Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Electrochemical Detection of Nucleic Acids Using Three-Dimensional Graphene Screen-Printed Electrodes.

Electrochemical approaches, along with miniaturization of electrodes, are increasingly being employed to detect and quantify nucleic acid biomarkers. Miniaturization of the electrodes is achieved through the use of screen-printed electrodes (SPEs), which consist of one to a few dozen sets of electrodes, or by utilizing printed circuit boards. Electrode materials used in SPEs include glassy carbon (Chiang H-C, Wang Y, Zhang Q, Levon K, Biosensors (Basel) 9:2-11, 2019), platinum, carbon, and graphene (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). There are numerous modifications to the electrode surfaces as well (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). These approaches offer distinct advantages, primarily due to their demonstrated superior limit of detection without amplification. Using the SPEs and potentiostats, we can detect cells, proteins, DNA, and RNA concentrations in the nanomolar (nM) to attomolar (aM) range. The focus of this chapter is to describe the basic approach adopted for the use of SPEs for nucleic acid measurement.

Bacterial microbiome diversity along poultry slaughtering lines: insights from chicken carcasses and environmental sources.

Introduction: This study aimed to determine the bacterial diversity of chicken carcasses and their surrounding environment at various stages along a poultry slaughter line. Material and Methods: Amplicon sequencing of the 16S rRNA gene was employed to assess the shifts in bacterial community diversity at both phylum and genus levels. Samples were collected from September to November 2021, targeting carcass surfaces at various operational stages (post-defeathering, post-evisceration, post-water chilling, and post-cooling), as well as from the internal environments and air of these units. The study took place in a vertically integrated poultry slaughterhouse in Konya, Turkey. Results: Microbial diversity increased after the chilling and storage stages as a result of redistribution of the microorganisms after the physical effect of the slaughtering stages. The final product sample taken after storage had the highest bacterial abundance. The abundance at this stage was found to be strongly correlated with that at other slaughtering stages, as well as with the abundance in chilling water and on the personnel's hands. The common genera in chicken carcasses during slaughter stages were Macrococcus, Acinetobacter, Enterococcus, Escherichia-Shigella, Psychrobacter, Streptococcus, Lactococcus and Ligilactobacillus. Microbiome data in environmental samples indicated that the genera in highest relative abundance were Bacillus, Anoxybacillus, Acinetobacter and Psychrobacter. In air samples, the storage room had the highest diversity and in this place Bacillus spp. and Staphylococcus spp. were in the majority. Conclusion: This study may provide some useful information to pinpoint the critical contamination sources in the poultry slaughtering process.

Comparison of meat quality characteristics of dry aged lamb loins and optimization of dry aging process.

The purpose of the present study was to investigate the physicochemical characteristics, meat quality, oxidative stability and sensory properties of lamb meat during 0, 7 and 14 day of the dry aging process. The M. longissimus lumborum (LL) and M. longissimus thoracis (LT) muscles from male Akkaraman lambs were used. The pH values of the LT and LL cuts were not changed during the aging periods. The LT cuts had significantly higher weight loss, a* and b* values, and lower shear force compared to the LL cuts. However, dry aging led to greater decreases in shear force in the LL cuts on 7th day of aging. The total mesophilic aerobic counts, total psychrophilic counts, Enterobacteriaceae counts, lactic acid bacteria, and yeast-mold counts were increased during the aging process. The sensory panel scoring showed a significant difference in the LL cuts and no significant difference in the LT cuts compared to the control group. There were significant changes in sensory panel scores for the LL cuts, whereas there were no significant changes for the LT cuts according to the non-aged samples. In conclusion, dry aging improved the quality of both cuts, however, the LL muscle of lamb was more suitable for dry aging. Moreover, 7 days were sufficient to produce the desired sensory properties in the lamb loins. Increasing the aging time from 7 to 14 days did not appreciably affect the sensory attributes or tenderness.

Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera.

Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.