Enteropathogenic (EPEC) and Shigatoxigenic Escherichia coli (STEC) in broiler chickens and derived products at different retail stores

Enteropathogenic (EPEC) and Shigatoxigenic Escherichia coli (STEC) are foodborne pathogens that cause potentially fatal infant diarrhea and hemolytic uremic syndrome, respectively. We investigated the presence of intimin and Shiga toxin encoding genes, as indicators of EPEC and STEC presence in cloacae and chicken products. The analyzed products were hamburgers, giblets and carcasses obtained from poultry and butcher shops. EPEC contamination predominated over STEC contamination in cloacae and chicken products, although some differences were detected when the kind of food or shop was taken into account. In particular, among chicken hamburgers we found a greater proportion of EPEC than STEC-positive samples at poultry shops, while in butcheries STEC was predominant. This finding could suggest cross contamination during handling at butcheries. The results indicate that it is necessary to improve hygienic measures both during slaughtering and manipulation of chicken products at retail stores, to provide a safe product to consumers.     

PCR and DHPLC methods used to detect juice ingredient from 7 fruits

To provide accurate and fast method for labeling regulation on fruit juice, conventional PCR, real-time PCR and DHPLC techniques were explored in this study to detect ingredient from 7 fruit species. ITS1-5.8S-ITS2, TrnL-TrnF from chloroplast genome and thaumatin-like protein gene from nucleus genome were targeted. Sensitivity of 6 primer (probe) pairs was determined to be 1-10 pg DNA. Orange and mandarin were universally amplified by the same primer pair and the 8 bp divergence of PCR products could be differentiated by DHPLC analysis. 30 Fruit samples collected from local market were tested and no mislabeling was discovered.     

Comparison of the RAPID-B® flow cytometer and the BAX® system for the detection of non-O157 shiga toxin-producing Escherichia coli (STEC) in beef products

The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B^®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B^® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX^® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B^® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX^® system and RAPID-B^® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B^® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX^® system and RAPID-B^® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B^® flow cytometer and USDA-FSIS reference method. The RAPID-B^® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B^® flow cytometer based on whole cell detection generated similar results as BAX^® system therefore the RAPID-B^® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products.     

Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

We developed a multiplex real-time (RTi) PCR method for the simultaneous detection of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus using zot, vmrA, and vuuA as the respective target genes. A set of primer pairs specific for those target genes was designed and employed in the SYBR Green-based multiplex RTi-PCR assay. Quantitative analyses with ten-fold serially diluted genomic DNA of each target organism resulted in a linear correlation between C_T values and the amount of each target genome per reaction, with a lower detection level of less than ten genome copies per reaction. Similar sensitivities were observed for Vibrio-spiked seafood samples (oyster, crab meat, and raw fish). After 8 h of enrichment culture of the seafood homogenate in alkaline peptone water, our optimized multiplex RTi-PCR was shown to achieve theoretical maximum sensitivity (ca. 10⁰ CFU/gram food homogenate). Our proposed method is simple, robust and readily adaptable in routine laboratories, allowing for high-throughput surveillance of pathogenic Vibrio species in seafood.     

Monitoring of genetically modified soybean events in sausage products in South Korea

The use of genetically modified (GM) ingredients in various types of food products has increased worldwide. In this study, qualitative real-time PCR assays targeting five GM soybean events (RRS, MON89788, A2704-12, A5547-127, and MON87701) were performed to monitor their use in sausage samples. Thirty sausage samples containing soybean ingredients on their label were collected from Korean food markets and analyzed. The endogenous lectin gene of soybean was used as an internal positive control. Real-time PCR showed that the threshold cycle (Ct) values ranged from 28.62 to 33.90 for the 30 sausage samples. Of the 30 sausage samples, 19 samples were negative for the presence of GM soybeans. In the positive samples, the results revealed three GM soybean events [Roundup Ready Soybean (RRS), A2704-12, and/or MON89788] in 11 of the 30 food samples tested. These results demonstrate that sausage products contain different GM soybean events.     

Codfish authentication by a fast Short Amplicon High Resolution Melting Analysis (SA-HRMA) method

Atlantic cod (Gadus morhua, L.) is one of the most consumed white fish with high gastronomic relevance in Europe occupying the 8th place in the most captured specie worldwide. G. morhua stocks are depleted configuring a framework favoring the fraudulent substitution of this specie by less valuable ones. The authentication of fish based products at the species level constitutes presently an important issue. Current methods for fish species identification are mainly based on sequencing, a technique that is time and resources consuming boosting the need for the development of more expedite methods.In this work we had developed a one-tube fast High Resolution Melting Analysis (HRMA) method for the identification of G. morhua adulteration with Gadus macrocephalus and Gadus chalcogrammus. HRMA of the ca. 600 bp COI fragment initially assayed proved to be inefficient in species discrimination which lead to the development of a new approach targeting small fragments (ca. 100 bp) containing sequence variations between the three species. Seven small polymorphic regions were identified and specific primer pairs were de novo designed. One region proved to be highly efficient in the species discrimination in HRMA mode. The technique was applied in marketed processed samples showing that 2 out of 33 samples (6%) were mislabeled as G. morhua (identified as G. macrocephalus). PCR and SA-HRMA was performed in fast mode allowing very short analysis time (less than 1 h) enabling therefore the possibility of high sample throughput.     

Identification of species in ground meat products sold on the U.S. commercial market using DNA-based methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.