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Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
Molecular Diagnosis - Prac. Microbiology
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Molecular Diagnosis - Prac. Microbiology

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  • 1. When? If the organism is:  Difficult/costly to culture  Slow grower  Present in low concentration in clinical samples  Hazardous to propagate in the lab
  • 2. PCR Definition: It is amplification of a specific DNA sequence using specific primers.
  • 3. PCR is:  Sensitive (1 copy → million copies)  Specific (specific primers)  Rapid, efficient, reproducible
  • 4. Primers are: 1- Specific 2- Single stranded 3- Short sequence 4- Complementary to the flanks of desired region of target DNA
  • 5. PCR Technique Extracted N.A. + 3 items: 1- Two primers (forward & reverse) 2- Taq polymerase 3- Nucleotide pool 3 steps: 1- Denaturation (95°C) 2- Primer annealing (40-60°C) 3- Primer extension (65-75oC) .
  • 6. Thermal Cyclers
  • 7. 3 methods for detection of amplified DNA : 1- Gel electrophoresis. 2- Probes 3- Sequencer
  • 8. Gel electrophoresis: • DNA fragments migrate in the gel • Migration rate depends on size of DNA fragments (length), smaller fragments → more rapid • A dye (in the gel) binds to DNA fragments → allows visualization by UV light
  • 9. Gel electrophoresis
  • 10. Gel electrophoresis
  • 11. In vitro Antibiotic Susceptibility Tests
  • 12. Antibiotic Susceptibility Tests Disc Diffusion Method: for routine use. Easy/rapid Test org. on surface of media + Antibiotic Discs incubation Zones of Inhibition = Susceptibility to antibiotic
  • 13. Disc Diffusion Method Sensitive to 5 ,3 ,2 ,1 1 2 Resistant to 3 4 5 4
  • 14. Questions 1-All of the following are characters of primers EXCEPT: a. Specific b. Double stranded c. Short sequence d. Complementary to the flanks of target DNA e. Used in PCR
  • 15. 2- The amplified DNA products may be detected by any of the following EXCEPT: a.Gel electrophoresis b.Disc diffusion method c.Sequencer d.Probes
  • 16. 3- Regarding the PCR technique all the following are included in the reaction EXCEPT: a.Two primers (forward & reverse). b.Taq polymerase. c.Extracted N. A. (target). d.Nucleotide pool. e.Antibiotic discs.
  • 17. 4- Formation of clear zones around antibiotic discs is due to: a.Resistance of the bacterium b.Short incubation period c.The expiry of the antibiotic discs d.Inhibition of bacterial growth around the discs e.Low concentration of the antibiotic
  • 18. 5- Regarding gel electrophoresis: a.It is a method of amplification of DNA. b.It is a method of testing electrical forces of microorganisms. c.Visualization of fragments is done by infrared light. d.Smaller fragments migrate faster. e.DNA visualization does not need a dye.
  • 19. Thank you

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