Molecular methods have progressed past the point of microorganism identification and now detect antibiotic resistance, fastidious bacteria, and unculturable microorganisms. To grasp what these methods are and how they relate to one another, consider the following:
The discovery of nucleic acid amplification techniques (NAAT) has led to many molecular typing techniques such as:
- Restriction analysis: DNA is cut with restriction enzymes
- Plasmid profiling: Plasmids - extrachromosomal DNA - are cut with restriction enzymes; used in outbreaks of Enterobacterales, Neisseria gonorrhoeae, Shigella
- Restriction fragment length polymorphism (RFLP): Uses spoligotyping (useful in tuberculosis outbreaks, parasitology, and Brucella)
- Pulse field gel electrophoresis (PFGE): DNA migration on agarose gel using electric current; used for detection of Listeria monocytogenes, Campylobacter
- PCR amplification of gene target: The basis of ALL types of PCR methodology. Target specific method using primers that amplify a unique region of the organism.
- Multiplex PCR: PCR using more than one pair of primers, allowing for panels of different pathogens to be run (respiratory, gastrointestinal, and meningitis panels are examples)
- Nested PCR: Two consecutive PCR reactions of 25 cycles each using the product of the first run in the second PCR with nested primers (bacteria, fungal, parasitic targets)
- Real-time PCR: Viewing the increase in the amount of DNA at the same time it is being amplified using a fluorescent component to report increased signal (numerous assays such as HIV, HBV, and other viruses)
- Direct sequencing: A most reliable method for detecting the sequences, used for fungal and parasitic differentiation at the species level. Direct sequencing methods include:
- Whole genome sequencing (WGS): First used in 2005 by Roche; CDC now uses WGS techniques for TB - wgSNP, wgMLST
- Target gene sequencing: Most common method (used in carbapenem resistance detection)
- Universal gene target: Particular gene targets are used for detecting genus and species levels; universal gene targets include:
- Ribosomal RNA (rRNA): Detection of fastidious or slow growers can be identified by amplifying DNA encoding ribosomal RNA genes followed by DNA sequencing. Three genes make up rRNA functionality in bacteria. 16S rRNA is used frequently. Used in identifying tuberculosis, Brucella, and Stenotrophomonas from cystic fibrosis patients.
- Heat shock proteins (HSP): Uses sequencing of heat shock proteins produced by microorganisms; all microorganisms respond to this stressor, enabling survival. Used for differentiation of Leishmania species as well as Trypanosoma cruzi.
- Antibiotic resistance gene: For detecting multidrug-resistant bacteria, such as rifampicin-resistant tuberculosis, chloroquine-resistant Plasmodium falciparum, and others.
