1. PARIOFORMA
Summary Report – September 2010
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 Since its introduction little more than 10 years ago, real-time PCR has become the main technical
platform for nucleic acid detection in research and development.
 The scope of real-time quantitative (qPCR) assays for gene-expression analysis in research
applications and for sequence-specific nucleic acid detection for diagnostic purposes continues to
grow.
 Growth drivers include:
◦ Speed
◦ Increasingly robust assays
◦ Automation
◦ Higher-throughput
◦ Multiplexed analytical strategies
◦ Increasingly affordable machines
 Technology trends have focused on:
◦ Increased throughput
◦ Faster analysis time
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3. PARIOFORMA
◦ Quantitative resolution
◦ Assay kits – specific assays for known targets – (now even with the probe and primer sequences to facilitate
compliance with evolving MIQE guidelines).
◦ Ready to use reagents
◦ Automation/ pre-analytical sample processing
◦ User friendly interfaces:
 Software driven inputs/ controls – “menu” driven devices
 Data output - analysis software providing amplification curves, color-coded heat maps, cycle threshold (Ct)
data, standard curves, etc. for each run.
◦ Point of test/ point of care testing
 Real-time PCR is becoming a mature technique. Instruments are very robust and reliable, and
optimised assays can be ordered from multiple vendors.
 Although the technique will be further refined and more niche products will be developed, main
future improvements are not expected within the real-time PCR technology itself.
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4. PARIOFORMA
 Real-time monitoring of PCR has simplified and accelerated PCR laboratory procedures and has
increased information obtained from specimens including routine quantification and differentiation
of amplification products.
 Fast, quick and reliable results are expected of molecular diagnostics - early diagnosis allows for
appropriate and timely clinical intervention.
 Rapid or "almost point-of-care" molecular tests take about a few hours versus days for a culture-
based test.
 Clinical diagnostic applications and uses of real-time PCR are growing exponentially, real-time PCR is
rapidly replacing traditional PCR, and new diagnostic uses continue to emerge.
 Real-time PCR was first introduced for infectious disease testing and kits are offered today from a
variety of suppliers: - e.g. Roche, Abbott, Qiagen, Biotools, Roboscreen and LightUp Technologies.
All assays are probe-based to minimize the rate of false positives.
 Assays for targets with high mutation rates, such as HIV and HCV are optimized for subtypes and
clades prevalent in developed countries, and may have poor sensitivity for the variants common in
the parts of the world were these tests are most needed.
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5. PARIOFORMA
 In vitro diagnostic tests may be segmented:
◦ Commercial in vitro diagnostic products
◦ Diagnostic assays developed by laboratories develop themselves - so called "home brew" assays .
 There is a high cost coupled with a considerable time lag for commercial kits to gain regulatory
approval and be available on the market. Consequently, for some infections there may be no
commercial interest in developing FDA-approved assays.
 Much initial diagnostic work is done with home brew assays - 2003 definition/ guidance from the
FDA:
◦ Assays incorporate “general purpose reagents [and] general purpose instruments [used] by a laboratory to set
up an in-house (“home brew”) test or laboratory testing service.
◦ While specimens can [and do] travel to the laboratory setting up this service, the test itself is not marketed
outside of the single laboratory setting up this service.
◦ It is the responsibility of the laboratory to develop a recipe for the [home brew] test at hand and to take
responsibility for establishing and maintaining performance.
◦ Although not part of the [ASR/home brew] rule, FDA has indicated that FDA is not requiring the in-house test to
go through premarket FDA review. See FDA, CDRH, Analyte Specific Reagents.
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6. PARIOFORMA
 In Europe, assay systems/ kits need to be CE marked under the European In Vitro Diagnostics
Directive – but in-house (home brew) assays fall outside of the scope of the Directive. However, if
use of the in-house assay becomes part of a commercial transaction then the Directive ”should”
apply.
 Market feedback demonstrated that in some countries home brew assays are reimbursed for clinical
use.
