Why Test for Legionella ?
Legionellosis is an infection caused by Legionella bacteria in a water system, such as fountains, misting apparatus and cooling towers, particularly in health establishments, public buildings, and recreational water systems. The bacteria can cause acute pneumonia, Legionnaires' disease and a milder form of pulmonary infection, Pontiac fever. The actual number of Legionnaires' cases is thought to be much higher than reported as many people with pneumonia may not be tested for Legionella infection. Legionella pneumophila is responsible for approximately 90% of the clinical cases. Regular control of the presence of Legionella in water supply systems is an important way of preventing the disease. One of the most sensitive and specific methods developed for Legionella pneumophila testing is PCR (polymerase chain reaction). To ensure accuracy of data, commercially available detection methods must comply with specific requirements and validations recommended by government agencies and professional organizations that have published guidelines not only for the design, but also the commissioning requirements for cooling towers and water systems.
Bio-Rad’s iQ-Check method has been used for over 10 years to monitor Legionella in cooling towers and has been demonstrated to be a very powerful tool for industry and government agencies in outbreak surveillance.
An official third party performance recognition
ISO (the International Organization for Standardization) is a worldwide federation of national standard bodies (ISO member bodies). The ISO TS 12869 describes the detection and enumeration of Legionella by PCR. The aim of this standard is to provide proof of performance of a Legionella PCR system that meets the highest level of quality management, from manufacturing of the product to the analysis of results. This helps build vendor credibility and buyer confidence by providing the marketplace with the assurance that performance claims are valid, credible and supported by high-quality, independent test data. This technical specification describes a method for the detection and quantification of Legionella spp. and L. pneumophila using PCR. It specifies general method requirements, performance evaluation requirements, and quality control requirements including inclusivity/exclusivity analysis, limit of detection, limit of quantification of PCR step, calibrating function, link to primary standard, and efficiency and robustness of extraction. The guidelines, minimum requirements and performance characteristics are intended to prove that the results are reliable and reproducible between different laboratories.
Only methodologies certified by an independent and recognized third-party validation body can claim the ISO certification.
iQ-Check Legionella spp. and iQ-Check Legionella pneumophila Kits are NF VALIDATION certified according to NF T90-471 and ISO/TS 12869. The MD 15161-2013 Control of Legionella in Mechanical Systems Standard published by Public Works and Government Standards Canada (PWGSC) recommends the use of methods that are in accordance with the ISO TS 12869.
The iQ-Check Legionella solution is an ISO/TS 12869 validated method based on amplification and detection of genomic sequences by real-time PCR. The Aquadien Kit is also an ISO/TS 12969 method that allows an optimal DNA extraction and purification from bacteria present in all types of water samples for PCR detection after a filtration step. The principle of the extraction is based on alkaline lysis of bacteria in presence of thermal shock and DNA purification using ultrafiltration. The Aquadien W2 Wash Solution is optimized for dirty water samples including those containing potential PCR inhibitors such as additives and chemicals used for water treatment. The iQ-Check Kit contains ready-to-use reagents required to perform the analysis of samples. Additional standards are provided for quantification. The iQ-Check method is based on the following steps:
- Direct quantification of samples (with provided standards in line with the International Standard Reference Material according to ISO TS 12869)
- Direct detection when screening for the presence or absence of Legionella according to ISO TS 12689
- Testing large water resources, including water supply systems, industrial water systems (cooling towers), health establishments, public buildings, and recreational water systems
- Management of complex (dirty) water samples with high-yield DNA extraction using the Aquadien Kit
- Easy implementation by service labs
- Detection of Legionella in biofilm, aerosol, and amoebae samples
- Outbreak investigation for rapid results during a crisis
- Real time monitoring of water systems with results in 4 hours
iQ-Check Free DNA Removal Solution
One of the primary challenges when performing PCR for environmental water testing is the potential presence of free DNA, which can lead to an overestimation of the level of targeted DNA sequences. When quantifying Legionella, culture and PCR levels can differ from each other by up to 2 logs. To improve Legionella testing in water, a supplemental step using the iQ-Check Free DNA Removal Solution (FDRS) can be easily integrated in the Aquadien DNA Extraction Kit Workflow. FDRS is a non-carcinogenic safe way to remove free DNA from dead cells in environmental water samples. According to the principle, free DNA is degraded by an enzymatic treatment of the sample prior to DNA extraction, and inactivated by the Aquadien Extraction Kit R1 solution allowing for the extraction of DNA from intact and living cells. The inclusion of FDRS in the iQ-Check workflow reduces the occurrence of false positives.
Standard Media and Confirmation Latex Test
Bio-Rad, as a key partner for water testing, also manufactures a large range of products and solutions for Legionella testing such as GVPC and BCYE medium for the enumeration of Legionella according to ISO 11731 and latex kits for colony confirmation and serotyping. Contact your Bio-Rad sales specialist for more information.
Water Safety and Quality Reference Guide
(PDF 4.4 MB)
Legionella Testing Solution Brochure
(PDF 717 KB)
iQ-Check Legionella Fact Sheet
(PDF 569 KB)
Legionella Aquadien Standard DNA Extraction Protocol
Legionella Aquadien Short DNA Extraction Protocol