Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex-based assay

Abstract

Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 10(3) (EAds, HBoV2, and RVA) and 10(4) (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

Fig 1

Analytic specificity of the multiplexed Luminex assay. The analysis of the specificity of the multiplexed Luminex assay was carried out with EV71, HPeV, PBV, rotavirus A, NoV GI, NoV GII, SaV, HAstV, EAds, and HBoV2. TE buffer was used as a blank control, and DEPC water was used as a negative control. Biotin-labeled PCR products were separated by probe-coupled beads and are presented in terms of dye signal median fluorescence intensity (MFI) in arbitrary units on the y axis. Each peak was identified by beads coupled with specific capture probes and is indicated on the z axis.

Fig 2

Multiple-target detection by the Luminex-based multiplexed assay. The analysis of the mixed-sample detection capacity of the Luminex-based multiplex assay was carried out using four sets of random mixed samples. Mix 1 included HAstV, rotavirus A, and EAds. Mix 2 included NoV GI, NoV GII, and sapovirus. Mix 3 included rotavirus A, NoV GII, and HBoV2. Mix 4 included HAstV, EAds, and HBoV2. DEPC water was used as a negative control. Biotin-labeled PCR products were separated by probe-coupled beads and are presented in terms of dye-signal average median fluorescence intensity (MFI) in arbitrary units on the y axis. Error bars represent 5% of the MFI value.