Molecular detection of viable bacterial pathogens in water by ratiometric pre-rRNA analysis

Abstract

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples.

FIG. 1.

Time course of nutritional stimulation of pre-rRNA in A. hydrophila (A) and M. avium strain 104 (B) cells. Pre-rRNA stimulation ratios are the ratios of pre-rRNA in stimulated samples relative to that in control samples, measured by RT-qPCR. Values are means and SDs of two or more experiments per time point. To conduct RT-qPCR on extracted RNA, cDNA was first generated using the Superscript III system (Invitrogen) and cleaned using a Qiagen PCR purification kit (catalog no. 28104). Amplification of cDNA was performed using the Applied Biosystems Power SYBR green mix. Reactions were conducted in triplicate at two different dilutions to ensure quantitative readouts. Amplifications were run in 96-well plates on an ABI Prism RT-7500 as follows: 10 min at 95°C and 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C using 9600 emulation. ABI SDS software was used to set cycle threshold values.

FIG. 2.

Correlation between the presence of viable A. hydrophila cells and the pre-rRNA stimulation ratio (A) or genomic DNA quantified by qPCR (B) in chlorine-treated laboratory suspensions. Pre-rRNA stimulation ratios (A) are the ratios of pre-rRNA in stimulated samples relative to that in control samples measured by RT-qPCR. Values are means of three measurements per sample. Numbers of genomic DNA copies (B) were quantified by qPCR normalized to a genomic DNA standard curve. DNA was measured in stimulated (open squares) and nonstimulated (open triangles) samples.