Application of quantitative PCR for the detection of microorganisms in water

Abstract

The occurrence of microorganisms in water due to contamination is a health risk and control thereof is a necessity. Conventional detection methods may be misleading and do not provide rapid results allowing for immediate action. The quantitative polymerase chain reaction (qPCR) method has proven to be an effective tool to detect and quantify microorganisms in water within a few hours. Quantitative PCR assays have recently been developed for the detection of specific adeno- and polyomaviruses, bacteria and protozoa in different water sources. The technique is highly sensitive and able to detect low numbers of microorganisms. Quantitative PCR can be applied for microbial source tracking in water sources, to determine the efficiency of water and wastewater treatment plants and act as a tool for risk assessment. Different qPCR assays exist depending on whether an internal control is used or whether measurements are taken at the end of the PCR reaction (end-point qPCR) or in the exponential phase (real-time qPCR). Fluorescent probes are used in the PCR reaction to hybridise within the target sequence to generate a signal and, together with specialised systems, quantify the amount of PCR product. Quantitative reverse transcription polymerase chain reaction (q-RT-PCR) is a more sensitive technique that detects low copy number RNA and can be applied to detect, e.g. enteric viruses and viable microorganisms in water, and measure specific gene expression. There is, however, a need to standardise qPCR protocols if this technique is to be used as an analytical diagnostic tool for routine monitoring. This review focuses on the application of qPCR in the detection of microorganisms in water.

Fig. 1

Mechanism of the TaqMan probe. The probes rely on the 5′–3′ nuclease activity of Taq DNA polymerase to cleave a dual-labeled probe during hybridisation to the complementary target sequence (adapted from [213])

Fig. 2

SYBR® Green I detection mechanism. SYBR® Green I is 1,000-fold more fluorescent in the bound state (green star) than in the unbound state (blue circle). The fluorescent signal increases proportionately as the PCR amplification increases (adapted from [214])

Fig. 3

Mechanism of molecular beacon chemistry. The molecular beacon includes a hairpin loop structure, the loop being complementary to a target sequence and the stem formed by the addition of internal complementary sequences. The molecular beacon hybridises to the target and the fluorophore and quencher are far enough apart to allow fluorescence to be detected (adapted from [213])

Fig. 4

Mean, range and 95 % confidence intervals of the mean for faecal indicator organisms (FIOs) (expressed as log₁₀ CFU/100 mL) and Bacteroidales marker concentrations (gene copies/100 mL) in crude sewage (blue circle), secondary treated sewage (red triangle) and UV-disinfected sewage (green dot). The secondary treatment sewage that was significantly different from the mean of the crude extract (*) and UV-disinfected sewage that was significantly different from the secondary treated sewage (**) are also indicated in the figure (adapted from [168])