Molecular Diagnostics in Microbiology

Question 1

Diagnosis of infection:

Answer 1

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

Question 2

Microbial identification

Answer 2

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

Question 3

Specimen collection

Answer 3

• 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

Question 4

What are the types of microbial tests?

Answer 4

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

Question 5

micorscopy different shapes:

Answer 5

coccus (sphere)

bacillus (rod shape)

vibro (rounded rod shape)

coccobacillius (oval shape)

spirillum (like spiral)

Question 6

what are the differents taining of bacteria?

Answer 6

Gram +ive and +ive

ACID-FAST = TB

Flurescence in fluresce microscopy > TB

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

Question 7

What is used for protozoan staining?

Answer 7

• iron hematocylin & trichrome

Question 8

Bacterial culture

Answer 8

multiplication of bacterial cells

Question 9

To enumerate microbes….

Answer 9

colony count

Question 10

To obtain pure cultures (isolate organisms)….

Answer 10

selecting 1 distinct colony

Question 11

How can you indicate a specific organism (culture)?

Answer 11

• colony morphology
• growth requirement

(temp, oxygen requirement, salt requirement)

Question 12

What is important after cultivation in cell cultures?

Answer 12

to isolate the virus

Question 13

Selective media

Answer 13

Suppress unwanted microbes and encourage desired microbes

Question 14

Differential media

Answer 14

Allow distinguishing of colonies of different microbes on the same plate

Question 15

Enrichment Culture

Answer 15

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

Question 16

What is biochemical profiling?

Answer 16

differential biochemical properites:
Growth requirment
Enzymatic activities

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

Question 17

Tests for biochemical profiling bacteria?

Answer 17

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

Question 18

Immunoassays

Answer 18

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

Question 19

What is a disadvantage of biochemical strips?

Answer 19

expensive

Question 20

When doing immunoassays and molecular techniques
3 main features:

Answer 20

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

Question 21

Monoclonal antibodies

Answer 21

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

Question 22

difference between direct and indirect diagnosis:

Answer 22

DIRECT DIAGNOSIS  to detect and identify microbes in clinical samples

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

Question 23

ELISA - immunoassays

Answer 23

darker shade - more of microorganisms

Question 24

Molecular tests based on nucleic acids

Answer 24

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

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

Question 25

Method of molecular tests based on nucleic acid

Answer 25

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

Question 26

Explain the process of nucleic acid extraction:

Answer 26

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

Question 27

basic steps of nucelic acid extraction;

Answer 27

- cell lysis - inactivation of cellular nucleases - separation of nucleic acid from cellular debris

Question 28

PCR

Answer 28

polymerase chain reaction

Question 29

what does PCR allow?

Answer 29

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

Question 30

PCR components?

Answer 30

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

Question 31

what are the 3 steps of PCR?

Answer 31

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.

Question 32

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

Answer 32

25 - 35 cycles

Question 33

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

Answer 33

1 million

Question 34

Reaction rate of PCR are affected by limiting factors list the 3:

Answer 34

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

Question 35

What is DNA electrophresis?

Answer 35

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

Question 36

DNA electrophoresis method:

Answer 36

- 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

Question 37

DNA visualisation

Answer 37

DNA of different sizes migrates at different speed size of PCR product compared with DNA ladder and mol. weight marker

Question 38

In microbial diagnosis - DNA visualisation

Answer 38

- 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

Question 39

PCR results: - ive

Answer 39

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

Question 40

PCR results: +ive

Answer 40

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

Question 41

Real-time PCR

Answer 41

qualititive technique uses florescent probs that measure amount of amplificated DNA generated in PCR determine copies of specific nucleic acids > in clinical saample