Data related to publication "Development and validation of field-compatible workflow for molecular diagnostics of microbial water quality"

General Information: This data is related to the publication "Development and validation of field-compatible workflow for molecular diagnostics of microbial water quality" by Piglmann L., Campostrini L., Kirschner A.K.T, Farnleitner A.H., Kolm C. and Reischer G.H. (2026) submitted to Water Research. The raw data of the qPCR and LAMP analyses are split up into six files: "File 1: Establishment of filter-based DNA extraction method for bacteria (qPCR analysis)", "File 2: Assessment of sample processing method with selected target bacteria (qPCR analysis)", "File 3: Validation of sample processing method with dilution series of selected target bacteria (qPCR analysis)", "File 4: Validation of sample processing method with various environmental water matrices (qPCR analysis)", "File 5: Validation of integrated simplified workflow – combining sample processing with LAMP detection (LAMP analysis)" and "File 6: Validation of integrated simplified workflow with environmental water matrices (LAMP analysis)". 

All qPCR analyses were carried out on a qTOWER G real-time thermocycler in triplicates, no-template controls were included in each run, and plasmid dilution series containing a known number of the target gene were used to generate a calibaration curve.  All LAMP analyses were carried out on a qTOWER G real-time thermocycler in triplicates and no-template controls were included in each run. As bacterial target strains Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145) and Enterococcus faecalis (ATCC 19433) were selected, grown overnight, cells were harvested and resuspended in isotonic saline solution. 

In File 1 the raw data of the establishment of the filter-based DNA extraction method are summarized. To this end, E. coli was used as a model for DNA extraction experiments and analysed by specific qPCR. In Experiment 1, a comparison of different DNA extraction methods was made between a commercially available QIAamp DNA Mini Kit (Qiagen), different volumes of the ionic liquid [C2mim][OAc] and different mixing approaches to ensure contact of the ionic liquid with the filter. Each DNA extraction method was performed with biological duplicates and with extraction controls in biological duplicates. In Experiment 2, the effect of additional filter rinsing was evaluated, either once, twice or three times with 10 mM Tris pH 8 buffer. Each DNA extraction method was performed with biological triplicates and with extraction controls in biological triplicates.

In File 2 the raw data of the assessment of the sample processing method with E. coli, P. aeruginosa and E. faecalis are summarized. The bacterial target strains were used for DNA extraction experiments and quantified using species-specific qPCR assays. As reference methods for DNA extraction, an ionic liquid-based extraction method without filtration and the QIAamp DNA Mini Kit were used. Each DNA extraction method was performed with biological triplicates and with extraction controls in biological triplicates. 

In File 3 the raw data of the validation of sample processing method with dilution series of selected target bacteria are summarized. The three bacterial strains and the human associated faecal marker HF183 were analysed in serial 10-fold dilutions and quantified using species-specific qPCR assays. As reference methods for DNA extraction, two commonly used laboratory reference protocols for filter-based DNA extraction (Phenol-Chloroform extraction and the Qiagen PowerSoil Kit) were used. Samples are named after their dilution step: ud (undiluted), 1 (first dilution step), 2 (second dilution step), ... , 7 (seventh dilution step = lowest dilution representing a condition in which the presence of the target organism is no longer statistically expected). Each DNA extraction method was performed with biological duplicates and with extraction controls in biological duplicates.

In File 4 the raw data of the validation of sample processing method with various environmental water matrices are summarized. For this, environmental water samples (wastewater, surface water, groundwater) were spiked separately with known concentrations of E. coli, P. aeruginosa, E. faecalis and HF183, respectively. DNA was extracted with our sample processing method, a phenol-chloroform protocol and the Qiagen PowerSoil Kit and quantified using species-specific qPCR assays. Additionally, the non-spiked water samples (named n.S. in file) were also used for DNA extraction and analysed by species-specific qPCR assays to obtain background values for the sample matrix. Each DNA extraction method was performed with biological duplicates and with extraction controls in biological duplicates.

In File 5 the raw data of the validation of integrated simplified workflow – combining sample processing with LAMP detection are summarized. Here the same dilution series as described for File 3 were used and analysed with their respective LAMP assays. 

In File 6 the raw data of the validation of integrated simplified workflow with environmental water matrices are summarized. Here the same spiked environmental water samples as described for File 4 were used and analysed with their respective LAMP assays.