Maria Teresa Bottero

A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products

Abstract A multiplex PCR able to identify cows’, goats’ and sheeps milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments of different length. The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism. The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheeps and cows’ milk in dairy products with a good sensitivity threshold (0.5%).

Animal species identification in food products: Evolution of biomolecular methods

Species identification in food has increasingly acquired importance due to public health, economic and legal concerns. Traditional methods have relied on the identification of morphological traits, but this does not lead to accurate identification of those species used in many types of processed food. As a result, laboratory techniques have been devised using electrophoretic and immunological methods focussing on protein profiles and, more recently, biomolecular techniques have been developed. However, these techniques also present problems and difficulties, especially in the case of matrices that are heterogeneous or have been subjected to severe treatments during processing.

Identification of Four Tuna Species by Means of Real-Time PCR and Melting Curve Analysis

Dalmasso, A., Fontanella, E., Piatti, P., Civera, T., Secchi, C. and Bottero, M.T., 2007. Identification of four tuna species by means of Real-Time PCR and melting curve analysis. Veterinary Research Communications, 31(Suppl. 1), 355–357

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis.

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.

Multiplex primer-extension assay for identification of six pathogenic vibrios

A multiplex Primer-Extension Reaction (PER) assay, was specifically designed for the identification, of the major human pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and V. fluvialis) in fishery products. The assay, directed towards the rpoA gene, was tested on a total of 287 samples representing six Vibrio species and ten non-Vibrio species. The primers used in the preliminary PCR, designed in well conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 284 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of these human pathogenic species in seafood samples and to evaluate the potential hazard for consumers.

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy.

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate). Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance. A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy. All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearsons correlation coefficients (r) showed close agreement between all three methods. Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.

A five year surveillance report on PFGE types of Listeria monocytogenes isolated in Italy from food and food related environments

Listeria monocytogenes can cause severe invasive disease in humans and has been isolated from a variety of foods. This study aimed to investigate type diversity and distribution across sources by subtyping via PFGE a set of 300 L. monocytogenes isolates collected in Italy from foods over a five year period (from 2003 to 2007). The most frequent serotypes were 1/2a (45%), 1/2c (22%), and 4b/4e (16%); 5% of the isolates were untypeable by conventional serotyping. Significant associations were observed between serotype 1/2a with dairy (O.R.=13.9) and 1/2c with meat (O.R.=33.3). All isolates were typeable, generating 164 combined PFGE profiles. Of these, 121 were unique, being displayed by only one isolate. The other 43 profiles grouped the remaining isolates and were shared between two (N=22), three (N=10), four (N=3) and five isolates (N=4). The remaining 4 profiles were shared between 7, 14, 17 and 46 isolates, respectively. Some profiles (N=7) were retrieved in samples collected in different years, indicating persistency in foods and processing plants. This research may pose the ground for designing a broad typing database which could ease the understanding of L. monocytogenes diversity and could be used for facilitating epidemiological investigations for the identification of listeriosis outbreaks. Data show how large subtype databases may facilitate the identification of common and source-specific types. More comprehensive databases may be needed to fully understand L. monocytogenes diversity and to provide useful data to be considered in epidemiological investigations.

Novel Multiplex Single Nucleotide Polymorphism-Based Method for Identifying Epidemic Clones of Listeria monocytogenes

ABSTRACT A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Ruminant Rhombencephalitis-Associated Listeria monocytogenes Strains Constitute a Genetically Homogeneous Group Related to Human Outbreak Strains

ABSTRACT Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cows milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Apoptosis in muscle-to-meat aging process: The omic witness

UNLABELLED Meat derives from a muscle that undergoes a great number of biochemical and physiological changes. The anoxic condition established from the moment of animal sacrifice forces muscle cells to a sort of reaction, resulting in methodical programmed cell death to avoid necrosis. The duality autophagy and/or apoptosis is at the center of the scientific debate about the biological processes driving the muscle to meat conversion. Here we report an omic time course overview carried on proteome, phosphoproteome and metabolome of Piedmontese longissimus thoracis muscle searching for clues helping us to extricate through the dilemma. The survey depicts a progressive physiological impairing and our evidences push towards the apoptotic behavior: the proteomic time course trend of annexin A2, RKIP, HSPB6, αB crystalline, adenylate kinase, DJ-1 and 31kDa actin fragment; the 0-1day increased phosphorylation of myosin 2 and synaptopodin and the metabolomic time course trend of key metabolic indicators, like GSH/GSSG ratio, taurine and nitrotyrosine. The employed techniques provide strong indications about the likely apoptotic behavior of aging meat in muscle-to-meat conversion process. BIOLOGICAL SIGNIFICANCE Our work underlines compelling evidences of the apoptotic behavior of Piedmontese beef muscle cells undergoing the muscle-to-meat process, whereas no autophagic clues are inferred from this omic investigation.