1. Molecular microbiology methods like PCR and hybridization have revolutionized clinical diagnostics by enabling fast and direct detection of pathogens from clinical samples. 2. PCR in particular has become a mainstay technique, allowing amplification of specific DNA sequences from small amounts of input DNA. Variations like real-time PCR, multiplex PCR, and broad-range PCR further expanded diagnostic capabilities. 3. Emerging technologies like DNA microarrays promise even greater multiplexing, with the ability to simultaneously genotype large genomic regions or measure expression of many genes, positioning them as promising future molecular diagnostic tools.

1. Molecular microbiology methods
1. Detection and identification of the causative infectious agent is a highly relevant issue in
microbiological diagnostics.
The mayor breakthrough in clinical and diagnostics microbiology was done by
introducing molecular methods in daily diagnostic routine.
2. Since the discovery of the secret of DNA helix which was fifty years ago, we witness the
flourishing of the method of diagnostic procedures in clinical microbiology
3. where is possible to apply molecular diagnostic methods in microbiology?
one of the main efforts is focused on - direct detection of microorganisms together
with identification of microorganisms extraordinary significance is in the field of
detection of genes that determine virulence and especially detection of genes that
determine resistance to antimicrobial agents last but not least are bacterial isolates
typing in epidemiological research
4. So, molecular diagnostic in clinical microbiology evolves in several directions
1. Correct identification of the organism isolated in pure culture
2. Fast detection directly from clinical specimens
3. detection of organisms from samples from which the pathogen did not isolate by
traditional cultivation (eg. endocarditis) because of
- A small amount of agents
- unviable microorganisms
4. Characterization of causative agents and genotyping for
- detection of resistance genes and other
- epidemiological markers
5. Particular importance should be put on detection of nutritionally demanding
bacteriaHACEK endocarditis (Haemophilus, Aggregatibacter, Cardiobacterium,
Eikenella, Kingella which are a normal part of the human microbiota, living in the oral-
pharyngeal region.
for slow growing bacteria – genus mycobacterium and for bacteria that require special
techniques for laboratory cultivation such as cell cultures (we should keep in mind the
importance of intracellular bacteria). it should be emphasized the possibility of etiological
diagnostic in clinical cases After already applied antibiotic therapy, when standard
methods are not useful, as well

2. 6. nowadays, there is an application of this diagnostic methods for:
detection of pandemic viruses
human immunodeficiency virus 1 (HIV-1), hepatitis B virus (HBV) and C (HCV);
causative agents of sexually transmitted infections as part of a women's health
monitoring program: Chlamydia trachomatis, Neisseria gonorhoeae, human
papillomavirus;
respiratory infections;
treatment of patients treated with
transplantation; neurotropic viruses in
cerebrospinal fluid sepsis
7. Unfortunately molecular diagnostic has some weaknesses
Disadvantages
1. susceptibility to inhibitors - false negative results
2. Sensitivity to contamination - false positive results
3. Inability to distinguish viabile from unviable cells
4. Inability to further characterize the strain (antibiotic susceptibility)
8. However, weaknesses are overwhelmed with numerous benefits Advantages
speed of the method (important for the success of the treatment)
uniqueness of the method (application in certain patient groups due to inability to apply
other methods)
quantitative determination of viruses (facilitated monitoring of therapeutic effect) typing
for the purpose of epidemiological analysis
9. although many methods are used today, some of the most important are: I. Nucleic
Acids Hybridization developed in the 1970's and still in use
based on the ability of 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/ Sonde = single-stranded nucleic acid (DNA, RNA) isolated from a known
identity organism
labeled with enzymes or chemiluminescent molecules
2. A TARGET single-stranded nucleic acid from an unknown organism to be detected or
identified.
PROBE can identifies organism and species level

