often used as a fluorophore in imaging (Campos et al., 2016). However, there are limitations due to its weak fluorescence. In this study, we sought to increase the sensitivity of styrylpyridine probes by connecting large numbers of fluorescent phosphor groups to the latex surface.

To evaluate the potential of pyridine-styrylpyridine Red dye 53 as a novel fluorescent probe, we synthesized and assessed the fluorescent dye-linked rapid diagnostic system in immunoassays.