molecular diagnosis - prac. microbiology
TRANSCRIPT
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When?If the organism is: Difficult/costly to culture Slow grower Present in low concentration in clinical
samples Hazardous to propagate in the lab
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Definition:
It is amplification of a specific DNA
sequence using specific primers.
PCR
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Sensitive (1 copy → million copies)
Specific (specific primers)
Rapid, efficient, reproducible
PCR is:
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Primers are:
1- Specific
2- Single stranded
3- Short sequence
4- Complementary to the flanks of desired
region of target DNA
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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) .
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Thermal Cyclers
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3 methods for detection of amplified DNA :
1- Gel electrophoresis.
2- Probes
3- Sequencer
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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
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Gel electrophoresis
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Gel electrophoresis
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In vitroAntibiotic Susceptibility Tests
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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
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Disc Diffusion Method
12
3
54
Sensitive to 1 ,2 ,3 ,5
Resistant to
4
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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
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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
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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.
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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
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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.
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