3. 10. there are different types of hibridization such as
"Dot-blot" hybridization - the isolated DNA is on the membrane in the form of a dot
Southern blot - DNA fragments separated with electrophoresis on nitrocellulose membrane
Northern blot - RNA blot, is a technique used in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA) in a sample.
also similar technique can be witness in Immunoblot or Western Blot Hybridization – whicih is
used for the detection of specific antibodies to electrophoresis-isolated viral antigens applied on
the nitrocellulose membrane according to 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 used as confirmation method of antibody specificity in serology (upon ELISA)
12. the second method and the most popular and used is Polymerase chain reaction method -
PCR
From a single copy of the DNA to several trillion copies which are obtained in the reaction
(2ndepending on the number of copying cycles)
PCR has popularized molecular methods and introduced them into a daily routine
13. The principle of PCR method is contained in a few rules: this is the amplification of a
particular DNA fragment in in vitro conditions
such multiplication process depends on a number of factors and
the conditions should be defined,
there is no single protocol,
and every reaction protocol must be optimized before
14. For the amplification of special nucleotide sequence among other conditions we need two
critical factors: 1. Primers (a pair of short nucleotides representing the boundaries: left and
the right of slicing DNA that is multiplied)
2.An enzyme - thermostable DNA polymerase
The success of the reaction depends on:
Choosing primers - the length ranges from 18 to 24 pb (to long - reduces the efficiency of
matching, too short - leads to multiplying the nonspecific product)

4. Annealing temperatures: 50-65 ° C and always 5 ° C lower than "melting point"
15. PCR in short looks like sheme in this figure: there are 3 steps
16. after 30 or 40 cycles from one DNA molecule we have 1 bilion of copies or amplicons
17. after step of amplification there is need of detection of amplicons or visualization one of the
most used method is gel electrophoresis - a well-known method for separation and analysis
of macromolecules (eg. DNA, RNA and proteins) and their fragments, based on their size
and charge in electric field through a matrix of agarose
18. the final step is documentation of gel with photo camera
19. the development of technology created conditions for fully automated RNA and DNA
amplification and detection system for routine diagnostic PCR such as this one presented
here in slide - system automates amplification and detection of target nucleic acids making
diagnostic PCR routine
20. variations of PCR have been developed on these foundations such as Reverse
transcription polymerase chain reaction: technique used to detect gene expression through
creation of complementary DNA (cDNA) transcripts from RNA
21. i 22. Completely different from RT-PCR and is often confused with separate and distinct
technique - real-time polymerase chain reaction also known as quantitative polymerase
chain reaction.
It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and
not at its end, as in conventional PCR reaction products are labeled with fluorescent dyes and
analyzed during the formation
Fluorescent colors for labeling the product real-time PCR and for determination of the number of
RNA copies in a sample by comparing with the standard curve are:
1. SYBER Green 1 – bind to newly created double-stranded DNA and emit fluorescence that is
measured
2. Fluorescence oligonucleotide - probe TaqMan
22. the analyzer measures fluorescence level during the reaction which rise during
amplification (if there are target sequence present)

5. 23. respect the RT-PCR and qPCR today one of the most used molecular diagnostics
methods based on is Multiplex PCR: an amplification of multiple targets in a single PCR
reaction tube this technique utilize multiple primer pairs in a reaction mixture giving a
multiplexing assay which means that more than one target sequence can be amplified during
same time.
24. Similar with Cobas Amplicor today on market can be found commercially available
multiplex PCR assay such as this one which has power for simultaneous identification of 25
most common bacterial and fungal pathogens directly from the blood of patients in short time
25. there are also different else PCR based method such as Broad range PCR which is
used for universal detection of microbial agents and PCR targeting the gene encoding 16S
ribosomal RNA - first used as a taxonomic tool, then in clinical microbiology
26. the future of molecular diagnostics method probably belongs to DNA microarray (also
known as DNA chip or biochip) which can be used to measure the expression levels of large
numbers of genes simultaneously or to genotype multiple regions of a genome. In DNA
microarray probes (or reporters or oligos) are utilized and they can hybridize a cDNA or cRNA
at the exact positions of the quartz-covered chip surface.
27. The applications of DNA chip are: gene expression analysis, transcription factor binding
analysis and genotyping with capability to detect single-nucleotide-polymorphism.
Which make this method the molecular method of the future.