MALDI Biotyper Quick Guide for Food Testing Edition 1, Innovation with Integrity MALDI-TOF

Transcription

1 MALDI Biotyper Quick Guide for Food Testing Edition 1, 2018 Innovation with Integrity MALDI-TOF

2 MALDI Biotyper Quick Guide for Food Testing Confirmation of foodborne pathogens, confirmation of quality indicators, and identification of bacterial isolates using the MALDI Biotyper The MALDI Biotyper identifies microorganisms using MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry to measure a unique proteomic fingerprint of an organism. Specifically, the MALDI Biotyper system measures highly abundant proteins that are found in all microorganisms. The characteristic patterns of these proteins are used to reliably and accurately identify a particular microorganism by matching the respective pattern with an extensive reference library. The Bruker MALDI Biotyper method is designed to: Rapidly confirm microorganisms Confirm the preliminary presumptive result of an alternative or a reference method Rapidly identify microorganisms Determine the identity of an analyte from a variety of agar plates The MALDI Biotyper is recognized as an Official Method of Analysis SM by AOAC INTERNATIONAL with the two following reference numbers and scopes: AOAC-OMA # (First Action): Confirmation and Identification of Salmonella spp., Cronobacter spp., Campylobacter spp. and other gram-negative organisms; AOAC-OMA # (First Action): Confirmation and Identification of Listeria monocytogenes and Listeria spp., and other gram-positive organisms. The AOAC-OMA # and AOAC-OMA # protocols are available on

3 The MALDI Biotyper is validated according to the ISO part 6 standard by MicroVal with the four following certificate numbers and scopes: Certificate #2017LR72: Confirmation of Cronobacter spp. from various agar plates; Certificate #2017LR73: Confirmation of Salmonella spp. from various agar plates; Certificate #2017LR74: Confirmation of Campylobacter spp. from various agar plates; Certificate #2017LR75: Confirmation of Listeria spp. and L. monocytogenes from various agar plates. The certificates and the reports of the ISO part 6 validation studies are available on This Quick Guide is designed to support your daily work with the MALDI Biotyper in food microbiology, and it is not a replacement for the full MALDI Biotyper user manual. It provides the list of materials, instruments, libraries and software included in the AOAC and MicroVal approvals, as well as an overview of the necessary workflow. If you have any further queries, requests or suggestions about the application, we will be pleased to assist you. Your MALDI Biotyper team

4 Steps in the analysis workflow (1) Add a target plate to a MALDI-TOF project list Select an isolated colony Transfer sample onto the target plate and add matrix Purpose of the analysis Confirmation of foodborne pathogens or quality indicators See also the flowcharts AOAC Official Method of Analysis and ISO validation by MicroVal Culture media recommended for isolation (1) Salmonella spp. 1. Xylose Lysine Deoxycholate (XLD) (2) 2. Brilliant Green Sulfa Agar (BGA) (2) 3. RAPID Salmonella Agar (Bio-Rad) 4. Brilliance Salmonella (ThermoFisher Scientific) 5. ASAP (biomérieux) 6. Non-selective agar, e.g. Tryptone Soy Agar (TSA) (2) Cronobacter spp. 1. Enterobacter Sakazakii Isolation Agar (ESIA) (2) 2. Cronobacter Chromogenic Isolation Agar (CCI) (2) 3. Non-selective agar, e.g. Tryptone Soy Agar (TSA) (2) BRUKER Bacterial Test Standard BRUKER Sample preparation BRUKER MBT Compass Software for analysis and report creation (4) Run minimum one Bacterial Test Standard (BTS) control per target plate and per run. It is mandatory to get at least one valid BTS control with a log(score) 2.0. Follow the BRUKER Direct Transfer Method. Only use the MBT Compass Library (version with 6903 Do not use self-created or third party reference patterns (MSPs) in the AOAC and MicroVal (ISO 16 The MBT Report for Food Microbiology activated in the MBT Compass Software provides the final flowcharts provided on the following pages. The log(scores) of the first- and second-best matches shall be 1.7 and provide both the same identification at the genus level. If the validation criteria are not met, repeat the analysis by Direct Transfer, and if it fails again, proceed by running the BRUKER procedure for Extended Direct Transfer and/or Extraction. (1) Follow the time and incubation temperature prescribed by the detection or enumeration methods, or described in the technical insert of the culture media. Select isolated colonies on selective agar plate(s). In case there is not enough biological material or no isolated colony(ies), proceed to a purification step. (2) Any standard formulation, including brand names. (3) mccda contains charcoal; this component may result in a lower quality of spectra. Pay particular attention not to transfer any mccda agar material when transferring the colonies onto the target.

