Monitoring of adenovirus serotypes in environmental samples by combined PCR and melting point analyses

Abstract

Background: Human adenoviruses are promising candidates for addressing health risks associated with enteric viruses in environmental waters. Relatively harmless but common, these DNA viruses persist within the population and are generally considered extremely stable, remaining infectious in water for long periods of time. Group-specific or single species detection of human adenoviruses in environmental samples is usually based on polymerase chain reaction assays. Simultaneous identification of specific species or serotypes needs additional processing. Here we present a simple molecular approach for the monitoring of serotypic diversity in the human adenovirus populations in contaminated water sites.

Methods: Diversity patterns of human adenoviruses in environmental samples, collected in an outdoor artificial stream and pond simulation system, were analyzed using a closed tube polymerase chain reaction method with subsequent melting point analysis.

Results: Human adenovirus serotype 41 was the most prominent adenovirus serotype detected in environmental water samples, but melting point analyses indicated the presence of additional adenovirus serotypes.

Conclusions: Based on investigations with spiked and environmental samples, a combination of qPCR and melting point analysis was shown to identify adenovirus serotypes in sewage contaminated water.

Figure 1

Stabilities of human adenovirus serotypes in processed water at 6°C. Concentrations were determined by qPCR in four-week intervals. Decreases of virus copy numbers at 6°C did not exceed 1.5 log₁₀ units for any tested serotype. Dashed lines represent calculated mean concentrations.

Figure 2

Decrease of human adenovirus serotype 41 in processed water at distinct temperatures over time. The decrease of virus copy numbers at room temperature exceeded the decrease at 6°C by about 2 log₁₀ units.

Figure 3

Melting curves of different human adenovirus serotypes. Melting curves were determined following qPCR amplification. Differences of only 1 to 3 bp in the amplified regions led to significant differences in melting temperatures.

Figure 4

Melting curves of environmental water samples. The melting curves of the human adenovirus serotype 41 reference and four environmental samples are shown. Each curve represents the mean value of two parallels. Equivalent results were obtained during several runs.

Figure 5

Melting curves of an adenovirus plasmid standard using different sets of primers. The plasmid contained an insert of the human adenovirus serotype 41 hexon gene. Three sets of primers were used for amplification of the insert, producing amplicons of different lengths. Melting temperatures correlated with the lengths of amplicons. The small blue peak at around 75°C corresponds to the primer peak of the neHex3deg/neHex4deg system.