Quantitative Real-Time PCR Approach

As an alternative to traditional based approaches, quantitative Real-Time PCR (or qPCR) provides a sensitive and rapid technique for quantifying the abundance of specific bacteria (i.e. Dehalococcoides spp., Dehalobacter sp., methanogens, etc).

Advantages include:

• Specific: Can be used to target organism at a variety of levels (Phylogentic group - Species)

• Rapid: Results for this approach are avialable in 7-10 days.

• Sensitive: Practical Detection Limimts (PDL) as low as 100 cells per sample with a dynamic range of over seven orders of magnitude

• Cost Effective: Cost comparable with traditional most probable number (MPN) based analyses

How does it work?

This approach relies on the principles of a technique called Real-Time PCR, whereby many copies of a target gene are made, and as each copy is made a fluorescent marker is released. The amount of fluorescence measured throughout the amplification process is used to determine the number of the target gene copies within a sample. Cell estimates of this target organism are determined by correlating the number of gene copies measured to that of a known standard of the target organism.

In essence, Real-Time PCR is like having a copy machine with a counter. During this process "primers" are used to select which pages of a book (or target bacteria) are copied and the counter keeps a running total of how many pages were copied (or how many Dehalococcoides are present?) during this process. This approach has a high degree of sensitivity and a wide dynamic range of over seven orders of magnitude.