5 MALDI-TOF spectrum automatically generated by the software LIBRARY Spectrum instantly matched against reference library to give identification Final review and result validation Campylobacter spp. 1. Modified Charcoal Cefoperazone Deoxycholate Agar (mccda) (3) 2. Campy Cefex Agar (2) 3. RAPID Campylobacter Agar (Bio-Rad) 4. CampyFood Agar (CFA) (biomérieux) 5. Non-selective agar, e.g. Columbia Blood Agar (CBA) (2) Listeria spp. and L. monocytogenes 1. Ottaviani & Agosti (O&A) (2) 2. PALCAM (2) 3. Oxford Agar (2) 4. Modified Oxford Agar (MOX) (2) 5. RAPID L.mono (Bio-Rad) 6. Non-selective agar, e.g. Tryptone Soy Yeast Extract Agar (TSYEA) (2) Identification of bacterial isolates AOAC Official Method of Analysis Any of the culture media mentioned for isolation of foodborne pathogens and quality indicators, or any equivalent non-selective culture agar, e.g. TSA with 5% blood. MSPs or higher) ) validated workflow. confirmation and identification results according to the rationale described hereafter and in the The log(score) of the best match shall be 1.7 for Listeria spp. confirmation. The MBT Subtyping Module enables the differentiation of L. monocytogenes from L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and starts automatically when the log(score) is 2.0. The species identification is displayed after the wording typed as described in the MBT Subtyping Manual. For the identification of L. grayi species, the log(score) of the first match shall be 2.0. Use the consistency category of the first- and second-best matches as described in the MBT Compass User Manual and in the MBT report for Food Microbiology. (4) Results should be reviewed by a trained microbiologist: final foodborne pathogen or quality indicator confirmation or final organism identification should be based on all relevant information available. Confirm or identify the number of colonies prescribed in your detection or enumeration method. In case of discrepancy between the result of the detection or enumeration method, and the result of confirmation with the MALDI Biotyper, it is the responsibility of the laboratory to investigate and use additional relevant tests. The cumulative identification results obtained during ISO part 6 studies and the AOAC-OMA studies have demonstrated that the MALDI Biotyper provides correct identification for more than 99% of foodborne isolates.

6 Confirmation of Listeria spp. and L. monocytogenes, and the identification of other isolates Isolated colonies on appropriate medium Perform Direct Transfer (DT) Analyze on MALDI Biotyper (MBT) Log(score) 1,7 for the best match, leading to Listeria spp. confirmation. Listeria species* identified with log(score) 2 Release the identification result Genus or species (other than Listeria) identification based on the consistency category table as described in the MBT Compass Manual and the MBT report for Food Microbiology Repeat by running DT, Extended Direct Transfer (edt) until Extraction (Ext) Release the identification result Genus or species identification based on the consistency category table as described in the MBT Compass Manual and the MBT report for Food Microbiology Streak onto non-selective agar and repeat the workflow *L. grayi is identified without the need for the MBT Subtyping Module. L. monocytogenes is differentiated from L. innocua, L. ivanovii, L. seeligeri and L. welshimeri with the MBT Subtyping Module. However, L. marthii cannot be differentiated from L. monocytogenes. Note that L. marthii isolates do not usually show a halo on Ottaviani & Agosti Agar and on RAPID L. mono (Bio-Rad). As only a few

7 The MBT report for Food Microbiology, activated in the MBT Compass software, provides the confirmation and identification results according to this flowchart Conclusive identification Release the results for Listeria spp. or L. monocytogenes confirmation Repeat by running DT, Extended Direct Transfer (edt) until Extraction (Ext) Conclusive identification Release the results for Listeria spp. or L. monocytogenes confirmation Streak onto non-selective agar and repeat the workflow strains of the recently described Listeria species are available, the MBT Library contains a limited number of reference spectra. Therefore, the identification is only guaranteed at the genus level.

