AOAC 2019 First Action Methods

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 ˊ end with fluorescein; the second probe is labeled at the 5ˊ end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template deoxyribonucleic acid (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 All items are available from bioMérieux, Inc. (Hazelwood, MO, USA) unless otherwise specified. (a)  GENE-UP ® ECO 2.— REF 423108 (192 tests). Store kit at 2–25°C. ( 1 )  PCR reagent strips.— Two pouches with 12 strips of eight tubes (2 × 96 tubes). ( 2 )  Optical caps.— Two pouches with 12 strips of eight tubes (2 × 96 caps). ( 3 )  Control buffer.— One pouch with control buffer vial (850 μ L). (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)  Buffered peptone water (BPW).— ( 1 )  6 × 225 mL.— REF 42043. ( 4 )  4 × 3 L.— REF AEB910303/4. ( 5 )  2 × 5 L.— REF AEB910305/2. (6) Acriflavin.— Major laboratory supplier. (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 2 L.— REF 415180. ( 3 )  SMASHER ® XL bag 4 L.— REF 415181. (i)  Plate centrifuge.— ( 1 )  MPS1000 mini PCR plate spinner 110 V.— REF 419196. ( 2 )  MPS1000 mini PCR plate spinner 230 V.— REF 414555. ( 2 )  6 × 90 mL.— REF 42042, 42729. ( 3 )  6 × 225 mL Mini bag.— REF 42729.

AOAC Official Method 2019.03 E. coli O157:H7 in Select Foods GENE-UP® E. coli O157:H7 2 (ECO 2) Kit First Action 2019

[Applicable to detection of E. coli O157:H7 in fresh raw ground beef (73% lean; 25 and 375g), fresh spinach (200g), raw milk cheese (25g), raw ground chicken (25g), raw beef trim (25 and 375g), raw ground pork (375 g), Romaine lettuce (375 g), raw sausage (25g), raw milk (25g), raw ground veal (25 and 375g), and processed water (25mL).] See Table 2019.03A for a summary of results of the interlaboratory study. Caution:  Dispose of used or unused reagents as well as any other contaminated disposable materials following procedures for infectious or potentially infectious products. It is the responsibility of each laboratory to handle waste and effluents produced according to their type and degree of hazardousness and to treat and dispose of them (or have them treated and disposed of) in accordance with any applicable regulations. E. coli O157:H7 is a Shiga toxin-producing E. coli strain. Strains of serotype O157:H7 are a demonstrated hazard to laboratory personnel. The infectious dose is estimated to be low, 10–100 organisms. Care must be taken when handling samples that may contain E. coli O157:H7. Strict compliance with biosafety level (BSL)‑2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or cultures. While BSL-2 containment is suitable for all other E. coli , BSL‑3 practices and equipment are recommended for activities likely to produce significant aerosols or for activities involving production quantities of this particular organism. Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for E. coli . Follow appropriate safety guidelines when handling potentially contaminated samples. Waste should be disposed of in compliance with local and national legislation. A. Principle The GENE-UP ® E. coli O157:H7 2 (ECO 2) kit is designed for use with the GENE-UP ® Thermocycler. The GENE-UP ® ECO 2 kit contains all of the necessary components for PCR, including sample-specific primers and probes and an internal amplification control. The GENE-UP ® Thermocycler detects fluorescence at several wavelengths (channels) to allow for multitarget detection in the same reaction vessel. The fluorescent signal from a sample is recorded in channel 640, while the fluorescent signal for an internal amplification control is recorded in channel 705. The software automatically interprets the results for the internal amplification control and determines the sample result based on the outcome of the control. Both the assay for the sample and the internal amplification control utilize dual fluorescence resonance energy transfer (FRET) hybridization probes. These probes consist of two different short oligonucleotides that hybridize to an internal

(j)  GENE-UP ® PCR tube holder.— REF 414573. (k)  GENE-UP ® lysis tube remover.— REF 414469. (l)  GENE-UP ® heavy rack holder.— REF 414571. (m)  GENE-UP ® lysis tube holder.— REF 414572. (n)  Troemner vortex mixer adaptor.— REF 421713. (o)  Biotix filter pipet tip.— 10 µL; REF 419194.

Confirmation materials.— All items are available from bioMérieux, Inc. (a)  Cefixime-Tellurite (CT) mixture.— REF 42606. (b)  SMAC CT agar.— REF 43391. (c)  CHROMID ® O157:H7 ID-F.— REF 42605/42630. (d)  CHROMID ® EHEC agar.— REF 413093/413697. (e)  Latex O157 , ex. SLIDEX ® E. coli O157.— REF 417407. (f)  VIDAS ® ESPT.— REF 30229.

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