W8 Molecular Diagnostics in Microbiology Flashcards
How can an infection be diagnosed?
- Clinical diagnosis
- Non microbiology investigation
▪ Radiology
▪ Haematology
▪ Biochemistry
What is the aim of microbial identification?
- Rapidly & accurately identify pathogens from clinical specimens that are
responsible for the infection OR confirm the absence of suspected microbes
▪ Possibility to identify specific therapeutic options
▪ Exclude other diagnosis
▪ Test antimicrobial susceptibility testing of these organisms (if isolated) - Microbiological quality control of pharmaceutical products
- Testing clinical specimens must follow a prescribed and standardised process
-Avoid contamination
-Proper waste
Specimen Collection:
What are the criteria? (6)
- Numerous methods used; choice of method depends on specimen.
▪ Specimen should represent the diseased area
▪ Sufficient quantity for tests
▪ Avoid contamination from the environment
▪ Proper container and promptly sent to laboratory
▪ Obtain specimen before antimicrobial treatment
▪ Accompanied by a putative diagnosis
What are the types of microbiological tests?
(all can be done on bacteria and fungi)
Microscopy
Culture
Biochemical testing
Molecular testing
Rapid tests and immunoassays e.g. ELISA
What is the purpose of Microscopy?
What are the different shapes and arrangements of bacteria?
- Preliminary identification of microbes
- Based on characteristic morphology of microbes in clinical specimens (bacteria, fungi and protozoa)
- Or indirectly identify typical cytopathic effects on the cells/tissues (viruses)
Shape:
Coccus, Bacillusm Vibrio, Coccobacillus, Spirillum, Spirochete
Arrangements:
Diplobacilli, Streptobacilli
Microscopy - staining
What are the differential staining? (3)
Bacteria:
* Differential staining
▪ Gram → Gram+ and Gram- presence of a bacterial infection and which species
▪ Acid-fast (Ziehl-Neelsen) → presence of TB
▪ Fluorescence (auramine) in fluoresce microscopy→ presence of TB
▪ To analyse specific cell components (capsule)/ characteristics (spores)
Protozoan staining
▪ Iron hematoxylin and trichrome
What is the purpose of culture? (2)
How can the specific organism be indicated?
- To enumerate microbes – e.g. colony count (e.g. bacteria in the urine to diagnose UTIs)
- To obtain pure cultures (to isolate organisms) –selecting 1 distinct colony
Indication of the specific organism by
1.Colony morphology
2.Growth requirement
* Temperature
* Oxygen requirement
* Salt requirement
Virus isolation after cultivation in cell cultures
What are the 3 types of culture?
Selective media
Differential media
Enrichment culture
What is selective media?
example?
- Suppress unwanted microbes and encourage desired microbes
E.g. Saboraud’s Agar: 5.6pH discourages bacterial growth. Used to isolate fungi
MacConkey agar: Growth of Gram- and inhibit most Gram+
What is differential media?
example?
- Allow distinguishing of colonies of different microbes on the same plate
E.g. Blood Agar: to distinguish bacteria that destroy red blood cells (haemolysis).
MacConkey agar: to differentiate lactose-fermenting and non-lactose fermenting bacteria
What is Enrichment Culture?
Encourages the growth of a desired microbe by increasing very small numbers of a desired organisms to detectable levels (without suppressing others)
Biochemical profiling - bacteria
What are biochemical properties based on?
- Growth requirement
- Enzymatic activities
-Example. Ability to ferment various sugars (acidity turns colour yellow)
-Ex. Oxidase and catalase enzymatic activity
- Kits and robotic automation of biochemical tests made identification of pathogens more efficient
- API strip with 20 microtubes with multiple biochemical tests
Immunoassays:
Why are they used instead of culture?
- Some microorganisms do not grow in culture, grow only in specific situations
(anaerobes) or grow very slowly (M.tuberculosis) - Culture is time consuming
- Biochemical strips are expensive
- Some results/identification could be misinterpreted
So– Immunoassays and molecular techniques (Without isolation of the microorganism) is done
Immunoassays and molecular techniques
What 3 main features does a good diagnostic system have?
- Sensitivity: a measure of how well a diagnostic test can correctly identify
individuals or samples that truly have a particular condition or substance (NO FALSE NEGATIVES) - Specificity: a measure of the test can correctly identify individuals or samples that do not have a particular condition or substance (NO FALSE POSITIVES)
- Simplicity: The test must be carried out efficiently at the level of routine
Immunoassays:
What is direct and indirect diagnosis?
- Based on the specificity of the antigen-antibody binding
- Monoclonal antibodies – recognising a specific epitope of an antigen- UNIQUE portion of a specific protein
- ELISA, Lateral-Flow tests (LTF), Hemagglutination, Western blot
DIRECT DIAGNOSIS= to detect and identify microbes in clinical samples
INDIRECT DIAGNOSIS= to detect the antibody response developed to specific microorganisms in clinical specimens
Nucleic acids extraction
Extraction of nucleic acids
* isolation 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
- The basic steps are :
- cell lysis
- inactivation of cellular nucleases
- separation of nucleic acid from cellular debris
Immunoassays - ELISA
- Quantitative (amount of substrate is correlates to the amount of antigen/antibodies in the clinical specimen)
- Protocols available to identify the most common bacteria and viruses
Molecular tests based on nucleic acids
Detection of specific nucleic acids
sequence of microorganisms in clinical
specimens (i.e. unique viral genes)
* Polymerase Chain Reaction (PCR)
* Hybridisation techniques
* Sequencing
- Used to identify new organisms (e.g. SARS-COV-2 in 2019)
What is PCR?
