Pre-analytical Sample Preparation for Microbiology
As the leading technology of controlled, focused, non-contact, isothermal mechanical disruption of cells and molecules, Covaris offers new and improved solutions to accelerate microbiology discovery workflows. Whether quantifying a specific bacterial gene, performing microbial whole genome sequencing (WGS), identifying pathogens and infectious disease agents, or building reference genomes from de novo NGS data for your bioinformatics databases, the Adaptive Focused Acoustics® (AFA®) technology will provide consistent and high-quality pre-analytical sample prep results.
When research involves evaluating proteins via MALDI TOF MS or LC-MS, AFA can be utilized to isolate target biomolecules. The table below contains additional information and selected publications, which provide further insight into the versatility AFA brings to the bench. As the gold standard in the field of nucleic acid mechanical shearing and molecular extraction techniques, Covaris provides the expertise needed to move genomic and proteomic-based applications to the next level.
Next Generation Sequencing (NGS)
Advanced high-throughput ultra-deep sequencing technologies continue to rapidly improve research discovery capabilities. Specifically, the shift from conventional 16S rRNA amplicon sequencing to NGS is becoming more pragmatic due to its affordability [1,2]. Metagenomic analysis via NGS enables researchers to gain a more comprehensive understanding the microbiome plays in regulating health and disease, drug metabolism, effective clinical interventions for oncology patients, and other key biological functions in a variety of hosts [3,4]. The Covaris AFA technology is the gold standard for providing unbiased and uniform sample fragmentation in the NGS library construction workflows.
Precise: Controlled delivery of focused high-frequency bursts of acoustic energy applies hydrodynamic shear forces to the DNA for random sample fragmentation
Reproducible: Random fragmentation with less insert size variation resulting in higher quality (uniform sequencing coverage, lower IQR values) data for rendering confident detections of nucleotide variants
Accurate: Minimizes bias and allows for the taxa and relative abundance to be determined rapidly with confidence at the species level
Controlled: Not dependent on experimental condition variables such as sample or enzyme concentrations, presence of EDTA, and reaction temperature
Matrix Assisted Laser Desorption Ionization (MALDI) Time of Flight (TOF) Mass Spectrometry (MS)
The extraction of peptides from Acid Fast Bacilli (AFB) is challenging because of the mycolic acids, complex glycolipids, glcyopeptidolipids, waxes, and other fatty acids that make up the cell walls of mycobacterium . Rapid and accurate species identification is critical for rendering patient diagnoses and for the selection of the most effective clinical antibiotic treatment plan. The extraction of high-quality peptides from recalcitrant mycobacterium can be circumvented by using the Covaris truXTRAC MALDI TOF MS Protocol that relies on the patented AFA ultrasonic technology and precisely engineered microTUBES with pre-loaded 0.5 mm glass beads.
Versatile: Extract mycobacterial peptides for rapid species identification from solid agar (Middlebrook, Lowenstein-Jensen) and liquid cultures (MGIT) for spot ready analysis on the Bruker Daltonics Biotyper
Efficient: Single processing vessel with fewer transfer steps for a more rapid and streamlined approach (less direct hands-on time) using the truXTRAC protocol with each sample needing <5 minutes for processing (manual, conventional protocol: >90 minutes)
Accurate: Achieve log scores for confident species identifications using most parts of the existing commercial MALDI TOF MS spectral databases without the need for augmentation
Quantitative PCR (qPCR)
Template DNA or RNA needs to be isolated prior to performing standard quantitative molecular biology assays such as qPCR, RT-qPCR, or TaqMan-based assays for accurate and sensitive quantifications of bacterial target genes . AFA treated bacterial samples such as Mycobacterium allows for the isolation of the targeted nucleic acids without relying on a harsh solvent-based extraction methods.
Precise: Extract stable DNA and labile RNA molecules from difficult to lyse bacteria such as mycobacterium via AFA
Controlled: Gently extract stable RNA with intact subunits with minimal fragmentation to perform quantitative PCR assays
Versatile: Mechanically disrupt samples to lyse and extract target molecules to develop your own in-house molecular assays
Liquid Chromatography Mass Spectrometry (LC-MS)
The use of harsh chemicals, probe sonicators, and bead beating techniques for cell lysis of bacterial and yeast samples may result in loss or damage to target proteins. As a result, missed identifications that may be critical for your biomarker discovery projects may be observed. The extraction of intact cellular proteins via AFA reduces overall degradation of targeted biomolecules to provide a more detailed understanding of the target proteins being studied .
Sensitive: Higher protein recoveries leading to improved unique identifications previously missed when using other extraction methods
Controlled: Precisely controlled ultrasonic energy processes samples in an isothermal environment to ensure rapid dissipation of heat introduced into the system during treatment that may damage native conformations and activity of target proteins
Reproducible: Improve repeatability from sample to sample and operator to operator with lower CVs
User-Friendly: The LC-MS approach is less labor-intensive compared to PFGE, no need for primers (PCR-based), or immunoassays that require the use of antibodies
These systems deliver ultrasonic acoustic energy in a highly tunable manner, enabling customized programmable control of peak incident power, duty factor, cycles per burst, duration and temperature for each specific sample application. Established applications and management controls are enabled.