Mycoplasma Detection Via MycoSensor QPCR Assay Kit

October 01, 2013

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Utah State University
ADVS
Research Associate

Company

Agilent Technologies

Catalog Number

302107

Our lab group is involved in researching cell homeostasis during stress, particularly in the endoplasmic reticulum. And we work with Drosophila and mammalian cell lines. My encounter with Mycoplasma occurred when transfection experiments in my lab using mammalian cells were not working. After repeatedly failed transfections and troubleshooting, I suspected the silent, invisible microbes – Mycoplasma contamination in our culture. As a lab rat, my cell culture definition for Mycoplasma is – the main cause for ruining the experiments. These are in reality notorious microbes that belong to the genus Mycoplasma – bacteria that lack cell wall. Unlike bacteria and fungi, their presence in cell culture is hard to notice with the naked eye (turbidity of culture media, fungal growth) or microscope, and hence, suspecting the presence of these microbes can be a grueling task. I have come across the MycoSensor QPCR Assay Kit in my quest to look for an efficient way to detect Mycoplasma contamination in mammalian cell cultures.

Experimental Design and Results Summary

Application

Mycoplasma detection in cell culture, routine cell mainteinance

Starting Material

Cells / cell culture supernatant

Protocol Overview

The MycoSensor QPCR Assay Kit is used to detect Mycoplasma contamination of cell cultures by real-time quantitative PCR, using SYBR Green dye detection. The kit comes with all the reagents you need for DNA extraction, amplification, fluorescence detection, and internal controls. Internal controls include a positive control, a negative control and an amplification control to confirm PCR amplification and mycoplasma genomic DNA. Setting up the reaction is simple and easy; you just have to follow the manufacturer’s instructions. Uses less sample volume. And, unlike the long testing time of the ‘gold standard’ conventional Mycoplasma detection techniques, this assay takes approximately 2h from extracting the DNA to analyzing the real time qPCR data.

Tips

I would suggest using the amplication control every single time you run the test

Results Summary

The results are indicated as amplification and dissociation curves in the real time PCR platform. In amplification curve, the cycle threshold (ct) clearly indicates the presence or absence of Mycoplasma. The results are further supported by the specific temperature peaks for each control in the dissociation curve. Though the assay is simple, specific and straightforward, it would not accurately address the most common and pivotal issue – the viability of mycoplasma contaminants. In addition to the high price of the kit, equipment expenses related to real time PCR are more costly compared to conventional PCR. I routinely use this assay several times in the lab to check the quality of the cells and it has worked efficiently every single time.

Additional Notes

None

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Summary

The Good

Simple, easy and time efficient, reliable.  Sensitive and specific detection of the eight most common Mycoplasma species:

  • Mycoplasma arginine
  • Mycoplasma fermentans
  • Mycoplasma hominis
  • Mycoplasma hyorhinis
  • Mycoplasma pirum
  • Mycoplasma salivarium
  • Mycoplasma orale
  • Acholeplasma laidlawi

The Bad

Expensive and needs technical expertise (for qPCR).

The Bottom Line

I would highly recommend this detection kit if you have access to a real time quantitative PCR platform.

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