Division 2.2 Group-specific real-time RT-PCR by Guthrie et al., 2013F1

Real-time RT-PCR using fluorescence resonance energy transfer (FRET) probes to detect nucleic acid of AHSV.

The AHSV RT-PCR assay described was designed using sequences from a wide variety of currently circulating field strains of AHSV (Quan et al., 2010 ^f2 ). It also incorporates a proprietary synthetic external control assay to verify proper functioning of the assay components.

Kits for the one-step real-time PCR are available commercially. Below are some basic steps as described by Guthrie et al. (2013), which can be modified depending upon local/case-specific requirements, kits used and equipment available.

Primer and probe sequences for the detection of AHSV species viruses:

— forward Primer

5′-AGA-GCT-CTT-GTG-CTA-GCA-GCC-T-3′

— reverse Primer

5′-GAA-CCG-ACG-CGA-CAC-TAA-TGA-3′

— MGB-TaqMan probe

5′-FAM-TGC-ACG-GTC-ACC-GCT-MGB-3′

1. 2.2.1.

   Primer and probe mix stock solutions are made up in a 25× concentration at 5 μΜ for the forward and reverse primers and 3 μΜ for the probe. A test plate layout should be designed and loaded into the real-time PCR machine software. Using the layout as a guide, 5 μl of RNA samples, including test samples and positive and negative controls, are added to appropriate wells of the plate following the layout.

2. 2.2.2.

   The RNA is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 3 minutes.

3. 2.2.3.

   An appropriate volume of real-time one-step RT-PCR master mix for the number of samples to be tested is prepared, following the manufacturer's instructions. 1 μl of 25× primer probe mix stock solution (from point 2.2.1 above) is included in the master mix to give a final concentration in each well of 200 nM for each primer and 120 nM of the probe. 20 μl of the master mix is distributed in each well on the PCR plate containing the denatured RNA.

4. 2.2.4.

   The plate is placed in a real-time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection as suggested by the manufacturers. Amplification conditions consist of, for example, a first reverse-transcription step at 48 °C for 10 minutes, followed by 10 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C and 45 seconds at 60 °C.

5. 2.2.5.

   Samples are considered positives, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold within 36 PCR cycles in all replicates of a sample.

   Samples are considered inconclusive, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold between 36 and 40 PCR cycles in any replicate of a sample.

   Samples are considered negative, if the normalised fluorescence for the AHSV RT-PCR assay did not exceed a 0,1 threshold within 40 PCR cycles in all replicates of a sample and if the normalised fluorescence for the proprietary synthetic external control assay exceeded a 0,1 threshold within 33 PCR cycles.