PCR: application in diagnostics

Components and general mechanism
Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction
5) Mg2+ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

General mechanism

Application
Diagnostics - The detection of the presence, or absence, of a pathogen and its subsequent identification and characterization.
Features:
Approach 1-> designs primers that are complementary to a DNA target that is specific for the microbe being assayed. For instance, by selecting unique regions of the Whipple bacillus' 16S rRNA gene, one can create primers that will amplify only the 16S rRNA gene from the Whipple bacillus, Tropheryma whippelii.
Approach 2-> multiplexing, in which multiple specific PCR assays are run simultaneously in the same reaction tube to test for multiple different DNA templates. In multiplex PCR, several sets of primers are added to the reaction in order to generate several different PCR products. For instance, one could have a PCR assay designed to detect bacterial DNA that uses five different specific PCR reactions in one tube, with primer pairs directed toward S. pneumoniae, N. meningitidis, H. influenzae, Listeria monocytogenes, and the group B Streptococcus.

Advantages/disadvantages
Advantages- High sensitivity and specificity (specific primer design), rapid test, ease of use, and robustness, capability to detect pathogens which are impossible to cultivate on media.
Disadvantages – Requirement of special conditions, high cost equipment, expensive reagents.

PCR in diagnostics
Assays are available for a variety of pathogens, including HIV, HSV, hepatitis B virus, hepatitis C virus, cytomegalo virus, ennterovirus, Chlamydia trachomatis, M. tuberculosis, T. whippelii, and Neisseria gonorrhoeae, Brucella sp. For the detection of RNA Viruses is applied RT-PCR method (Reverse Transcriptase PCR). Reverse transcriptase is enzyme capable to synthesize DNA strand from RNA template.
Generally the principle of detection is based on the detection of pathogen’s specific DNA/RNA region, amplification of that sequence and analyzing the presence or absence of detection amplicons on electrophoretic agarose gel )

Procedure
The detection of Brucella sp. and strains (cause of brucellosis) using a PCR assay
Isolation of DNA
PCR
Gel electrophoresis
Analysis of results

Isolation of DNA

Isolation of DNA

Isolation of DNA

PCR
*Reference Material - positive (DNA of certain strain isolated from pure culture) and negative controls (no DNA)

PCR
Composition of PCR Buffer 10x

PCR
Primer sets

PCR
Primer sets sequences

PCR
Regime

Gel electrophoresis
A 1,5% agarose gel stained with ethidium bromide is used
10 μl of the product is loaded with 2 μl loading buffer
2 μl of a 100 bp DNA molecular weight marker is loaded with 2 μl loading buffer a single outside well
Gel electrophoresis is performed at 100 to 120V for 30 min
*The composition of LOADING buffer was not mentioned in manual, but on practice it is possible to use loaders like bromphenol blue and xylene cyanol, or cresol red.

Analysis of results
Visually