Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

Oakley, Brian B.; Line, J. Eric; Berrang, M. E.; Johnson, Jessica Marie; Buhr, R. J.; Cox, N. A.; Hiett, Kelli L.; Seal, Bruce S.

doi:10.1016/j.diagmicrobio.2011.11.001

Cited by 8 publications

(6 citation statements)

References 47 publications

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“…To follow specific foodborne pathogens along the food supply chain, we used qPCR to quantify the abundance of two of the most important pathogenic genera, Campylobacter and Clostridium , across our longitudinal samples using previously validated primer sets [7], [53]. Because of the importance of C. jejuni as a human pathogen, we also designed a novel TaqMan qPCR assay specific and sensitive for C. jejuni targeting the beta-subunit of the cytolethal distending toxin gene ( cdtB ).…”

Section: Resultsmentioning

confidence: 99%

“…Quantitative PCR for 16S rRNA genes was performed with SYBR green chemistry (ABI, Foster City, CA, USA) using primer sets previously validated as specific for Clostridial Group I [53] and the genus Campylobacter [7] with thermal cycling protocols as previously described [53]. PCR products were obtained from Clostridium perfringens ATCC 13124 and Campylobacter jejuni NCTC 11168 and purified with Qiaquick spin columns (Qiagen, Valencia, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The poultry microbiome is of particular interest as a major source of foodborne infections both worldwide and in the U.S., where foodborne pathogens are estimated to have accounted for 47 million episodes of illness at an economic cost of $77 billion in 2011 [4], [5]. In the course of commercial poultry production, chickens typically progress in a single age cohort from a microbiologically depauperate gastrointestinal (GI) tract in newly-hatched chicks to market age adults at 6–8 weeks harbouring up to 10 11 bacterial cells g -1 of intestinal contents [6], representing hundreds [7], [8] to thousands [9] of distinct taxa. The poultry GI microbiome differs ecologically from mammals in that colonization occurs primarily from the surrounding environment and individuals of the same age reared in close proximity rather than through direct contact with adults [10], [11], [12], [13], [14], [15].…”

Section: Introductionmentioning

confidence: 99%

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The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

et al. 2013

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Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2–4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

et al. 2013

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“…Kogut et al . data are unpublished, collected, and analyzed as previously described (Oakley et al ., , ) representing 20 birds and c . 20 000 sequencing reads.…”

Section: Spatial Variabilitymentioning

confidence: 99%

The chicken gastrointestinal microbiome

Oakley

Lillehoj

Kogut

et al. 2014

FEMS Microbiol Lett

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579

484

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The domestic chicken is a common model organism for human biological research and of course also forms the basis of a global protein industry. Recent methodological advances have spurred the recognition of microbiomes as complex communities with important influences on the health and disease status of the host. In this minireview, we provide an overview of the current state of knowledge of the chicken gastrointestinal microbiome focusing on spatial and temporal variability, the presence and importance of human pathogens, the influence of the microbiota on the immune system, and the importance of the microbiome for poultry nutrition. Review and meta-analysis of public data showed cecal communities dominated by Firmicutes and Bacteroides at the phylum level, while at finer levels of taxonomic resolution, a phylogenetically diverse assemblage of microorganisms appears to have similar metabolic functions that provide important benefits to the host as inferred from metagenomic data. This observation of functional redundancy may have important implications for management of the microbiome. We foresee advances in strategies to improve gut health in commercial operations through management of the intestinal microbiota as an alternative to in-feed subtherapeutic antibiotics, improvements in pre- and probiotics, improved management of polymicrobial poultry diseases, and better control of human pathogens via colonization reduction or competitive exclusion strategies.

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“…In all cases, once a set of specific primers has been designed, the PCR assay must be checked for cross-reaction and false positives with nontarget microorganisms. The HTS approach has been successfully used to validate a Campylobacterspecific PCR assay and to demonstrate the specificity of the PCR assay against a complex set of untargeted bacteria in a natural environment (27).…”

Section: Hts Applications In Foodmentioning

confidence: 99%

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

Ercolini

2013

Appl Environ Microbiol

415

229

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Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.

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Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

Cited by 8 publications

References 47 publications

The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

The chicken gastrointestinal microbiome

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

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