Procedurally, the food or environmental sample is briefly enriched, then
heat-killed for user safety. An aliquot of
the heat-killed enrichment is placed in
the MICT device. As the sample flows
through the device, antibody-coated
supermagnetic nanoparticles bind the
target antigen if present. Bound nanoparticles are subsequently captured by
additional affixed antibodies in a downstream stage of the device, creating a
classic immunoassay “sandwich.” When
the device is placed into the reader or
magnetometer, the presence of bound
nanoparticles is detected, resulting in a
positive result. System software enables
test results to be downloaded into an
Excel format to the user’s laptop or laboratory information management system
for further data management.
An AOAC “Performance-Tested
Method” for E. coli O157:H7 in the
MICT format (Method #060902) is currently available in North America. In
terms of its critical performance attributes, the following data have been published by the AOAC Research Institute.
Internal and independent studies of E.
coli O157:H7 detection via MICT at 1
CFU/g in 25-g and 375-g ground beef
samples compare well with both cultural
and molecular reference methods.
The MICT format requires a 6-hour
sample enrichment at 39 °C in commercially available media. Finished results
are available within an hour of sample
enrichment and at sensitivity and specificity levels greater than or equal to 98%.
The procedure appears to require technical expertise no greater than standard
Good Laboratory Practices (GLPs). It is
believed that the method’s limit of detection (LOD) is 100 to 1,000 times better than current immunoassay or
molecular offerings. This lower LOD
would account for the reduced sample
enrichment time required, enabling the
generation of an accurate test result
within a single production or lab shift.
The improved time-to-result may benefit
both operations and quality assurance
(QA) by accelerating raw and finished
product release, reducing product hold
times and providing an earlier indication
of emerging process control issues.
Published pricing data indicate instrument and consumables costs are in the
neighborhood of $10,000 and $10, respectively. This represents a sharp decrease in system instrument cost versus
many automated test systems, while vol-ume-dependent cost-per-test appears
consistent with other rapid methods. Superior speed, comparable accuracy, ease-of-use and reduced overall cost would
support further consideration of the diagnostic innovation afforded by MICT
technology and its potential impact on
food processing. MICT-based rapid assays for additional foodborne pathogens
are currently under development, with
anticipated completion dates in 2010.
LAMP represents a process innovation in DNA-based testing methodologies, specifically in the area of
polymerase chain reaction (PCR), the
technology basis for most current molecular methods. To achieve exponential
DNA amplification and enable rapid, accurate detection of the target analyte,
current PCR-based methods utilize ther-mocycling. As the temperature of the enriched sample aliquot is increased, DNA
strands within it denature. As the temperature is reduced, specific primers bind
the single target DNA strands and amplification occurs. This process is repeated
hundreds of times to amplify the target
DNA to a detectable level.
The temperature cycling and DNA
amplification process, enabled by PCR,
is conducted within a thermocycler. The
process innovation that LAMP technology provides is the amplification of target DNA at a static temperature (63 °C).
This is achieved by the utilization of
multiple primers, including loop primers
and Bst polymerase, resulting in an amplification rate of 109 target nucleotides
(i.e., 1 billion DNA copies) within 15–60
minutes at a single, static temperature.
Because the process is isothermal, a
simple, inexpensive heating block, accommodating 32 sample tubes simultaneously, replaces the thermocycler.
Figure 2: Turbidometer and Laptop
A by-product of LAMP amplification
is magnesium pyrophosphate, resulting
in increased sample turbidity. Optical
sensors within the turbidimeter (Figure
2) monitor sample turbidity at 6-second
intervals. As the turbidity threshold is
exceeded, indicating amplification of the
target DNA, the LAMP-based system reports a positive result, often in as little as
15 minutes. If there is no turbidity increase within an hour, the system reports
a negative result. The system software resides in a dedicated laptop, providing additional data management capabilities.
As with all test methods, sample enrichment is required prior to running the
1-hour amplification/detection procedure. Independent LAMP technology
performance studies conducted in 2009
report an LOD of 104 CFU in the enriched sample, comparable to other molecular methods, and statistically
equivalent or better sensitivity and specificity levels. One LAMP-based system
manufacturer has an enrichment optimization project under way to reduce
sample enrichment times to less than 8
hours. LAMP-based assays for E. coli
O157:H7, Salmonella, L. monocytogenes
and Campylobacter are currently available.
Several tests have been awarded Japanese
Ministry of Health approvals. Others are
also currently involved in the AOAC approval process.
In terms of critical performance attributes, much can be said for LAMP’s
molecular method innovation. The amplification/detection process has been reduced to less than 1 hour. Whatever the
manufacturer’s enrichment optimization
project achieves will ultimately determine LAMP’s bottom-line speed equivalence or advantage. Independent studies
and Japanese Ministry of Health approvals support equivalent accuracy to
cultural and molecular reference methods, while the pending AOAC approval
process will further reinforce those findings. Multiple independent research papers presented at the International
Association for Food Protection’s 2009
Annual Meeting support the technology’s viability.
The LAMP test procedure indicates
no technician requirement beyond
GLPs, consistent with all molecular
methods. Replacement of the PCR ther-