This document discusses various molecular diagnostic methods used in clinical microbiology. It describes techniques such as nucleic acid hybridization, polymerase chain reaction (PCR), and real-time PCR which can directly detect microorganisms, identify pathogens, and detect antibiotic resistance genes. The advantages of these molecular methods include speed, ability to detect fastidious or non-culturable bacteria, and quantitative analysis. However, limitations include possible false negatives from inhibitors and inability to determine bacterial viability. Overall, molecular diagnostics provide crucial information to improve patient treatment and conduct epidemiological analyses.

1. Improved Medical Education and Basic
sciences
for Better Medical practicing
ImproveMEd
Molecular methods and clinical microbiology
domagoj Drenjancevic

2. Thymine Adenine
Cytosine
Guanine
Adenine
Guanine
Thymine
Cytosine

3. The use of molecular diagnostic methods in
microbiology
• Direct detection of microorganisms
• Identification of microorganisms
• Detection of genes which determine the virulence of
• Detection of genes controlling resistance to antibiotics
• Typing of bacterial isolates in epidemiological studies

4. 4
MOLECULAR DIAGNOSTICS
1. true identification organism
isolated in pure culture
2. fast detection directly from clinical specimens
3. detection organisms from a sample from which the traditional cultivation
is not isolated a cause (endocarditis)
- A small amount of pathogens
- nonviable microorganisms
4. Characterization agents, typing
- resistance genes
- epidemiological markers

5. 5
When to perform Molecular detection and identification?
• In nutritically demanding bacteria-HACEK endocarditis
• In slow-growing bacteria - mycobacteria
• For bacteria that need special techniques for laboratory breeding -
cell cultures
• After already applied antibiotic therapy

6. ... the need for the application of molecular
technology ......
Diagnostics:
• pandemic viruses, ie human immunodeficiency virus 1 virus
(HIV-1), hepatitis B virus (HBV) and C (HCV);
• the causative agents of sexually transmitted infections as
part of the program of monitoring women's health
(Chlamydia trachomatis, Neisseria gonorhoeae, human
papillomavirus);
• respiratory infections;
• Treatment of patients treated with transplantation;
• neurotrophic viruses in the cerebrospinal fluid
• Sepsis

7. molecular diagnostics
Shortcomings
1. sensitivity inhibitors -Lažni negative results
2. sensitivity contamination -Lažni positive
results
3. inability distinction viable from non viable
cells
4. The inability to further characterization
fourth strain (Antibiotic sensitivity?, typing?)
The inability to further characterize the strain (antibiotic susceptibility, typing?)

8. Advantages of use of molecular methods in
clinical microbiology laboratory
• speed of the method (important for the success of
treatment)
• the uniqueness of the method (application in certain
patient groups due to the inability to apply other
methods)
• quantitative determination of the virus (facilitated
monitoring of the effect of therapy)
• typing of strains for the purpose of epidemiological
analysis

9. 9
I. Hybridization nucleic acid
• developed in the 1970's and is still in use
• is based on the feature that two single-stranded
nucleic acids have complementary sequences and bind
to each other by forming a double-stranded nucleic
acid = duplex or hybrid.
• The test needs:
• 1.PROBE / Sondu = single-stranded nucleic acid (DNA,
RNA) isolated from a known identity organism
• labeled with enzymes or chemiluminescence
molecules
• 2. A TARGET single-stranded nucleic acid from an
unknown organism to be detected or identified.
• PROBAMA can be identified by organism and species
level

10. The principle of molecular hybridization
ABC
cytosine
guanine
thymine
adenine
target DNA
DNA probes
DNA isolation DNA denaturation - Molecular hybridization -
the sample separation of DNA strands binding of labeled DNA probes
the target DNA

