Molecular detection of pathogens (molecular microbiology) is a new, dynamic and progressive spinoff of classic microbiology. It plays an important role in those clinical situations when standard microbiology (relying on the successful cultivation of potential pathogens) produces suboptimal results or completely fails. OR Modern approach for identification and quantification of microorganisms (pathogens) in the diagnostics of infections or foodborne illness using molecular microbiology. Broadest range of available tests and tailor-made packages.

2. Introduction :
• Identification and characterisation of microorganisms is a
key part of the management of food safety and quality,
tracing contaminants and troubleshooting problems such as
spoilage.
• Identification of microorganisms – provides the name of the
organism (to genus or species level), which can help in
determining whether it is a safety or spoilage concern or is
likely to be heat resistant, for example.
• Characterisation of microorganisms (typing) – this groups
together organisms that share similar DNA fragment
patterns or antigenic profiles, to assist with tracking or
tracing contamination.

3. Introduction :
• Molecular detection of pathogens (molecular microbiology)
• is a new, dynamic and progressive spinoff of classic microbiology.
It plays an important role in those clinical situations when
standard microbiology (relying on the successful cultivation of
potential pathogens) produces suboptimal results or completely
fails.
• OR
• Modern approach for identification and quantification of
microorganisms (pathogens) in the diagnostics of infections or
foodborne illness using molecular microbiology. Broadest range
of available tests and tailor-made packages.

4. Identification of Microorganisms
• How to identify unknown specimens ??????
• The methods microbiologist use fall into three
categories:
• Phenotypic- morphology (micro and macroscopic)
• Immunological- serological analysis
• Genotypic- genetic techniques
•

5. Identification of Microorganisms
• Phenotypic Methods
• ‘Old fashioned’ methods via biochemical,serological and
morphological are still used toidentify many
microorganisms.
• Phenotypic Methods
• Microscopic Morphology include a combination of cell
shape, size, Gram stain, acid fast reaction,special
structures e.g. Endospores, granule and capsule can be
used to give an initial presumptive identification.

6. Identification of Microorganisms
• Phenotypic Methods
• Macroscopic morphology : are traits that can be accessed with
the naked eye e.g. appearance of colony including texture, shape,
pigment, speed of growth and growth pattern in broth.
• Physiology/Biochemical characteristic : are traditional
mainstay of bacterial identification. These include enzymes
(Catalase, Oxidase,Decarboxylase), fermentation of sugars,
capacity to digest or metabolize complex polymers and sensitivity
to drugs can be used in identification.

7. Identification of Microorganisms
• Immunological Methods
Immunological methods involve the interaction of a
microbial antigen with an antibody (produced by
the host immune system).
• Testing for microbial antigen or the production of antibodies is
ofte easier than test for the microbe itself.
• Lab kits based on this technique is available for
• the identification of many microorganisms.

8. Immunological Methods
ELISA - Enzyme-linked immunosorbent assay
• The enzyme-linked immunosorbent assay (ELISA) has become
one of the most widely used serological tests for antibody or antigen
detection. This test involves the linking of various “label” enzymes to
either antigens or antibodies.
• Enzymes used in ELISA include Alkaline Phosphate, Peroxidase
and ßGalactosidase.
• During indirect ELISA the Ag is trapped between two Ab molecules
(sandwich ELISA).

9. Immunological Methods
ELISA
• The specimen is added to a well with
attached Ab.
• If the Ag (microbe) is present it will
attached to the Ab.
• After washing away unbound material,
a second Ab with a conjugated
enzyme is added.
• The second Ab is specific for the Ag.
A substrate is added which reacts with
the enzyme to give a coloured
reaction.
• ELISA tests are available for the
detection of many organisms including
Staphylococcus aureus, E. coli and
Salmonella.

10. Problems With Traditional Methods
• Cultivation-based methods insensitive for detecting some organisms.
Cultivation-based methods limited to pathogens with known growth requirements.
• Poor discrimination between microbes with common behavioral features.
Failure to detect infections caused by uncultivated (e.g., novel) organisms, or
organisms that fail to elicit a detectable host immune response.
Visual appearance of microorganisms is nonspecific.
Examples of Failures With Traditional Approaches :
Detection and speciation of slow-growing organisms takes weeks
(e.g., M. tuberculosis).
A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus).
Diseases presumed to be infectious remain ill-defined with no detected
microorganism (e.g., abrupt fever after tick bite).

