hygiena KIT 2300 49 Salmonella Detection Kit User Manual

June 10, 2024
Hygiena

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hygiena KIT 2300 49 Salmonella Detection Kit User Manual

hygiena KIT 2300 49 Salmonella Detection Kit.jpg

foodproof®
Salmonella Detection Kit

Order No. KIT 2300 49
Kit for 96 reactions for a maximum of 94 samples

Order No. KIT 2300 50
Kit for 480 reactions for a maximum of 470 samples

Store kit at -25 °C to -15 °C For testing of food and environmental samples Approvals:

Approvals:

1. OVERVIEW

1.1 General Information
Number of Reactions
The kit is designed for 96 reactions or 480 reactions respectively with a final reaction volume of 25 µl each. Up to 94 samples or 470 samples (single sample preparation) plus positive and negative control can be analyzed per run.

Storage and Stability
Store all components at -25 °C to -15 °C. They are guaranteed to be stable through the expiration date printed on the label. Opening of the kit does not shorten the expiration date.

1.2 Applicability
The kit described in this Instruction Manual has been developed for real-time PCR instruments with a FAM and a VIC/HEX detection channel. The performance of the kit was tested with the following real-time PCR instruments: LightCycler® 480, LightCycler® 96 (Roche Diagnostics), Applied BiosystemsTM 7500 Fast (Thermo Scientific), Mx3005P® (Agilent), and others.

The foodproof® Salmonella Master Mix is sequence-specific for a highly conserved gene found in all subgroups of Salmonella. Inclusivity has been tested in several internal and external studies (AOAC-R and MicroVal) with more than 700 strains of Salmonella enterica (all subspecies) and Salmonella bongori comprising members of all O-serogroups according to White-Kauffmann-Le Minor scheme 2007. Exclusivity was determined during the above mentioned studies using more than 50 species of closely related organisms or organisms occurring in the same habitat.

A relative detection limit of 1 to 10 cells per 25 g sample can be achieved with all kinds of foods. foodproof® Salmonella Detection Kit detects down to 103 – 104 cfu/ml in enrichment cultures (depending on the sample preparation kit used: foodproof® ShortPrep I Kit, foodproof® StarPrep One Kit or foodproof® Magnetic Preparation Kits I or IV).

1.3 Kit Contents
A schematic representation of the foodproof® Salmonella Detection Kit with all its components.

KIT 2300 49:

FIG 3 KIT 2300 49.JPG

OVERVIEW
KIT 2300 50:

FIG 4 KIT 2300 50.JPG

FIG 5 KIT 2300 50.JPG

2. INSTRUCTIONS

2.1 Required Material
Most of the required equipment and reagents are available through HygienaTM.
Please contact us for further information.

**** Use a real-time PCR cycler suitable for detection of respective probes.

Material

FIG 6 Material.JPG

2.2 Precautions and Preparations
The kit provides all reagents required for the PCR. However, in order to achieve reliable results, the entire assay procedure must be performed under nuclease-free conditions.

Follow the instructions below to avoid nuclease, carry-over, or cross- contamination:

  • Keep the kit components separate from other reagents in the laboratory.

  • Use nuclease-free labware (e.g., pipettes, pipette tips, reaction vials).

  • Wear gloves when performing the assay.

  • To avoid cross-contamination of samples and reagents, use fresh aerosol barrier pipette tips.

  • To avoid carry-over contamination, transfer the required solutions for one experiment into a fresh tube, rather than directly pipetting from stock solutions.

  • Physically separate the workplaces for DNA preparation, PCR setup, and PCR to minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps.

  • Sample Material: Use any sample material suitable for PCR in terms of purity,
    concentration, and absence of inhibitors.

  • DNA Extraction: We provide sample preparation kits suitable for all kind of food
    samples and primary production stage samples.

  • Positive Control: Always run a positive control with the samples. Use the provided
    control DNA (Control Template) or a positive sample preparation control.

  • Negative Control: Always run a negative control with the samples. To prepare a negative control, replace the template DNA with PCR-grade water. Include a negative control during sample preparation to monitor reaction purity and cross contamination.
    This extraction control can be used as an additional negative control reaction.

  • Confi rmation: If required, positive results may be confi rmed by appropriate methods
    (e.g., reference method).

