Public health laboratories were the first to provide the reagents and procedures for the reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed by the Centers for Disease Control and Prevention (CDC) under the Emergency Use Authorization (EUA). As information was shared between laboratories, other facilities implemented RT-PCR procedures to detect and differentiate the H1N1 "swine" strain from previously encountered seasonal strains.
Although many facilities utilized laboratory-developed procedures, the FDA did grant emergency approval to a handful of commercially developed methods. One example was Prodesse's ProFlu-ST™ assay, which became available in October 2009. Employing real-time methodology, the kit was also optimized for use with automated extraction platforms, such as Roche's MagNA Pure Systems and Biomerieux's NucliSENS® easyMAG®.
The ProFlu-ST™ assay was a multiplex RT-PCR assay utilizing fluorogenic hydrolysis (Taqman) probes for use on the SmartCycler platform. As a multiplex assay, it includes primers and probes for seasonal H1, seasonal H3, and 2009 H1 strains of influenza A. Targets are as follows:
- Seasonal H1: conserved area of A/H1 hemagglutinin (HA) gene
- Seasonal H3: conserved area of A/H3 hemagglutinin (HA) gene
- 2009 H1/N1: conserved area of the 2009 nucleoprotein (NP) gene
RNA extraction from patient samples was followed by a one-step multiplex reverse transcription of RNA targets into complementary DNA (cDNA), which is subsequently amplified in a real-time thermocycler. The probe anneals specifically to the template in this process, followed by primer extension and amplification. The assay utilizes the 5'–3' exonuclease activity of the Taq polymerase, which cleaves the probe, thus separating the reporter dye of the fluorogenic probe from the quencher. This generates an increase in fluorescent signal. Each cycle cleaves additional reporter dye molecules from their respective probes, increasing the fluorescent signal.
The ProFlu ST test was a less sensitive subtyping assay. Other manufacturer developments occurred rapidly and included platforms for multiplex testing and reporting of FluA, FluB, and 2009 H1/N1.
