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6 EDVO-Kit # 953 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Multiplex PCR Analysis for Water Contaminants Background Information As both point and nonpoint pollution eventually contaminate water sources used for drinking water, water treatment must effectively remove these impurities. While drinking water in U.S. cities is generally safe, several outbreaks still occur each year, making monitoring still necessary. Additionally, private well owners should also test water regularly for harmful chemicals, algae, and bacteria. Waterborne microbes such as Giardia and Cryptosporidium can cause severe, life-threatening illness if ingested in sufficient quantities (these are not actually bacteria but single-celled organisms called protists). Giardia and Cryptosporidium exist in the environment as cysts, hard, biologically inert spheres that begin to divide when ingested. Similarly, Salmonella and Shigella are two other (bacterial) waterborne pathogens that can cause community-wide infectious disease. In particular, elderly people or those afflicted with AIDS, cancer, and other diseases can have weakened immune systems that make them more vulnerable to such infections. Because testing for multiple organisms is time-consuming and prohibitively expensive, water is typically analyzed for the presence of a group of relatively harmless bacteria, known as coliform. Coliform bacteria are often used to indicate the presence of more harmful organisms and include bacteria such as Escherichia Coli (E. Coli), Klebsiella, Enterobacter, and Serratia. These organisms normally inhabit the intestine and gut of animals and humans. If a water sample is negative for coliform bacteria, it is assumed that more harmful organisms are also absent, and the water is deemed safe for drinking or swimming. In addition to the coliform test, a separate second test is often performed for E. Coli, whose presence could indicate fecal contamination. If a sample tests positive for E. Coli or coliform, it is often sent to a wellequipped laboratory that can perform more complex (and more expensive) tests to determine the specific (possibly pathogenic) organisms that might be present. One laboratory method that is now universally used to detect specific microorganisms is the polymerase chain reaction (PCR). PCR, invented in 1986 by Dr. Kary Mullis at Cetus Corporation (for which he later received a Nobel Prize), has become invaluable for the amplification of specific DNA sequences. The basis for PCR is a heat-stable DNA polymerase (usually Taq polymerase) that can withstand near-boiling temperatures. Applications of PCR include genetic disease diagnosis, forensic identification, paternity testing, and detection of specific microorganisms (usually bacteria and viruses) in body fluids, soil, and water. Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2008, 2009 EDVOTEK, Inc., all rights reserved EVT 090205K The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants EDVO-Kit # 953 7 Multiplex PCR Analysis for Water Contaminants The basic PCR reaction is shown in Figure 2. The first step involves denaturation of the DNA sample to be tested (e.g., an unknown water sample). Denaturation involves separation of the two DNA strands at near boiling, generally around 95° C, at which the Taq polymerase remains stable. The temperature is then lowered to 45° to 60° to allow binding (annealing) of sequence-specific primers, short DNA molecules (Figure 1, small red and blue bars), to the source DNA (template). The primers are designed to bind only to the sequence to be amplified, called the target sequence (Figure 1, light green bar). An example of a target PCR sequence would be a gene found only in a specific water bacterium. Following primer annealing during the second PCR step, the temperature is raised to 72°C. At this temperature, the polymerase can add new bases to the primers in a process known as extension, creating new DNA molecules between each primer-binding site. These three steps: denaturation, annealing, and extension, together constitute one PCR cycle. The entire process is then repeated, resulting in a doubling of the target sequence following each cycle. The PCR reaction is generally repeated for 30 - 40 cycles, resulting in an ideal amplification of 230 - 240 fold of the original DNA target; in reality, however, the exact amplification is lower, due to imperfect reaction conditions. Background Information For water microbe detection, PCR can be used to sensitively and exclusively detect specific aquatic organisms. For example, given a positive result for total coliform, one may further test the water sample, using PCR, for pathogenic organisms such as Cryptosporidium. For specific detection, primers are designed to correspond to gene sequences distinct to those species. In this experiment, PCR tests will be performed to concurrently detect specific bacteria, including E. coli, Bacillus subtilis, and Serratia marcescens. A multiplex PCR reaction is one in which multiple amplifications are performed simultaneously using several primer pairs in a single tube. This exercise will simulate follow-up testing that occurs subsequent to a positive coliform test. The experiment will thus demonstrate concurrent detection of specific water pathogens, using specific primers for unique, organism-specific genes, in a single PCR reaction. Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2008, 2009 EDVOTEK, Inc., all rights reserved EVT 090205K The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Page 1 and 2: The Biotechnology Education Company
Page 3 and 4: Water Quality Testing III: Multiple
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Page 27 and 28: Appendix A PCR Experimental Success
Page 29 and 30: Appendix C Preparation and Handling
Page 31 and 32: Appendix E 1.0% Agarose Gels - Quan
Page 33 and 34: Appendix G Staining and Visualizati
Page 35 and 36: Material Safety Data Sheets Full si

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