PathogINDICAtor Cq to CFU Equations

Transcription

1 PathogINDICAtor Cq to CFU Equations Overview: State regulations for microbial thresholds use nomenclature from culture- based methods to set acceptable thresholds. With this in mind, it was necessary for Medicinal Genomics (MGC) to convert the quantification cycle (Cq) value from the PathogINDICAtor qpcr Assay to colony forming units (CFU) which is reported when using culture- based methods. This document demonstrates the initial development of the equations used to calculate Cq to CFU when using the SenSATIVAx DNA Extraction Kit, and the PathogINDICAtor qpcr Master Kit and Detection Assays. Verification Samples Using the SenSATIVAx DNA Extraction Kit, the PathogINDICAtor qpcr Master Kit, and PathogINDICAtor qpcr Detection Assays, the Cq to CFU equations were built using the following species: S.cerevisiae (commonly known as Ale yeast) DH10B E.coli The analysis and testing was completed using a serial dilution from 1:10 through 1:10M (7 serial dilutions) 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 PetriFilm and SimPlate plating. The summary of the data for the verification is shown on Table 1-4 for Total Yeast & Mold, Total Aerobic Count, Total Coliform, and Total Enterobacteriaceae respectively.

2 Table 1, Total Yeast & Mold Detection Assay: 1:10 Dilution - > 1:5000* 500,000 60, :100 Dilution - > 1:500* 78,000 23, Common Threshold 10,000 10, :1,000 Dilution - > 1:50* 4,100 4, :10K Dilution 250 > :100K Dilution :1M Dilution 25 2 <40 1:10M Dilution 5 0 <40 1:100M Dilution 2 0 <40 Table 2, Total Aerobic Count Detection Assay: 1:10 Dilution - > 1:5000* 415,000 1,010, Common Threshold 100, , :100 Dilution - > 1:500* 70, , :1,000 Dilution - > 1:50* 5,400 12, :10K Dilution 408 > :100K Dilution :1M Dilution :10M Dilution 0 0 <40

3 Table 3, Total Coliform Detection Assay: Petrifilm CFU SimPlate CFU** Cq Value 1:10 Dilution - > 1:5000* 300, :100 Dilution - > 1:500* 65, :1,000 Dilution - > 1:50* 4, Common Threshold 1,000 1, :10K Dilution :100K Dilution :1M Dilution :10M Dilution Table 4, Total Enterobacteriaceae Detection Assay: 1:10 Dilution - > 1:5000* 300, :100 Dilution - > 1:500* 65, :1,000 Dilution - > 1:50* 4, Common Threshold 1,000 1, :10K Dilution :100K Dilution :1M Dilution :10M Dilution *These CFU numbers were calculated by using the CFUs obtained with the intermediate dilutions due to the detection limitations of the plating methods. ** The Coliform/E.coli SimPlate did not change color, which signifies no growth from the DH10B E.coli. This was confirmed with a repeat experiment.

4 Equations From the data in the above tables, a line of best fit was created. The coefficient of determination, or R 2, for the line of best fit for all detection assays is above 93%, with three of the four detection assays above 98%, the outlier being Total Yeast & Mold. The slightly lower R 2 value was analyzed more thoroughly to explain the differences between plating and qpcr in Total Yeast & Mold in two peer- reviewed publications 1,2. The derived equations can be found below or with in the protocols for MGC PathogINDICAtor Data Analysis for Agilent Real- Time PCR System or MGC PathogINDICAtor Data Analysis for BIO- RAD CFX96 Touch Real- Time PCR Detection System. Equation for Total Yeast & Mold Detection Assay: CFU/g = 10 [( Cq Value)/3.6916] (+ Cq of 0.25) Equation for Total Aerobic Count Detection Assay: CFU/g = 10 [( Cq Value)/3.044] (- Cq of 1.5) Equation for Total Coliform Detection Assay: CFU/g = 10 [( Cq Value)/2.7849] (+ Cq of 1) Equation for Total Enterobacteriaceae Detection Assay: CFU/g = 10 [( Cq Value)/3.0432] (+ Cq of 0.5) Conclusions 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 factors known to affect the growth of bacterial and fungal species. All thresholds and equations were determined based on the results using the BIO- RAD CFX96 Touch Real- Time PCR Detection System and verified on Agilent AriaMX Realtime PCR System. When using a different qpcr machine ramp rates and temperature thresholds can alter the values and thus alter the equation. It is recommended that thresholds be calibrated for each specific laboratory setting. Disclaimer This test was developed and its performance characteristics determined by Medicinal Genomics Company, for laboratory use. Any deviations from this protocol are not supported by MGC. The results may vary based on laboratory conditions. Altitude and humidity are among factors known to affect the growth of bacterial and fungal species. All thresholds were determined based on the results using the BIO- RAD CFX96 Touch Real- Time PCR Detection System. It is recommended that thresholds be calibrated for each specific laboratory setting.

5 References 1. McKernan K, Spangler J, Helbert Y et al. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests [version 1; referees: 3 approved, 1 approved with reservations]. F1000Research 2016, 5:2471 (doi: /f1000research ) 2. McKernan K, Spangler J, Zhang L et al. Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers [version 2; referees: 2 approved]. F1000Research 2016, 4:1422 (doi: /f1000research )