Under the Microscope
Trends in instrumentation in diagnostic molecular microbiology
Sepehr N Tabrizi Department of Microbiology The Royal Children’s Hospital Parkville, Vic. 3052, Australia Email:
[email protected]
In recent years, replacement of conventional microbiological culture-based diagnostic testing with molecular detection of bacterial, fungal and viral pathogens has become routine across many laboratories. This increase in adopting molecular methods has been primarily due to the availability of automated instrumentation that can provide results in a timely and efficient manner, without the need for in-depth knowledge of molecular biology. The use of molecular methods is therefore translating into better treatment, care and management of patients. Advancement in instrumentation has occurred across all stages of molecular testing from preto post-analytical stages of testing. This short summary is by no means exhaustive of all systems available or soon to become available; however, it gives a brief overview of the most common instruments in use and also some of the advancements that are in the process of becoming available. An increasing number of molecular instruments are available for use in laboratories. The utility ranges from pre-analytic sample handling and pipetting to nucleic acid extraction, amplification and postanalytic processing (Table 1). Included among these are a number of platforms that have integrated primary sample processing, testing and reporting using a bidirectional laboratory information management system (LIMS) connection. This allows diagnostic results to be reported faster and with more accuracy. Most of the integrated systems are closed platforms that only allow use with specific assays manufactured by their respective manufacturers. An exciting advancement is integration of pre-analytic pipetting and hit (cherry)picking platforms including Tecan Freedom Evo, which allows
Figure 1. The Roche FLOW System, soon to become available, allows integration of all steps in sample handling and PCR set-up using realtime amplification technologies. 18 2
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LIMS enquiry with automated picking from a range of samples on board for appropriate tests of choice. However, such open systems require some optimisation and adaptation to the users’ applications and integration with other platforms used in the laboratory. An upcoming advancement in instrument integration by Roche, called Roche FLOW, combines a few of the existing Roche platforms to allow a truly open platform for pre-analytic, analytic and postanalytic integration (Figure 1). FLOW allows flexible primary tube loading using a new primary sample handling platform, nucleic acid extraction using MagNA Pure 96, a new PCR set-up platform and amplification on LightCycler 480. The process uses reagent and specimen tracking through a common software with ability to link to LIMS. This system allows high-throughput testing with the ability to process >384 samples and generate over 1000 qPCR results per 8-hour workday. Such an open system platform allows laboratories to enhance their automation, minimising the risk of transcription error and improving result reliability. It also allows economic use of reagents and disposables with variable batch sizes. Advancement in the field of nucleic acid sequencing and large scale analysis will continue at a fast rate with continual improvement in bioinformatics as well as automation. Increased speed and accuracy will make the prospects of personalised medicine in the area of infectious diseases detection together with determination of virulence factors and antimicrobial resistance a reality in the near future. Mass spectrometry can provide rapid identification of multiple organisms, although cost of such instruments are prohibitive in implementation in most laboratories. However, with increase use, cost of instrumentation will naturally be lowered. Acquiring a molecular testing system can eliminate a significant volume of costly send-out tests. Simplification of molecular assays and instruments will be critical to foster significant movement into laboratories with medium and small testing volumes and limited laboratory space. The advancement in automation and improvement of instrumentation will simplify integration of molecular assays and foster wider implementation of more efficient systems. The future is very exciting for faster, more accurate and sensitive detection of analytes using advancement in molecular detection technologies.
Biography Sepehr Tabrizi is a molecular microbiologist with considerable expertise in the development and validation of molecular diagnostic methods for detection of bacterial, viral and fungal pathogens. He is currently the senior scientist in charge of the Molecular Microbiology Laboratory at the Royal Women’s Hospital and an Associate Professor at the University of Melbourne, Department of Obstetrics and Gynaecology. His research has had a strong focus on using molecular tools for better understanding disease transmission and clinical translation of research findings, which has led to improvements in the diagnosis, management and prevention of infections. MICRO BIOLO G Y A UST R ALIA NOV EMBE R 20 13 *
Under the Microscope Table 1. List of instruments and manufacturers used in molecular microbiology. Process
Name of platform
Manufacturer
Pre-analytic
epMotion M5703
Eppendorf
COBAS p 630
Roche/Hamilton
Nimbus/Starlet
Seegene/Hamilton
Freedom EVO
Tecan
m2000 sp, m24sp
Abbott
Nuclisens EasyMAG, Nuclisens MiniMAG
bioMe´rieux
MagMax, iPREP, BenchPro 2100, Gene Catcher
Life Technologies
QIAsymphony, EZ1 series, QIAcube, QIAxtractor
Qiagen
MagNA Pure LC, MagNA Pure LC 2.0, MagNA Pure 96, MagNA Pure Compact
Roche
Seeprep 12
Seegene
Versant K-PCR Sample prep Module
Siemens
m2000rt
Abbott
MX3005p
Agilent Technologies
Nuclisens EasyQ
bioMe´rieux
QX100 Droplet Digital PCR; CFX series: CFX Connect, CFX96, CFX384
BioRad
SmartCycler
Cepheid
Mastercycler nexus
Eppendorf
3M Integrated Cycler
Focus
StepOne (plus), 7500 (Fast), ViiA 7, QuantSudio series
Life Technologies
Illumigene
Meridian
Nucleic acid (NA) extraction
Amplification (real-time)
Rotor-GeneQ 2-plex, Rotor-GeneQ 5-Plex
Integrated NA and amplification instruments
Analyte analysis
LightCycler 1.5, 2.0, 480, 1536, cobas z 480, LightCycler 96
Roche
m2000/Real time
Abbott
Easy-Plex
AusDiagnostics
BDMax
BD
Genexpert
Cepheid
Panther, Tigris TDS
Gen-Probe
QIAsymphony RGQ
Qiagen
cobas 4800, COBAS Ampliprep/Taqman
Roche
Versant K-PCR AD module
Siemens
Tapestation – 2200 electrophoresis system
Agilent Technologies
Bruker Maldi Biotyper
BD
Diversilab strain typing platform
bioMe´rieux
Caliper Labchip DX -fragment analysis
Caliper
MAGPIX, Luminex 100/200, FLEXMAP 3D
Luminex
Pyromark, QIAxcel
Qiagen
Seaplex
Seegene
MassARRAY
Sequenom
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Qiagen
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