Molecular biology techniques

Polymerase chain reaction (PCR)
- The idea is to amplify a piece of DNA of interest (for example a piece of DNA from B. pertussis, B. parapertussis or B. holmesii)
- How it works:
   - It needs among other: two primers, DNA polymerase and the building blocks
   - A cycli consists of: heating to denature the DNA --> slow temperature decrease allow primers to attach      to target single stranded DNA --> elongation --> final hold.
   - This cycli is repeated many times.
- Controls:
  - internal control: in each cuvette, to see whether the PCR process works (to check that negative results are really negative and not false negative).
  - negative control: usually to put in the middle, so when this is positive, contamination is happened due to pipetting.
   - positive control: a piece of DNA from the targeted pathogens.
- The pre-PCR room should be 'clean', for example that lab coat from outside the room does not allow to enter, because of possible DNA contamination.
- It is faster than culture of especially fastidious bacteria. Thus this is useful for example in detecting: Bordetella, Mycoplasma.

Real time PCR
- Instead of using agar electrophoresis to separate the DNA bands, it displays curves on the computers.
- We get a curve of our control and we know after how many cycli the J form of the curve start to appears (this is referred as crossing point (CP)).
- When our samples have higher CP than this control --> weak.
- When CP falls within the range of control: the result can be given at species level.
- Weak results can be repeated, when it is weak again --> results can be given at genus level only (for example Bordetella spp.)
- Example of the machine: LightCycler (Roche)