Real-time PCR technology simultaneously amplifies and detects the DNA of fecal indicator organisms in water
The PCR reaction is visualized in real-time on an amplification plot
The presence of an amplification curve indicates the presence of the indicator organism
The absence of an amplification curve indicates the absence of the indicator organism
Variations in DNA sequences between living organisms makes it possible to distinguish organisms from one another through molecular biology techniques. In our microbial source tracking laboratory, Polymerase Chain Reaction (PCR) technology is used to identify the presence of microorganisms in water samples based on the unique genetic sequence of that organism. Water samples delivered to Source Molecular are first filtered to capture microorganisms and coliform in water. These organisms are then lysed (broken down) and the DNA (RNA in some viruses) is extracted and purified in preparation for downstream PCR analysis in total coliform.
Real-Time Polymerase Chain Reaction
PCR allows for the exponential amplification and simultaneous quantification of short DNA templates. It entails the use of short oligonucleotides called primers and a fluorescent reporter molecule called a probe. These oligonucleotides are synthesized to be complimentary to a DNA sequence that is unique to the bacteria of interest. To initiate the PCR process, the starting double stranded DNA template must first be separated by raising the temperature of the reaction mixture to 95˚C. The temperature of the reaction mixture is then lowered to around 55˚C-65˚C to allow the primers and probe to bind to a defined location on the now single stranded DNA target. The primers are extended by DNA polymerase, an enzyme that catalyzes the addition of bases complimentary to the bases of the exposed template. The probe is cleaved by the DNA polymerase as the DNA strand is extended. This results in an increase in the fluorescent signal of the fluorescent reporter molecule which is detected by the PCR instrument and displayed by the software as an amplification plot of fluorescence intensity vs. cycle number. This completes the first cycle of the PCR reaction. The cycle is repeated 30-40 times with the number of DNA copies (and fluorescent signal) from the previous cycle doubled with each cycle. In our laboratory, we use the Applied Biosystems StepOne Real- Time PCR system (Applied Biosystems, Foster City, CA).
Real-time PCR detects the accumulation of PCR product over time. The PCR reaction can be divided into four phases that are displayed in the amplification curve, the linear-ground phase, the exponential phase, the linear phase and the plateau phase. During the ground-linear phase,only background fluorescence is detected. The cycle at which the amplification fluorescence exceeds a chosen threshold above the background fluorescence is called the Cycle threshold or Ct value and this marks the early exponential phase. It is important to quantitate the initial DNA copy number at this time as it will be the most accurate. During the exponential phase, the amount of DNA is theoretically doubled with every cycle. The reaction begins to slow as PCR reagents become consumed and the products begin to degrade. The DNA is no longer doubled at each cycle and the reaction enters the linear phase. A plateau phase is then observed once all of the reagents are consumed and no additional product is made. These phases can be seen in an amplification plot below.
Presence or Absence of Fecal Indicator Organisms
Determining the presence or absence of a fecal indicator organism in an environmental sample is dependant on the detection of a genetic sequence that is unique to that organism. Real-time PCR assays are designed to amplify and detect only these specific sequences. If the organism is absent in the sample, no amplification curve will be seen in the amplification plot. For quality control purposes, a positive control, consisting of the organism’s genomic DNA that is known to give a signal, is always run alongside the samples to ensure a properly functioning reaction and reveal any false negatives. If the organism is present, its unique genetic sequence will be amplified and detected. If no marker is present no amplification will occur.