A primary requirement for the widespread acceptance of novel detection systems for food-borne pathogens is convenience and ease of use, and a clear indication of benefit to both the producer and the consumer. Convenient immunoassays have been developed at the NDC for the detection of food borne pathogens, Listeria and E.coli 0127:H7. These assays are applied to food homogenates after an enrichment period, and can be used to identify suspect samples which are then subjected to traditional confirmation procedures. Immunoassays have also been developed to detect antibodies to Salmonella species in meat juices (both pork and chicken), indicating Salmonella contamination of specific animal population and highlighting where control measures require evaluation.

Food borne assays developed at NDC have been combined with DNA probe hybridisation into a colormetric membrane-based detection system to offer increased specificity. The test has a turn around time of 24 hours and a throughtput of up to 40 samples per test run. The system may also be adapted to simultaneously screen a sample for the presence of a range of bacterial pathogens. The assays are currently used to test samples at various stages of broiler production process for the presence of Salmonella and Campylobacter, and are being adapted for monitoring environmental samples from poultry houses.

Rapid molecular identification and typing of micro-organisms is extremely important in efforts to monitor the geographical spread of virulent or epidemic pathogens. In this area, the NDC’S focus is on standardising molecular typing methods, in particular AFLP (Amplified Fragment Length Polymorphism) for food pathogens for epidemiological studies, and in European networks to validate common guidelines for these research techniques.

NATIONAL FOOD BIOTECHNOLOGY CENTRE (NFBC)
SNaPIT TM
– Single Nucleotide Polymorphism Identification Technology -
A novel process for analysis of genetic variation

Patrick Vaughan

Overview of SNaPITTM Technology

SNaPIT TM is a technology which permits the reliable, accurate and robust detection of SNPs in DNA. The system exploits the use of highly specific DNA glycosylase enzymes to excise specific substrate bases incorporated into amplified DNA. It may be applied to both fragment size analysis (e.g. gel analysis) and solid phase/immobilised (e.g. microtitre plate) formats. More importantly, SNaPIT permits the scanning of genes for new SNPs not previously characterised (SNP discovery), and permits the high throughput genotype analysis of those SNPs in target populations (SNP genotyping). This allows a direct comparison between the genomes of different population groups, such as diseased and healthy individuals.

Some of the key features of SNaPIT-check (SNP genotyping) and SNaPIT-scan (SNP discovery) which set SNaPIT apart from other SNP discovery and genotyping techniques are:

1. Its speed, accuracy and reliability.

2. The ability to automate the platform on either gel-based or non-gel-based formats means that SNaPIT may be used for the simultaneous detection of multiple different polymorphisms, thus greatly reducing capital investment, labour costs and time.

3. A similar platform is used for both SNP discovery and genotyping.

Fig 1.  General features of SNaPITTM

(Click on  graphic for expanded view )

By employing uracil DNA glycosylase (UDG) and deoxyuridine tri phosphate (dUTP), SNaPIT-SCAN and SNaPIT-check can detect 10 out of the 12 possible base substitution polymorphisms (83%) – i.e. Guanine (G) to Adenine (a), G to Thymine (T), A to G, A to T, A to Cytosine (C), T to G, T to A, T to C, C to A and C to T – since they involve the gain or loss of A/T or T/A base pairs, in addition to all deletion and insertion mutations. In reality, >95% of all single base substitutions are detectable with SNaPIT, since most polymorphisms and disease causing mutations are C to T (or G to A) transitions. This is due in part to the high incidence of deamination which occurs at cytosine and 5-methyl cytosine residues in DNA to yield uracil and thymine residues respectively, which increases the incidence of C to T (or G to A) base substitutions in DNA.

