MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses

Abstract

The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.

FIG. 1.

Schematic of the major steps in the MultiCode-PLx respiratory virus detection system. For amplification, after RT, the target regions are amplified by PCR with one of the primers containing a single iC base at the 5′ end for subsequent site-specific labeling. For TSE, tagged, target-specific extenders are site specifically labeled with biotin-di-iso-GTP when the correct amplicon is present by primer extension. For capture, the tagged TSE products are captured by color-addressable microspheres through hybridization of each tag to its complementary, virus-specific oligonucleotide code conjugated to the microsphere surface. Both the tags and codes also contain the iC and iG bases. After the addition of streptavidin-phycoerythrin, the fluorescent signal associated with each microsphere is analyzed and decoded by using a Luminex 100 instrument.

FIG. 2.

Sample data output from the MultiCode-PLx respiratory virus detection system. The dot plots show the MFIs for 187 patient specimens and 5 negative controls for the INF A target (A) and the corresponding DNA (B) and RNA (C) IPCs. Forty-seven samples had MFI values above the threshold for the INF A test. MFI values for the DNA IPC exceeded the threshold for all samples. The RNA IPC was not detected in three (1.6%) of the patient specimens.