AOAC 2019 First Action Methods

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 into 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 products 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 The following items are available from bioMérieux, Inc. (Hazelwood, MO, USA): ( a )  GENE-UP Cronobacter .―Stock No. 421920 (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 μL). ( b )  GENE-UP Lysis Kit. ―Stock No. 414057. ( c )  GENE-UP Thermocycler .―Stock No. 414056. ( d )  Laboratory paddle blender .—For sample homogenization. ( 1 )  SMASHER ™.―Stock No. AESAP1064. ( 2 )  SMASHER XL .―Stock No. AESAP1100. ( e )  Buffered peptone water (BPW) .—( 1 )  6×225 mL .―Stock No. 42043. ( 4 )  4×3 L .―Stock No. AEB910303/4. ( 5 )  2×5 L .―Stock No. AEB910305/2. ( 6 )  Novobiocin .―Stock No. AEB184150/10. ( f )  Advanced digital heavy-duty vortex mixer .— (1) 120 V .― Stock No. 9456TAHDUSA. ( 2 )  230 V .―Stock No. 9456TAHDEUA. ( g )  GENE-UP lysis rack adaptor .―Stock No. 414570. ( h )  Sterile blender bags.—(1) Type P filter bag .―Stock No. AES400P/50G. ( 2 )  SMASHER XL 2L bag .―Stock No. 415180. ( 3 )  SMASHER XL 4L bag .―Stock No. 415181. ( i )  Plate centrifuge.—(1) MPS1000 Mini PCR plate spinner 110V .―Stock No. 419196. ( 2 )  MPS1000 Mini PCR plate spinner 230V .―Stock No. 414555. ( j )  GENE-UP PCR tube holder .―Stock No. 414573. ( k )  GENE-UP lysis tube remover .―Stock No. 414469. ( l )  GENE-UP heavy rack holder .―Stock No. 414571. ( m )  GENE-UP lysis tube holder .―Stock No. 414572. ( n )  Troemner vortex mixer adaptor .―Stock No. 421713. ( o )  10 µL Biotix filter pipet tip .―Stock No. 419194. Confirmation materials: ( p )  Enterobacter sakazakii isolation agar (ESIA ™ ) .―Stock No. AEB20010. ( 2 )  6×90 mL .―Stock Nos. 42042 and 42729. ( 3 ) 6×225 mL mini bag. ―Stock No. 42729.

AOAC Official Method 2019.01 Cronobacter species in Select Foods and Environmental Samples GENE-UP ® Cronobacter Test Method First Action 2019

[Applicable to detection of Cronobacter ( C. sakazakii , C. dublinensis , C. muytjensii , C. malonaticus , C. turicensis , and C. universalis ) in milk-based powered infant formula (PIF) with probiotics (25 and 375 g), milk-based PIF without probiotics (25 and 375 g), soy-based PIF (25 and 375 g), nonfat dry milk (100 g), soy ingredients (100 g), and stainless-steel and plastic environmental samples.] 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. Cronobacter is a Gram-negative facultative aerobic and anaerobic rod-shaped bacterium. Care must be taken when handling samples that may contain Cronobacter . Strict compliance with biosafety level 2 (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 a Cronobacter , 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 Cronobacter . Follow appropriate safety guidelines when handling potentially contaminated samples. Waste should be disposed of in compliance with local and national legislation. See Table 2019.01A for a summary of results of the interlaboratory study. See tables in J. AOAC Int . for detailed results of the interlaboratory study. A. Principle The GENE-UP ® Cronobacter kit is designed for use with the GENE-UP Thermocycler. The GENE-UP Cronobacter 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 nm, while the fluorescent signal for an internal amplification control is recorded in channel 705 nm. The software automatically interprets the results for the internal amplification control and determines the sample result based on the outcome of the control. The assay, for both the sample and the internal amplification control, utilizes dual fluorescence

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