Evaluation of a multiplexed PCR assay for detection of respiratory viral pathogens in a public health laboratory setting

Abstract

There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered "true positives," the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

FIG. 1.

Schematic of the PLx-RVP system. Four major steps are required to perform the PLx-RVP assay. PLx is enabled by one additional base pair constructed from the complementary bases isoguanosine and 5′-methyl-isocytosine (iC). These additional bases are used in steps 2 to 4. The average time required for each step is shown on the right. (Step 1) Reverse transcription (RT). Random 6-mer primers are used to produce cDNA from extracted RNA. (Step 2) Multiplexed PCR. PCR primers are designed to be target specific, and at least one primer of each pair contains a single iC on its 5′ end. (Step 3) TSE. The iC-containing strands of the PCR products act as labeling templates for the TSE step. During this step, labels attached to 2′-deoxy-iso-GTP are incorporated site-specifically onto EraCode-tagged target-specific extenders directed by the complementary iC contained in the template. (Step 4) Capture. EraCode tags are short sequences (typically 8 bases) assembled using a mix of natural and nonnatural bases. The tags are designed to hybridize only to their perfect complements encoded on spectrally addressed microspheres. The TSE reaction products are decoded by room temperature hybridization to complementary codes on spectrally addressed microspheres, followed by reading of the reporter signal.