Identification and Detection of Microorganism

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.

1. Molecular detection By Esraa Alaouden Abdo Alazez AUC : Food Hazard Fundamentals , Microbial Contamination of Food Course

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 Meth used to separate DNA fragments generated by restriction endonucleases . 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 .