What are Cq to CFU Conversions?

Regulations for total count tests, such as 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 total count microbial tests, such as total yeast and mold and total aerobic bacteria count, currently use nomenclature from culture-based methods to set acceptable thresholds or allowable amounts. 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 allows the accuracy and speed of a qPCR platform to be utilized for tests that require reporting in CFUs.

This article demonstrates the initial development of the equations used to calculate Cq to CFU when using the SenSATIVAx® DNA Purification Kits and the PathoSEEK® qPCR Detection Assays.

The Method for the generations of Cq to CFU conversion equations is described below

1) Flower Curve Generation

Day 0  - Culture Growth

  1. Prepare a single o/n culture in 25 mls TSB, in a 50 mL conical tube.  If doing this for total assays, you need to grow a multitude of organisms to get an accurate curve.  We used the lists of inclusivity organisms supplied by AOAC for help in knowing what organisms we needed in the construction of our curve and tested it against ten different organisms.

Day 1

For qPCR of overnight cultures

  1. Prepare 6, 15ml conical tubes with 9 mls of TSB or water for each organism.  Labeled these 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000.
  2. Mix cultures well by vortexing.
  3. Add 1ml of culture to the first tube labeled 1:10.
  4. Mix well by vortex and then add 1ml of the 1:10 to the tube labeled 1:100.
  5. Continue this all the way out to 1:1,000,000.
  6. Prepare three 1.5ml tubes for each dilution.  
  7. Perform the flower extraction on each sample and ran the qPCR. 

8. Analyze the qPCR and use the average of the three replicates to use on the graph for the data (this is the Cq data).

2) Non-flower Curve Generation

Day 0 - Culture growth

  1. Prepare a single o/n culture in 25 mls TSB, in a 50 mL conical tube.  If doing this for total assays, you need to grow a multitude of organisms to get an accurate curve.  We used the lists of inclusivity organisms supplied by AOAC for help in knowing what organisms we needed in the construction of our curve and tested it against ten different organisms.

Day 1 - Dilution of organisms

  1. Prepare 6, 15ml conical tubes with 9 mls of TSB or water for each organism.  Labeled these 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000.
  2. Mix cultures well by vortexing.
  3. Add 1ml of culture to the first tube labeled 1:10.
  4. Mix well by vortex and then add 1ml of the 1:10 to the tube labeled 1:100.
  5. Continue this all the way out to 1:1,000,000.
  6. Remove 2.4ml of the diluted culture and add to 4.6ml of MIP Solution A.  Mix well.
  7. Prepare three 1.5ml tubes for each dilution.  
  8. Perform the MIP extraction on each sample and ran the qPCR. 
  9. Analyze the qPCR and use the average of the three replicates to use on the graph for the data (this is the Cq data).

3) Plating

  1. Plate 100ul of each dilution made above in triplicate (Do not use the sample mixed with solution A, use the dilution made beforehand).  For instance, for 1:10, plate 3 plates, for 1:100 plate 3 plates, and so on.
  2. Count the colonies of growth on the plate after the time has passed to allow for proper growth (depending on plate type, that could be anywhere from 24 hours to 5 days).  If samples are Too Numerous to Count, extrapolate the data from farther out dilutions.  Use the average of the replicates to graph the data (this is the cfu data).

4) Cq to CFU Equation Creation

Graphing Data

  1. Convert all the cfu data into log10.
  2. Put the Average Cq data on the x axis, and the log10 cfu data on the y axis of a scatterpojnt graph, making sure the dilutions match up between plating and qPCR.
  3. Have the program plot the best fit line in the sample, which gives you a formula of y = mx + b.
    1. Y = log10 cfu
    2. M = slope
    3. X = average Cq
    4. B  = y intercept
  4. To solve for y, you need to plug in the data points, and then take the inverse log10 of the sample (10^y).

Summary

These equations were developed for each of our enumeration, or total count, assays and can be found within the user guides and validation documents here: 

https://www.medicinalgenomics.com/product-literature/

https://www.medicinalgenomics.com/validation-documents/

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 the User Guide for each product are not supported by MGC.

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.