VIABILITY PCR

Introduction to Viability PCR (v-PCR)

Reliable detection of living microbes and viruses is of widespread interest across numerous sectors including basic research, food safety, agriculture, and public health. However, traditional methods of measuring microbial viability rely on time-consuming culturing, colony counting, or microscopic observation. In addition, certain organisms cannot be cultured with current technology. Consequently, these viable but non-culturable (VBNC) species require molecular measures of viability. Viability PCR (v-PCR) is a reliable and culture-free method that offers sensitive and rapid detection of viable microorganisms. Since 2006, Biotium has pioneered the field of v-PCR with the development of PMA (Propidium Monoazide) and the improved PMAxx™ v-PCR dye. These photoreactive, membrane-impermeant, DNA binding dyes offer superior dead cell selectivity over traditional culture-based methods and the previous EMA (Ethidium Monoazide) v-PCR dye.

An Industry Leader in v-PCR

PMA is the v-PCR dye of choice among industry leaders, and has been validated in hundreds of peer-reviewed publications and numerous strains of bacteria, fungi, and viruses. Early studies focused on v-PCR in bacteria, validating the procedure in dozens of different bacterial strains such as E. coli, Listeria, Legionella, Lactobacillus, M. tuberculosis, Chlamydia, H. pylori, and many other strains with relevance to human health. v-PCR has been most widely used in bacteria, but there are also dozens of publications using v-PCR in yeast and fungi, viruses, as well as a variety of other cell types such as archaea, amoeba, and parasites.

Measuring Bacterial Viability in Environmental Samples?

Sign up to our e-newsletter to download our Literature Digest: Monitoring Viable Bacteria in the Environment with PMA & PMAxx™.

In this digest we have provided summaries and takeaways for three publications on the use of v-PCR with PMA and PMAxx™ for monitoring bacterial viability in samples taken from wastewater, fresh water, and drinking water treatment plants.

v-PCR is a proven method for monitoring capsid integrity for dozens of viral strains, including SARS-CoV-2.

The figure below displays the genus and strain number of viruses investigated for using v-PCR to monitor capsid integrity in peer-reviewed studies in the last decade (2009 to 2021) in the fields of food safety and environmental virology.

v-PCR Workflow

The basic workflow for v-PCR is shown in the infographic below (detailed protocols can be found on product pages). It is adaptable for many different sample types and experimental set-ups. For a small number of cultures, the samples can be prepared in microcentrifuge tubes, and exposed to light in the PMA-Lite™ 2.0 LED Photolysis Device (See lower on this page for more information).

PMA is validated in hundreds of journal articles for a wide variety of strains

Since Biotium first developed PMA, there have been hundreds of publications on the use of the dye in many sample types including dozens of bacterial strains, biofilms, yeast, fungi, viruses, and eukaryotic cells. It has been used in such applications as food and water safety and environmental testing, and has been used in conjunction with qPCR, NextGen Sequencing (NGS), Sanger sequencing, and Loop-mediated Isothermal Amplification (LAMP).

PMAxx™ is a superior alternative to PMA

While PMA is generally effective at differentiating between live and dead bacteria by qPCR, it does not completely eliminate PCR products from dead cell DNA. This could potentially give false positive results. PMAxx™ was designed by Biotium scientists to be a superior alternative to PMA. PMAxx™ is much more effective at eliminating PCR amplification of dead cell DNA, and therefore provides the best discrimination between live and dead bacteria. Learn more about viability PCR with PMA and PMAxx™ with links to our flyer and our full list of references below.

The customizable Viability PCR Starter Kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and qPCR. These kits can be used with any cell type of your choosing (see the PMA/PMAxx™ Reference List and Validated Bacterial Strains List for published validated cell types). All of the kits contain Forget-Me-Not™ EvaGreen® qPCR Master Mix, and your choice of PMA or PMAxx™ dye. You also have the choice of selecting a kit with or without the Enhancer for Gram Negative Bacteria (not to be used with other cell types). The user will need to supply their own primers to amplify their species of interest (see this blog post for primer design considerations). Each kit contains reagents sufficient to treat 80 bacterial cultures and perform 200 PCR reactions.

PMA Enhancer for Gram Negative Bacteria was designed to improve discrimination between live and dead gram-negative bacteria during viability PCR. PMA Enhancer is provided as a 5X solution, and is added to a sample before the addition of viability dye. When a sequence from a gram-negative bacteria is amplified by PCR, samples pre-treated with Enhancer show a decrease in the signal from dead cells, with no change in the signal from live cells. PMA Enhancer is compatible with PMAxx™ as well as PMA. PMAxx™ plus Enhancer is the optimal way to perform viability PCR on gram-negative bacteria. PMA Enhancer is available as a stand-alone product, and is also a component of our PMA Bacterial Viability Kits (gram-negative strains only) (see below).

The mechanism by which Enhancer improves live/dead discrimination has not been determined. It may improve passive permeability of dead cell walls or membranes to the dye, and/or improve access of the dye to the dead cell DNA.

For Microcentrifuge Tubes

PMA-Lite™ 2.0 LED Photolysis Device Features:

• Uniform illumination to up to 18 microcentrifuge tubes
• Efficient activation of v-PCR dyes such as PMAxx™ and PMA
• Timer for 5-45 minutes of photoactivation
• Long-lasting LED lights

Strain-specific v-PCR kits are designed for selective detection of viable bacteria from a specific strain using PMAxx™ or PMA dye and real-time PCR. The kits contain PMA dye OR PMAxx™ dye, Forget-Me-Not™ EvaGreen® qPCR Master Mix, and validated PCR primers for detection of selected strains of bacteria that are of widespread interest to food safety, public health, and/or antibacterial research.

Don’t see your favorite bug? Let us know at techsupport@biotium.com

Kits include:

• PMA dye OR PMAxx™ dye
• Forget-Me-Not™ EvaGreen® qPCR Master Mix
• ROX reference dye
• PMA Enhancer (gram-negative strains only)
• Validated PCR primers for specific bacterial strain