Public health laboratories were the first provided with the reagents and procedures for the reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed by the Centers for Disease Control (CDC) under the Emergency Use Authorization (EUA). As information was shared between laboratories, other facilities implemented RT-PCR procedures that provided for the detection and differentiation of 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 is 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
Extraction of RNA from patient samples is followed by a one-step multiplex reverse transcription of RNA targets into complementary DNA (cDNA), which is subsequently amplified in a real time thermocycler. In this process, the probe anneals specifically to the template, 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. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing the fluorescent signal.