Bacterial Identification Techniques Quiz

Advances in Bacterial Identification

• Molecular diagnostics have been hugely beneficial for viral diagnostics, but have had limited impact on bacterial diagnostics.
• Cost and sensitivity are limitations of PCR compared to traditional culture methods.
• Nucleic acids are relatively stable, ubiquitous in nature, and have purification processes that are virtually identical regardless of the organism.

Polymerase Chain Reaction (PCR)

• PCR produces a large number of identical molecules of nucleic acids in vitro.
• It is critical for amplifying DNA in various situations, such as epidemiological investigations (e.g., determining separate Ebola outbreaks in Africa in 2014).
• Typical steps in PCR include 16S rDNA sequencing.

16S rDNA Sequencing

• 16S rDNA is the gene that codes for 16S rRNA, a component of prokaryotic ribosomes.
• The 16S rDNA sequence is used as a target for PCR primers, which anneal to conserved regions and amplify all bacterial DNA present.
• The PCR reaction is designed to produce an amplicon containing several variable regions, allowing for identification of the bacteria.

Advantages of 16S rDNA Sequencing

• No need for viable organisms, as PCR can be performed directly on specimens.
• Detection and identification of organisms in "partially treated" patients.
• Detection and identification of non-growing/slow-growing organisms.

Disadvantages of 16S rDNA Sequencing

• Only useful for normally sterile sites.
• Only useful for mono-microbial infections.
• Contamination during specimen collection can be a significant problem.
• May take a week to obtain identification.

Gel Electrophoresis

• Separates DNA molecules based on electrical charge, size, and shape.
• Allows scientists to isolate DNA of interest.
• Negatively charged DNA is drawn towards the positive electrode.
• Agarose acts as a molecular sieve, and smaller fragments migrate faster and farther than larger ones.
• DNA is stained with ethidium bromide, which fluoresces when exposed to UV.

Real-time PCR/RT-PCR

• Detection during PCR/RT-PCR allows for faster results, as no electrophoresis is required.
• Fluorescence is proportional to the quantity of DNA produced.
• Many different chemistries can be used (e.g., SYBR green, Taqman, FRET).
• Can be used for qualitative or quantitative analysis.

16S rRNA Gene

• Common to all bacteria.
• Essential gene that codes for the small ribosomal subunit.
• Evolves at an extremely slow rate, making it a "molecular clock".

Test your knowledge on molecular and rapid techniques used for bacterial identification and outbreak investigations. This quiz covers advances in bacterial identification, including molecular diagnostics like PCR.