What does it allow?
- PCR allows the rapid amplification of a designed (SPECIFIC) fragment of DNA
- Continuous DNA Replication focused on a specific short DNA fragment= Like “A DNA photocopier in a test tube”
- Each strand of DNA is used as a template to create a replicon of the DNA target– amplification to make it easily detectable
- Many applications, including the detection of microorganisms
-Microbial detection - Amplification of a microbe-specific nucleic acid sequence
A basic PCR set-up requires:
A) DNA template (microbial genome – what we are looking for)
- Each strand is used as template to generate copies
- If the target DNA, is present= PCR will lead to a specific amplification of the DNA target
- If properly conducted, Non-specific DNA will not be amplified
B) A heat-resistant DNA polymerase (Taq polymerase),
- Enzyme that polymerizes new DNA strands and can work up to 100°C
- Adding nucleotides to the new growing chain, respecting the complementarity of the DNA template (forming phosphodiester bonds 5’-3’)
C) Pair of primers (forward and reverse), short DNA fragments that are
complementary to the 3’ ends of each strand of the DNA target (base pair rule)
Necessary to determine the sequence to be amplified (based on sequence
complementarity) determines the specificity of the PCR
Essential to start DNA polymerase providing a free 3’-OH group)
Primers sequence and PCR protocols are available for most microbes
D. Deoxynucleoside triphosphates (dNTPs – A, C, G, T)
Used by the DNA polymerase to synthesise a new DNA strand
E. buffer solution
providing a suitable chemical environment, stabilising the enzyme (pH, salinity, etc)
F. Mg 2+ ions or other bivalent cations
Essential co-factor of DNA polymerase
Polymerase Chain Reaction (PCR)
PCR cycle consists of what 3 steps?
- Denaturation
* The two DNA strands are separated by heating (DNA denaturation -breaking H-bonds) at 95°C - Annealing
The reaction is rapidly cooled to allow primers to bind specifically to the target
sequences, on a SINGLE-stranded DNA (if present!) 50-65°C - Extension
* The reaction is heated (to the DNA polymerase optimum temperature) for efficient DNA synthesis 72°C
PCR Process:
- Double-stranded DNA template (25°C)
- Denaturation- separates DNA strands (95°C)
- Primers anneal to single-stranded DNA strands (50-65°C)
- Taq adds complementary nucleotides to extend the primer (72°C)
- Double-stranded DNA created, process repeats
*The 3 steps are repeated 25-35 cycles to amplify the DNA:
Exponential amplification after the first cycles
-if primers binds to the template sequence
In 20 cycles, PCR can generate 1 million (220) copies of a single DNA target
Polymerase Chain Reaction (PCR)
Reaction rate of PCR are affected by which limiting factors? (3)
a) Exponential amplification phase (initial cycles): Initially, after every cycle, the amount of product is doubled
b) Linear phase: The amplification slows (DNA polymerase loses activity, reagents are depleted, unwanted products accumulate)
c) Plateau phase: No more amplification due to reagents/enzyme exhaustion
Breakdown of the word PCR (for info)
POLYMERASE CHAIN REACTION
Polymerase= DNA polymerase activity
Chain Reaction= DNA is repeatedly replicated
What is DNA electrophoresis?
PCR products (amplified DNA) are visualised in agarose gel (forming a three-dimensional matrix)
- After thermal cycling, sample tubes are collected
- Tubes’ contents are loaded onto one end of an agarose gel
- DNA is negatively charged and 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
What is DNA visualisation?
- DNA of different sizes migrates at different speed
- The size of the PCR product is compared
with a DNA ladder, a molecular weight
marker (DNA with known sizes), which runs
alongside the PCR products to determine
the size
DNA visualisation:
microbial diagnosis (for info)
In microbial diagnosis,
- 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
What is a negative control?
What us a positive control?
PCR is a very sensitive test
In diagnosis, PCR requires the use of appropriate controls
A negative control:
* 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
A positive control:
* 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
What is Real-time PCR?
PCR (end-point PCR) is a qualitative technique * Most of the PCR techniques used in microbial diagnosis are quantitative PCR→Real-Time PCR
▪ Real-Time PCR uses a fluorescent probe that measures the amount of amplificated DNA generated in the PCR
▪ Determine the copies of specific nucleic acids (e.g. microbial gene) in a clinical sample
➢E.g. SARS-CoV-2 diagnosis (molecular test)
What is the main role of primers in PCR?
A) Serve as templates for DNA amplification
B) Facilitate DNA denaturation through hybridization
C) Provide a complementary sequence for DNA polymerase to initiate replication and define the target DNA
D) Stabilising Taq polymerase
E) They provide a pool of nucleotides
= C
