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Transfer and Optimization of a Single-tube FMDV Real-time RT-PCR Assay to a 96 Well Format for High-throughput Diagnosis

Heather Engleking1, A.J. Eberling2, Mary A. Kenney2, T.S. McKenna2 , T.R. Beckham2.

1Texas A&M University, College Station, TX; 2Plum Island Animal Disease Center

Objective:  To transfer and optimize a portable, single tube real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay (Tetracore, Gaithersburg, MD) for the detection of Foot and Mouth Disease Virus (FMDV) to multiple 96 well platforms (MX4000, ABI7000, ABI 7700)

Sample Population: 80 viral isolates from the Foreign Animal Disease Diagnostic Laboratory repository at Plum Island Animal Disease Center, and 24 field samples from Afghanistan were used to demonstrate equivalency between the wet and dry chemistries and across platforms.  A representative virus from each FMDV serotype was used to determine the limit of detection (LOD) on each individual PCR machine. Intra-assay precision for each platform was examined using three different RNA concentrations that spanned the linear range of the assay.

Procedure: RNA was extracted using the RNeasy Minikit from Qiagen (Valencia, CA).  PCR reactions, regardless of platform, were performed in a final volume of 25 Ál using 2.5 Ál of extracted RNA as template.  Thermocycling conditions for the Cepheid Smart Cycler were 60░ C for 10 minutes, followed by 55 cycles at  95░ C for 2 s, and 60░ C for 1 minute.  Thermocycling conditions for the 96 well platforms were 60░ C for 10 min, followed by 45 cycles at 95░ C for 30 s and 60░ C for 1 min.

Results:  The LODs and diagnostic sensitivity of each 96 well platform was equivalent to or better than that of the Cepheid SmartCycler II.  The precision of the Applied Biosystems ABI7700 and ABI7000 was equivalent to that of the Cepheid Smart Cycler II.  The MX4000 performed in a less precise manner than the other machines tested. 

Conclusions/Clinical Relevance: A single-tube FMDV assay has been successfully transferred and optimized on three 96 well platforms. This assay can now be performed in a 96 well format, thus providing reference and regional laboratories the capability for high-throughput processing of samples in the event of an outbreak.