On June 16th, 2022 MGC released an updated workflow for TYM testing. This included a new User Guide and a new Validation document. Why?
This update is a result of months of torture testing our TYM process, during which:
- Six different internal lab users tested the same samples to understand the process variance
- A beta-testing site ran the updated protocol on real-life samples and achieved concordance with plating
- Inclusion and exclusion testing was expanded to 30 and 50 organisms respectively as seen in other AOAC SMPRs
New PT Recommendation for flower
In order for your lab to validate the new PathoSEEK® Total Yeast & Mold Detection Assay with SenSATIVAx® Extraction protocol, we recommend the following standard from NSI:
- PT Express Catalog Number CMPT-085B from NSI Lab Solutions
This new CRM includes both yeast and mold organisms, which more accurately represents what labs encounter on real-life samples and achieves better concordance when comparing qPCR and plating results.
Important information regarding Cq to CFU conversion equations for TYM Testing:
The Medicinal Genomics Total Yeast and Mold assay targets ITS (Internal Transcribed Spacer) regions in the yeast and filamentous fungi genomes. The prevalence of these regions can vary by 10-100x between organisms.
Mathematically, The equation assumes 1 genomic copy = 1 PCR target. Yeast have ~20 ITS regions per genome and molds have 50-100 ITS regions which means when amplifying these organisms in situ, you’re not amplifying 1 copy, but 20-100 copies per copy of the genome. This increases the sensitivity of qPCR when targeting a region present in multiple copies per genome.
Plate enumerations for yeast and mold is highly variable due to 4 variables involved that can vastly affect results
- Time the plate is incubated
- Carbon source
- Temperature used
- Organisms that do not culture
Using an assay that targets a genomic region present in 20-100 copies is less variable than juggling the 4 previously mentioned growth factors. In addition, having 10X higher sensitivity or 100X higher sensitivity isn't a deficit when you are comparing it to a ‘gold standard’ of plating which also accepts that many microbes are unculturable.