techniques of bacterial infection diagnosis and identification of bacteria - laboratory tests - PCR - real time PCR - serologic tests - western blot

1. Detection of bacterial infection using
methods other than culture
Presented by:
Siham Moubayed
Submitted to:
Dr. Hiam Al-soufi

2. 1- PCR : Polymerase Chain Reaction

3. Why use PCR ?
Some bacteria are very hard to cultivate in the
lab… others are late grower bacteria. So , while
time is the most important factor in the progression
of a disease, PCR was the fastest and most accurate
and sensitive method for bacterial identification.

4. What is PCR ?
PCR is an amplification of a specific gene in the
DNA or RNA.
PCR involves 2 oligonucleotide primers ,usually
17‐30 nucleotides in length, which flank the
DNA target sequence that is to be copied.
One of the primers is the same sequence as one
strand of the DNA, while the other primer is the
same sequence as the other DNA strand.
PCR sample is an extremely pure DNARNA
sample… We can take it from any specimen,
than DNA is extracted from nucleated cells
(WBCs).

6. The PCR reaction is split into three separate stages,
each of which is performed at a different
temperature.
The cycle of denaturation– annealing–extension is
repeated 20–30 times in order to achieve
satisfactory amplification of a specific DNA
Sequence.
Theoretically, after 30 cycles of PCR, we go from 1
DNA into billion copies of DNA.

7. 1- Denaturation
During this initial step, heat (90-95◦C)
separates double-stranded DNA into two single
strands. This process is called "denaturation."
Denaturation is possible because the hydrogen
bonds linking the bases to one another are weak.
The hydrogen bonds break at high temperatures,
whereas the bonds between deoxyribose and
phosphates, which are stronger covalent bonds,
remain intact.

8. 2- Annealing
-Happens at 50-55 ◦C
-One of the primers recognizes and binds to
one of the target DNA strands, and the other
primer recognizes and binds to the
other strand.
-Primers anneal at 3’ ends only and so DNA
synthesis proceeds on both strands through the
region between the two primers.

9. 3- Extension
-A DNA polymerase binds to the free 3'‐end of
each of the bound oligonucleotides and uses
dNTPs to synthesize a new DNA
strand in a 5' to 3' direction.
-The DNA polymerase used in the PCR reaction
is a heat stable enzyme, it’s mostly the Tac
polymerase enzyme extracted from the
Thermus Aquaticus bacteria.

10. Inside the PCR
tube:
-Primers
- Taq polymerase
- DNA sample
- dNTPs
(nucleotides)
- MgCl2 needed
for the Taq
polymerase
activity.

11. While amplification of a gene from a RNA, an
additional step is done:
First, the RNA must be transformed into a DNA
by using the REVERSE TRANSCRIPTASE
ENZYME, then the PCR protocol may be
continued as previously described.

12. After PCR reaction…
DNA ELECTROPHORESIS is done :
-Agarose gel is prepared
-DNA is dyed with intercalated
ETHIDIUM BROMIDE that provides DNA
Fragments fluorescence.
-Into the first well, we pour a known
DNA sequence. Into the other wells,
we pour our prepared sample.
-Run the electrophoresis:
DNA(negatively charged) will migrate to
the positively charged electrode.
DNA fragments will be separated
according to their size so the smaller
fragments will be ahead the bigger ones.

13. After the gel electrophoresis is done, the
Gel is taken to be read under UV light :
-If there hasn’t been amplification, nothing is
observed (same image of negative control)
patient doesn’t have the target gene  patient is
not infected by that bacteria.
-If amplification has been done DNA fragments
are observed (same image as positive control) 
 patient is infected by the bacteria.

14. Example:

15. Real time PCR
Quantitative real‐time RT–PCR is an accurate, precise,
and relatively easy to perform reaction that allows the quantitation
of reaction products for each sample in every cycle.
Real‐time PCR systems rely upon the detection and quantitation of a
fluorescent reporter, whose signal increases in direct proportion to
the amount of PCR product in a reaction.

16. Benefits of real time PCR
1- Sensitive, specific, simple and rapid.
2- Powerful quantitative tool.
3- Real time detection and quantification of pathogen
load.
4- Closed system no need for post amplification
processes.
5- know the effectiveness of a treatment.
6- know the progression of a disease through the
quantity of pathogen load.

17. Anothermolecular method for bacterial identification
2-Plasmid profiling:
This method involves the purification of all plasmids from
a bacterium followed by their separation on an agarose
gel where the plasmid profile can be compared in a
collection of bacterial isolates. It is a relatively easy
technique to perform and was used to successfully identify
different serotypes of salmonella .
Application of this method may be limited as only some
percentage of bacteria such as campylobacter may
contain plasmids. However, plasmid profiling is important
when characterizing genetic markers associated with
antibiotic resistance.

18. 3- Identification through serological tests
3 main methods  agglutination
 ELISA
 western blot
Agglutination:
The use of LATEX tests: the latex contains antibodies against a
protein or any substance that the bacteria produce. Example:
Rapid agglutination test for S.Aureus:
90% of S.Aureus have protein A and receptor for fibrinogen.
So, the latex contains an IgG Ab capable of binding to protein A
and a receptor for fibrinogen.
+ agg  presence of S.Aureus.

19. ELISA
-Based on interaction
between antigen and
antibody.
-The ELISA test is a
commonly used method
to detect and identity,
but not to quantify,
bacterial pathogens
-Antigen covering wells.
-Antibody obtained –if
present- from patient’s
serum.
The ELISA tests have been developed
for: Salmonella, E. coli, S. aureus enterotoxin, Vibrio
cholerae, Mycobacterium tuberculosis,
Mycobacterium leprae, Legionella pneumophila,
Borrelia burgdorferi, Treponema pattidum,
Candida, E. c. subsp. atroseptica, E.
c. subsp. carotovora

20. Western blot analysis can detect your protein of interest from a
mixture of a great number of proteins. Western blotting can
give you information about the size of your protein (with
comparison to a size marker or ladder ), and also give you
information on protein expression (with comparison to a
control).
Mostly used in the diagnosis of LYME disease caused by a bacterium
SPIROCHETE (borrelia species).
 Western Blot

21. 4-Dipstick rapiddiagnostic test
Used for the detection of many bacteria: strep A , S.flexneri (stool) ,
V.cholera (stool)…
Example: Rapid strep A test
Sample: throat swabs.
The rapid test is based on immunochromatography to detect group A
streptococcal antigens. The dipstick contains a membrane strip that has
been coated with colored anti-Strep A antibodies. If the specimen being
tested contains Group A Strep bacteria, the Strep A antigens extracted
from the throat swab will react with the antibodies, producing a colored
line. (Note: A control line will always appear on the dipstick to
demonstrate the test is working properly).