Has anyone used q-PCR for cell counting? How is its accuracy compared to colony formation assay and other methods?
Theoretically, I can insert unique genetic markers/barcodes in the target genomes of microorganisms and count the quantities of them to represent the cell density in the medium. I think it is better than any absorbance-based and flowcytometric method for live cell counting.
Can somebody share their experience for using real time PCR for cell counting?
P.S. My target culture is a mixed culture of very closely related bacteria which may only differed by the inserted markers.
See:
Quantitative real-time PCR assisted cell counting (qPACC) for epigenetic - based immune cell quantification in blood and tissue
However a direct count with stain or dye will be vastly more efficient and cheaper.
To Craig,
I thought about direct counting before but abandoned the idea due to the smaller size of bacterial cells. When you have a mixture of a group of closely related bacteria, I do not think it is easy to stain and count each of them.
Theoretically that is possible. That would be similar to the barcoding system that is used for NGS. To design the barcode may not be easy. I have not done this before but I would think it increases the error rates especially in a PCR reaction.
This paper goes into the limitations of barcode system:
https://www.nature.com/articles/srep43249.pdf
I would have gone for flow cytometry because it is simplistic and fairly reliable. For determining density, I would just ensure the fluorophore emission are all equal and do individual stains. Is there a reason why you specifically do not want to use this? I don't work with bacteria, I do more molecular biology and immunology so I'm curious.
This might also help: Article PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture
To Darellynn,
The true reason for not using flowcytometry is actually about money. We do not have a flow cytometer in our university and our lab cannot afford to get one. Some other universities near us do have the device, but I am not sure if frozen samples would work and I need to get the counts in almost real-time for my purpose.
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