Fig 2.    General features of SNaPITTM

(Click on  image for expanded view )

The remaining two single base substitutions, i.e. G to C and C to G substitutions, which occur at a very low frequency, can be detected by first converting the C residues to U residues by bisulfite treatment, thereby making them susceptible to excision by uracil DNA glycosylase. They can also be detected by screening for single stranded conformational variations of the SNaPIT-SCAN cleavage products when separated on a non-denaturing polyacrylamide gel. This technique has the advantage that all mutations can be detected with just one glycosylase. Alternatively, the use of an additional glycosylase/modified precursor nucleotide which substitutes for G or C can be used for this purpose, in this way allowing the detection of 100% of mutations. SNaPIT readily detects all other types of mutations, namely insertion and deletion mutations.

NATIONAL PHARMACEUTICAL BIOTECHNOLOGY CENTRE (NPBC)

The National Pharmaceutical Biotechnology Centre, BioResearch Ireland’s centre on the campus of Trinity College, Dublin, comprises 35 staff in 10 different departments working on projects supervised by 15 Principal Scientists. The four major areas of research are Neurobiology & Nutrition, Inflammation & Cancer, Pharmacy, and Vaccine Development.

In the Vaccine Development area, NPBC scientists, led by Professor Tim Foster of the Microbiology Department in the Moyne Institute of Preventive Medicine, have been working for several years on the discovery and characterization of cell surface protein adhesins from Staphylococcus aureus and coagulase-negative staphylococci. One application of the research is the prevention and treatment of Staphylococcal hospital-acquired infections which account for 40% of all deaths from such diseases and which add considerably to the length of time a patient spends in hospital and hence the cost to health services. The problem is compounded by the emergence of strains of S.aureus that are resistant to treatment by all available antibiotics. Prof. Foster and his group have discovered several novel adhesins, cell surface proteins which allow bacteria to colonise the host and initiate infection by binding to proteins present in plasma or in the extracellular matrix, or by sticking to deposits on indwelling medical devices. Two fibrinogen binding proteins have been characterized which are members of a larger family of structurally-related surface proteins with a very unusual domain consisting of serine-aspartate dipeptide repeats.

The figure shows that the surface of the S. aureus bacterial cell is decorated with a number of different proteins that interact with components of the host extracellular matrix. The binding domains are usually located in the circular segments furthest from the cell. The proteins have flexible (dark lines) and sometimes additional rigid (cylinders) stalks that project the binding domains further away from the surface. Candidate antigens for vaccines are the circular binding domains which have been cloned and expressed in recombinant form.

In collaboration with scientists in Gothenburg, Sweden, and at Inhibitex Inc., Alpharetta, Georgia, USA, it has been shown that immunization of animals with a single protein antigen corresponding to part of one of the surface adhesins will protect against subsequent challenge with S.aureus. Recently BRI has signed an exclusive licensing agreement with Inhibitex Inc. (www.inhibitex.com), who will develop vaccines for staphylococcal infections of hospital patients. Initially, rare human blood donors with high antibody levels against particular S.aureus antigens will provide plasma which will be pooled, and the immunoglobulin (IgG) purified and then administered to at-risk patients. Later, volunteers will be immunized with antigens to provide IgG with much higher levels of specific antibody. Inhibitex will then expand its vaccine portfolio to include the other major causes of hospital acquired sepsis, Staphylococcus epidermidis and Enterococcus faecalis. The former project will also be in collaboration with BRI.

Another project is to develop a vaccine to prevent bovine mastitis, a disease of dairy cattle of great economic importance world-wide.

NATIONAL CELL AND TISSUE CULTURE CENTRE (NCTCC)

Biocontrol – the future of horticulture and the food industry

Elizabeth Morris & Owen Doyle

With increasing consumer demand for improved food safety standards, EU directives, along with regular national testing, ensure that maximum residue levels (MRLs) in fruit and vegetable produce are not exceeded, and that Irish food produce remains safe. However, the pests that we wish to control have begun to develop levels of resistance to our diminishing list of crop protection products. This sets a new challenge to both the food producer and the scientist. How do we control pests without pesticides? A "green" solution – the use of biological control agents – is one possibility.