Kit for the qualitative evaluation of the human mycobiota by PCR amplification of the ITS1 region of fungal ribosomal DNA (rDNA) and NGS sequencing.
Detailed Description
Operating Principle
Mycobiota Solution is based on the PCR amplification of the hypervariable ITS1 region of the fungal rDNA, using primers that allow the identification of most fungal populations present in the sample. The generated amplicons are subsequently sequenced by Next Generation Sequencing (NGS).
The protocol includes eight stages: DNA extraction and quantification, Target PCR, purification of Target PCR products, Index PCR, purification of Index PCR products, normalization and pool preparation, sequencing, and data analysis.
Clinical Applications
The kit is intended for the qualitative evaluation of the human mycobiota given its relevance in multiple physiological conditions and pathological states (for example, mechanisms of colorectal carcinogenesis and fungal infections in immunocompromised patients).
Benefits
Qualitative, rapid (~8 hours), and highly sensitive DNA-based evaluation of the mycobiota, without relying on morphological analysis by culture.
Automated post-sequencing analysis with dedicated software that generates reports on the fungal species identified in each sample and comparisons between samples selected by the user.
Technology Used
PCR amplification of the fungal rDNA ITS1 region followed by NGS.
Target Audience/User
This product is aimed at clinical microbiology or research laboratories that require a solution based on Next-Generation Sequencing (NGS) for the characterization of the human mycobiota. It is especially oriented towards users who need a protocol from library preparation to bioinformatic analysis for the study of fungal communities without relying on culture.
Considerations or Limitations
The kit is intended for manual use; any automation must be validated by the user.
Data analysis with MicrobAT provides taxonomic assignment down to the species level for most reads, along with descriptive statistics such as rarefaction curves, alpha-diversity, and dysbiosis index.
Other Relevant Technical Information
Recommended extraction methods: QIAamp PowerFecal Pro DNA Kit or QIAamp Fast DNA Stool Mini Kit for feces, and DNeasy PowerSoil Pro Kit for other samples; other methods may be used following user validation.
Requires a DNA input of 5–50 ng/µl.
Compatibility with Illumina MiSeq™, iSeq 100™ and MiniSeq™ platforms.
For optimal coverage of the amplified region, it is recommended to obtain at least 50,000 reads per sample.
Key Aspects
Qualitative evaluation of the human mycobiota via ITS1 PCR + NGS.
Library preparation ~8 hours.
Applicable to samples such as feces, rectal, vaginal, skin and oral swabs, saliva, sputum, and urine.
Avoids the culture phase and reduces the bias associated with laboratory culture conditions.
Dedicated software for per-sample and comparative reports, with descriptive diversity metrics.
Compatible with Illumina MiSeq™, Illumina iSeq 100™ and Illumina MiniSeq™.
Presentation Details
Format and References
| AD-005.048 | 48 reactions |
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