A rapid, standardized protein extraction method using adaptive focused acoustics for identification of mycobacteria by MALDI-ToF MS

Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI- based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2 min. Using a log confidence cut-off of ≥2.0, overall
agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall
agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M.mucogenicum. Addition of spectra froma single clinical strain of each species to the existing database increased overall agreement to 93.1%.

Automated NGS Library Preparation with Beckman Biomek FXP and Covaris E220

Genomics Group at Biogemma has developed an automated workflow for NGS library preparation that combines high versatility with robustness. Covering all steps of NGS library preparation, it dramatically reduces hands on time while maintaining a high level of flexibility in DNA input and type of library prepared.

For the DNA fragmentation step, the combination of the Covaris E220 with Beckman FXP liquid handler enables an automated DNA transfer both before and after the mechanical DNA shearing step. Combined with the high level of performance in precision, reproducibility and accuracy provided by Covaris Focused-ultrasonicators, it robustly integrates DNA fragmentation into the overall
workflow.

AFA-based MALDI Biotyper™ Sample Prep for MGIT™ Positive Mycobacteria Cultures

At the ASM 2016 meeting, Johns Hopkins Medical Institutes, Baltimore, MD reported (4) on preliminary results to improve MGIT positive cultures identified with Covaris AFA sample preparation for the MALDI Biotyper. The authors noted that longer incubation of the MGIT tube after initially signalling positive by the instrument improved results. The laboratory tested longer incubation of spiked samples by 24, 48 and 72 hours. It was theorized that slowgrower Mycobacteria would require longer re-incubation, whereas fast-grower Mycobacteria could require less re-incubation. By allowing additional replications of the Mycobacteria in liquid, the contributions of both media components and human proteins would be in a lower ratio to the peptides available for extraction by Covaris AFA.

AFA-based Sputum Liquification to Enable Downstream Microbial Analysis

Adaptive Focused Acoustics is well established for shearing DNA for Next Generation Sequencing and for extraction of peptides prior to MALDI-TOF MS. These applications are optimized by considering the initial starting material concentrations, volumes and desired outcomes. Then, a specific software-controlled program allows the researcher to conduct experiments with Peak Incident Power (PIP), Duty Factor (DF), Cycles per Burst (CPB), temperature control and process duration. Following recommendations from Covaris, a research Lab can optimize an extraction protocol by measuring changes in results while controlling a single variable. After each variable is considered, a final set of conditions can be locked in for reproducible results.

AFA-based MALDI Biotyper Sample Preparation for Mycobacteria Colonies

MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) continues to be adopted worldwide as a highly effective instrumentation method for microbial identification. MALDI-TOF MS measures the unique molecular fingerprint of an organism. This Application Note updates the use of Adaptive Focused Acoustics® (AFA™) specifically for use with the MALDI Biotyper® for identification of mycobacteria. Thousands of bacteria, yeasts and fungi have been

AFA-based Efficient DNA Extraction and Optional Mechanical Shearing from Mycobacteria smegmatis

We demonstrate efficient extraction and shearing of DNA from M. smegmatis prior to Next Generation Sequencing. Extraction of DNA from a strain of Mycobacterium smegmatis using the Covaris M220 Adaptive Focused Acoustics AFA instrument with a specific chemistry and set of energy conditions is shown to optimize DNA recovery.

AFA-based RNA Extraction from Mycobacteria smegmatis

We demonstrate that intact RNA can be efficiently extracted from Mycobacteria smegmatis using the Covaris Adaptive Focused Acoustics® (AFA®) M220 or ME220 Focused-ultrasonicators. By applying precise control of AFA power conditions, RNA can be differentially extracted to favor intact subunits or shorter fragments. AFA technology can effectively replace bead-beating protocols for extraction of RNA. AFA extraction methods are more consistent, provide high quality RNA and are easy to perform.

Microbiome Sample Preparation: Efficient DNA Extraction and Optional Mechanical Shearing

Ranjan et. al., (1) described the process for mechanically shearing DNA to 300-600 bp fragments with a Covaris® S220 Focused-ultrasonicator. The shearing process was done on DNA previously
extracted and frozen. To achieve high level of reproducible shearing, the Covaris S220 Focused-ultrasonicator with SonoLab software controls critical parameters such as Peak Incident Power
(PIP), Duty Factor (DF), Cycles Per Burst (CPB), temperature and duration of treatment.