Quantitative Polymerase Chain Reactions (qPCR) – an Analytical Tool for Detection and Enumeration of Health-Threatening Pathogens in Waste Water and Biosolids
Traditionally, detection and enumeration of water borne pathogenic bacteria (e.g., E. Coli and Salmonella) have been based on time-consuming, laborious and monospecific culture methods (e.g., USEPA 1682, 1602, ISO 2000 and APHA 2005), which are prone to a large number of methodological drawbacks. Most of these conventional methods merely determine presence or absence of microbes in the sample. On the other hand, PCR and real-time qPCR methods are sensitive, specific, rapid, cost effective, and considered to be fit-for-purpose for detection, quantification and verification of waterborne pathogens in wastewater and biosolids. Furthermore, they also differentiate between viability and nonviability of cells as they make use of various biomarkers at the gene level. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification facilitates pathogen detection, determination and automation. Availability of kits minimizes the need of trained personnel.
Detection and enumeration of pathogens in waste water and biosolids using PCR and qPCR are based on the separation and concentration of specific pathogen-derived DNA. In the sequencer, amplification is quantified by measuring fluorescent signals emitted by specific dual-labeled probes or intercalating dyes. These fluorescence intensities are directly proportional to the amounts of PCR products formed.
Key Learning Objectives
1. Compliant sampling of waste water and biosolids. Overview of methods – recipes. References to documented reports.
2. State of art instrumentation – the big players
3. General principles and concepts of PCR and qPCR – DNA sequencing. Definition of terms and mechanisms. What is (Real Time) qPCR?
4. qPCR is a technique in which fluoro-probes bind to specific target regions of amplicons to produce fluorescence. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt spectrometer.
5. Amplification: Cyclic denaturation (unwinding of DNA duplex to single-stranded DNA), annealing (specific primers anneal to DNA strands), and finally thermostable DNA polymerase carries out the polymerization process.
6. Analysis using target-specific kits – TaqMan and SYBR, primers, probes – differences, comparisons, advantages and disadvantages.
7. Multiplex qPCR enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.
8. Quantification – detection vs. absolute quantification. Relative calibration, using a dilution series, reference DNA with known DNA concentrations.
9. Validation – QC checks – duplicates, positive and negative controls.
10. Future developments – biomarkers for viruses.
Dr. Joe Brenner is a forerunner in the implementation of new instrumentation and methodologies for analysis of emerging contaminants in environmental samples.
He obtained his PhD in Geochemistry at the Hebrew University and was a senior scientist in the Application and R and D laboratories in Jobin Yvon in Longjumeau, France and Varian-Ginston Research Center at Palo Alto, California. He was a product and application adviser for Atomic and Mass Spectrometry at Thermo Fisher Scientific in Madrid and in the UK. During his career he spent significant periods of time in national and international institutes and academic establishments worldwide mainly in the field of compliant environmental analysis.
At the present time he is a pioneer of the pathogen qPCR laboratory in Egodan Tel Aviv, a national authority for recycling waste water and sludge for land use in Israel.