AOAC 2019 First Action Methods

utilize dual Fluorescence Resonance Energy Transfer (FRET) hybridization probes. These probes consist of two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the same assay is labeled at the 3’ end with fluorescein; the second probe is labeled at the 5’ end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, which forms a real-time amplification curve, is how the amplified target is detected by the GENE-UP ® Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software interprets data for each sample and gives a positive, negative, or inhibited result. B. Apparatus and Reagents Items available from bioMérieux, Inc. (Hazelwood, MO, USA). (a) GENE-UP ® Listeria monocytogenes 2 (LMO 2). ― REF 423107 (192 tests) – Store kit at 2–25°C. (1) PCR Reagent Strips. ― Two pouches with 12 strips of 8 tubes (2 x 96 tubes). (2) Optical Caps. ― Two pouches with 12 strips of 8 tubes (2 x 96 caps). (3) Control Buffer. ―One pouch with control buffer vial (850 μL). (b) GENE-UP ® Lysis Kit. ― REF 414057. (c) GENE-UP ® Thermocycler. ― REF 414056. (d) Laboratory paddle blender for sample homogenization . (1) SMASHER ® . ― REF AESAP1064. (2) SMASHER ® XL . ― REF AESAP1100. (e) LPT Broth. (1) Six x 225 mL. ― REF 410848. (2) Six x 100 mL. ― REF 410846. (3) Ten x 225 mL mini bag. ― REF 410847. (4) Four x 3 L. ― REF 410749. (5) Twenty x 10 mL. ― REF 410845. (f) Advanced Digital Heavy-Duty Vortex Mixer. (1) 120 V. ― REF 9456TAHDUSA. (2) 230 V. ― REF 9456TAHDEUA. (g) GENE-UP ® Lysis Rack Adaptor. ― REF 414570. (h) Sterile blender bags. (1) Type P filter bag. ― REF AES400P/50G. (2) SMASHER ® XL Bag 2L. ― REF 415180. (3) SMASHER ® XL Bag 2L. ― REF 415181. (i) Plate Centrifuge.

(1) MPS1000 Mini PCR Plate Spinner 110V. ― REF 419196. (2) MPS1000 Mini PCR Plate Spinner 230V. ― REF 414555.