Regulations for microbial threshold tests like TYM currently use nomenclature from culture-based methods to set acceptable thresholds. It is necessary to convert the quantification cycle (Cq) value from the qPCR assay to colony forming units (CFU).
State regulations for microbial thresholds currently use nomenclature from culture-based methods to set acceptable thresholds. With this in mind, it was necessary for Medicinal Genomics to convert the quantification cycle (Cq) value from the PathoSEEK® qPCR Assay to colony forming units (CFU), which is reported when using culture-based methods.
This article demonstrates the initial development of the equations used to calculate Cq to CFU when using the SenSATIVAx® DNA Extraction Kits and the PathoSEEK® qPCR Detection Assays.
This article is an excerpt from our manufacturers validation document and outlines our development of the conversion equation for our Total Yeast and Mold assay.
S. cerevisiae culture was used to determine the Cq to CFU conversion equation for the Total Yeast and Mold assay. DH10B E. coli culture was used to determine the Cq to CFU conversion equation for the Total Aerobic Count, Total Coliform, and Total Enterobacteriaceae assays. The analysis and testing was completed using a serial dilution of 1:10 for each species for each of the four detection assays. Due to the limited dynamic range of Petri dishes and culture-based methods, additional intermediate dilutions are required to measure the colony forming units (CFU); 3 intermediate points at 1:5000 from the 1:10 dilution, 1:500 from the 1:100, and 1:50 from the 1:1000. The quantification cycle (Cq) data for each sample was plotted against the CFU from 3M® PetriFilm™ plating. For MIP/extract, initial studies showed gummy behaved differently compared to other MIP samples. Therefore, a separate validation study was performed for gummy only. The summary of the data for the verification is shown on Figures 28-30 for Plant, Extract and Gummy respectively.
Cq vs Log CFU, Plant:
Cq vs. Log CFU, Hemp Oil Extract:
Cq vs. Log CFU, Gummy:
Cq to CFU Equations
From the data in the above tables, a line of best fit was created. The coefficient of determination, or R2, for the line of best fit for all detection assays is above 92%. The derived equations can be found below or within the protocols for MGC PathoSEEK® Data Analysis for Agilent Real-Time PCR System or MGC PathoSEEK Data Analysis for BIO-RAD CFX96 Touch Real-Time PCR Detection System.
Please reference https://www.medicinalgenomics.com/product-literature/ for our most up to date SOPs and conversion equation guidance.
Cq to CFU/g equations for Flower, MIP/Extract and Gummy
Based on these findings, the Cq to CFU equations developed by Medicinal Genomics Company (MGC) and provided to its partner laboratories, meet all internal specifications and are approved for laboratory use. Any deviations from this protocol are not supported by MGC. The results may vary based on laboratory conditions. For example, altitude and humidity are known to affect the growth of bacterial and fungal species. All thresholds and equations were determined based on the results from the BIO-RAD CFX96 Touch™ Real-Time PCR Detection System and verified on Agilent AriaMX Real Time PCR System. When using a different qPCR machine ramp rates and temperature thresholds can alter the values and thus alter the equation.
Additional Data Supporting Cq/CFU Conversions
Colony forming units (CFU) are reported when using the 3M™ Rapid Yeast and Mold PetriFilm™ product. The Cq to CFU equation was tested and compared to plating using multiple yeast and mold species listed below. The following species in Table 58 were tested on both 3M™ Rapid Yeast and Mold PetriFilm™ and PathoSEEK Yeast and Mold Detection Assay. The data in this appendix included the decontamination step that precedes the qPCR set-up step of the PathoSEEK workflow.
Species evaluated in Cq/CFU comparison experiments:
Organisms were reconstituted and incubated according to the ATCC product documentation. Cultures of ATCC strains were then grown in 5mL Tryptic Soy Broth (TSB) for 5 days at room temperature and checked visually for turbidity. TSB was chosen because it is a general-purpose liquid enrichment medium that has minimum interference with the MGC SenSATIVAx DNA Extraction Kits. The broth recommended by 3M is Butterfields phosphate buffered dilution water. The phosphates in this liquid medium interfere with the magnetic particles in the MGC SenSATIVAx DNA Extraction Kits. 3M states, “Do not use diluents containing citrate, bisulfite or thiosulfate with the 3M PetriFilmTM Coliform Count Plates; they can inhibit growth.” TSB does not contain any of the above ingredients. The undiluted culture, 1:10, 1:100, and 1:1000 dilutions were plated in triplicate on 3M™ PetriFilmTM Rapid Yeast and Mold Count Plates, incubated at room temperature, and counted after 3–5 days. An aliquot of the undiluted culture was extracted using the MGC SenSATIVAx DNA Extraction Kits. The DNA was then tested using PathoSEEK Yeast and Mold detection assay. The Cq value obtained from the qPCR results was plugged into the Cq to CFU/g equation and compared to the CFU obtained from the 3M™ PetriFilm™.
The summary of the validation samples is shown below:
A graphical representation is shown here:
The concordance was measured between CFU derived from the PathoSEEK qPCR Cq and 3M™ PetriFilm™ CFU pictured in chart above. The Aspergillus species CFU counts are approximately three orders of magnitude lower than expected based on Cq estimates that were developed and optimized by plating cultured cells of other species. Excluding the two Aspergillus species and Candida glabralta, the correlation between CFU/g and Cq is greater than or equal to 0.71. The correlation between CFU per gram of plant material and Cq is 0.99 for all fungal species except Candida glabralta.
Aspergillus has been proven to grow poorly in culture media, and is therefore severely under-reported by current culture-based platforms. This phenomenon has been published in two peer reviewed papers. The differential growth of other toxigenic fungi, depending on the companion species present, further influences the results. Bacterial pathogens are not uncommon, and beneficial bacteria are also capable of influencing the growth or inhibition of other flora. This document and the associated peer reviewed demonstrate that the verification samples processed using PathoSEEK Total Yeast and Mold Detection Assay meet the standards set forth in the PathoSEEK Cq to CFU Equations document.