Rapid and sensitive real-time assay for the detection of respiratory syncytial virus using RT-SIBA®

A total of 40 nasopharyngeal (NP) swab specimens were obtained from Discovery Life Sciences Biobank (Discovery Life Sciences, Inc., CA, USA). The specimens were previously determined to be positive (n = 25) or negative (n = 15) for RSV using the Alere BinaxNOW RSV , a rapid immunoassay test (Alere Inc, USA). These specimens were used to evaluate the performance of RT-SIBA in comparison with previously published RT-PCR for the detection of RSV [15]. Viral RNA was extracted from the specimens using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Thereafter, 2 μl of purified RNA was added to the RT-SIBA or RT-PCR RSV assay. A set of NP swab specimens was also subjected to a quick and crude specimen preparation protocol instead of RNA purification. To this end, 5 μl of the NP swab specimen was added to 45 μl of lysis buffer (2% Triton X-100 and 125 mM magnesium acetate; Sigma-Aldrich, USA), and 2 μl of the crude lysate was subsequently added to the RT-SIBA reaction.

RNA was purified from RSV-A (ATCC VR-1540) and RSV-B (ATCC VR-1400) American Type Culture Collection (LGC Standards, Germany) viral particles using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The purified RNA was quantified using the Genesig® RSV RT-PCR kit (Primerdesign™ Ltd., UK). The kit includes RSV RNA standards with pre-determined copy numbers. These standards were used to generate standard curves for the quantification of RSV-A RNA and RSV-B RNA. RT-PCR was performed on a Bio-Rad qPCR CFX95 instrument (Bio-Rad Laboratories, CA, USA). The quantified RSV RNA was subsequently used to establish the analytical sensitivity of the RT-SIBA RSV assay in comparison with a previously published RT-PCR assay for the detection of RSV [15].

The assay was designed to detect all known human RSV strains including subtypes A and B [14]. RSV sequences were retrieved from the Virus Pathogen Resource [16] as well as the National Center for Biotechnology Information database and aligned. The assay was designed to amplify ~70 nucleotides within the highly conserved region of the RSV N-protein gene. The assay used the same target region within the RSV-A and RSV-B genomes. The RSV-A and RSV-B genomes share about 90% homology within the chosen target region. A set of primers and invasion oligonucleotides (IOs) were designed to amplify this target region. The RSV assay used one forward primer, one reverse primer, two IOs, and one probe. The use of multiple IOs facilitated improved detection of RSV-A and RSV-B in the same reaction tube, but could not differentiate between the two subtypes of RSV. The RT-SIBA assay was also developed to detect the bacteriophage MS2. Target-specific probes for both the RSV and MS2 assays were designed as previously described [17]. This allowed the RSV and MS2 assays to be multiplexed in the same reaction tube. The MS2 assay served as an internal control for monitoring sample-derived inhibition [18].

RT-SIBA reactions were performed using the SIBA® reagent kit (Orion Diagnostica Oy, Finland) with the addition of 16 units of GoScript™ Reverse Transcriptase (Promega, UK). The RSV forward and reverse primers were each used at a concentration of 400 nM. RSV-A, RSV-B, and MS2 IOs were used at concentrations of 200, 200, and 100 nM, respectively. The RSV and MS2 probes were each used at a concentration of 100 nM. The sequences of the oligonucleotides used for the RT-SIBA RSV and MS2 assays are shown in Table 1. In total, 2000 copies of MS2 RNA (Sigma-Aldrich) were added to the reaction. The UvsX and gp32 enzymes were used at a concentration of 0.25 mg/ml. In total, 2 μl of the sample or RNA template was used in a total reaction volume of 20 μl. RT-SIBA was detected using fluorophore-labeled probes and SYBR Green 1 (1:100,000 dilution). The reactions were incubated at 41 °C for 60 min, and fluorescence readings were taken at 60 s intervals using Bio-Rad CFX 96 (Bio-Rad Laboratories). A melting curve profile was generated after amplification at 40–95 °C to verify that the reaction products were specific.

The performance of the RT-SIBA assay was compared with a previously published RT-PCR assay for the detection of RSV-A and RSV-B [15]. Both RT-SIBA and RT-PCR reactions were performed at the same time. 2 μl of the sample or RNA template was used in a total RT-PCR reaction volume of 20 μl. The concentrations of the probes and primers were previously described [15]. RT-PCR was performed using EXPRESS qPCR SuperMix (Thermo Fisher Scientific, USA). A thermal program of 50 °C for 15 min, 95 °C for 2 min, and 45 cycles of 95 °C for 30 s and 45 °C for 1 min was used with Bio-Rad qPCR equipment (Bio-Rad Laboratories).