Molecular characterization of microbial diversity

Question 1

APPLICATION OF MOLECULAR METHOD

Answer 1

• Detecting and identifying specific genes (GM)

• Application to Food Authenticity and Legislation

• Detection of microbial contamination of foods

• Species identification

• Detection of food constituents (ingredients or contaminants)

• Halal and Kosher certification

Question 2

COMMON MOLECULAR METHODS

Answer 2

• PCR (Real Time-PCR, Multiplex), Restriction Fragment
Length Polymorphism (RFLP), Single Strand Confirmation
Polymorphism (SSCP) and sequencing

• Plasmid profiling, ribotyping, macrorestriction analysis by
pulsed-field gel electrophoresis (PFGE)

• Newer techniques, which use fluorescent dyes, DNA
microarrays, protein chemistry, and mass spectrometry

• Random Amplified Polymorphic DNA Analysis
(RAPD)

• Amplified Fragment Length Polymorphism (AFLP)

• Biosensors

Question 3

PCR STEPS

Answer 3

• Isolation of DNA from the food

• Amplification of the target sequences by PCR separation of the amplification products by agarose gel electrophoresis

• Estimation of their fragments size by comparison with a
DNA molecular mass marker after staining with ethidium
bromide

• Verification of the PCR results by specific cleavage of the
amplification products by restriction endonuclease, transfer of separated amplification products onto membranes (Southern Blot) followed by hybridization with a DNA probe specific for the target sequence.

Question 4

REAL TIME PCR

Answer 4

• Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR

• The fluorescence, measured in real time, is detected in a PCR cycler with an inbuilt filter flurometer

Question 5

MULTIPLEX PCR

Answer 5

• Several primers pairs with similar annealing requirements can be added to a PCR mixture to simultaneously detect several target sequences

• Saves times and minimize the expense on detection of food borne pathogens

• Primers should have some melting temperature

• Must not interact with each other

• The amplified fragments of same length cannot be detected

• Standard PCR-unable to differentiate viable and non-viable microorganism

• Ethidium monoazide can be used to separate dead and viable bacteria

• Real-time PCR using RNA as template is more aunthentic since the RNA is present only in viable microbes

• RNA is the first reverse transcribes to cDNA and then used for amplification

Question 6

BIOSENSORS

Answer 6

• Self-contained integrated device that is capable of providing specific qualitative or semi qualitative analytical information using a biological recognition element, which is a direct spatial contact with a transduction element

• In a biosensor, the sensing element, which respond to the substances being measured is biological in nature, such as enzymes,

• So, a biosensor can be defined as a device that has a
biological sensing agent (enzymes, cell antibody, nucleic
acid) and an electronic transducer that converts a biological reaction into a signal that can be processed and displayed.