3. When?
If the organism is:
 Difficult/costly to culture
 Slow grower
 Present in low concentration in clinical
samples
 Hazardous to propagate in the lab

11. PCR
Definition:
It is amplification of a specific DNA
sequence using specific primers.

12. PCR is:
 Sensitive (1 copy → million copies)
 Specific (specific primers)
 Rapid, efficient, reproducible

13. Primers are:
1- Specific
2- Single stranded
3- Short sequence
4- Complementary to the flanks of desired
region of target DNA

14. 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) .

15. Thermal Cyclers

19. 3 methods for detection of amplified
DNA :
1- Gel electrophoresis.
2- Probes
3- Sequencer

20. 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

21. Gel electrophoresis

22. Gel electrophoresis

23. In vitro
Antibiotic Susceptibility Tests

24. 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

25. Disc Diffusion Method
Sensitive to
5 ,3 ,2 ,1
1
2
Resistant to
3
4
5
4

26. 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

27. 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

28. 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.

29. 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

30. 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.

31. Thank you