Multiplexing
 There has been a trend to increase the multiplexing capabilities of qRT-PCR analysis.
 Instrument manufacturers were aware that limitations on the number of channels capable of
measuring different mRNA targets had to be addressed in order to allow for qRT-PCR to analyze the
full spectrum of interacting mRNA signals resulting from a cellular response.
 New instruments have been brought to market in the past 5 years installed with many more filter
channels capable of complex multiplexing.
 Market feedback suggests that multiplexing is not without its difficulties in terms of experimental
design, setup and in particular reproducibility. Nevertheless, end users increasingly demand
machines with multiplex capabilities even though more often than not, they are only using duplex
functionality.
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7. PARIOFORMA
 In the R&D industry (includes academic but primarily the private sector of pharmaceutical/
biotechnology companies), qRT-PCR has become a valuable tool utilized to understand the response
to different treatment options by monitoring the various signalling pathways present in cells.
 An early inhibitor to the utility of qRT-PCR in the R&D sector was that end-users were being hindered
by the time and effort necessary to design and optimize their experiments.
 However, this has been overcome in the main by the development of:
◦ Sophisticated software solutions aimed at experimental design and setup (e.g. primer design)
◦ Higher quality reagents
◦ “Off-the-shelf” kits
 Today the major concerns surround:
◦ Standardisation of sample management
◦ Data management
 This is a very high throughput and high-volume segment - in addition, this is a crowded segment that
attracts the largest of market players each chasing the R&D “cash cow.”
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8. PARIOFORMA
 The global market for food safety testing can be broken down by contaminant type and includes:
◦ Pathogens
◦ Genetically modified organisms (GMOs)
◦ Toxins
◦ Residues
◦ Other contaminants / ingredient verification
 Pathogen testing has the largest share of the market followed by GMO testing.
 Drivers for food testing include:
◦ Recurring food-related public health issues
◦ Consumer expectations
◦ Mounting regulatory food safety requirements
◦ Industry competitive advantage
◦ Cost of product recall
◦ Protection of brand value
◦ Avoid litigation
 Many companies do not recover financially from a food-borne illness outbreak associated with their
product.
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9. PARIOFORMA
 Historically, pathogen reduction efforts focused on the pre-release screening of finished product –
however, this approach does little to prevent processing failure or improve production efficiency.
 Today the focus is on the entire processing continuum.
 This shift in focus has not only resulted in a measurable reduction in the incidence of food-borne
pathogens but has also significantly reduced the need for extensive finished product screening and
costly release holds.
 Drivers for adoption of real time PCR in food testing:
◦ The main driver for PCR systems over other methods is in time saving, both in the total time from sampling to
result and in the technician time needed to set up and run the assay.
◦ High degree of automation built into PCR systems means that staff time and training is effectively minimised.
◦ High specificity generally means fewer repeat tests are required.
◦ Availability of commercial kits with AFNOR and/or AOAC validation.
◦ Multiplex capabilities allowing for several pathogens to be assayed in a single run.
◦ Real-time PCR assays can also be made quantitative without adding additional stages or operations.
◦ Cost benefits in rapid test results.
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10. PARIOFORMA
 Drivers for GMO testing:
◦ Controversies surrounding human and environmental safety
◦ Labeling and traceability
◦ Consumer choice
◦ Intellectual property rights
◦ Food security
 Efficient detection of genetically modified organisms is becoming increasingly important to comply
with more restrictions and increased awareness in food safety.
 The presence of transgenic crops in the food and feed market has given rise to different regulations
in different countries – for example:
◦ US: food labelled “GM free” if <5% GM
◦ EU: food labelled “GM” If > 1% GM
◦ Japan: food labelled “GM” If > 5% GM
 PCR is the favoured method for detecting and quantifying GMOs.
 However, criticism of the technique remains in that it is costly, highly technical, time consuming, and
often wavers in accuracy and precision.
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