11. dentification of Microorganisms
Genotypic Methods
• Genotypic methods involve examining the genetic
material of the organisms and has revolutionized
bacterial identification and classification.
• Genotypic methods include PCR, (RT-PCR, RAPDPCR),
use of nucleic acid probes, RFLP and plasmid fingerprinting.
• Increasingly genotypic techniques are becoming the sole means of
identifying many microorganisms because of its speed and accuracy

12. Genotypic Methods
• Genotypic methods of microbe identification include
the use of :
• Nucleic acid probes
• PCR (RT-PCR, RAPD-PCR)
• Nucleic acid sequence analysis
• 16s rRNA analysis
• RFLP ( Restriction Fragment Lengeth Polymorphism )
• Plasmid fingerprinting.

13. APPLICATIONS OF MOLECULAR METHOD IN FOOD INDUSTRY
• Detecting and identifying specific genes (GM foods)
• Application to Food Authenticity and Legislation
• Detection of microbial contamination of foods
• Species Identification
• Detection of Food Constituents (Ingredients or
Contaminants)
• Detection of antibiotics, pesticides residues etc.
• Halal and Kosher certification

14. Polymerase Chain Reaction (PCR)
• PCR is an in vitro method of the DNA synthesis with which a particular segment of DNA is
amplified by being delimited with a pair of flanking primers. Copying is achieved
exponentially through repeated cycles of different incubation periods and temperatures in the
presence of a thermostable DNA polymerase enzyme.
• In this way, millions of copies of the desired DNA sequence can be obtained in a couple of
hours. This is a highly specific, fast, sensitive, and versatile molecular biology
technique to detect the smallest amounts of a specific DNA, fostering its easy
identification and avoiding the use of radioisotopes
• Despite the benefits that the PCR technique offers in comparison to culture for the detection
of some microorganisms, the commercially available techniques are scarce and are limited
to research laboratories or to reference laboratories specialized in molecular diagnoses,
among other causes, due to their high cost , sensitivity and specificity of the used
diagnostic techniques .

15. Polymerase Chain Reaction (PCR)
• PCR is widely used for the
identification of microorganisms.
• Sequence specific primers are
used in PCR for the amplification
of DNA or RNA of specific pathogens.
• PCR allows for the detection even
if only a few cells are present and
can also be used on viable nonculturables.
• The presence of the appropriate
amplified PCR product confirms
the presence of the organisms.

16. Polymerase Chain Reaction (PCR)

17. Polymerase Chain Reaction (PCR)
• End point PCR
• is the analysis after all cycles of PCR are completed
which allows quantification as template is doubling
(exponential phase), end point analysis is based on the
plateau phase of amplification.
Gel electrophoresis for detecting PCR products
1. Meth used to separate DNA fragments generated by
restriction endonucleases .
2. separates molecules on the basis of size , charge and
shape

18. Polymerase Chain Reaction (PCR)
3 . Electromotive force
moves molecles through the
matrix at different speeds
bused on size and charge .
4 . Agarose gel
elecrtophoresis can resolved
DNA fragments that range
roughly from 500 t0 30.000
base pairs .

19. Polymerase Chain Reaction (PCR)

20. Real-time PCR
• Rapid detection and identification of several bacterial strains.
• Promising tool for distinguishing specific sequences from a
complex mixture of DNA and therefore is useful for
determining the presence and quantity of pathogen-specific or
other unique sequences within a sample.
• Facilitates a rapid detection of low amounts of bacterial DNA
accelerating therapeutic decisions and enabling an earlier
adequate antibiotic treatment.

21. Real-time PCR

22. Real-time PCR
Real Time detection of PCR products :
• No gels required. Recent method. Relies on the
ability of a dye, SYBR Green, to interact with
double stranded amplicons produced during PCR,
to produce fluorescence which is detected in a
flurometer.

23. Plasmid fingerprinting
• Plasmid fingerprinting identifies microbial species or similar
strains as related strains often contain the same number of
plasmids with the same molecular weight.
• Plasmid of many strains and species of E. coli, Salmonella,
Camylobacter and Psseudomonas has demonstrated that this
methods is more accurate than phenotypic methods such as
biotyping, antibiotic resistance patterns , phage typing and
serotyping.

24. Plasmid fingerprinting
• The procedure involves:
• The bacterial strains are
grown, the cells lysed and
harvested.
• The plasmids are separated by
agarose gel electrophoresis
• The gels are stained with EtBr
and the plasmids located and
compared .