APPLICATIONS OF

MOLECULAR BIOLOGY
TECHNIQUES TO MEDICAL
MICROBIOLOGY

Two principle molecular

techniques used in detection of
microorganisms
1- Nucleic acid hybridization( Southern
Blotting)
2- Polymerase chain reaction (PCR)

Polymerase chain reaction

The benefits of PCR based diagnostic
testing:
Rapid diagnosis
Detection
Same day result
High accuracy, high specifity,high
sensitivity

The role of PCR in diagnosis

of infectious diseases
Fastidious and slow growing microorganism
Detect antimicrobial resistance
Detect microorganism cannt be cultivated
Measurment value of viral load

Polymerase chain reaction

PCR- Amplify minute amounts of target DNA
within a few hours
Develobed by Nobel laureate biochemist
Kary Mullis in 1984
Discovered of thermostable polymerase work
at 100 c
Taq polymerase

PCR methodology
Materials
Target DNA
Taq polymerase
Four DNA nucleotides
Tow primerS
Reaction buffer
Temperature cycles

PCR methods
1- Denaturation: doble stranded DNA
eprated into two single strands 90-95c
2- Cooling at 30-60c
3- Annealing: Primers attached
complementary region of target DNA
4- Heating at 70c
5- Extention :The primers extended to form
a new strand of DNA

Defferentversions of PCR
1-Nested PCR: Increased sensitivity and
specifity
2-Reverse transcriptase PCR: (RT-PCR)
PCR also applied to amplification of RNA
3-Amplified fragment length
polymorphism(AFLP) replaced southern
blotting
4-Inverse PCR : Amplifies unknown DNA

Ideal applications for PCR

PCR applied in the research setting to
hundreds of microbes:
1- Routine culture are limited -microbe cannt
grow e.g.,Mycobacterium lepra, HCV
2-Grow slowly- M.tuberculosis
3-Diffecult to culture - Brucella, HIV

Diagnosis of infectious diseases

Examples of infection agents that detected
by PCR:
Chlamydia trachomatis
C. pneumonia
Mycobacterium tuberculosis
Mycoblasma pneumoniae
Neisseria gonorrhoeae
Herbes simplex virus

Detection of antimicrobial
resistance
Table 2: PCR-based nuclic acid amplification for detection of antimicrobial resistance
Organism
Methicillin-resistanct
Staphylococcus aureus
coagulase-negative
staphylococci
Vancomycin-resistanct
Enterococcus spp.
Streptococcus pneumonia

Antimicrobial resistance
and

Enterobacteriaceae-producing
extended-spectrum B-lactamase
Mycobacterium tuberculosis
Herpes simplex virus
Cytomegalovirus
HIV

Methicillin and all other Blactam antibiotics

Vancomycin
Pencillin
Extended-spectrum penicillins
and cephalosporins
Isoniazid
Rifampin
Acyclovir
Ganciclovir
Reverse transcriptase inhibitors
Protease inhibitor

Gene targets for nucleic acid

amplification
MecA

VanA,vanB,vanC1,vanC2,vanC
-3
Pbp1A
SHVand TEM-Blactamse gene
sequence
KatG.inhA,ahpC
RpoB
Thymidine kinase gene
Viral phosphotransferase gene
Reverse transcriptase gene

Southern blotting
Named after Edward M. Southern whodeveloped this procedure in 1975
Allow investigators to detrmine the
molecular weight of a restriction fragment
To measure relative amounts in different
samples
To locate a particular sequence of DNA
within a complex mixture

Southern blotting methodology

1- Digest DNA with appropriate restriction
enzyme
2- Run digest on a agrose gel
3- Denaturate the DNA
4- Transfer the denaturated DNA to the
membrane
5- Add labeled probe to the membrane
6-Detection

Just a few application of southern

blotting
To look for or to confirm the presence of a
gene often in conjugation with PCR
To test for the presence of a specific allel of
a gene
To aid in restriction fragment analyses
RFLP