  • Waste Disposal: All contaminated and potentially infectious material, like enrichment
    cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations. For more information, e.g., proper disposal of unused chemicals, please refer to the appropriate safety data sheet (SDS).

**** Keep the PCR mix away from light.

For more information, please refer to the appropriate safety data sheet (SDS). The SDS is
available online at www.bc-diagnostics.com.

2.3 Enrichment and DNA extraction
The foodproof® Salmonella Detection Kit is intended for the rapid detection of Salmonella
DNA isolated from enrichment cultures prepared by valid methods and inoculated with all kinds of foods that are potentially contaminated with Salmonella. This method is intended for use in a laboratory setting. The kit must not be used in diagnostic procedures.

General procedure: Pre-enrichment broth and temperature according to ISO 6579 or BAM (Chapter 5) or USDA for 20 +/- 2 h. Sub-cultivation 1/10 in pre-warmed brain heart infusion broth (e.g. 1 ml sample + 9 ml broth) for 3 h at 37 °C is recommended. Other suitable, validated enrichment procedures can also be used.

Recommended DNA extraction kits:

  • KIT 2301 75 / 76 – StarPrep One Kit / large (suitable for most matrices)
  • KIT 2301 79 – Magnetic Preparation Kit I (for an automated extraction)
  • KIT 2301 84 – Magnetic Preparation Kit IV (for an automated extraction)
  • KIT 2301 70 – ShortPrep I Kit (for a very quick extraction)

2.3.1 Certified Methods
The kit is AOAC-RI Performance Tested MethodsSM program validated with the following
foods: chicken breast, cocoa powder, coconut, cumin, dough, dry pet food, egg powder, food dye, Frankfurter sausage, ice cream, Lyoner sausage, milk chocolate, milk powder, minced meat, pasta, sliced cabbage, smoked fish, watermelon, wet pet food, white pepper and environmental samples (AOAC certificate No. 120301). The validation was done on a LightCycler 1.5 from Roche Diagnostics. AOAC-RI (certificate no. 120301)

Validation with the foodproof® ShortPrep I Kit, S 400 01 for DNA extraction according to BAM/FDA:

  • White pepper and cumin: enrichment in TSB for 24 h +/- 2 h at 35 °C, sub-cultivation
    in 1/10 BHI, 3 h at 37 °C

  • Milk powder: enrichment in brilliant green water for 24 h +/- 2 h at 35 °C, sub-cultivation in 1/10 BHI, 3 h at 37 °C

  • Cocoa: enrichment in low fat milk for 24 h +/- 2 h at 35 °C, sub-cultivation in 1/10 BHI,
    3 h at 37 °C

  • Egg powder, coconut, sausages, smoked fish, ice cream, watermelon, sliced cabbage,
    food dye, pasta, dough, wet and dry pet food: enrichment in lactose broth for 24 h +/- 2 h at 35 °C, sub-cultivation in 1/10 BHI, 3 h at 37 °C

In a harmonized MicroVal / AOAC-RI validation (MicroVal projects 2011LR40 and 2011LR42, AOAC no. 120301), the kit is validated in combination with the foodproof® Magnetic Preparation Kit I (KIT 2301 79) for automated sample preparation and the foodproof® StarPrep One Kit (KIT 2301 75) for manual sample preparation. The validation was done with the following food categories: beef meat, chocolate and bakery products, egg products, feed samples, meat and meat products, milk and dairy products and primary production stage samples (PPS). The validation was done with the following PCR instruments: LightCycler® 480 II (software version 1.5.1) from Roche, CFX96TM from Bio-Rad (software version CFX Manager 2.0) and Mx3005P from Agilent (software version MX4.1d). To be in compliance with the actual standards a renewal of the MicroVal projects 2011LR40 and 2011LR42 was done according to ISO 16140-2. The table below shows an overview how the samples were processed within the latest study.

Protocols MicroVal projects 2011LR40 / 2011LR42 – Renewal study

FIG 7 Protocols MicroVal projects.JPG

The foodproof® Salmonella Detection Kit is MicroVal validated according to ISO 16140-
2:2016, certificate number 2011LR39.

