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M201 > W8 Molecular Diagnostics in Microbiology > Flashcards

W8 Molecular Diagnostics in Microbiology Flashcards

1
Q

How can an infection be diagnosed?

A
  • Clinical diagnosis
  • Non microbiology investigation
    ▪ Radiology
    ▪ Haematology
    ▪ Biochemistry
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2
Q

What is the aim of microbial identification?

A
  • 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
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3
Q

Specimen Collection:
What are the criteria? (6)

A
  • 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
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4
Q

What are the types of microbiological tests?
(all can be done on bacteria and fungi)

A

Microscopy
Culture
Biochemical testing
Molecular testing
Rapid tests and immunoassays e.g. ELISA

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5
Q

What is the purpose of Microscopy?

What are the different shapes and arrangements of bacteria?

A
  • 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

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6
Q

Microscopy - staining
What are the differential staining? (3)

A

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

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7
Q

What is the purpose of culture? (2)
How can the specific organism be indicated?

A
  • 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

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8
Q

What are the 3 types of culture?

A

Selective media
Differential media
Enrichment culture

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9
Q

What is selective media?
example?

A
  • 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+
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10
Q

What is differential media?
example?

A
  • 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
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11
Q

What is Enrichment Culture?

A

Encourages the growth of a desired microbe by increasing very small numbers of a desired organisms to detectable levels (without suppressing others)

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12
Q

Biochemical profiling - bacteria
What are biochemical properties based on?

A
  1. Growth requirement
  2. 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
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13
Q

Immunoassays:
Why are they used instead of culture?

A
  • 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

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14
Q

Immunoassays and molecular techniques
What 3 main features does a good diagnostic system have?

A
  1. 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)
  2. Specificity: a measure of the test can correctly identify individuals or samples that do not have a particular condition or substance (NO FALSE POSITIVES)
  3. Simplicity: The test must be carried out efficiently at the level of routine
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15
Q

Immunoassays:
What is direct and indirect diagnosis?

A
  • 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

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16
Q

Nucleic acids extraction

A

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
17
Q

Immunoassays - ELISA

A
  • 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
18
Q

Molecular tests based on nucleic acids

A

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)
19
Q

What is PCR?
What does it allow?

A
  • 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
20
Q

A basic PCR set-up requires:

A

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

21
Q

Polymerase Chain Reaction (PCR)
PCR cycle consists of what 3 steps?

A
  1. Denaturation
    * The two DNA strands are separated by heating (DNA denaturation -breaking H-bonds) at 95°C
  2. 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
  3. Extension
    * The reaction is heated (to the DNA polymerase optimum temperature) for efficient DNA synthesis 72°C
22
Q

PCR Process:

A
  • 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

23
Q

Polymerase Chain Reaction (PCR)
Reaction rate of PCR are affected by which limiting factors? (3)

A

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

24
Q

Breakdown of the word PCR (for info)

A

POLYMERASE CHAIN REACTION
Polymerase= DNA polymerase activity
Chain Reaction= DNA is repeatedly replicated

25
Q

What is DNA electrophoresis?

A

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
26
Q

What is DNA visualisation?

A
  • 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
27
Q

DNA visualisation:
microbial diagnosis (for info)

A

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

28
Q

PCR results
What is a negative control?
What us a positive control?

A

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

29
Q

What is Real-time PCR?

A

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)

30
Q

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

A

= C

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