ECCMID 2015 POSTER NUMBER EV0463
Four Minute Mycobacterium spp Sample
Preparation Process for MALDI-TOF Identification
Philip Onigman , Gary Smejkal ,
2
2
Carl Beckett , Bill Skea ,
2
2
Srikanth Kakumanu , Jim Laugharn
1
2,
Grand Street Consulting, Reading, Massachusetts USA
2
Covaris, Inc., Woburn, Masschusetts USA
1
Covaris, 14 Gill Street, Unit H,
Woburn, Massachusetts, 01801-1721, USA
www.covarisinc.com
INTRODUCTION AND PURPOSE
METHODS
RESULTS
Implementation of MALDI-TOF for routine identification of
M. tuberculosis and NTB is simplified and accelerated with Covaris
Adaptive Focused Acoustics® (AFA™).
M. smegmatis ATCC 19420 was cultured using Middlebrook 7H10 agar plates at 37 °C for 48-72
hours. The Bruker MycoEX conventional tube extraction protocol was used to prepare samples
for the Bruker Microflex LT MALDI-TOF with the BDAL and Bruker Mycobacterium database. Bioburden Reduction
MALDI-TOF Scores
Scores of >2.0 result in correct identification of genus/species.
Many Microbiology labs delay validation of MALDI-TOF for identification of
Mycobacterium spp, due to the time required and the inconvenience of
the sample preparation protocol. We have developed an alternative to the
heating and tube-extraction protocol. Using Covaris AFA technology, we
demonstrate a safe, rapid, easy-to-use, and robust protocol that delivers
the equivalent or higher MALDI-TOF scores. Covaris AFA is a unique, multipurpose platform with proven performance in sample preparation for NGS,
PCR, LC-MS, and other pre-analytical applications. Capable of singlesample through automated, 96-sample batch processing, a computerized
format enables extremely precise and reproducible sample preparation.
M. smegmatis samples were also processed using the Covaris protocol. The Covaris M220
Focused-ultrasonicator delivers highly controlled, isothermal, mechanical energy to each
sample. Software controlled acoustic parameters include peak incidence power, cycles per
burst, duty factor, temperature and time. The Covaris Acoustical Cuvette (microTUBE, 130 µL
capacity) containing 0.5 mm diameter beads was used as a process vessel for each sample.
A solvent mixture of acetonitrile/formic acid/water (50%:35%:15%) up to 110 µL was added.
A 1 µL loop was used to transfer colonies into each microTUBE with beads and solvent. The
Covaris microTUBE is a closed vessel that enables non-contact processing and prevents aerosol
formation.
The Covaris protocol at 18 °C for 2 minutes was equivalent to
conventional protocol heating at 100 °C for 30 minutes for
bioburden reduction. Both protocols were effective in
bioburden reduction prior to samples being applied to the
MALDI-TOF target plate. The Covaris protocol reduced the
M. smegmatis concentration from 108 cfu/microTUBE to <101
cfu/microTUBE. This is a significant benefit, because heating
each sample for 30 minutes prior to MALDI-TOF analysis is a
major inconvenience.
M220
• Single-sample
processing
BEFORE
Each microTUBE was processed for two minutes using AFA on the M220. Temperature was held
consistent at 18 °C. After the AFA treatment, each microTUBE was centrifuged for 2 minutes at
18,000 RCF to clarify. A 1 µL sample was applied directly to the MALDI-TOF target plate.
Samples were compared for bio-burden reduction, preparation time, and score consistency.
Each sample preparation protocol was performed in duplicate and five replicates were applied
to the MALDI target plate.
Control
AFTER
Covaris protocol - treated
CONCLUSIONS
Covaris Protocol 4 minute / 4 step / 0 open-close
microTUBE™
E220
STEP 1:
Centrifuge microTUBE
prefilled with beads
STEP 2:
STEP 3:
Add reagent
STEP 4:
Add biomass
AFA
Centrifuge
18,000 RCF
for 2 min
• Closed vessel
• Non-contact processing
Centrifuge at 3,000 RCF
For 10 seconds to
pellet beads
• Automated, 96-sample,
batch processing
Add 100 μL of
Extraction Solvent
Add 1 μL inoculation
loop with biomass
AFA at 40W PIP,
50% DC, 200 CPB
for 120 sec
MALDI-TOF
4.4 x 10 cells
8
Spot 1 μL of supernatant
on MALDI target.
Conventional Protocol 45 minute / 10 step / 3 open-close
MALDI-TOF
Reagent kits
• Pre-mixed solvents
• Acoustical Cuvettes
Add colony
+ H20
Heat 100°C
30 min
Add
EtOH
Vortex
Spin
2X
Decant,
dry pellet
Add
beads, ACN
Vortex
Add formic acid
Vortex
Spin
Spot 1 μL of supernatant
on MALDI target.
8 - log reduction
The Covaris protocol provides safe, effective, rapid sample preparation for
identification of Mycobacterium spp by MALDI-TOF. This protocol
eliminates the heat inactivation step and consolidates transfer,
centrifugation, and wash steps into a closed vessel, making it easier with
less hands-on time for laboratory personnel. By reducing send-outs and
delays, laboratories will manage cases more effectively, utilize existing
capital, and reduce operating costs. The Covaris AFA technology-based
protocol enables laboratories already using MALDI-TOF to add routine
identification of Mycobacterium spp to their service.