Biological Water Testing
Thu 11 Mar, 2021
Improving Water Monitoring for Pathogens and other Microorganisms
Water quality and purity is essential for public and environmental health but monitoring drinking water and wastewater for microbial orgamisms can be difficult and time consuming. In this article we want to highlight the Real-time PCR-based detection methods for some major pathogens and microorganisms that increases analysis speed and quality significantly. You will be able to do an analysis run in a few hours, instead of weeks.
Save Time and Improve Reliability at the Same Time
A conventional cultivating method takes up to 14 days, but only a few hours with Analytik Jena‘s PCR-based detection method for the highly specific and reliable microbiological parameters.
Overall Bacterial Load
The total bacterial load is a key indicator for the hygienic quality of water. It can indicate potential fouling of both, drinking and industrial water.* The innuDETECT Bacteria Quantification Assay is a semiquantitative assay which detects universal bacterial target genes simultaneously with bacterial standard DNA included in the assay, too. Internal Controls, co-amplified during the PCR, give maximum assurance on results and validate negative findings. To demonstrate the excellent quality of the innuDETECT Bacterial Quantification Assay, it was used to analyze the general bacterial load in DNA extracts obtained from a 1 mL water sample. The results are shown in Figure 1. Table 1 demonstrates bacterial loads obtained from Real-time PCR, based on exact ct values. Detected bacterial targets can be quantified upon correlation with standard curves, in an unknown sample.
* Deutsche Trinkwasserverordnung (2001) and 42. Bundesimmisionsschutzverordnung (2017) require determination of culturable microorganisms according to DIN EN ISO 6222:1999-07
Figure 1 / Table 1: Co-amplification of bacterial standard DNA (red) allows semi-quantification of an unknown sample (blue) via ct values. Negative control is shown in black.
| Sample | Ct value | General bacterial load / reaction |
| Standard 1 | 21.5 | 1 x 106 |
| Standard 2 | 24.8 | 1 x 105 |
| Standard 3 | 28.4 | 1 x 104 |
| Standard 4 | 32.6 | 1 x 103 |
| Standard 5 | 36.1 | 1 x 10² |
| Unknown sample | 31.7 | 2.93 x 103 |
| Negative control | 40.2 |
Hygiene Control via Detection of Indicator organisms
The presence of specific gastrointestinal microorganisms in water are frequently used as indicator for fecal contamination. Escherichia coli , shiga toxin 1 and/or shiga toxin 2-producing E. coli (STEC) as well as shiga toxin-producing Shigella dysenteriae can get into water, e.g. via fecal contamination orfertilizers. As this bears the risk of causing severe dysentery, hemorrhagic colitis, and hemolytic uremic syndrome water quality evaluation regarding E. coli is obligatory**. Several assays, including innuDETECT E. coli O157 are designed for the highly specific detection of such pathogens. Figure 2 and Table 2 show a sample application with the combination of three innuDETECT Assays for the characterization of EHEC O104:H41.
** Deutsche Trinkwasserverordnung (2001) requires determination of coliforms and E. coli according to DIN EN ISO 9308-1:2017-09 & DIN EN ISO 9308-2:2014-06 1 strain causing the HUS epidemic in Germany 2011
Figure 2 / Table 2: Characterization of E. coli O104:H4 by real time PCR via combination of innuDETECT Assays for E. coli O104 (red), Shiga Toxin 1 (green), and Shiga Toxin 2 (blue). Shown are signals detected in FAM channel and corresponding results (positive/negative).
| innuDETECT Assay for | Signal FAM channel |
| E.coli 0104 | positive |
| Shiga Toxin 1 | negative |
| Shiga Toxin 2 | positive |
Differential Pathogen Detection – Co-Identification of Most Relevant Subspecies
The presence of Legionella needs to be tested in drinking and process water, whereby the subspecies L. pneumophila plays a major role. Legal regulations such as the 42. BImSchV require the evaluation of this Legionnaires’ disease-causing pathogen and aims to prevent its emission from evaporation coolers and cooling towers. Identification of Legionella by conventional methods is highly challenging and hampered by co-presence of other microorganisms. The innuDETECT Legionella Assay overcomes these issues and allows the simultaneous detection of total Legionella spp. and L. pneumophila according to the ISO/TS 12857 within a single reaction. Figure 3 shows a Legionella detection and differentiation within water samples from industrial cooling towers. The presence of L. pneumophila could only be detected in sample 4.
Figure 3 / Table 3: Differential detection of Legionella in water samples obtained from cooling towers. Legionella spp. (left) are detected in the Cy5 channel and Legionella pneumophila (right) in the FAM channel.
| Legionella spp. | Legionella pneumophila | |
| Water sample 1 | (weak) positive | negative |
| Water sample 2 | negative | negative |
| Water sample 3 | positive | negative |
| Positive control (PCR) | positive | positive |
| Negative control (PCR) | negative | negative |
Advanced Pathogen Detection
Clostridia spp. are bacteria generally found in all environmental habitats including water. Its detection is obligatory according to drinking water regulations***. The detection via conservative cultivation entails several critical issues. That is why PCR-based assays offer an indispensable alternative. Moreover, Yersinia enterocolitica is widely present, especially in animal reservoirs and contaminations are observed in drinking and surface water bearing a severe risk to health. Both innuDETECT Clostridium perfringens Assay and innuDETECT Yersinia enterocolitica Assay offer the perfect solution for fast and sensitive detection of respective pathogens whereby included internal controls ensure result reliability. This is clearly shown in Figure 4 and Table 4.
*** Deutsche Trinkwasserverordnung (2001) requires determination Clostridium perfringens according to DIN EN ISO 14189:2016-11
Figure 4 / Table 4: Detection signals and ct values in FAM channel during real time PCR were obtained from C. perfringens DNA dilutions using the innuDETECT Clostridium perfringens Assay in FAM channel.
| DNA amount | Ct value Clostridium perfringens (FAM) |
| 1 ng | 18.44 |
| 100 pg | 21.69 |
| 10 pg | 25.28 |
| 1 pg | 28.99 |
| 100 fg | 33.56 |
| Negative control (PCR) | No Ct |
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