Using PCR and qPCR in Water Treatment

Using Polymerase Chain Reaction (PCR) and Real-Time PCR (qPCR) in Water Treatment

The list of waterborne diseases, many life threatening, one can contract by not only drinking contaminated water, but also coming in contact with polluted water is alarming. According to the Center for Disease Control (CDC), the presence of contaminants in water can lead to adverse health effects, including gastrointestinal illness, reproductive problems and neurological disorders. Infants, young children, pregnant women, the elderly, and people whose immune systems are compromised because of AIDS, chemotherapy, or transplant medications, may be especially susceptible to illness from some contaminants.

To protect human health and safety and fulfill regulatory requirements, water treatment plants 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.

PCRmax Alpha Cyclers Theraml Cyclers
Easily create, modify and access programs with the PCRmax® Alpha Cyclers.

As technology continues to advance, so do testing methods and equipment. Arguably, the most substantial breakthrough came with the invention of PCR to test biological contaminants in water. PCR or polymerase chain reaction allows a scientist to take a small amount of sample DNA from a source, such as water, and replicate it exponentially.

The PCRmax® Alpha Cyclers are compact thermal cyclers ideal for use in wastewater treatment plants. These units deliver reproducible results every run—with little to no training needed. Features include a clear responsive touch screen, secure user-specific programming, adjustable heated lid, and active sample cooling for sharper amplification. A Program Wizard generates a protocol specific to the sequence in seconds—a quick way to optimize new assays. Units also retain approximately 1000 reports for reviewing later and run on an intuitive, HD Android™ tablet interface.

Testing for biological contaminants in water using PCR

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

  1. Denaturation—Usually occurs at around 95°C. This separates the two strands of the target DNA. This step lasts 10 to 15 seconds but may be longer for more complex targets.
  2. Annealing—Specific primers bind to the target. This temperature can significantly vary 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 Hold time is around 30 seconds.
  3. Extension—The new DNA is synthesised 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. 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.

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.

Real-time or qPCR

While PCR provides excellent qualitative date, it does not indicate the quantity of the bacteria or virus in the water. Quantitative results required further downstream processing, until development of a new kind of PCR that provides qualitative data in real time.

Real-time PCR, also known as qPCR, not only detects the presence of know 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 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.

PCRmax Eco 48 Real-Time qPCR System
PCRmax® Eco-48™ compact thermal cycler provides fast cycling—runs 40 cycles in 40 minutes, shorter when optimized.

Whether it is for primary testing or simply a check of current processes, real-time PCR, using the PCRmax Eco-48™ thermal cycler, 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” (Haramoto et all., 2018). Knowing how much of a virus or bacterium is present allows for the wastewater scientist to proceed in the most effective way to eradicate the pathogen in the sample.

The PCRmax Eco-48 accommodates a 48-well polypropylene PCR plate utilizing the same geometry as standard 384 well plates, but only 1/8 of the size. Because of this, one can dramatically reduce the qPCR reagent volumes compared to traditional 96-well instruments, saving precious samples, while still producing a strong fluorescence signal. Suitable for both single labs and core facilities, the thermal cycler’s fast cycling enables several experiments per day. A specially designed thermal block delivers a unique heating and cooling system that provides accurate ±0.1°C temperature control and quickly cycles from one temperature to the next. Also featured is an advanced high-performance optical system that delivers precise and sensitive fluorescence detection, facilitating all four-color multiplex applications with only a six second read time for all four colors across the entire plate. The unit is MIQE compliant and the high-resolution melt (HRM) functionality is standard. Easy-to-use software provides instrument control, data collection, and data analysis. An ergonomic plate loading dock allows for quick loading of the plate and is backlit for clear visibility. This qPCR unit is ideal for wastewater treatment.

PCRmax detection and reagent kit for PCR
Quickly and accurately detect the presence of microbial pathogens.

As with PCR, reagent kits with primers are readily available for the most common viruses and bacteria found in wastewater. However, the creation of primers can be used to detect different strains of pathogens and combat newly emerging diseases by identifying them in the sample.

Detecting pathogens with qPCR

qPCR can detect and quantify 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 (Hellmér et all., 2014). Further investigation into models like these across the world could allow for better preparedness for an outbreak and lead to a 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 just furthered the wastewater plants efficiency, by allowing to take real time quantitative measurements. This is crucial in the development of disease treatment and discovery, as well as for testing the water before and after treatment, to prevent outbreaks and prepare people for possible outbreaks, decreasing mortality from waterborne diseases.

Resources

Detection of Pathogenic Viruses in Sewage Provided Early Warnings of Hepatitis A Virus and Norovirus Outbreaks

Maria Hellmér, Nicklas Paxéus, Lars Magnius, Lucica Enache, Birgitta Arnholm, Annette Johansson, Tomas Bergström, Heléne Norder

Appl. Environ. Microbiol. Oct 2014, 80 (21) 6771-6781; DOI: 10.1128/AEM.01981-14

A review on recent progress in the detection methods and prevalence of human enteric viruses in water

Eiji Haramoto Masaaki Kitajima Akihiko Hata Jason R.Torrey Yoshifumi Masago Daisuke Sano Hiroyuki Katayamagh