Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay

Abstract

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

FIG. 1.

Detection and identification of TSPE reaction products captured onto microsphere beads containing anti-tag oligonucleotides (oligo) that hybridize to TSPE products containing a complementary tag oligonucleotide. The microbeads are sorted with the Luminex 100 flow cell instrument, which identifies spectrophotometrically colored beads with one laser and a phycoerythrin signal on the beads with a second laser.

FIG. 2.

RVP test results obtained for 11 NP specimens showing TSPE signals recorded for all microbeads. The 11 positive specimens are indicated across the bottom, with the signals for each of the 21 beads indicated by colored bars. The first sample on the left shows a positive signal for the influenza A virus bead (matrix gene positive) and for the influenza A virus subtype H1 hemagglutinin gene. All of the other beads for this sample were negative. ADENO, adenovirus; H1, H3, and H5, influenza A virus, subtypes H1, H3, and H5, respectively; PARA-1 to PARA-4, parainfluenza virus types 1 to 4, respectively; 229E, HKU1, NL63, and OC43, CoVs 229E, HKU1, NL63, and OC43, respectively; SARS, SARS-CoV; Flu-A and Flu-B, influenza A and B viruses, respectively; RHINO, rhinovirus; ENTERO, enterovirus; RSV-A and RSV-B, RSV types A and B, respectively; HMPV, human MPV; RSV-detect, bead 6 (a common RSV bead detecting both RSV type A and RSV type B).