Title: TBD
Learner Objectives:
To inform stakeholders on how to appropriately assess qPCR technologies for Legionella monitoring by highlighting the requirements of ISO/TS 12869 and illustrating the process using a real-world example.
Content/Topic Outline:
- Review the criteria by which a qPCR assay designed for the monitoring of Legionella should be assessed as it pertains to the requirements of ISO/TS 12869.
- Describe the importance and application of validation methods including linear regression calibration, LOD, LOQ, inclusivity/exclusivity, and reproducibility using real-world data.
- Explain how the transparency of validation data is essential for the assessment of any Legionella qPCR detection method.
Presenter:
Michael Waud, PhD, LuminUltra Technologies Ltd.
Presentation Description:
The ability to rapidly and reliably detect bacteria belonging to the genus Legionella is critically important to operators of water distribution systems and cooling water circuits in order for them to quickly assess and react to the presence of these potentially pathogenic microbes. This requirement for quick results is often hindered by the need to send collected samples for analysis at off-site laboratory facilities rather than generating results in the field. Although the "gold standard" and widely accepted regulatory method of detecting Legionella species remains culture-based, qPCR is an established detection and screening method that can be designed to target DNA unique to Legionella in order to quantitatively measure the bacterial population present within a water system in hours, rather than the typically required days. It must be noted that when dealing with organisms dangerous to public health, the consequences of false positive or negative results can be extremely serious, which necessitates robust and continual assessment and validation of any detection method, and the availability of validation data to an informed user-group able to adequately assess methodology.
In this study we present the validation of a quantitative polymerase chain reaction (qPCR) assay for the detection of bacteria belonging to the genus Legionella in either laboratory or field settings in accordance with International Standards Organization (ISO) Technical Specification 12869:2019. The results of this study are presented with special emphasis on end-user assessment of qPCR assays designed for the detection of Legionella and methods of validation applicable to any qPCR-based detection method. These methods include the determination and verification of a linear regression curve used to convert raw qPCR results into a concentration of genome units (GU) of Legionella, inclusivity and exclusivity of the designed qPCR primers, limit of detection (LOD) and limit of quantification (LOQ). Since many risk management plans offer advice on treatment and cleaning according to CFU/mL results, it is important to understand how qPCR results relate to these established guidelines. Towards this purpose, the correlation between culture-based enumeration and qPCR testing is reviewed and data presented.
Presenter Bio:
Michael Waud, PhD: A molecular ecologist by training, Michael obtained his doctorate from KU Leuven (Belgium) and now applies his understanding of microbial communities and their potential influences towards unravelling the complex communities present within environmental and industrial systems and then designing qPCR methods for the detection of specific microbes of interest.