8 Confirmation of Salmonella spp., Cronobacter spp., Campylobacter spp., and the identification of other isolates The MBT report for Food Microbiology, activated in the MBT Compass software, provides the confirmation and identification results according to this flowchart Release the identification result Genus or species identification based on the consistency category table as described in the MBT Compass Manual and the MBT report for Food Microbiology Repeat by running DT, Extended Direct Transfer (edt) until Extraction (Ext) Release the results for pathogen confirmation Log(score) 1,7 for the two best matches, leading to Salmonella spp., Cronobacter spp. or Campylobacter spp. confirmation Release the identification result Genus or species identification based on the consistency category table as described in the MBT Compass Manual and the MBT report for Food Microbiology Streak onto non-selective agar and repeat the workflow

9 Isolated colonies on appropriate medium Perform Direct Transfer (DT) Analyze on MALDI Biotyper (MBT) Log(score) 1,7 for the two best matches, leading to Salmonella spp., Cronobacter spp. or Campylobacter spp. confirmation Release the results for pathogen confirmation

10 MALDI Biotyper for Food Testing Overview Instrument, library and software Accessories and consumables # microflex LT/SH MALDI-MS System, or # / MALDI Biotyper smart System # MBT Compass Software, Build or higher The activation of the MBT Report for Food Microbiology in the MBT Compass Software directly provides the confirmation and identification results according to the flowcharts provided on the previous pages. # MBT Explorer Module # MBT Compass Library (version with 6903 MSPs or higher) # MBT Subtyping Module for the confirmation of Listeria spp. and L. monocytogenes # spot polished steel target with bar code, or # spot polished steel target with bar code, or # MBT Biotarget 96 with # MSP adapter for MALDI Biotarget # HCCA Matrix, or # MBT Galaxy HCCA Matrix GPR # Bacterial Test Standard (BTS) Recommended accessories can be used to facilitate sample preparation: # bar code scanner # Holder for bar code scanner # MBT Pilot System # MBT Galaxy System

11 Manuals and IFUs Chemicals and small lab equipment # microflex LT/SH smart User Manual for use with the MALDI Biotyper smart System # microflex User Manual for use with the MALDI Biotyper System # MBT Explorer Module User Manual # MBT Compass User Manual # MBT Subtyping Module User Manual # IFU for Bruker Bacterial Test standard (BTS) # IFU for MBT Biotarget 96 # IFU for Bruker Matrix HCCA, portioned or # IFU for MBT Galaxy HCCA Matrix GPR # MALDI Biotyper Protocol Guide For best results, use freshly prepared solutions and chemicals of HPLC or MALDI compatible grade: Standard Solvent (acetonitrile 50%, water 47.5% and trifluoroacetic acid 2.5%) [Sigma-Aldrich #19182 or equivalent] Acetonitrile HPLC/MS-grade water Formic acid Absolute ethanol Colony transfer device, e.g. tooth pick or micropipette tip for 2-20 µl Sterile 1 μl inoculation loops Micropipette tips μl, μl, μl [Biopure or PCR grade] Suitable pipettes for volumes from 1 μl to 1000 μl Eppendorf plastic tubes, 1.5 ml Screw-cap micro tubes [P/N: , Sarstedt or equivalent] and screw caps [P/N: , Sarstedt or equivalent] Bench-top microcentrifuge capable of 13,000 to 15,000 rpm Vortex mixer Additionally, trifluoroacetic acid or guanidine hydrochloride, 70% aqueous ethanol, as well as a lint-free tissue, are required to clean the polished steel targets.

12 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. BDAL , MALDI Biotyper Support Thank you for choosing the MALDI Biotyper System. Whilst every effort has been made to include as much information and help as possible into this guide, there is a dedicated team of application specialists available to assist you. For additional support, please contact your local support team or alternatively contact us: For application support via EMEA: Biotyper.Appl.Support.EMEA@bruker.com US: Applications.MBT.Americas@bruker.com For technical support about hardware and software, call our support team at: EMEA: +49 (0) US: +1 (877) Or send an to: Biotyper.Support@bruker.com IFUs and SDS sheets can be downloaded from: MALDI Biotyper is a registered trademark of Bruker Daltonik GmbH in the European Union and USA. For General Purpose Use (In the United States, Japan and Taiwan: For Research Use Only (RUO)) This product has no declared clinical intended purpose and is not for clinical diagnostic use. Any clinical diagnostic use is at the user s own risk and responsibility. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) ms.sales.bdal@bruker.com -