For this study, all SYBR green assays were modified from the previously described assays (5), with the exception of the reverse primers for the newly designed PB1 and NS segments (Table 1). The subtype H1N1 swine influenza viruses isolated in Hong Kong during the past few years were mainly derived from the Eurasian avian-like swine lineage (6,7). To generate more precise genotyping data for our ongoing surveillance, the NA segment–specific assay was specifically designed to react with the pandemic (H1N1) 2009 virus and a portion of Eurasian avian-like swine viruses that are circulating in southeastern China ( Technical Appendix Figure 1). To avoid overlapping the emission spectrum of SYBR green, we labeled all pandemic (H1N1) 2009 virus–specific hydrolysis probes (Integrated DNA Technologies, Inc., Coralville, IA, USA) with cyanine 5 (Cy5) and Black Hole Quencher-2 dyes at their 5′ and 3′ ends, respectively (Table 1). To enable use of short oligonucleotide sequences without compromising the annealing temperature of these probes, we modified the probes with locked nucleic acids (7). RNA extraction and complimentary DNA synthesis were identical to the protocols described (5,8). One microliter of 10-fold diluted complimentary DNA sample was amplified in a 20-µL reaction containing 10 µL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and the corresponding primer probe set (0.5 µmol/L each). All reactions were optimized and performed simultaneously in a 7500 Sequence Detection System (Applied Biosystems) with the following conditions: 20 s at 95°C, followed by 30 cycles of 95°C for 3 s and 62°C for 30 s. SYBR green and Cy5 signals from the same reaction were captured simultaneously at the end of each amplification cycle. The expected PCR results of virus segment derived from different swine viral lineages are shown in Table 2.

The dissociation kinetics of PCR amplicons were studied by a melting curve analysis at the end of the PCR (60°C–95°C; temperature increment 0.1°C/s). We also tested various probe and SYBR green concentrations under different PCR conditions. The condition described above gave the most robust and consistent DNA amplification (data not shown). We tested 31 human pandemic (H1N1) 2009 and 63 human seasonal influenza viruses (33 subtype H1N1, 30 subtype H3N2) as controls. As expected, all human pandemic influenza viruses were double positive (i.e., positive with SYBR green and Cy5) and all seasonal influenza samples were double negative in all 8 assays.

To evaluate the sensitivity of the assays, we tested serial diluted plasmid DNA of the corresponding segments of influenza A/California/4/2009 virus as a standard. The fluorescent signals generated from the SYBR green reporter dye in all assays were highly similar to those previously reported (5), and the modified assays had a linear dynamic detection range from 10² to 10⁸ copies/reaction (Technical Appendix Figure 2). As expected, the threshold cycle values deduced from the Cy5 reporter signal were generally higher than those from the SYBR green reporter (Technical Appendix Figure 2) (9). This finding can be partly explained by the nature of these 2 kinds of real-time PCR chemistries: a single Cy5 fluorophore of the hydrolysis probe was released from quenching for each amplicon synthesized while multiple SYBR green dyes bound to a single amplicon (10). After 35 PCR amplification cycles, the linear dynamic detection range of Cy5 signals generated from these reactions was 10² to 10⁸ copies/reaction (data not shown). However, to avoid nonspecific SYBR green signals, we purposely limited the number of amplification cycles to 30.

Using these assays, we tested 41 swine virus isolates collected during January 2009–January 2010. In all 8 reactions, 10 pandemic (H1N1) 2009 virus samples transmitted from humans to pigs (6) were double positive (Technical Appendix Figure 1, pink). In these assays, gene segments of another 31 swine isolates were either SYBR green positive/Cy5 negative (Technical Appendix Figure 1, yellow) or double negative (Technical Appendix Figure 1, green), indicating that these virus segments were derived from the sister clade of pandemic (H1N1) 2009 virus or other swine lineages (except NA), respectively. For example, the reassortant of pandemic (H1N1) 2009 virus (A/swine/Hong Kong/201/2010 [H1N1]) was double positive for NA, double negative for HA and matrix protein, and SYBR green positive/Cy5 negative for PB2, PB1, PA, NP, and NS (Figure; other data not shown). All genotyping results of the studied viruses were consistent with results of previous phylogenetic analyses (5,6), indicating that our modified probes and SYBR green assays can provide more accurate genotyping results. With these genotyping data, viruses with atypical positive signal patterns might suggest a novel viral reassortment event and can be highlighted for investigation with sequencing-based methods.

To demonstrate the potential use of these assays in studying swine viruses circulating in other geographic locations, we tested 7 recent swine isolates (1 pandemic influenza subtype H1N1, 4 subtype H1N2, and 2 subtype H3N2) collected in the United States. Genotyping results agreed 100% with data deduced from sequence analyses (Technical Appendix Table 1). We also analyzed all 436 contemporary (2008–2010) US swine virus segments available from the National Center for Biotechnology Information influenza virus sequence database. On the basis of the in silico analysis of sequences targeted by our primers and probes, 95% of the sequences (n = 413) are predicted to yield the expected results (Technical Appendix Table 2).

The emergence of pandemic (H1N1) 2009 has highlighted the need for global systematic influenza surveillance in swine. Our results demonstrated that the addition of locked nucleic acid hydrolysis probes specific for pandemic (H1N1) 2009 virus into previously established SYBR green assays can help differentiate segments of pandemic (H1N1) 2009, Eurasian avian-like, and triple reassortant virus lineages. These assays might provide a rapid and simple genotyping method for identifying viruses that need to be fully genetically sequenced and characterized. They may also help provide better understanding of the viral reassortment events and viral dynamics in pigs. Although at present, genes derived from human seasonal viruses cannot be characterized with our modified assays, the performance of our assays warrants similar investigations for genotyping human influenza viruses.