PCR: application in diagnostics

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

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.

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.

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 )

brucellosis) using a PCR assay
Isolation of DNA
PCR
Gel electrophoresis
Analysis

of results

from pure culture) and negative controls (no DNA)

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.