In this project the foodproof® Enterobacteriaceae plus Cronobacter Detection Kit for prescreening, followed by the foodproof® Salmonella Detection Kit, were validated; both in
combination with the foodproof® Magnetic Preparation Kit IV (KIT 2301 84) and Reagent
D (KIT 2300 01) for automated sample preparation. The validation was done according to section 2.4 “MicroVal Protocol including the semi-automated DNA extraction” in the manual “R_302_15-1_L_20_foodproof_Eb_plus_Cronobacter” for the foodproof® Enterobacteriaceae plus Cronobacter Detection Kit.

Manual DNA extraction was performed with the foodproof® StarPrep One Kit (KIT 2301 75 / 76) and Reagent D (KIT 2300 01), according to section 2.5 “Protocol including the manual DNA extraction with the foodproof® StarPrep One Kit” in the manual “R_302_15-1_L_20_foodproof_Eb_plus_Cronobacter_BPZ_V2-3” for the foodproof® Enterobacteriaceae plus Cronobacter Detection Kit.

The alternative method was validated to be applicable to the scope: infant formula and infant cereals, probiotics containing products, ingredients, and environmental samples for the manual extraction procedure. For the semi- automated DNA extraction procedure, the alternative method was validated to be applicable to the scope: infant formula and infant cereals as well as probiotics containing products. The validation was performed in comparison to the ISO method for Salmonella (ISO 65791:2017). For the validation the LightCycler® 480 II (software version 1.5.1) from Roche Diagnostics was used.

If the foodproof® Salmonella Detection Kit is used in combination with the foodproof®
Enterobacteriaceae plus Cronobacter Detection Kit for pre-screening, please refer to the
manual of the foodproof® Enterobacteriaceae plus Cronobacter Detection Kit (KIT 2300 43).

Cultural Confirmation
Positive PCR results were confirmed with the ISO 6579 reference method for MicroVal validations and with the BAM (Bacteriological Analytical Manual Online, Chapter 5: Salmonella, Andrews H.W., Hammack S.T., December 2007 Edition) for AOAC-RI validations, e.g., serologically by latex agglutination (Salmonella Test Kit, Oxoid DR1108A) and biochemically by using API 20E strips (bioMerieux 20100). For further information please visit the following web addresses: www.iso.org or www.cfsan.fda.gov/~ebam/bam-5.html.

Within the MicroVal study LR39, all Salmonella-positive PCR results were culture confirmed as described in ISO 6579-1:2017.

Within the MicroVal study 2011LR40 / 2011LR42, all Salmonella-positive PCR results were culture-confirmed as described in EN ISO 6579/A1 (2007) for PPS samples and according EN ISO 6579:2002 for food and feed samples.

Details of the confirmation procedure within the MicroVal studies In the context of the MicroVal validations, all presumptive positive samples must be confirmed. For this, 0.1 ml of the first enrichment in BPW was transferred into 10 ml RVS (incubate at 41.5 °C for 24 h +/- 3 h) and 1 ml enrichment was transferred into 10 ml MKTTn (incubate at 37 °C for 24 h +/- 3 h). 10 μl from MkTTn and RVS were spread onto XLD and a chromogenic agar. For the PPS samples MRVS was inoculated with the BPW enrichment (incubate at 41.5 °C for 24 h +/- 3 h or optionally additional 24 h +/- 3 h). Isolations were done onto XLD and a chromogenic agar. For all samples: Incubate the plates according to the manufacturer’s instruction. Confirm 1 to 5 characteristic colonies using the ISO 6579 reference confirmation tests.

For the automated protocol the isolation and confirmation steps were only done for RVS and XLD. For the PPS samples the confirmation was additionally done by streaking 10 μl MOSSEL broth onto XLD and a chromogenic agar, followed by confirmation using the ISO
6579 reference confirmation tests.

2.4 Procedure
This protocol describes how to perform the analysis of DNA extracts by real- time PCR.

2.4.1 Workflow
Thaw the solutions, mix by flicking the tubes four to five times, and briefly spin vials in a
microcentrifuge before opening.

FIG 8 Workflow.JPG

FIG 9 Workflow.JPG

2.4.2 Program Setup
Program your real-time PCR instrument before setting up the PCR reactions. Select the
following channels:

FAM (Salmonella), and VIC (Internal Control).

As an alternative to VIC, HEX can be used. For the PikoReal® 24, Yakima Yellow has to be
selected.

