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Laboratory of food microbiology 2 - Coggle Diagram

- - - - BIOLOGICAL REPLICATES are different samples measured across multiple conditions that show biological variation
        TECHNICAL REPLICATES are repeated measurements of the same sample that represent independent measures of the random noise associated with protocols or equipment
      - sample preparation, isolation of enumeration technique, selective medium, T and time of incubation
      - planning experimental activity--> Selected FERMENTED PRODUCT and REFERENCES. SAMPLES to be analysed (included BIOLOGICAL and TECHNICAL REPLICATES). Expected MICROBIAL COUNT range of YEASTS/LAB. SAMPLE PREPARATION. ISOLATION and ENUMERATION TECHNIQUE. SELECTIVE MEDIUM, T° and TIME of INCUBATION
  - - - PCR (POLYMERASE CHAIN REACTION)1993 - Kary Mullis--> microorganism genome is stored inside DNA molecules, but analyzing this genetic information requires quite a large amount of DNA
        
        Kary Mullis invented the process known as
        Polymerase Chain Reaction (PCR), in which a small amount of DNA can be copied in large quantities over a short period of time.
        
        3 steps about DNA--> Denaturing – when the doublestranded template DNA is heated to separate it into two single strands (92-98 °C 30”-60”). Annealing--> when the temperature is lowered to enable the DNA primers to attach to the template DNA(40-60 °C 30”-60”). Extension--> when Taq polymerase synthesizes the new DNA strands from the primers (70-75 °C 30”-120”)
        
        REACTION MIXTURE:
        PCR primers are short fragments of single stranded
        DNA that are complementary to DNA sequences
        that flank the target region of interest. The purpose
        of PCR primers is to provide a “free” 3'-OH group
        to which the DNA polymerase can add dNTPs
        
        pre-PCR area
        Preparation of the sample: DNA EXTRACTION, PCR area. Preparation of the reaction: ALIQUOTING of reagents,REACTION in thermal cycler post-PCR area. Detection of amplified DNA: ELECTROPHORESIS in agarose gel
- - - - The RSA technique is based on the polymorphismof
        the 16S-23S spacer region of the rRNA operon and allows to hypothesize which genus or species the lacticbacteria strains under study belong to. rRNA operon is highly conserved in terms of nucleotide sequence. Lactic bacteria belonging to different spacers in terms of molecular weight and number
      - The use of master PCR mixes ensures a high degree of consistency. the fewer pipetting steps involved also means fewer opportunities for contamination. Using a PCR master mix also reduces the chance for a preparation error
        
        PCR master mix is a premixed solution that has all of the components for a PCR reaction. master mix usually contains a Taq polymerase, dNTPs, MgCl2 primers and additives in a buffer optimized for PCR.
        
        ANALYSIS of ITS (INTERNAL TRANSCRIBED SPACER)RFLP--> Differences among fungi in the lengths of DNA fragments cut by enzymes. Amplification with ITS1 and ITS4 primers
        
        Restriction enzymes--> They break the phosphodiester bond, There are 2 types of enzymes that cut with different mechanisms, Identification based on
        restriction sites within the PCR product
        
        ASYMMETRIC cut: they cut the two chains in different positions. They form the sticky ends. SYMMETRIC cut: they cut the two chains in the same position. They form the blunt ends. RUNNING CONDITIONS: 2% agarose concentration, 120Vfor 30 min
        
        Biotyping makes use of the pattern of metabolic activities expressed by an isolate, colonial morphology and environmental tolerances. Strains are referred to as "biotypes".
        
        Disadvantages:
        Stability/variability of the phenotype
        Time consuming and expensive
        
        Repetitive Element Polymorphic DNA (REP)--> Molecular technique capable of producing strainspecific fingerprintings in relation to the presence of repeated sequences of nucleotides, randomly distributed in the genome. A single primer anneals at this repeated sequences of the genome
- - - - shotgun sequences--> metagenome--> profile of the potential activities during fermentations and spoilage. Rapid detection of foodborne. metatranscriptome--> profiling of the complex genes expressed during fermentation and spoilage, pathogens response mechanism to food processing conditions or to antimicrobials
        
        shotgun metagenomic sequences--> higher costs, data analysis more computationally intensive and difficult. Detection of microbial function directly in food matrix. No need of PCR (avoiding PCR bases, pimer design), detection of the whole microbial population (bacteria, fungi, virus, etc.) at the same time
      - pangenome--> exploring strain diversity during fermentation and spoilage. Epidemiological biotype matching of pathogens in foodborne illness outbreaks. Strain selection for technological purposes--> entire gene set of a microorganism. It includes genes present in all strains (core genome) and genes present only in
        some strains of a species (variable or accessory genome).
      - disadvantages--> bioinformatic skills, overall cost and scalability to the food industry. advantages--> screening of many food samples at the same time. Reliable identification of the majority of microbes in raw materials and during food production/storage safer bench work compared to other molecular approaches. Possible insights on both taxonomy and activities in case of microbiome studies