Introduction

Question 1

Why are some bacteria pathogenic to humans?

Answer 1

Due to the:
- Ability to adhere to and colonize skin or mucosal surfaces
- Ability to cross skin or mucosal surfaces
- Ability to cause tissue destruction
- Ability to produce toxins (endotoxins, exotoxins, superantigens)
- Ability to induce inflammation
- Ability to withstand or avoid immune defences

Question 2

In what order is bacteria classified?

Answer 2

1) Kingdom (Bacteria)
2) Phylum (Proteobacteria)
3) Class (Gammaproteobacteria)
4) Order (Enterobacterales)
5) Family (Enterobacteriaceae)
6) Genus (Escherichia)
7) Species (Escherichia coli)

Question 3

Which parts does the bacterial genome consists of?

Answer 3

1) The core genome: genes present in “all” bacterial strains in a strain collection / species
2) Accessory genome: genes present in some, but not all, bacterial strains in a strain collection / species
3) Pangenome: all genes present in all bacterial strains in a strain collection / species

Question 4

What is the difference between monomorphic bacteria and polymorphic bacteria?

Answer 4

Monomorphoc bacteria (Mycobacterium tuberculosis) has a high degree of homology.
Polymorphic bacteria (Staphylococcus aureus, Escherichia coli) has a high degree of diversity, and there is a high rate of mutations / homologous recombination and/or horisontal gene transfer

Question 5

What three mechanisms are there for exchange of genetic materials?

Answer 5

1) Conjugation: transfer of bacterial DNA by plasmids
2) Transformation: uptake of short fragments of naked DNA by naturally transformable bacteria
3) Transduction: transfer of bacterial DNA by bacteriophages

Question 6

What is conserved regions and variable regions?

Answer 6

Conserved regions has unspecific applications.
Variable regions has group or species-specific applications

Question 7

What is Shiga Toxin Genes (Stx)?

Answer 7

Stx is genes encoded on a bacteriophage which may infect E.coli and some other bacteria and integrate in the bacterial chromosome. E.coli containing Stx genes are termed Enterohaemmorrhagic E.coli (EHEC). EHEC can infect humans through food or drink contaminated with animal faecal content.

Question 8

How does the amplification through PCR occur?

Answer 8

1) Denaturation, where the DNA template is heated to 94-96°C, breaking the hydrogen bonds between base pairs and resulting in single-stranded DNA.
2) Annealing, where the reaction mixture is cooled to 50-65°C, allowing specific primers to bind to their complementary sequences on the single-stranded target DNA.
3) Extension, where the temperature is raised to the optimal working temperature for the DNA polymerase (usually Taq polymerase, 72°C) to synthesis new DNA strands complementary to the template, starting from the primers.

Typically, 25-40 cycles are performed.

Question 8

What are the two types of Stx genes?

Answer 8

1) Stx1: rarely associated with severe diseases like HUS, and is therefore classified as low virulent.
2) Stx2: some subtypes can cause HUS / are highly virulent and some subtypes do not cause severe diseases

Question 8

What is the fundamental principle of PCR?

Answer 8

The principle of PCR is the enzymatic amplification of a specific DNA sequence, typically a gene or a portion of a gene unique to the pathogen of interest.

Question 8

Why is Taq polymerase used in PCR?

Answer 8

Taq polymerase is crucial to the PCR process due to its ability to withstand the high temperatures of the denaturation step. This heat-stable enzyme allows for the automation of PCR, as the reaction mixture can be subjected to repeated heating and cooling cycles without the need to add new polymerase after each cycle.
Taq polymerase possesses 5´-3´ DNA polymerase activity and 5´-3´ exonuclease activity, but lacks 3´-5´ exonuclease (proofreading) activity

Question 8

What is the key to PCR´s power?

Answer 8

The key lies in its exponential amplification: theoretically, the amount of target DNA doubles with each cycle. This means that after 30 cycles, a single copy of the target sequence can be amplified to over a billion (10^9) copies.

Question 8

What components are required in a PCR reaction?

Answer 8

1) A sample containing the target DNA sequence to be amplified
2) Two primers: a forward primer and a reverse primer
3) Taq DNA polymerase
4) dNTPs
5) A buffer that maintains suitable chemical environment for DNA polymerase activity

Question 8

How is the specificity of PCR determined?

Answer 8

The specificity is primarily determined by the primers, which are designed to be complementary to sequences flanking the region of interest. The high temperature of the denaturation step ensures that only true complementary binding persists, while the lower annealing temperature allows for efficient primer binding.

Question 9

What is conventional PCR?

Answer 9

In conventional PCR, the amplification products are detected after the PCR amplification is finished (post-PCR), typically by gel electrophoresis.

Question 10

What is Real-Time PCR (qPCR)?

Answer 10

qPCR, which uses fluorescent dyes or probes, allows for monitoring of amplification as it occurs. It enables quantification of the initial amount of target DNA in the sample, and also reduces contamination risk by eliminating the need for post-PCR processing.

Question 11

What is advantages and disadvantages of using fluorescent dyes for visualization of amplification products?

Answer 11

Advantages is that it is simple and cost-effective, and that it can be used with any dsDNA product.
Disadvantages are that non-specific binding to any dsDNA can happen, including primer-dimers and non-target sequences, which can lead to false positives.

Question 12

How does FRET work?

Answer 12

In FRET PCR (fluorescence resonance energy transfer), two probes are designed to hybridize in close proximity to each other on the target DNA. If that happens, the donor dye on one probe is excited and transfers energy to the acceptor dye on the second probe and the acceptor dye emits fluorescence.

Question 13

How does reverse transcriptase PCR (RT-PCR) work?

Answer 13

RT-PCR is used to amplify RNA targets by first converting target RNA to complementary DNA (cDNA) using reverse transcriptase, followed by standard PCR amplification.

Question 14

What is nested-PCR?

Answer 14

Nested PCR is a two-step process using two sets of primers, which can increase both sensitivity and specificity but can also increase the risk of contamination.

Question 15

What is important to consider when designing PCR primers?

Answer 15

1) Primers should be 18-22 nucleotides long
2) The GC content should be 40-60%
3) They should have similar melting temperatures (ideally within 5°C of each other)
4) One should avoid secondary structures in the target DNA sequence, complementarity between the two primers, and self-complementarity between copies of each single primer.

Question 16

Which factors is involved in optimizing a PCR assay?

Answer 16

Optimization of reaction conditions includes determining the optimal annealing temperature, correct MgCl2 concentration, and other cycling parameters.