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PMP-201 > Molecular Diagnostics in Microbiology > Flashcards

Molecular Diagnostics in Microbiology Flashcards

1
Q

Diagnosis of infection:

A

Clinical diagnosis
* Non microbiology investigation
- Radiology
- Haematology
- Biochemistry

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2
Q

Microbial identification

A
  • identify pathogens
    > specific therpeutic options
    > exclude another diagnosis
    > test antimicrobial suceptibility
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3
Q

Specimen collection

A
  • 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
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4
Q

What are the types of microbial tests?

A
  1. rapid tests and immunoassays
  2. microscopy
  3. Culture
  4. Biochemical tests
  5. Molecular testing
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5
Q

micorscopy different shapes:

A

coccus (sphere)

bacillus (rod shape)

vibro (rounded rod shape)

coccobacillius (oval shape)

spirillum (like spiral)

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6
Q

what are the differents taining of bacteria?

A

Gram +ive and +ive

ACID-FAST = TB

Flurescence in fluresce microscopy > TB

analyse specific cell components (capsule)/ charicteristics (spores)

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7
Q

What is used for protozoan staining?

A
  • iron hematocylin & trichrome
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8
Q

Bacterial culture

A

multiplication of bacterial cells

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9
Q

To enumerate microbes….

A

colony count

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10
Q

To obtain pure cultures (isolate organisms)….

A

selecting 1 distinct colony

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11
Q

How can you indicate a specific organism (culture)?

A
  • colony morphology
  • growth requirement

(temp, oxygen requirement, salt requirement)

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12
Q

What is important after cultivation in cell cultures?

A

to isolate the virus

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13
Q

Selective media

A

Suppress unwanted microbes and encourage desired microbes

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14
Q

Differential media

A

Allow distinguishing of colonies of different microbes on the same plate

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15
Q

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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16
Q

What is biochemical profiling?

A

differential biochemical properites:
Growth requirment
Enzymatic activities

> Ex. Ability to ferment various sugars (acidity turn colour yellow)
Ex. Oxidase and catalase enzymatic activity

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17
Q

Tests for biochemical profiling bacteria?

A
  1. Kits and robotic automation of biochemical tests
    made identification of pathogens more efficient
  2. API strip with 20 microtubes with multiple
    biochemical tests
18
Q

Immunoassays

A
  • some microorganisms DO NOT grow in culture
  • culture is time consuming
  • some results can be misinterpreted
19
Q

What is a disadvantage of biochemical strips?

A

expensive

20
Q

When doing immunoassays and molecular techniques
3 main features:

A
  1. 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)
  2. Specificity: This test should give a positive result only in the presence of the
    target molecule (NO FALSE POSITIVES)
  3. Simplicity: The test must be carried out efficiently at the level of routine
21
Q

Monoclonal antibodies

A

Recognising a specific epitope
of an antigen > UNIQUE portion of a specific protein
> ELISA, Lateral-Flow tests (LTF), Hemagglutination, Western blot

22
Q

difference between direct and indirect diagnosis:

A

DIRECT DIAGNOSIS  to detect and identify microbes in clinical samples

INDIRECT DIAGNOSIS  to detect the antibody response developed to
specific microorganisms in clinical specimens

23
Q

ELISA - immunoassays

A

darker shade - more of microorganisms

24
Q

Molecular tests based on nucleic acids

A

1) detection of specific nucleic acids sequence of microorganisms in clinial specimens

  1. polymerase chain reaction (PCR)
    hybridisation techiques
    sequencing
  2. used to identify new organisms
25
Q

Method of molecular tests based on nucleic acid

A

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

26
Q

Explain the process of nucleic acid extraction:

A

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

27
Q

basic steps of nucelic acid extraction;

A
  • cell lysis
  • inactivation of cellular nucleases
  • separation of nucleic acid from cellular debris
28
Q

PCR

A

polymerase chain reaction

29
Q

what does PCR allow?

A

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

30
Q

PCR components?

A

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

31
Q

what are the 3 steps of PCR?

A
  1. Denaturation - two DNA strands of separated heating 95C
  2. Annealing - reaction rapidly cooled and allow primers to bind specifically to target (50-65C)
  3. extention - heated, DNA pol.
32
Q

he 3 steps are repeated ___-____ cycles to amplify the DNAT

A

25 - 35 cycles

33
Q

n 20 cycles, PCR can generate _______ (220) copies of a single DNA target

A

1 million

34
Q

Reaction rate of PCR are affected by limiting factors

list the 3:

A
  1. Exponential amplification phase (initial cycles): Initially,
    after every cycle, the amount of product is doubled
  2. Linear phase: The amplification slows (DNA polymerase loses
    activity, reagents are depleted, unwanted products accumulate)
  3. Plateau phase: No more amplification due to
    reagents/enzyme exhaustion
35
Q

What is DNA electrophresis?

A

PCR products (amplified DNA) are visualised in
agarose gel (forming a three-dimensional matrix)

36
Q

DNA electrophoresis method:

A
  • 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
37
Q

DNA visualisation

A

DNA of different sizes migrates at different speed

size of PCR product compared with DNA ladder and mol. weight marker

38
Q

In microbial diagnosis - DNA visualisation

A
  • 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
39
Q

PCR results: - ive

A

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

40
Q

PCR results: +ive

A

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

41
Q

Real-time PCR

A

qualititive technique

uses florescent probs that measure amount of amplificated DNA generated in PCR

determine copies of specific nucleic acids > in clinical saample

PMP-201 (34 decks)

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