Inorganic pyrophosphatase-based detection systems. I. Detection and enumeration of cells.
A novel technique, useful for detection and enumeration of both pro- and eukaryotic cells, has been developed. The method relies on the detection by a very sensitive assay of an enzyme, the inorganic pyrophosphatase, which is constitutively expressed in all cells. This technique has been used to demonstrate detection and enumeration of both Escherichia coli and Saccharomyces cerevisiae cells. Cells were incubated, for a chosen time period and at a specific temperature, in a specific lysis buffer which contained both lysing activity and inorganic pyrophosphate. The activity of the inorganic pyrophosphatase released from the cells after lysis was determined by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509). The amount of PPi hydrolyzed was proportional to the logarithm of the cell number. The sensitivity of the assay was dependent on several factors, such as cell type, incubation time, and incubation temperature. E. coli cells at concentrations of 1 x 10(4) cells/ml could be detected in an assay performed at 58 degrees C for 10 min and S. cerevisiae cells at concentrations of 2 x 10(4) cells/ml at 49 degrees C within 10 min. The assay could be designed both as a present/absent test, e.g., for determination of bacteriuria, or as a quantitative assay. Determination and enumeration of all types of cells is in principle possible by the appropriate design of the experimental parameters. Possible applications for the approach in a wide variety of areas, such as fundamental and medical sciences, clinical laboratories, food and dairy industry, pharmaceutical industry, water sanitary plants, and biomass determination in general, are discussed.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
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Edwin
D004926:Escherichia coli / Q000201:enzymology*; D005057:Eukaryotic Cells / Q000201:enzymology; D043564:Inorganic Pyrophosphatase; D011387:Prokaryotic Cells / Q000201:enzymology; D011755:Pyrophosphatases / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012680:Sensitivity and Specificity
D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase