- Real-time PCR technology simultaneously detects and quantifies the concentration of fecal indicator organisms in water
- The PCR reaction is visualized in real-time on an amplification plot
- The starting concentration of the indicator organism is extrapolated from reference
standard curves of known concentration
Variations in DNA sequences between living organisms makes it possible to distinguish between these organisms through molecular biology techniques. In our microbial source tracking laboratory, Real-Time Polymerase Chain Reaction (real-time PCR) technology is used to identify the presence of microorganisms in water samples based on the unique genetic sequences. Water samples delivered to Source Molecular are first filtered to capture microorganisms. These organisms are then lysed (broken down) and the DNA (RNA in some virus) is extracted and purified in preparation for downstream PCR analysis.
Quantitative 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.
In order to quantitate the amount of DNA present, a standard curve of known amounts of DNA (or RNA) must be produced. This is done by making a series of 10-fold dilutions of a reference sample that contain the sequence or marker of interest. For example, when quantitating the amount of human specific Bacteroidetes present, it is crucial to use genomic DNA from the human specific Bacteroidetes strain or a plasmid containing the human specific marker as the reference standard. Real-time PCR is carried out on both the experimental sample and the reference standard. Since the values of the reference standards are known, the value of the marker present in the experimental sample can be extrapolated from the standard curve generated by the reference standards. The value is typically expressed as copy numbers. A bacterial genome may contain one or more copies of the marker. The copy number for Bacteroidetes and Enterococcus indicator organisms are generally provided as a percentage of the human-specific strain present in all Bacteroidetes or Enterococcus detected.