ERP Micro December 2019

1618  B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018

( e ) When the identification run is started, the sample positions to be measured appear as white circles in the target display. ( f ) During the run, the appearance of the sample positions and BTS control positions in the target display reflects the success of the measurement and identification process of each test organism. ( g ) If spectrum measurement is successfully completed, the left half of the sample position is colored green. If measurement fails, the left half of the sample position is colored orange. ( h ) The coloring of the right half of the sample position indicates the score value of the identification (green, yellow, or red). The legend display explains the color coding used to indicate the status of sample positions in the target display. ( i ) The MBT Subtyping Module supports the identification of the most-encountered Listeria species in food, feed, and related environmental samples: L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri . This module is not required for L. grayi identification. The Subtyping Module starts automatically and assesses the quality of the profiles before checking the specific biomarkers for species identification. The other genera and related species are identified using the Consistency Category of the MBT Compass Manual. Scores >2.0 will be presented in green and are determined to be acceptable with high confidence identification (both Listeria species and non- Listeria organisms). Results presented in yellow will have values between 1.70 and 1.99 and are determined to be acceptable with low confidence identification (both Listeria species and non- Listeria organisms). Results presented in red are considered not acceptable for identification. The consistency category of the identification is based on the confidence level of the best and second-best matches as described in Table 2017.10E . Tip : Point to the information button in the top-right corner of the target display to show the color-coding legend. ( j ) When the identification procedure is completed, the organism identification result will appear in the result table. ( k ) The result table of the MALDI Biotyper System window gives a real-time overview of the identification results for the active test run. This table contains only the best-matching reference pattern for each test organism and is a summary of the complete Run Results Report.

Tip : APDF result report can be generated at any time by clicking the View Results Report button or View > Results on the menu bar. ( l ) When measurement and identification of all sample positions have been completed, the status bar displays the status message Finished Successfully . ( m ) If the identification is not conclusive, the DT procedure should be repeated. For 5–10% of the isolates, an eDT or EXT may be required. ( a ) If the identification score received is below 2.00, the isolate should be reanalyzed following the eDT. Note: After inoculation of bacteria on recommended isolation media, colonies are stable for up to 12 h when held at room temperature (20–25°C). If testing is not done within 12 h, subculture the test organism before testing on the MALDI Biotyper System. ( b ) Using a sterile colony-transfer device, smear an isolated colony of bacteria as a thin film directly onto a sample position on a cleaned reusable steel target or disposable Biotarget. ( c ) Overlay the sample spot with 1 μL 70% aqueous formic acid and allow to dry at room temperature (20–25°C). ( d ) Select a minimum of one BTS control position on a target plate to inoculate with BTS solution. It is mandatory to get at least one valid BTS control per target plate and per run. Therefore, it is advised to select two BTS control positions. Note: After samples and BTS have dried, HCAA Matrix portioned solution must be added within 30 min, or the target must be cleaned and the inoculation of sample and BTS must be repeated. ( e ) Pipette 1 μL of reconstituted BTS solution onto the selected positions and dry at room temperature (20–25°C). Note: Make sure that the screw-cap tube containing HCCA Matrix is tightly closed after use tominimize solvent evaporation. ( f ) Overlay each of the sample position and BTS control positions with 1 μL of HCCAMatrix. Use a new pipet tip to add matrix to each inoculated sample position. ( g ) Dry the inoculated target at room temperature (20–25°C). ( h ) The inoculated target is now ready for use. Note: An inoculated target must be processed within 24 h of preparation, or the target must be cleaned and the inoculation of samples and BTS must be performed again. J. Formic Acid/Ethanol (Tube) Extraction Sample Preparation ( a ) If the identification score received is still not conclusive after the eDT, the isolate should be reanalyzed following the EXT procedure. ( b ) Transfer 300 μL HPLC grade water to a tube. Transfer colonies from the plate agar into the water to create a cell ( d ) Centrifuge the suspension for 2 min at 15871–21130 g , equivalent to 13 000 to 15 000 rpm for Eppendorf tube centrifuged with a 5424R rotor). Decant the ethanol. ( e ) Repeat step (d) to remove all residual ethanol. Avoid contact with the pellet. ( f ) Air-dry the pellet for a minimum of 5 min at room temperature (20–25°C). suspension of approximately 1.5–2 McFarland. ( c ) Add 900 μL ethanol and mix suspension. I. extended Direct Transfer (eDT)

Table 2017.10E. Identification consistency category descriptions

Identification consistency category

Description

High

The best match is a high-confidence identification. The second-best match is: - a high-confidence identification in which the species is identical to the best match. - a low-confidence identification in which the genus is identical to the best match. - a nonidentification. The requirements for high consistency are not met. The best match is a high- or low-confidence identification. The second-best match is: - a high- or low-confidence identification in which the genus is identical to the best match. - a nonidentification. The requirements for high or low consistency are not met.

Low

None