MPD Enhanced Food Quality Diagnostics
ABSTRACT: We developed new, supersensitive and reliable methods for quantitation of very low levels (a few copies) of Salmonella typhimurium and pathogenic E. coli. We have achieved excellent results using our proprietary MPD enhanced quantitative PCR (qPCR/MPD) technology described above. We developed a high sensitivity/high throughput method using microtiter format. Both the limit of quantitation (less than 10 copies of Salmonella or E. coli DNA) and reproducibility are much better than using prior art methods. We also simplified the sample handling by developing new specifity methods permittting DNA quantitation without DNA extraction.
BioTraces has developed and applied qPCR/MPD for the InvA gene of Salmonella that can measure less than 10 copies to a few thousands copies from a single dilution of sample. Similar sensitivity have been achieved for pathogenic E. coli O157:H7. These assays has been adapted to streptavidin capture of biotinylated PCR products on 96 wells microtiter plates. We implemented a capture technique which uses two distinct labels for mimic and for target, respectively. It allows measurement of target and mimic simultaneously in the same well. Labeling by hybridization may permit decreasing the amount of target that can be detected leading to methods for direct detection of DNA at less than 1,000 copies.
Significance: In the U.S., nearly 5 million illnesses and more than 9,000 deaths are attributable to food poisoning each year. The majority of cases are caused by bacteria such as Salmonella or enteropathogenic Escherichia coli that cause food borne enteric infections. The problem is global, e.g. in 1997 within a few days, over 1,000 persons became ill due to tainted food in Japan. Food borne diseases are a continuing threat to all persons, regardless of age, sex, lifestyle, ethnic background and socioeconomic status. The food borne diseases cause suffering and death and impose a financial burden on society. In the USA, the annual national costs and charges associated with food borne bacteria range from 2.9 to 6.7 billion dollars. This range is for the combined direct and indirect costs associated with food borne illnesses caused by six pathogens: Salmonella, pathogenic E. coli, Campylobacter jejuni or coli, Clostridium perfringens, Listeria monocytogenes, and Staphylococcus aureus.
State of the art: The traditional methods for food pathogen detection are labor intensive and time consuming. These methods can take up to four days to detect a food pathogen (a 24-hour pre-enrichment, 24-hour selective enrichment, 24-hour selective plating followed by biochemical testing for identification). More rapid technologies currently available to the food testing laboratories can detect low levels of food pathogens within 18 to 24 hours. Most of these methodologies involve an overnight pre-enrichment/selective enrichment followed by a detection step. Two "rapid" methods are currently available and claim detection of 1 cfu of E. coli O157:H7 organism in 25 grams of ground beef in 6-8 hours. The Reveal test from Neogen Corporation involves 8 hours of pre-enrichment followed by a 20 minute presence or absence test using colloidal gold labeled antibodies specific to E. coli O157:H7. Another rapid test developed by USDA and IGEN International uses electrochemiluminescence technology. This methodology, currently under evaluation, uses magnetic beads coated with anti- E. coli O157:H7 antibodies and ruthenium-labeled antibodies.
Goals/Methods: We addressed the need for a test system that provides the most sensitive, cost effective and rapid test to be used in food quality monitoring. This new methodology has been implemented as tests for contamination by Salmonella and E. coli O157:H7. MPD permits the developement of two techniques: a fast and ultra sensitive MPD enhanced quantitative PCR (qPCR/MPD); and an ultrafast and very sensitive MPD enhanced direct detection DNA assay (dqDNA/MPD).
We developed methodologies that translate the superior sensitivity of MPD into speed and reliability of food tests. These new methods are generic and can be used for all important food pathogens and uses MPD's capability for: exquisite low end sensitivity, improved assay speed and specificity; simultaneous multicolor analysis and concurrent multi sample measurement, resulting in improved reliability and increased throughput. These advantages make possible the instruments and diagnostic tests with performance capabilities which far exceed current technologies. Ultra-sensitive MPD has already been used to detect sub-zeptomole amounts (< 10-21 M) of biological macromolecules such as DNA, RNA and proteins. Our prototype MPD Imager has enabled the development of super-sensitive tests using both immuno techniques and DNA probes.
