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34 Cards in this Set
- Front
- Back
microbial characterisation
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pure culture techniques: for selecting for particular organisms (diff. plates)
agar based plates DNA fingerprinting generated by digestion/ electrophoresis allows distinguish/ compare between different samples antigenic typing: ID strains at a fine level by typing Next-gen DNA seq single species or complex mix Metabolic Typing: different reactions |
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16S rRNA gene
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was used by Woese and Fox to discover Archaea
can investigate taxonimc relations variable and hypervariable regions evolve in a faster rate (mosaic structure) total length 1500bp long variable region red colours high variation blue is low variation varaible regions have different lengths laerger teh size the more variaton (information) microbial characterisation -> specific species -> complex communities primers to constant regions (should bind to most organisms) amplify variable ones -> seq/ fingerprinting to identify design primers that bind to variable (specific species) -> either get a band or not, depending on whether they are present or not. case study: each band corresponds to a diferrent species badn corresponds to |
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food poisoning
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salmonella 10,000 per anum
campylobacter 70,000 per anum e. coli 1,000 norovirus (enteric disease) |
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gram negative pathogens
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campylobacter jejuni
salomnella enterica e. coli vibrio cholerae yersinia enterolitica shigella spp (e.coli like) |
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gram positive pathogens
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staphylococcus aureus
clostridium perfringes clostridium botulinum bacillus cereus listeria monocytogenes |
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non bacterial pathogens
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aspergillus
aflatoxins viruses |
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bacterial strategies
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non invasive: 0157 e'coli attach to intestin. cells rearragnge cell surface structure=> allows them to persist
intracellular pathogens=> hide from competitin and avoid phagocytosis |
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detection
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homogenise food (buffere and broth)=> enrich homogenate for organisms being tested for (pathogens) , =? differ. plating + presumptive identification (colour change on plate) => more specific typing (serotyping etc)
bypass all culture steps => sequence directly form sample |
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techiques and level of accuracy
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typing => species level
important to ID on subspecies level (pathogenic strains) reference strains |
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Detection
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determination of presence or absence of a particular food poisoning bacterium in sample
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Identification
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determination of a detected species as belonging to a known group (genus, species, strain) by any technique
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Typing
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the use of techniques to distinguish isolates at the strain/ subspecies level
reference strain can be very useful |
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Typing techinques
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serotyping
phage typing antibiograms (resistance) mol. characterisation - plasmid dna - genotyping - physical/ exterior/ specific (Fla typing, Penner and Lior typing) - physiology (secretion systems) - metabolism of the cell (capacity) species level - DNA based methods => strains core genome, dispensable genome (strain level) small SNPs and 16S rRNA (PFGE, AFLP), whole genome sequencing |
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Traditional and modern methods
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selective media, gram stains, colorimetric
=> genus level visual methods ( fluorescent microscopy biochemical rests Immunological rests (immunomagnetic separation, serotyping) DNA based PCR, RFLP, AFLP (amplified) nex gen - need for one that does not require known bit of genome (for identifying unknown) |
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commercial pathogen detection systems
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ELISA,
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API 20E
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standard ID system
campylobacter dehydrated substrate ion tube grow organism colour change, depending on hat can/cannot be metabolised good for gram-ive not so useful for capmylobacter due to limited metabolic capacity 20 markers! +/- points added up for groups of three score is read and compared to table limitations: uncharacterised metabolisms |
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MAST scheme
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for Camylobacter jejuni
narrow down to species level 3 markers |
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Campylobacter serotyping
Penner scheme |
O-serotypes
capsular polysacharhyde being recoginsed uses HEAT STABLE antigen (present after heat treatment) Guilian Barre syndrome is related to Campylobacter (some seroptypes) Heat up suspension extract proteins (expose to EDTA) 42 serotypes of C. jejuni and 18 for C.coli different sugars inner and outer portion of lipopolysaccharides step 1) extract heat stable polysaccharides by exposing to saline solution at 100 oC or EDTA 2) treated cells are exposed to erythrocytes (have lipoglycoprotein receptor which binds to the antigen) 3) antigen-sensitised erythrocytes dispensed into wells containing antiserum, in two-fold dilutions *specific antibodies for each serum type. antibodies bind to polysaccharides (which are also bound to the erythrocytes) causing the haemaglutination, signalling the detection of the specific serotype 4) incubated overnight at 37C => highest dilution showing haemaglutination is recorder as the titre |
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underlying genetic variation
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genes responsible for production of lipopolysaccharydes vary in number and type
-> result in different structure genetic variation can be detected by serotyping scheme |
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Campylobacter serotyping
Lior scheme |
heat-Labile antigens. 21 serotypes recognised
now, more than 100 serotypes! rapid slide agglutination technique 1) loop of live bacteria mixed with drop of antiserum on glass slide 2) asses agglutination after 30-45 sec (bacteria clump together if the antigen is recognised by the antiserum) |
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Molecular typing
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PCR, RFLP, AFLP, PFGE, MLST, microarray, protein array
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PCR
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y
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RFLP
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variation in patterns after cutting by a specific RE
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AFLP
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amplify a specific gene followed by RFLP
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PFGE
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pulse field gel electrophoresis
large fragments at seperation cut up entire chromosome with enzyme that cuts rarely=> separate large fragemtns on special gell |
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MLST
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multilocous sequence typing
sequence specific regions and look for variation in them |
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microarray/ protein array, next gen seq
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y
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PCR
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standard
nested : second seet of primers amplify first fragments more sensitive, detects rarer pathogens multiplex PCR: multiple primers in same tube, diff. bands according to organisms present quantitative: product threshold level. |
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RFLP on Salmonella
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enteriditis and typhimurium
give different patterns in closely related species must be a variable region (like 16S) |
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RFLP with fla gene
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flaA or flaA and flaB
picks up different strains of Campylobacter detect proteins with antibodies or on DNA level |
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MLST
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house keeping genes
evolve slower than virulence genes allocate SNPs at different groups evolutionary relationships and clonality extent evolution of chromosome (scattered) changes are usually silent (changes on the last position of the codon) different disease related to different marker choice criteria: -chromosomal location - suitability for primer design - sequence diversity |
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Campylobacter jejuni MLST
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Dingle et al 2001
7 loci, 194 isolates => 155 sequence types ==> 62 clonal lineages selection against amino acid change |
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next gen seq
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more sequences
no need to clone, start from mix and separate out cost per base is much less (human genome for 1000$) in microbiology +++ high trhoughput (many food samples at the same time) +safer bench work +insights on taxonomy and activities |
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rareaction
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steeper-> more species
community less diverse as meat spoilt-> gives insight in process |