Can I use the Grim Reefer Free DNA Removal Kit with the PathoSEEK Aspergillus 5-color Assay?

Yes. You can use Grim Reefer with the Aspergillus 5-color qPCR assay by making a slight modification to the published protocol.

Important Note:

The following instructions provide a workaround not included in our PathoSEEK® 5 Color Aspergillus User Guide. The reason it is not included, and instead listed here in our help center, is because we do not believe a free DNA removal step is necessary with a method such as this that includes an enrichment step. 

Dead DNA or naked DNA is more vulnerable and exposed to DNase and other factors that could degrade it and therefore make it undetectable after enrichment. On the other hand the DNA of live organisms is protected by intact cell membranes. 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 proving these claims, showing that extracellular DNA is detected before enrichment, but not after. 

This disclaimer serves only to educate the user and explain why we've not included our Grim Reefer protocol as part of our PathoSEEK® User Guides where an enrichment step is performed. Due to popular demand for an option to run a free DNA removal step with our assays that require enrichment (presence/absence) we have added this article to our help center.

Grim Reefer Free DNA Removal with Aspergillus 5 Color

The Grim Reefer Free DNA Removal Kit eliminates free (extracellular) DNA from dead cells via an enzymatic reaction prior to lysing live cells. Lysis buffer deactivates the Grim Reefer® Enzyme so that it will not eliminate DNA from the newly-lysed cells.

The Grim Reefer® Positive Control, which contains a known amount of free DNA, is added after the lysis step to ensure that the Grim Reefer Enzyme was properly deactivated. If the Grim Reefer® Positive Control appears in the qPCR results, you know the Grim Reefer® Enzyme was properly deactivated.

The Grim Reefer qPCR Assay uses the Cy5 channel to detect the Positive Control. However, the PathoSEEK Aspergillus 5-Color Detection Assay also uses Cy5 to detect Aspergillus flavus. That means the Grim Reefer Positive Control would show up as a positive A. flavus on the Aspergillus 5-color Assay. Therefore, The Grim Reefer Positive Control should not be added to any samples that will be tested with the Aspergillus 5-Color Assay. 

Instead, we recommend you create a blank extraction that will serve as a control for the entire run. If there is a Cy5 signal for the blank extraction, you know the Grim Reefer® Enzyme was properly deactivated.

How to Modify the SenSATIVAx Extraction 

When setting up your initial flower extractions, create an additional "Grim Reefer" control tube with just 1mL of Tryptic Soy Broth (TSB)  that has been enriched in a separate Whirl-pak bag overnight.  Perform the enzymatic treatment and incubation steps as stated in the Grim Reefer SOP (link below) for each of the samples and the TSB in the Grim Reefer control tube.

However, do NOT spike the diluted Grim Reefer positive control DNA into the sample extractions. Only add the Grim Reefer Positive Control DNA to the TSB Blank Extraction.  If the Grim Reefer control is spiked into your samples, they will all show positive results for Aspergillus flavus as this probe contains the same Cy5 fluorophore as the Grim Reefer Assay probe.   

qPCR Setup

It’s important to add the Grim Reefer Assay to your master mix as stated in the SOP (link below).  This will ensure the mix is consistent between all the samples and controls. Cy5 amplification should be observed on the TSB blank sample, indicating the Grim Reefer enzyme was completely deactivated with the addition of the MGC lysis buffer. Any Cy5 amplification observed in the sample wells indicated a positive A. flavus result. 


Aspergillus 5 Color Assay with Grim Reefer - Supplemental Guide