Research Terms
Introduction: Infection by Shiga toxin-producing Escherichia coli (STEC) strains results in bloody diarrhea, hemolytic uremic syndrome, and renal failure. STECs are broadly divided into E. coli serotype O157 and non-O157 serogroups. In the last decade, the non-O157 serogroup has emerged as a major food-borne pathogen of concern worldwide. To date, a large number of STEC serogroups have been identified. The frequency of infections of STEC serogroups is variable, with six non-O157 STEC serotypes being most commonly reported: O26 (26%), O103 (22%), O111 (19%), O121 (6%), O45 (5%), and O145 (4%), leading to their classification by the USDA as adulterants (zero tolerance) in non-intact raw beef products. The food industry, especially the red meat industry, is constantly threatened by outbreaks associated with STEC serogroups.
Problem: E. coli belonging to the seven adulterants STEC serogroups are comprised of both virulent and avirulent strains. That is, many isolates are positive for the serogroup. However, they lack the virulence (stx and eae) genes and are considered avirulent strains. A major limitation of this official testing method and other commercially available assays for STEC testing is that they result in the diversion of meat products due to avirulent strains of E. coli serogroups (i.e., false-positive test results). According to a USDA, FSIS Office of Public Health Science 2018 report, the false-positive rates of potential screening were 93% for beef manufacturing trimming, 81% for ground beef, 100% for bench trim, and 94% for other components. This high false-positive rate results in an annual loss of approximately $47 million for the beef industry.
Technology: The present invention provides real-time PCR methods for the specific detection and differentiating of strains testing positive into virulent and non-virulent strains. The assay was validated using a large number of pure culture strains, spiked food samples, and DNA samples obtained from the federal meat inspection program. The assay shows greater than 90% sensitivity, specificity, positive predictive value, negative predictive, and test accuracy value for the Escherichia coli of O26, O111, and O121 assays.
The FDA's DNA barcoding method is the gold standard method for identifying seafood species. This method involves shipping samples to testing laboratories. The testing can take from 5 – 10 days and cost $100 - $200 per sample. This method is expensive, time-consuming, highly susceptible to tissue contamination during sample collection and provides detailed information regarding fish species. However, those in the wholesale/retail fish market don’t necessarily wants all the information generated by the DNA barcoding, they only care about whether or not the particular seafood species they’ve purchased or are interested in purchasing is the same species as mentioned on the label (a simple yes/ no answer). There is a need in the industry for a low-cost, rapid assay to discern whether a certain sample is the same seafood species as mentioned on the label or not. Furthermore, there is a need for a method that can be performed onsite, at an in-house food processing facility, in a resource limited setting, using a minimally trained labor.
Our assay is a method that rapidly determines if a specific food product is present or not, the method comprising: providing a sample comprising at least one target sequence; placing the sample into at least one container; using reagents to amplify a sample; amplifying a sample using a small footprint nucleic acid amplification device, wherein the sample is amplified by exposing it to different sets and types of primers (conventional and rhPCR primers) in conditions suitable for nucleic acid amplification, where each set of primers comprises of a forward and reverse primers; exposing the amplified sequence to a means of detection, wherein the means of detection provides a present/not present result; and identifying whether the food or ingredient is present or not, based on the results.
Current methods of estimating the concentrations of microorganisms are based on the most probable number (MPN) methods. The culture based MPN method is a slow, and laborious that requires two or more days for completion. Commercially available molecular methods generates presence and absence results for the detection assays. Target bacterial concentration in the test samples can be estimated by these detection assays can be extrapolated using external standard curve. As each sample varies in composition, microbial load, and presence of natural inhibitor (i.e., sample matrix effect for each sample type), a separate standard curve is needed for each food product.
Pathogen detection assays rely on the enrichment of samples for increasing the number of target pathogens in the biological sample (i.e., food, meat, blood, urine, tissue, swabs). DNA isolated from these enrichments is used for amplification of specific DNA sequences in a PCR reaction. These commercially available PCR assays generate only the presence or absence of results and are not geared for estimating initial pathogen levels in the test samples. Knowing initial target organism concentration in the biological sample is critical for food industries and clinicians.
What is needed is a method which not only specifically detects the presence of a target organism, but assay can estimate the initial number of organisms present in the test sample.
This invention describes a novel detection approach which specifically detects the target and estimates the initial target load in the test samples. The method incorporates a novel PCR amplification approach in which each DNA samples is amplified using a combination of multiple primer set with varying amplification efficiencies. A high assay specificity is achieved by the incorporation of a highly specific dual-labeled probe. The primer-pair with higher amplification efficiency detects samples with lower concentration. Whereas the primer-pair with lower amplification efficiency only detects samples with higher concentration. In this way, by knowing which primer-pair applied and which did not, target pathogen concentration in the test sample can be estimated.
