Top: PCRmax Eco 48 Real-Time qPCR System and PCRMax human microbial pathogen detection kits for qPCR
Bottom: PCRmax Alpha Cycler 4 Thermal Cycler, Quad 96-Well Capacity

By Michael Steinert

To protect human health and safety and fulfill regulatory requirements, water treatment plants must routinely test water quality—sometimes up to 100 times per day. Various types of water testing methods can be performed using different types of equipment, depending upon the application, results desired and required water quality regulations.

A real breakthrough for water treatment testing came with the technology of polymerase chain reaction (PCR). Before this technology was available, pathogen detection in a water sample was very limited. Most of the time, the bacteria would need to be cultured from a sample on a petri dish, allowed to grow, then stained and examined under a microscope. This was a time-consuming process that could take days and ineffective for types of bacteria and viruses extremely hard to grow and detect outside of the human host. PCR allows a wastewater scientist to detect pathogens in a water sample in a matter of hours, greatly reducing the time required to test for certain types of viruses and bacteria.

Steps for PCR testing
PCR requires a target DNA to be replicated, a pair of primers specific for the target, nucleotides, buffer and Taq polymerase enzymes.

  1. The first step is denaturation, which usually occurs at around 95°C. This serves to separate the two strands of the target DNA and typically lasts 10 to 15 seconds but may be longer for more complex targets
  2. The second step is the annealing step, where the specific primers bind to the target. This temperature can vary quite significantly and is dependent on the primer sequence; too high and the primers will not bind, too low and they may bind an incorrect region of the DNA. Hold time is typically about 30 seconds.
  3. The final step is the extension step where the new DNA is synthesized and extended from the primer. This is usually programmed to 72°C, the optimal temperature of the Taq polymerase. The hold time will depend on the length of the product. For example, if targeting a larger DNA sequence, the scientist would want a longer hold time, as it may take longer for the binding of all the necessary nucleotides to complete replication. This could be anything from 20 to 30 seconds up to several minutes.

These three steps are then repeated 30 to 40 times, giving an exponential increase in the amount of initial target. Then using gel electrophoresis, the scientist can discern whether a certain bacteria or virus might be present in the sample.

Essentially, PCR allows for the rapid cloning of a target DNA strand, such as a sequence of E. coli. The original DNA is unzipped and primers are annealed to the now separate segments of DNA. The replication is completed by the addition of nucleotides. At the end of the run is a high concentration of the target, making detection of the bacteria significantly easier. If there is no amplification, E. coli is not present in the sample. Previously scientists did not have this option. They would need to take the small concentration of pathogen that may be present in the water sample and grow it to confirm its presence. PCR simply targets a specific DNA sequence and detects the absence or presence of a pathogen, thus eliminating the need to physically grow the species one is trying to detect. It also allows for detection of extremely hard-to-grow diseases in a laboratory setting.

PCR has been a huge breakthrough in the scientific community, especially in the areas of virus and bacteria detection. Primer sets can be designed to target a specific sequence of DNA, which allows for the study of different strains of a virus, investigation into possible mutations and helping researchers to combat a disease most effectively. While PCR provides excellent qualitative data, it does not indicate the quantity of the bacteria or viruses in the water. Quantitative results require further downstream processing, until development of a new kind of PCR that provides qualitative data in real time.

Real-time PCR
Real-time PCR, or qPCR, not only detects the presence of known pathogens, but also the concentration of the bacteria in the sample. It is very similar to PCR but has one very important distinction: qPCR utilizes a reporter molecule or probe that is specific for a targeted DNA template that contains a fluorescent dye. By using fluorescence, the amount of DNA replicated can thus be detected. The temperature uniformity required for qPCR makes the data extremely accurate and repeatable. It has become easier to operate in wastewater laboratories as more probes and primer kits for the most tested microorganisms become available.

Whether for primary testing or simply a check of current processes, real-time PCR is a very valuable tool in any wastewater plant’s arsenal. (“qPCR-based methods have become the standard for detection of viral genomes in concentrated water samples.”1 ). Knowing how much of a virus or bacterium is present allows for the wastewater scientist to proceed in the most effective way to eradicate it in the sample. As with PCR, reagent kits with primers are readily available for the most common viruses and bacteria found in wastewater. The creation of primers, however, can be used to detect different strains of pathogens and combat newly emerging diseases by identifying them in the sample.

Detecting pathogens with qPCR
One of the more interesting applications qPCR allows for is the detection and quantification of pathogens immediately after the refuse is collected at the water treatment plant. If a scientist can run a test from an untreated sample, they can determine the load of the pathogen. For example, if higher than normal levels of a virus are found in the wastewater compared to what is normally measured, it can help predict that an outbreak is likely, as most infections are spread through fecal contamination. This would allow the wastewater plant to inform the CDC about a possible outbreak, as symptoms can start weeks after the virus is detectable in human fecal matter. This kind of modeling has been shown to be effective with Hepatitis A and Norovirus.2 Further investigation into models like these across the world could allow for better preparedness for an outbreak and lead to fewer deaths associated with waterborne diseases.

Summary
It is clear to see why PCR has replaced the old detection process of growing out colonies on a petri dish. It is faster, more reliable and relatively inexpensive. qPCR significantly improves wastewater plant efficiency, by allowing real-time quantitative measurements. This is crucial in the development of disease treatment and discovery, as well as for testing water before and after treatment, to prevent outbreaks and prepare people for possible outbreaks, decreasing mortality from waterborne diseases.

References

  1. Eiji Haramoto Masaaki Kitajima Akihiko Hata Jason R.Torrey Yoshifumi Masago Daisuke Sano Hiroyuki Katayamagh. A review on recent progress in the detection methods and prevalence of human enteric viruses in water. https://www.ncbi.nlm.nih.gov/pubmed/29471200.
  2. Maria Hellmér, Nicklas Paxéus, Lars Magnius, Lucica Enache, Birgitta Arnholm, Annette Johansson, Tomas Bergström, Heléne Norder. Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks. Appl. Environ. Microbiol. Oct 2014, 80 (21) 6771-6781; DOI: 10.1128/AEM.01981-14.

About the author
Michael Steinert is Product Marketing Manager for Biosciences at Cole-Parmer, responsible for creating and executing marketing strategy for products in the biosciences business unit including PCR and qPCR equipment. He also spent time at Cole-Parmer as an applications specialist, providing technical expertise to customers. Prior to joining the company, Steinert spent several years behind the lab bench as a development scientist in forensics. He also conducted research on circadian rhythms at Rush University Medical Center and has a BA Degree in biology from The University of Chicago. He can be reached at michael.steinert@cole-parmer.com.

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