When?If the organism is: Difficult/costly to culture Slow grower Present in low concentration in clinical

samples Hazardous to propagate in the lab

Definition:

It is amplification of a specific DNA

sequence using specific primers.

PCR

Sensitive (1 copy → million copies)

Specific (specific primers)

Rapid, efficient, reproducible

PCR is:

Primers are:

1- Specific

2- Single stranded

3- Short sequence

4- Complementary to the flanks of desired

region of target DNA

PCR TechniqueExtracted N.A. + 3 items:1- Two primers (forward & reverse)2- Taq polymerase3- Nucleotide pool

3 steps:1- Denaturation (95°C)2- Primer annealing (40-60°C) 3- Primer extension (65-75oC) .

Thermal Cyclers

3 methods for detection of amplified DNA :

1- Gel electrophoresis.

2- Probes

3- Sequencer

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

Gel electrophoresis

Gel electrophoresis

In vitroAntibiotic Susceptibility Tests

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

Disc Diffusion Method

12

3

54

Sensitive to 1 ,2 ,3 ,5

Resistant to

4

Questions1-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

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

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.

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

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.

Thank youThank you