11. Hybridization nucleic acid
1. "Dot-blot" hybridization - the isolated DNA is
fired on the membrane in the form of a dot
2. Southern blot DNA fragments separated
electrophoresis on nitrocellulose membrane
3. Immunoblot or Western blot hybridization - for
the detection of specific antibodies to
electrophoresis-isolated viral antigen applied on
the nerve-cell membrane by molecular weight
Ag complex and antibody-detection by enzyme-
labeled anti-mouse antibodies
Reading - a colored line on the membrane portion
where Ag and Ab bind
Frequently Certified Antibody Specification Method
in Viral Medicine (After ELISA)

12. II. Chain reactionpolymerase - PCR
• from one copy DNA was obtained by reacting several trillion copies
(2n-cycle depending on the number duplication)
• To carry out the reaction of copying we need to know the sequence
DNA or sequence nucleotides DNA sequence which can be
reproduced

13. 13
PCR
The principle - the amplification of a particular DNA
fragment in in vitro conditions,
• multiplication depends on a number of factors,
• the conditions should be defined,
• there is no single protocol,
• the reaction must be optimized beforehand,

14. PCR
• The nucleotide sequence serves to select Primer (a pair of
short nucleotides representing the boundary-the-right and
the right-sliced ​​DNA that is multiplied)
• Enzyme - thermostable DNA polymerase
• The success of the reaction depends on:
• Selection of INITIAL - length ranges from 18 to 24 pb (which
reduce the efficiency of matching, too short - lead to
multiplication of unspecified product)
• Annealing temperatures 50-65 ° C and always 5 ° C lower than
"melting point" / melting point

15. Amplification

17. Detection -
electrophoresis
2 1
3 4
5 6 7

18. Photography electrophoretic gel

19. TMB
COBAS AMPLICOR Analyzer
first amplification
2. Denaturing
4. Conjugation
5. Catalysis
substrates
6.Fotometer
3. Hybridization
magnetic
microparticles

20. RT-PCR

21. 21
III. Real-time PCR
• for a more precise quantification of the polymerase
chain reaction in real time
• reaction products are labeled with fluorescent dyes
and analyzed during the formation
• Fluorescent colors for labeling a product of real-time
PCR:
• 1.SYBER Green 1 - is for newborn double-stranded
DNA and emits fluorescence that is measured

22. 22
Real-time PCR
2. Fluorescence oligonucleotide probe TaqMan =
specific POINTS are unmarked and specifically labeled
probe
Real-time PCR Absolute Quantification - Determine
the number of RNA copies in a sample by comparing
with the standard curve

23. 23
Real Time PCR
camera
measure
the level of
fluorescence
the reaction
time

24. 24
Real- time PCR

25. 25
Multiplex PCR IV
• A method in which one or more pair of
primers and two or more DNA molds
are included in one tube are
simultaneously targeted, so it is
relatively easy to detect several
different bacteria in a single PCR
reaction.
• Initial pairs must be specific to the
target gene and the products must be
of different sizes

26. 26
SeptiFast (Roche)
• based on the ITS (Internal Transcribed Spacer) region of
bacterial or fungal genoma-identification based on the analysis
melting point (Melting point)
• At the same time DENTIFICATION 25 most common bacterial
and fungal pathogens
• Processing up to seven blood samples
• To Test duration 6 hours
• Instrument: LightCycler® 2.0

27. 27
V. Broad range PCR (broad ranged PCR)
•is used for the universal detection of microbial pathogens
•primers are selected from conserved regions of certain genes, which
share the organisms in the taxonomic grouping therefore is an
important choice really a wide range of primers

28. 28
VI. DNA-chips (DNA microarray)
• They consist of a large number of short DNA sequences of 25
nucleotides (Probe) that are chemically synthesized in precise
positions quartz chip surface covered

29. DNA-chips (GeneChip, Affymetrix)
• They consist of a large number of
short DNA sequences of 25
nucleotides (Probe) that are
chemically synthesized in precise
positions quartz chip surface covered
• DNA microarray (DNAčip) allows
analysis of the transcripts present in
the sample at the time of extraction
of mRNA and analyze both the
activity of the gene in the sample that
are related to the probe
komplemantarne chip