Academic journal article Journal of Environmental Health

Rapid Fluorometric Measurement of Trace Amounts of Contaminant DNA in Drinking Water

Academic journal article Journal of Environmental Health

Rapid Fluorometric Measurement of Trace Amounts of Contaminant DNA in Drinking Water

Article excerpt

Introduction

Contamination of drinking water by pathological organisms is a critical issue in the field of environmental water quality. Because an enormous number of pathological species exist on earth, it is impractical to implement an initial water quality test to identify each pathogen. Methods for evaluating the bacteriological quality of water are often based on cultivation of bacteria in a sample (e.g., standard plate count or heterotrophic plate count); however, this approach has the disadvantage that a long incubation time is required for colony growth (1). Moreover, the species of bacteria grown depends on the type of isolation media used, and some bacteria species and nonbacterial organisms are difficult to grow in an ordinary medium (2). Demand is growing for a rapid water quality test that covers a broad spectrum of pathological organisms. Because all living organisms contain nucleic acids, detection of DNA and RNA in water may serve as a comprehensive screening test for water quality. In this paper, we introduce a rapid and simple-to-use methodology for measuring DNA content in water and report findings of significant DNA contamination in some bottled drinking water.

Materials and Methods

Materials

The following materials were purchased from suppliers:

* salmon sperm DNA,

* SYBR DX,

* nitrocellulose and nylon membranes,

* RNase-free DNase I,

* rRNA,

* DNase-free RNase, and

* 24-well and 96-well plates.

The suppliers are listed in Table 1. Various brands of bottled water were purchased at a local supermarket in Irvine, California.

Methods

Various concentrations of DNA were suspended in autoclaved double-distilled water (dd[H.sub.2]O) or the following solution: 10 millimoles per liter (mM) Tris - pH 7.6, 1 mM EDTA - pH 8.0, 25 mM NaCl (TEN). The resulting solutions were vacuum-filtered through nylon membranes with the aid of a filter holder. The nylon membranes were 13 millimeters (mm) in diameter and had 0.45-micrometer ([[micro]meter]) pores. In some experiments, small volumes of DNA were simply spotted onto the membranes, then air dried.

The membranes were placed into 24-well plates with the DNA side up and were exposed to ultraviolet light (Stratalinker) for crosslinking. Each membrane was then incubated with 200 [[micro]liter] of 1:1000 dilution of SYBR DX or 0.5 micrograms per milliliter ([[micro]gram]/mL) of ethidium bromide in 0.5X the following solution: 45 mM Tris-borate, i mM EDTA - pH 8.0 (TBE). Incubation was at room temperature for five to 30 minutes. Each membrane, after it was washed with 1 mL of dd[H.sub.2]O three times, was placed DNA side down, and the fluorescent signal of the SYBR DX was determined in a fluorescent plate reader (CytoFluor 2300). For SYBR DX, excitation and emission wavelengths were 485 nanometers (ntu) (bandwidth, 20 nm) and 530 nm (bandwidth, 25 nm), and for ethidium bromide, they were 485 nm (bandwidth, 20 nm) and 630 nm (bandwidth, 20 nm), as described in previous studies (3-5).

In some experiments, immobilized DNA was incubated with 10 units of DNase I in 200 [[micro]liter] of the following solution: 50 [[micro]molar] Tris - pH 8.0, 10 mM Mn[Cl.sub.2], 50 [[micro]gram]/mL bovine serum albumin (BSA). Immobilized RNA was incubated with 0.5 [[micro]gram] of RNase in 200 [[micro]liter] of 1X the following solution: 40 mM Tris-acetate, 1 mM EDTA - pH 8.0 (TAE). The DNA and RNA were incubated at 37 [degrees] C for three to five hours before the SYBR DX assay was conducted.

Results

The first series of experiments was conducted to find suitable membranes for DNA quantification. As shown in Figure 1, two different sources of nitrocellulose membranes failed to demonstrate any positive DNA signals over fresh DNA-free membranes in both SYBR DX and ethidium-bromide staining. Although one nylon membrane (Brand a) expressed positive DNA signals after staining with ethidium bromide, DNA signals were not detected in other membranes [ILLUSTRATION FOR FIGURE 1B OMITTED]. …

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