About

The current gold standards of microbial genotyping are pulse-field gel electrophoresis (PFGE), multilocus variable number of tandem repeat analysis (MLVA, a.k.a. microsatellite analysis), and multilocus sequence typing (MLST), . However, these methods have disadvantages:

  • PFGE:  High cost due to requirement for highly trained and experienced personnel; portability issues that require strict standardization

  • MLVA:  Low portability due to standardization issues; high cost due to to expensive equipment and personnel needs

  • MLST:  High cost due to requirement for sequencing ca. 7 genes; modest resolution due to focus on conserved housekeeping genes

 

At MicrobiType, we recognize the need for affordable outsourcing of microbial genotyping. Our research team, with over 40 years experience in microbiology, genetics, and informatics, has developed an uncompromising, cost-effective alternative: Polymorphic Locus Sequence Typing (PLST). This technology exploits the numerous genome sequences that are currently and increasingly available, and applies powerful algorithms to identify the most informative and reliable typing targets. Validation based on published studies and exhaustive analysis of available genome sequences is provided for each microbe. Our optimized genome targets and amplification/sequencing primers provide maximum strain resolution with the full portability that only sequence-based typing can provide.

The MicrobiType Advantage

 

Beyond the affordable price tag, high resolution, and full portability,  MicrobiType offers:

  • Simple isolate submission: Start with a free Isolate Submission Kit. Then just add the isolated colony to the provided tube, heat to inactivate the microbe, and mail to MicrobiType. No costly packaging required!

  • Quick turnaround: Results typically in 3 to 4 business days after we receive your isolates.

  • Easy to interpret results: the dendrogram output will clearly illustrate the relatedness of your submitted isolate to previously or concurrently submitted isolates from your lab and to a large database of strains.

 

The MicrobiType Process

In your lab:

  1. Begin by requesting a free, no obligation Isolate Submission Kit (allow 1 week for delivery)

  2. Consult the list of microbes for which typing services are currently available (the list will be frequently updated, and requests are welcome)

  3. Prepare agar plates with isolated microbial colonies of interest and save for short periods in your refrigerator

  4. Inoculate tubes from kit with colonies of interest, recording the three letter code from the tube (e.g., ABC) in your notebook

  5. Heat inactivate microbe as per instructions in kit and on the Isolate Submission Guidelines page

  6. Submit now, or store tubes in freezer for later submission with additional isolates (there are substantial discounts for submission of 2, 3, or 4 or more isolates for a given service)

  7. Email us (support@microbitype.com) with your tube codes, requested typing service(s), and shipping date.

  8. Package tubes in a provided bubble bag, and ship overnight to MicrobiType

At MicrobiType:

  1. DNA is isolated from submitted heat-inactivated bacteria by methods that minimize risk of contamination

  2. Genomics-optimized primers are used to PCR amplify your selected targets

  3. Amplified DNA is sequenced by dideoxy methods using conditions that optimize readability and length

  4. Chromatograms are rigorously checked, ambiguities resolved, and sequences edited to defined lengths or endpoints

  5. BLASTN searches of GenBank databases are conducted to identify identical or closest matches

  6. Matched and representative sequences for that species are aligned with the submitted sequence using Clustal-Omega

  7. Alignments are submitted for phylogenetic analysis using dnapars (DNA parsimony) which weighs both base changes (SNPs) and insertions/deletions (indels)

  8. Phylogenies are presented as dendrograms that illustrate the relatedness of the submitted strain to (a) representative strains in GenBank databases and (b) previously or concurrently submitted samples from your lab

  9. Results are reported to you by email, typically within 3 to 4 business days of isolate receipt

 

Troubleshooting failed PCRs

 

The most likely reason for PCR failure is incorrect speciation, followed by insufficient or degraded DNA.  When such failures occur, MicrobiType will perform species identification using the appropriate Ribo-ID service on your sample at no extra cost.  If this also fails to yield a PCR product, you will be asked to submit a fresh sample of your isolate, again at no extra cost.

Founder
Tom Edlind, Ph.D.

Co-inventor of ribotyping and PCR-ribotyping; pioneered phylogenetic analysis of eukaryotic pathogens; expertise in antimicrobial agents and resistance; former Editor of ASM journal; > 80 peer-reviewed publications; avid bicyclist and hiker.

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