|Pyrosequencing in Microbiology|
Pyrosequencing in Microbiology - As Reliable as Sequencing, as Easy as PCR
Together with PCR, Pyrosequencing is an essential addition to any modern clinical microbiological laboratory.
Jeanne A. Jordan, Ph.D., HCLD
Associate Professor in Pathology, University of Pittsburgh Director-Microbiology, Virology, Molecular Diagnostics and Immunology at Magee-Women's Hospital
Associate Director, Magee-Women's Research Institute
Identify species and detect resistance from the DNA sequence
New technologies are enabling DNA sequence information to become the new gold standard for microbial species identification and resistance detection. By sequencing well-characterized hypervariable regions of genes such as 16S rRNA or rnpB, sequence data provides, either on its own or with supporting biochemical data, unambiguous and discriminatory information for microbial identification.
Pyrosequencing technology is highly suited to providing reliable sequence data in clinical situations. In roughly the time it takes to run a PCR reaction, Pyrosequencing technology reads a discriminatory stretch of DNA of up to 96 samples in parallel. Depending on the assay design, the sequence can be used to discriminate microbial species, types and strains, or detect genetic mutations that confer resistance to antibiotics and anti-viral drugs.
The obtained sequences are rapidly matched against a local database using software algorithms optimized for Pyrosequencing data. The raw data together with the matched hits are presented in identification reports.
The PyroMark ID system comprises:
- Pyrosequencing Assay Design Software
- Pyrosequencing Vacuum Prep Workstation for preparing 96 samples for sequencing in less than 30 minutes
- PyroMark ID instrument, a platform for sequencing 96 samples in 1 hour
- Pyro Gold chemistry, optimized for longer sequencing read lengths
- IdentiFire™ software for alignment of obtained sequences to a local user database.
How microbial identification by PyroMark ID compares to traditional sequencing
With PyroMark ID, sequencing becomes a practical reality since it is far easier to perform compared to traditional sequencing. PyroMark ID disposes of gels, dyes and cleanup steps to shorten the preparation, sequencing and analysis process to less than 2 hours for 96 specimens.
How PyroMark ID works for microbial identification
This animation illustrates the principle of microbial identification by PyroMark ID from microbial sample to identified sequence. QuickTime is required to view this animation. To download QuickTime click here
Benefits for the microbiologist
Pyrosequencing delivers the microbial sequence in 1 hour. For the clinical microbiologist, this means:
- True DNA Sequence data in a clinically-relevant timeframe, not just a yes/no answer
- Ideal for rapid identification, epidemiological studies and resistance detection
- Species identification and resistance characterisation with 1 system
- Fulfils 2 fundamental applications with 1 simple method
- Mutation-Tolerant Assays
- Unlike hybridisation, sequencing yields sequence data despite unpredicted mutations
- Quantification of multi-copy genes
- Antibiotic resistance characterization, measuring viral and fungal loads
- Built-in Quality Control
- Seeing the mutation in the context of the DNA sequence ensures a correct analysis
Broad-range identification strategies using signature sequences in the 16S rDNA gene and the rnpB gene have been described, as well as more specific assays for Bordetella pertussis, Mycobacteria spp., Neisseria spp., Helicobacter pylori, and Streptococcus spp.
Pyrosequencing is a proven technology for sequence identification of species as well as in typing applications. A variety of assays, broad-range as well as targeting specific species, are available using discriminatory genetic regions for Salmonella, Staphylococcus, Neisseria, Mycobacteria, Nocardia, Helicobacter, Lactobacillus, or to identify bacteria in e.g. neonatal sepsis, rapidly and accurately, alive or dead. Pyrosequencing uses a single PCR reaction to discriminate among a variety of species.
Read about Dr. Jordan’s application of Pyrosequencing in neonatal sepsis
Broad-range identification strategies of yeasts and molds using signature sequences in the ITS2 region have been described, capable of speciation of Candida spp., Aspergillus spp., Penicillium spp., Fusarium spp., and many others with a single assay.
Pyrosequencing technology identifies clinically important fungal species, dramatically shortening the time from sample collection to identification. Pyrosequencing technology is more efficient in identification than hybridization techniques, since a number of species can be discriminated from a single PCR product. One sequencing primer identifies a large number of species, instead of using one dual-labelled probe for each species. Pyrosequencing technology therefore works elegantly in combination with routine PCR screening. Fungal identification by Pyrosequencing technology offers major benefits of speed, ease and accuracy.
Read about Dr Klingspor’s application of Pyrosequencing in fungal identification
Identification of viruses including influenza, Human Papilloma Virus and Herpes Simplex Virus.
With proven applications for the typing of Human Papilloma Virus, Herpes Simplex Virus and detection of resistance in Influenzavirus, Pyrosequencing technology provides accurate sequencing data for discriminatory purposes.
Several genetic methods based on Pyrosequencing for resistance detection have already been described, including adamantane resistance in influenzavirus; ciprofloxacin resistance in Neisseria gonorrhoeae, isoniazid, rifampicin, ethambutol and quinolone resistance in Mycobacterium tuberculosis; macrolide resistance in Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium avium, Campylobacter jejuni and Haemophilus influenzae; clarithromycin resistance in Helicobacter pylori; and linezolid resistance in Enterococcus spp.