FIG 10 Program Setup.JPG

For some real-time PCR instruments the probe quencher as well as the usage of a passive
reference dye has to be specified. This kit contains probes with TAMRA as quencher and no
passive reference dye.

For users of the Agilent Mx3005P instrument: Click “Instrument” and “Filter Set Gain Settings” to open the Filter Set Gain Settings dialog box in which the gain settings may be
viewed and modified. For FAM the Filter Set Gain Setting has to be modified to “x1”.

2.4.3 Data Interpretation
Verify results of positive (Control Template) and negative controls (H2O), before interpreting
sample results. Always compare samples to positive and negative control. Review data from
each channel and interpret results as described in the table.

FIG 11 Data Interpretation.JPG

2.5 Troubleshooting

FIG 12 Troubleshooting.JPG

FIG 13 Troubleshooting.JPG

2.6 Support
If you have questions or experience any problems with our products, please contact us:

www.hygiena.com/technical-support-request

Our aim is to provide you with a solution as quickly and effectively as possible. We would
also like you to contact us if you have any suggestions for improving the product or in case
you would like to use our product for a different application. We highly value your feedback.

3. ADDITIONAL INFORMATION

3.1 Testing Principle

The foodproof® kit provides all necessary reagents and a control template for reliable interpretations of results. To ensure maximum reliability of the kit and to prevent misinterpretation of negative results due to inhibition of the amplifi cation, an Internal
Control (IC) is included. A hydrolysis probe was designed to bind specifi cally the IC, allowing detection in the respective channel, whereas the target DNA is detected in another channel. In case of a negative result due to inhibition of the amplifi cation by the sample DNA of interest, the amplifi cation of the IC is suppressed as well, whereas a negative result for the sample DNA of interest and amplifi cation of the IC clearly indicates the absence of parameter in the sample. The real-time PCR kit minimizes contamination risk and contains all reagents (except for template DNA) needed for the detection of target DNA. Primers and probes provide specifi c detection of target DNA in food and environmental samples, including primary production stage samples. The described performance of the kit is guaranteed for use only on the real-time PCR instruments listed above.

Step-by-Step Procedure

  1. Using the kit‘s sequence-specific primers in a polymerase chain reaction (PCR), the
    PCR instrument and the supplied reagents amplify fragments of specific sequences for target DNA.

  2. The PCR instrument detects these amplified fragments in real time through fluorescence generated by cleavage of the hybridized probe due to the 5´-nuclease activity of the Taq DNA polymerase. The probe is labeled at the 5´-end with a reporter fluorophore and at the 3´-end with a quencher.

  3. During the annealing/elongation phase of each PCR cycle, the probe hybridizes to an internal sequence of the amplicon and is cleaved by the 5’ nuclease activity of the Taq DNA polymerase. This cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal.

  4. The PCR instrument measures the emitted fl uorescence of the reporter dye.

Prevention of Carry-Over Contamination
The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing carry-over contamination between PCR’s. This technique relies on the incorporation of deoxyuridine
triphosphate (dUTP) during all amplifi cation reactions, and the pretreatment of all successive
PCR mixtures with the heat-labile UNG. The UNG cleaves DNA at any site where a deoxyuridine residue has been incorporated. The resulting abasic sites are hydrolyzed due to the high temperatures during the initial denaturation step, and can no longer serve as PCR templates. The heat-labile UNG is inactivated during the initial denaturation step. Native DNA (e.g., the isolated target genomic DNA) does not contain uracil and is therefore
not degraded by this procedure. Since dTTP is replaced with dUTP and UNG is included in this kit, decontamination can be achieved with the provided reagents.

3.2 Trademarks
foodproof®, microproof®, vetproof®, ShortPrep®, RoboPrep®, and LyoKit® are trademarks of BIOTECON Diagnostics GmbH.

HygienaTM is a registered trademark of Hygiena.
Other brand or product names are trademarks of their respective holders.

3.3 Reference Number
The reference number and original BIOTECON Diagnostics article numbers:
R 302 27 and R 302 27 L.

3.4 Change Index
Version 5, February 2022:
Information regarding the MicroVal validation has been included.
Rebranding, new document layout and updated content.

Hygiena LLC
CA 93012 Camarillo
USA
www.hygiena.com/technical-support-request

Manufactured by
BIOTECON
Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
bcd@bc-diagnostics.com
www.bc-diagnostics.com

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