Food Safety Magazine, April/May 2010

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.