We developed a unique technique which permits more rapid, sensitive, and less costly food quality testing. The ultrasensitive MPD technology will revolutionize food quality testing by: significantly reducing the turnaround time to report test results; substantially lowering the cost per test for screening food products; and providing a fast, supersensitive methodology for assuring process quality (e.g., identifying and monitoring potential sites of entry for contamination during processing).
We addressed the need for an integrated diagnostics system that provides the most sensitive, low cost and rapid tests. This new methodology has been implemented in the tests for contamination by Salmonella and pathogenic E. coli. We applied Multi Photon Detection (MPD) technology to food diagnostics to accomplish the following:
Results: We have developed a reliable, MPD enhanced qPCR method for quantitating rare DNAs (qPCR/MPD), i.e. for less than 10 copies of a given target. An ultra-sensitive qPCR/MPD assay for the Salmonella and E.coli O157:H7 has been developed, and adapted to microtiter format. A dual label streptavidin-capture qPCR/MPD has been demonstrated.
Our results suggest that the use of about 20 PCR cycles improves the quantitation. We achieved quantitation of a few copies of Salmonella, E. coli, HIV-1, CMV, RSV and kanamycin resistance gene in the presence of up to 10 micrograms of background human genomic DNA. Using MPD readout, standard curves were generated reliably over the range of 6 to 6,000 copies of target DNA sequences using a single concentration of mimic. Thus, MPD enhanced quantitative PCR with internal controls permits reliable quantitation of DNA at a level ten fold lower than prior-art methods.
It is important to stress that the excellent sensitivity, linearity and specificity of qPCR/MPD for Salmonella and E.coli O157:H7 are all due to the same fact: the unsurpassed sensitivity of MPD. Even for a few copies of DNA, a relatively small number of cycles of PCR are used. The main advantage of using MPD is that qPCR/MPD never approaches saturation of the PCR process.' qPCR/MPD for Salmonella and E. coli O157H:7 has been described in the sections concerned with qPCR/MPD. We stress that both a factor 10 higher sensitivity, and a factor of few higher throughput have been achieved. Also, due to the miniaturization allowed by MPD, a somewhat lower reagents cost is expected.
Simplified sample handling: Food quality testing is typically more difficult to perform than the standard biomedical applications of PCR. Foodstuffs inherently contain resident proteases and other non-specific PCR inhibitors. One of the main uncertainties is introduced by the current need for complicated and costly DNA extraction procedures. These procedures may also lead to sample loses thereby compromising reliability of results. Extraction, especially of trace amounts of DNA can lead to variability of recovery and thus loss of quantitative accuracy. Since most inhibitors are deleterous only at high concentrations, we minimized the effect of inhibitors by simple dilution. In brief, milk (2% fat) was spiked with 500 copies per microliter of target DNA and varying amounts were added to 50 microliter PCR reactions. If 20 microliters of milk was used, no target or mimic bands were observed, but the same amount of milk diluted ten-fold gave good amplifications. Thus, the inhibitory effect of milk on PCR is overcome by ten fold dilution. Because the qPCR/MPD is fully quantitative down to a few copies of DNA, the use of ten-fold dilution permits quantitation without DNA extraction down to about 50 copies/ml of nonprocessed material, e.g. milk.
Under development: We are developing a composite method which, through the combination of steps of culture growth and qPCR/MPD, permits quantitation of the number of viable Salmonella or pathogenic E. coli in 25 gram of material in about 3 hours. We are also developing the qPCR/MPD in microtiter format for other food quality targets, e.g. Listeria.
Most importantly, we are developing methods of MPD enabled direct quantitation of DNA (dqDNA/MPD) and its applications in food quality.
SUMMARY: We developed a highly sucessful MPD-enhanced quantitative PCR assay; both gel based and microtiter formatted assays permit reliable quantitation at a few copies of DNA. Not only the sensitivity, but also the ease of sample preparation and improved rejection of biological matrix artifacts, makes qPCR/MPD a superior method for food quality diagnostics.