It is not recommended that Free DNA Removal protocols be used with assays that require an enrichment such as Aspergillus, Salmonella and STEC. There is a simpler more cost effective alternative.
Grim Reefer is not necessary due to sample enrichment
DNA from dead cells or "naked DNA" is more vulnerable and exposed to DNase and other factors that could degrade it and render it undetectable after enrichment. Cannabis samples contain naturally occurring DNAse's that will eliminate extracellular DNA. The DNA inside live organisms is protected by intact cell membranes or cell walls. The DNA of live organisms is detectable after enrichment because the DNA is not being liberated until lysis buffer is added during the extraction process. We have performed experiments internally showing that extracellular DNA is detected before enrichment, but not after.
This is why we've not included our Grim Reefer protocol as part of our PathoSEEK® User Guides where an enrichment step is performed. It is an unnecessary step that only adds time and cost to the assay.
The following experiment demonstrates this.
- Approximately 500,000 copies of Aspergillus niger genomic DNA (serving as free floating or "dead" DNA) was added to TSB
- Live Aspergillus flavus organism grown internally was also added to the same container
- An "initial" aliquot was removed and taken through the PathoSEEK protocol
- An "18 Hour" aliquot was removed after 18 hours of incubation, or enrichment, and taken through the PathoSEEK protocol
Results:
Pre - Enrichment
The "initial" pre-enrichment time point showed signal for Aspergillus flavus at an average Ct of 22.24 and Aspergillus niger at an average Ct of 27.21. (See above)
Post Enrichment
The 18 hour time point showed a signal for Aspergillus flavus at an average Ct of 21.11 and Aspergillus niger was not detected. The cannabis internal control "SCCG" was detected at both the "initial" and "18hr" timepoints showing that DNA extraction was successful.
Alternative method to discern live vs dead DNA with the Aspergillus assay
Another solution to the live-dead problem is to perform a second DNA isolation and qPCR run after a short incubation. If the microbes are living, they should replicate their genomes in 20 minutes (bacteria) to 4 hours (mold). Therefore, qPCR results post incubation would show growth. The shift in signal is easy to detect even if the cells are partially replicating. This method is often referred to as Before-After enrichment qPCR.
Most labs that employ this technique, only perform the 2nd PCR on a sample that initially fails. This second, post enrichment live/dead re - test only needs to be performed on failures or approximately 10-20% of the samples tested.
Already using Grim Reefer with Aspergillus?
Medicinal Genomics never intended the Grim Reefer Free DNA removal kit to be used with assays that included an enrichment because of the reasons stated above. The Grim Reefer product was also never designed to work with the 5 color Aspergillus assay due to the shortage of required optical channels. The Grim Reefer assay control is detected on the same channel as A. flavus (Cy5)
However, due to the popularity of remediation techniques such as irradiation and the demand on labs from cultivators for free DNA removal techniques, we published a workaround for using our Grim Reefer with the 5 color Aspergillus assay earlier this year. In pursuit of adding Grim Reefer to our Aspergillus AOAC PTM, we began extensive internal testing in matrix. The data were not conclusive that Grim Reefer should be implemented in the Aspergillus 5 color assay. At this time we are not recommending it's use with any assay requiring an enrichment.
The more cost effective and simpler approach of an additional enrichment to discern live from dead Aspergillus positive hits is the method that we recommend for use with the PathoSEEK® 5 Color Aspergillus Detection Assay.