We have developed a nucleic acid-based assay that is rapid, sensitive, and specific and can be used for the simultaneous detection of five common human respiratory pathogens, including influenza virus A, influenza virus B, parainfluenza virus types 1 and 3, respiratory syncytial virus (RSV), and adenovirus groups B, C, and E. Typically, diagnosis on an unextracted clinical sample can be provided in less than 3 h, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs and therefore allow implementation of infection control measures and the timely administration of antiviral therapies. We present here a summary of the assay performance in terms of sensitivity and specificity. The limits of detection are provided for each targeted respiratory pathogen, and result comparisons were performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the University of California-Davis Medical Center hospital. Overall, the use of the multiplexed reverse transcription-PCR assay reduced the rate of false-negative results by 4% and reduced the rate of false-positive results by up to 10%. The assay correctly identified 99.3% of the clinical negatives and 97% of the adenovirus, 95% of the RSV, 92% of the influenza virus B, and 77% of the influenza virus A samples without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on unextracted samples.

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Multiplex RT-PCR suspension array assays provide a powerful tool for identifying the causative agent(s) of respiratory infections. These assays are time consuming and laborious on a time-per-sample basis if only a few samples require processing. To address this shortcoming and provide an automated solution for fast detection and identification of viral pathogens, we developed the first automated multiplex RT-PCR suspension array instrument capable of handling unprepared clinical samples. The instrument requires less than 3 minutes of hands-on time for a result generated in approximately 2.5 hours. In analytical studies, the instrument performed as well as manually performed assays. The performance of the instrument and loaded multiplex viral detection assay was then tested using unprepared nasopharyngeal samples. The instrument-performed assay detected 61 of 71 RSV positive samples, for a sensitivity of 85.9%. Adenovirus (n = 5) and influenza B (n = 3) were less prevalent in the sample set, but detected to similar levels, 80% and 75%, respectively. The same sample set was also tested using FDA approved immuno-assay rapid tests, and the instrument was found to be more sensitive than the rapid tests with the sole exception being influenza A (n = 16), which was poorly detected due to significant sequence mismatches between the influenza A primer/probe set included in the multiplex mixture and the circulating influenza A strains. Overall, these data demonstrate the developed prototype platform performs multiplex array assays as well as hand-performed assays, and that the instrument's sensitivity and specificity are dictated by the quality of the loaded multiplex assay.

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized