ERP Micro December 2019

1612  B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018

The subtyping module starts automatically to differentiate closely related Listeria species: L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. L. grayi does not require the use of the subtyping module.

was analyzed by the Bruker MALDI Biotyper using the DT technique and by the eDT or the EXT if necessary to obtain a reliable result. Growth from each TSA/YE plate was also analyzed by conducting a catalase test, determining hemolysis reaction from a blood agar plate, conducting a motility test, and by conducting biochemical confirmation using API Listeria (bioMérieux). Prior to testing, all isolates were well characterized by either 16S rDNAsequencing or by biochemical analysis. Statistical analysis .—Each collaborator reported results for each organism on the data sheets provided. The data sheets were submitted to the study director at the end of testing for statistical analysis. Data for each organism was analyzed to present the percentage of correct confirmations and identifications for each target ( L. monocytogenes, non- monocytogenes Listeria species, and Gram-positive organisms) by dividing the number of correct confirmations or identifications by the total number of isolates tested for that target.

B. Apparatus and Reagents

Items (a)-(k) are available from Bruker Daltonik GmbH (Bremen, Germany). ( a )  Microflex LT/SH MALDI-MS System (Cat. No. 8269956) or MALDI Biotyper Smart System (Cat No. 1853665). ( b )  MBT library .—Version MSP 6903 or higher. ( c )  MBT Compass software .—Cat. No. 1843241. ( d )  MBT Explorer module .—Cat. No. 1828476. ( e )  MBT Compass software .—Cat. No. 1829023. ( f )  Subtyping Module software .—Cat. No. 1842250. ( g )  Barcode scanner .—Cat. No. 8268821, optional. ( h )  Holder for barcode scanner .—Cat. No. 8276754, optional. ( i )  MSP 96 target polished steel BC (Cat. No. 8280800) or MSP 48 target polished steel BC (Cat. No. 8281817), or MBT Biotarget 96 (Cat. No. 1840375) with the MSP adapter MALDI Biotarget (Cat. No. 8267615). ( j )  HCCA (α-cyano-4 hydroxycinnamic acid) Matrix, portioned (Cat. No. 8255344) or MBT Galaxy HCCA Matrix GPR (Cat. No. 1823405). ( k )  Internal positive E. coli DNA: Bacterial Test Standard (BTS) .—Cat. No. 8255343. Additional Items Required ( l )  Incubators. —Capable of maintaining temperatures of 34–38ºC for optimal growth. ( m )  Adjustable, variable volume pipets .—Capable of sampling and delivering 1–1000 μL. ( n )  Micropipette tips .—Aerosol-resistant. ( o )  Refrigerator .—Capable of maintaining 2–8°C. ( p )  Freezer .—Capable of maintaining –20°C. ( q )  TSA/YE (standard formulation) . ( r )  OXA (standard formulation). ( s )  MOX (standard formulation). ( t )  Ottaviani and Agosti Agar (O&A, standard formulation). ( u )  RAPID’L.mono Agar (RLM, Bio-Rad). ( v )  PALCAM (standard formulation). ( w )  Vortex mixer. ( x )  Bench-top microcentrifuge .—Capable of 15 871– 21130 g , equivalent to 13000 to 15 000 rpm for Eppendorf tube centrifuged with a 5424R rotor. ( y )  Standard solvent (acetonitrile 50%, water 47.5%, and trifluoroacetic acid 2.5%). ( z )  HPLC grade water. ( aa )  Formic acid. ( bb )  McFarland Standards. ( cc )  Absolute ethanol. ( dd )  8MGuanidineHydrochloride(GdnHCl).—Commercially available. ( ee )  Trifluoroacetic acid.

AOAC Official Method 2017.10 Confirmation and Identification of Listeria monocytogenes, Listeria Species, and Other Gram-Positive Organisms Bruker MALDI Biotyper Method First Action 2017

[Applicable to identification and confirmation of L. monocytogenes, Listeria species, and other Gram-positive organisms from select media types.] See Tables 2017.10A and 2017.10B for a summary of results of the interlaboratory study. See Tables 2017.10C and 2017.10D for detailed results of the interlaboratory study.

A. Principle

The Bruker MALDI Biotyper method is intended to be used for the automated confirmation and identification of bacteria isolated onto agar plates. Classification and identification are based on proteomic fingerprinting using MALDI-TOF MS. First, the MALDI process transforms the proteins and peptides from the isolated microorganisms into positively charged ions. This is achieved by irradiating the matrix–sample composite with a UV laser. The matrix absorbs laser energy and transfers protons to the intact proteins or peptides in the gas phase. These ions are electrostatically accelerated and arrive in the flight tube at a mass-dependent speed. Because different proteins/peptides have different masses, ions arrive at the detector at different times (TOF). The MALDI Biotyper System measures the time (in the nanosecond range) between pulsed acceleration and the corresponding detector signal of the ions, and the time is converted into an exact molecular mass. The highly abundant microbial ribosomal proteins result in a mass spectrum with a characteristic mass and intensity distribution pattern. This pattern is species specific for many bacteria, yeasts, and molds, and can be used as a “molecular fingerprint” to identify a test organism. The mass spectra are transformed into peak lists by the MALDI Biotyper software and are compared with the patterns in the reference library.

( ff )  Colony-transfer device. ( gg )  Sterile inoculation loops. ( hh )  1.5 mL microcentrifuge tubes.

( ii )  Fiber-free cloth .—For cleaning MSP 96 target polished steel BC (Cat. No. 8280800) or MSP 48 target polished steel BC (Cat. No. 8281817) only.