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The L.p.SG1 DETECT Kit is a rapid, quantitative method for the detection and enumeration of Legionella pneumophila serogroup 1 (L.p. SG1) bacteria from different water matrixes. The method is based on a combination of immunomagnetic separation (IMS) and flow cytometric (FCM) quantification. To this end, the method employs magnetic particles conjugated to anti-L.p. SG1 antibodies for the IMS of the target bacteria from environmental matrices and fluorescently labeled anti-L.p. SG1 antibodies for subsequent quantification by FCM. The IMS can be performed either manually with a magnetic rack (rqmicro.MIMS) or automated with the rqmicro.STREAM sample preparation instrument. Compared to the reference method ISO 11731:2017, which is based on culturing and enumeration of colony forming units (CFU) on agar plates, and can take up to 10 days until results are available, analysis with the L.p. SG1 DETECT Kit is culture-independent and delivers results within 2 h. This Performance Tested Method validation study demonstrates a robust method with recoveries exceeding 69%, inclusivity of 100%, exclusivity of 97.2%, and a shelf life of at least 6 months at 4°C or 40 days at 25°C. The Limit of Detection (LOD) was determined at 21 CFU/L and the Limit of Quantification (LOQ) at 80 CFU/L for potable water using the rqmicro.STREAM. The matrix study across three different types of water matrixes (potable, surface, and industrial process water), demonstrates superior repeatability and reproducibility, as well as equivalent or even superior detection of L.p. SG1 bacteria compared to the standard ISO 11731 method.