Molecular Diagnostics in Microbiology Flashcards
Diagnosis of infection:
Clinical diagnosis
* Non microbiology investigation
- Radiology
- Haematology
- Biochemistry
Microbial identification
- identify pathogens
> specific therpeutic options
> exclude another diagnosis
> test antimicrobial suceptibility
Specimen collection
- Specimen should represent the diseased area
- Sufficient quantity for tests
- Avoid contamination from environment
- Proper container and promptly sent to laboratory
- Obtain specimen before antimicrobial treatment
- Accompanied by a putative diagnosis
James Redfearn/McGraw Hill
What are the types of microbial tests?
- rapid tests and immunoassays
- microscopy
- Culture
- Biochemical tests
- Molecular testing
micorscopy different shapes:
coccus (sphere)
bacillus (rod shape)
vibro (rounded rod shape)
coccobacillius (oval shape)
spirillum (like spiral)
what are the differents taining of bacteria?
Gram +ive and +ive
ACID-FAST = TB
Flurescence in fluresce microscopy > TB
analyse specific cell components (capsule)/ charicteristics (spores)
What is used for protozoan staining?
- iron hematocylin & trichrome
Bacterial culture
multiplication of bacterial cells
To enumerate microbes….
colony count
To obtain pure cultures (isolate organisms)….
selecting 1 distinct colony
How can you indicate a specific organism (culture)?
- colony morphology
- growth requirement
(temp, oxygen requirement, salt requirement)
What is important after cultivation in cell cultures?
to isolate the virus
Selective media
Suppress unwanted microbes and encourage desired microbes
Differential media
Allow distinguishing of colonies of different microbes on the same plate
Enrichment Culture
Encourages the growth of a desired microbe by
increasing very small numbers of a desired organisms
to detectable levels (without suppressing others)
What is biochemical profiling?
differential biochemical properites:
Growth requirment
Enzymatic activities
> Ex. Ability to ferment various sugars (acidity turn colour yellow)
Ex. Oxidase and catalase enzymatic activity
Tests for biochemical profiling bacteria?
- Kits and robotic automation of biochemical tests
made identification of pathogens more efficient - API strip with 20 microtubes with multiple
biochemical tests
Immunoassays
- some microorganisms DO NOT grow in culture
- culture is time consuming
- some results can be misinterpreted
What is a disadvantage of biochemical strips?
expensive
When doing immunoassays and molecular techniques
3 main features:
- Sensitivity: a measure of the ability of the test to detect very small amounts of
the target in the presence of other molecules (NO FALSE NEGATIVES) - Specificity: This test should give a positive result only in the presence of the
target molecule (NO FALSE POSITIVES) - Simplicity: The test must be carried out efficiently at the level of routine
Monoclonal antibodies
Recognising a specific epitope
of an antigen > UNIQUE portion of a specific protein
> ELISA, Lateral-Flow tests (LTF), Hemagglutination, Western blot
difference between direct and indirect diagnosis:
DIRECT DIAGNOSIS to detect and identify microbes in clinical samples
INDIRECT DIAGNOSIS to detect the antibody response developed to
specific microorganisms in clinical specimens
ELISA - immunoassays
darker shade - more of microorganisms
Molecular tests based on nucleic acids
1) detection of specific nucleic acids sequence of microorganisms in clinial specimens
- polymerase chain reaction (PCR)
hybridisation techiques
sequencing - used to identify new organisms
Method of molecular tests based on nucleic acid
a) sample processing
b) nucleic acids extrection and pruification
c) exposure to probe in liquid or solid phase
d) detection system of recognition between probe and target
= +ive and -ive
Explain the process of nucleic acid extraction:
solation of DNA and/or RNA from a tissue or cell
* it must meet two main requirements:
> the yield, i.e. the amount of nucleic acid in solution
> the purity, i.e. the absence of contaminants
basic steps of nucelic acid extraction;
- cell lysis
- inactivation of cellular nucleases
- separation of nucleic acid from cellular debris
PCR
polymerase chain reaction
what does PCR allow?
rapid amplification of a designed fragment of DNA
continuous DNA rep focused on sepcific short DNA fragment > DNA photocopier in test tube
each strand of DNA used as a template to create a replicon of DNA target > amplification to make it easily detectable
PCR components?
A. DNA template
- each strand used as template to generate copies
- target DNA present? PCR will lead to specific amplification of DNA targer
- non-specific DNA will NOT be amplified
B. a heat-resistant DNA polymerase
- enzyme that polymerizes new DNA strands and can work up to 100C
- enzyme polymerizes new DNA strand and work up to 100C
- adding nucletides to new growing chain, respecting the complementarity of DNA template (forming bonds 5’ > 3’)
c. pair of primers (forward and reverse)
short DNA FRAGMENTS THAT ARE COMPLEMENTARY to 3’ ends of each strand of DNA target
- determine sequence to be amplified
- essential to start DNA polymerase > free 3’-OH group
- primers sequence and pCR protocols are avail. for most microbes
d. deoxynucleoside triphosphates - used by DNA pol. to synthesis a new DNA strand
e. buffer solutioon ( stable enviroment for enzyme)
f. Mg2+ ions or other bivalent cations - essential co-factor of DNA polymerase
what are the 3 steps of PCR?
- Denaturation - two DNA strands of separated heating 95C
- Annealing - reaction rapidly cooled and allow primers to bind specifically to target (50-65C)
- extention - heated, DNA pol.
he 3 steps are repeated ___-____ cycles to amplify the DNAT
25 - 35 cycles
n 20 cycles, PCR can generate _______ (220) copies of a single DNA target
1 million
Reaction rate of PCR are affected by limiting factors
list the 3:
- Exponential amplification phase (initial cycles): Initially,
after every cycle, the amount of product is doubled - Linear phase: The amplification slows (DNA polymerase loses
activity, reagents are depleted, unwanted products accumulate) - Plateau phase: No more amplification due to
reagents/enzyme exhaustion
What is DNA electrophresis?
PCR products (amplified DNA) are visualised in
agarose gel (forming a three-dimensional matrix)
DNA electrophoresis method:
- After thermal cycling, sample tubes are collected
- Tubes’ contents are loaded onto one end of an agarose gel
- DNA is negatively charged moves toward the positive pole
when an electric current is applied
- DNA fragments separate as they migrate through the gel
pores - Smaller fragments move faster and farther than larger ones
- DNA bands of different fragment sizes, become visible
after staining with a dye and exposure to UV light
DNA visualisation
DNA of different sizes migrates at different speed
size of PCR product compared with DNA ladder and mol. weight marker
In microbial diagnosis - DNA visualisation
- the primers and PCR protocol are designed to amplify a portion of a specific
microorganism’s genome/gene (suspected to be responsible for the infection) - Nucleic acids extracted and purified from a clinical specimen are subjected to PCR
amplification and the product is loaded into the agarose gel. - The presence of a band at the expected molecular weight confirms the presence of the
microbe in the clinical specimen
PCR results: - ive
A sample expected to result in no
amplification of DNA
> Alternative result indicates a nucleic acid
contamination and the amplification in the
test samples cannot be reliable, giving
FALSE POSITIVE
PCR results: +ive
A sample expected to result in the PCR
amplification of DNA
> Alternative results indicates some reagents
did not work and the lack of amplification in
the test samples cannot be reliable, giving
FALSE NEGATIVE
Real-time PCR
qualititive technique
uses florescent probs that measure amount of amplificated DNA generated in PCR
determine copies of specific nucleic acids > in clinical saample
