Rapid Automated Detection of influenza A, influenza B and RSV A/B by multiplex real time RT-PCR using two new methods and novel primer/probe chemistry
Session III – Kelly Henrickson – Poster Only
Title of Contribution: Rapid Automated Detection of influenza A, influenza B and RSV A/B by multiplex real time RT-PCR using two new methods and novel primer/probe chemistry
K.J. Henrickson1, L. Jurgens1, M. Bose1, E. Beck1, L. Witt1, T. Patitucci1, P.Darga1, S. Kumar1, S. Kehl1, J. Fan1
1Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
Introduction: Rapid, sensitive and specific automated extraction, amplification, and detection of multiple respiratory viruses is a goal of our program and a number of research laboratories around the world. We report significant steps in this direction by developing a semi-automated protocol for the multiplex detection of influenza A (FA), influenza B (FB), and RSV A/B [easyMAG (bioMérieux, Durham, NC)-Raider (HandyLab Inc., Detroit, USA.)] then adapting this into a fully automated assay [Jaguar (HandyLab)].
Material and Methods: The assay protocols were developed in our laboratory and are run using two strategies. One utilized automated extraction (easyMAG) with manual transfer to a real-time thermocycler (Raider) vs. a single machine (Jaguar) that performed fully automated extraction, amplification and detection. 1) Development of multiplex PCR primers/probes: Novel highly conserved primers/probes were designed targeting the M gene of FA and FB, the L gene of RSV A and B and the phage MS2 (internal control) utilizing proprietary “superbases” and 5’-minor groove-binding probes (Nanogen Inc., Bothell, USA) 2) Nucleic acid (NA) extraction and amplification: Specimen volumes of 400 ul and 475 ul were spiked with 10 uL of MS2 (5x10^5 pfu/mL) each and eluted in volumes of 25 ul and 10 ul respectively using the easyMAG and Jaguar systems. Subsequent one step real time reverse transcription (RT) - PCR was performed by manual or robotic transfer of target NA into the Raider (5.3ul) and Jaguar (10ul) systems respectively. Cycling protocol used was: 20 min @ 50°C (RT), 2 min @ 95°C (denature), & 45 cycles of 15 sec @ 95°C, 30 sec @ 56°C and 15 sec @ 76°C (PCR). Discrimination of targets was accomplished by performing a subsequent melt analysis at 45°C to 85°C. Targets were considered to be positive if the cycle threshold (Ct) value was ≤ 40, the amplification curve shape was appropriate and the melt profiles yielded a Tm ± 2° of the expected Tm. (RSV: 75°C, FA: 62°C, FB: 68°C (FAM), MS2: 75°C (Cal red)), indeterminate if Cts >40.0 with an appropriate melt Tm and amplification curve shape, and negative if Ct >over 40.0, incorrect Tm, no melt curve or an abnormal amplification curve was seen. Assay calls were determined using an in house developed “workbook” (computational algorithm) that imports Raider or Jaguar raw data. 3). Analytical sensitivities and specificities: Limits of detection (LOD) were determined by testing NA extracted from serial dilutions of quantitated whole virus (TCID50 and copies/ml). Analytical specificity was determined by testing 25 respiratory organisms as well as by cross reactivity amongst the assay pathogens. 4) Clinical sensitivities and specificities: Frozen nasopharyngeal (NP) specimens that were tissue culture (TC) positive (20 RSV, 20 Flu A, 20 Flu B) and negative (39) were blindly tested. Results: 1. Analytical assay characteristics: The LODs for easyMag/Raider (and Jaguar) systems were: 0.01 (1), 0.1 (0.01), 0.1, 0.1 and 0.01 TCID50/ml for RSV A, RSV B, FA (H1N1), FA (H3N2), and FB respectively; Specificity testing showed no cross reactivity amongst the assay pathogens and none of the 25 non-specific targets tested positive on either system. 2) Clinical assay characteristics: 20/20 RSV positive specimens were positive for RSV and negative for other targets on both systems, and the 39 TC negative specimens were negative for all targets with 58/59 positive internal control signals detected. Clinical sensitivity for RSV was 100% (95% CI: 83-100%) and clinical specificities were 100 % for RSV (95% CI: 91-100% 39/39 neg) and 100% (95% CI: 94-100% 59/59 neg) each for FA and FB. Further clinical testing of influenza is ongoing and will be reported. The expected reagent costs per sample are $25-35 or as low as $8 per virus tested. Highly efficient run times of 3.0 and 2.5 hrs for a full (24 sample) and half (12 sample) load run allow 3-4 runs/shift.
