Patent EP-2402456-A1 (EP)
Inventor: WOHLER CHRISTIAN
IPC: C12Q1/68
Filed: 2010-06-30
Abstract: Determining organisms in water, preferably drinking water, comprises filtering the water sample by a filter, recovering cells of organisms from the filter using a solvent, lysing the cells of organisms and determining and/or quantifying the organisms by quantitative real time polymerase chain reaction (RT-qPCR), where the solvent and contact time and -conditions are selected such that the filter is dissolved in the solvent and the cells of organisms remain intact. Independent claims are included for: (1) a kit for determining organisms in water, preferably drinking water, comprising at least a solvent and at least a filter, where the filter is dissolved in the solvent and the cells of organisms are essentially intact; (2) a forward/reverse primer pair comprising primers according to SEQ ID NO. 2 (comprising cctttgctcattgacgttacccgcagaag), SEQ ID NO. 3 (comprising caagcttgccagtatcagatgc), SEQ ID NO. 4 (comprising gactcaagcttgccagtatcag), SEQ ID NO. 6 (comprising ggaatcttcggcaatggacg), SEQ ID NO. 7 (comprising tcagttactaacgtccttgttcttc), SEQ ID NO. 8 (comprising ctcaacctgggaactgcatc), SEQ ID NO. 9 (comprising caacctgggaactgcatctg), SEQ ID NO. 10 (comprising gaaatccccgggctcaac), SEQ ID NO. 11 (comprising gtattcctccagatctctacgc), SEQ ID NO. 12 (comprising ctctacgcatttcaccgctac), SEQ ID NO. 15 (comprising gtgtatgaagaaggccttcgg), SEQ ID NO. 16 (comprising cttcttctgcgggtaacgtc), SEQ ID NO. 18 (comprising gccttcgggttgtaaagtac), SEQ ID NO. 19 (comprising cttctgcgggtaacgtca), SEQ ID NO. 21 (comprising gaaggccttcgggttgtaaagtac), SEQ ID NO. 22 (comprising gtgtatgaagaaggccttcgg), SEQ ID NO. 23 (comprising cttcttctgcgggtaacgtc), SEQ ID NO. 24 (comprising cggggaggaagggagtaaag), SEQ ID NO. 26 (comprising catctgacttaacaaaccgcctg), SEQ ID NO. 27 (comprising cacatctgacttaacaaaccgc), SEQ ID NO. 28 (comprising caagcttgccagtatcagatgc), SEQ ID NO. 29 (comprising gactcaagcttgccagtatcag), SEQ ID NO. 31 (comprising cgataagcgtcggtaaggtg), SEQ ID NO. 32 (comprising ggttcgcctcattaacctatg), SEQ ID NO. 34 (comprising atgaaccgttataaccggcg), SEQ ID NO. 36 (comprising ggaatcttcggcaatggac), SEQ ID NO. 37 (comprising tcagttactaacgtccttgttcttc) and SEQ ID NO. 39 (comprising ggaatcttcggcaatggacg), preferably PCR primer pairs of SEQ ID NO. 2 and 3, SEQ ID NO. 2 and 4, SEQ ID NO. 6 and 7; (3) a PCR fluorescent probe according to SEQ ID NO. 1 (comprising cctttgctcattgacgttacccgcagaag), SEQ ID NO. 5 (comprising cgcgtgagtgaagaaggttttcggatc), SEQ ID NO. 13 (comprising ctggcaagcttgagtctcgtagagg), SEQ ID NO. 14 (comprising cggggaggaagggagtaaagttaatac), SEQ ID NO. 17 (comprising cggggaggaagggagtaaagttaatacc), SEQ ID NO. 20 (comprising cagcggggaggaagggagtaaagttaatacc), SEQ ID NO. 25 (comprising cctttgctcattgacgttacccgcagaag), SEQ ID NO. 30 (comprising aaacccagtgtgattcgtcacacta), SEQ ID NO. 33 (comprising cgaatggggaaacccagtgtgattcgtc), SEQ ID NO. 35 (comprising tgagtgaagaaggttttcggatcgta) and SEQ ID NO. 38 (comprising cgcgtgagtgaagaaggttttcggatc), where the probe is modified at its one end, preferably at 3'-end with a fluorescence quencher including carboxytetramethylrhodamine or black hole quencher-1, and is modified at its other end, preferably at 5'-end with a fluorescent dye, preferably 6-carboxyfluorescein or Yakima yellow(RTM: Fluorescent dye); and (4) a primer/probe kit for PCR, including the above primer pair and fluorescent probe.