A Culture Independent Method for the Detection of sp. from Water Samples.

The genus is present in a wide variety of water environments and is recognised as potentially pathogenic to humans and animals. Members of this genus are often confused with when using automated, commercial identification systems that are culture-dependent. This study describes a polymerase chain reaction (PCR) detection method for that is culture-independent and that targets the glycerophospholopid-cholesterol acyltransferase () gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCAT-PCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 µL to 4 mL. The proposed molecular method is much faster (5 or 29 h) than the culturing method (24 or 48 h) whether performed directly or after a pre-enrichment step and it will enable the fast detection of in water samples helping to prevent a possible transmission to humans.