The updated TAC protocol/user guide includes a new document format, extraction workflow, and sample prep using qPCR Master Kit v3.
In September of 2021 we published an updated Total Aerobic Count Assay (TAC) protocol. In short, we are updating the document format, the ratio of sample to TSB used in the extraction protocol, and PCR setup protocol.
Changes to the document structure are part of a larger effort to meet the format requirements set by AOAC for certification. Over time, we plan to make the following updates to all our PathoSEEK Assay documents:
- Protocols will now be referred to as "User Guides" to align with the AOAC's preferred nomenclature
- Each assay will have its own User Guide, containing instructions for extraction, sample prep, and data analysis
- Each assay will have its own validation document, and The SenSATIVAx® and PathoSEEK® Manufacturer Validation Document, which covers our past validation work on all assays, will be phased out.
The new TAC user guide will include a change to the amount of Tryptic Soy Broth (TSB) used in the SenSATIVAx® extraction workflow. If you are running multiple PathoSEEK® Assays, this may require a second, separate, Whirl-Pak bag homogenization per sample.
For the TAC and PathoSEEK® Total Yeast & Mold Detection Assay, a ratio of 1 gram to 9 mLs TSB is required. For other enumeration assays such as BTGN and Total Coliform, a ratio of 1 gram to 14.2 mLs of TSB is still required. This means that there will be additional day-1 extraction when running TAC with certain microbial tests. If performing presence/absence assays as well, we recommend enriching the bag containing the 1 to 9 ration of sample to TSB.
This discrepancy will be eliminated in the near future as Medicinal Genomics continues to update and standardize all User Guides and method validations. This change is a necessary step and will align the TAC extraction workflow with all other assays over time.
PCR Setup Updates
The qPCR sample setup workflow in the new TAC user guide will require qPCR Master Kit v3 in place of qPCR Master Kit v2. The qPCR Master Kit v3 is already required for the PathoSEEK® Total Yeast & Mold Detection Assay and the PathoSEEK® 5-Color Aspergillus Multiplex assay. qPCR Master Kit v3 is also recommended for all other presence/absence pathogen detection assays including our FemINDICAtor Plant Gender Detection Assay.
The Medicinal Genomics team developed the v3 kit to include more enzyme in order to accommodate our growing number of multiplexed presence/absence assays. Over the next several months, all PathoSEEK® user guides will be updated to require qPCR Master Kit v3, in order to streamline workflows and consolidate inventory for our customers. This transition will include proper notice to all existing customers as well as necessary updates to our user guides.
Data Analysis Update
With the use of a updated sample to TSB ratio and an updated, more robust master mix (v3) will also bring updated Cq to CFU conversion equations. Please be aware of this change and update any conversion equations you currently use.
Please reach out to firstname.lastname@example.org with any questions!