Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination

Abstract

Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F؉ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.It is important that sources of fecal pollution are rapidly and accurately identified so that environmental managers can develop strategies to eliminate the source of the pollution in an efficient and cost-effective manner. The identification of fecal sources also allows the potential risk to human health following the consumption of or exposure to contaminated food or water to be estimated. For this purpose, numerous fecal source tracking methods have been developed. Among these are library-dependent methods such as antibiotic resistance testing or molecular fingerprinting (e.g., terminal restriction length fragment polymorphism [T-RFLP] or ribotyping) and library-independent approaches, including the detection of human-specific bacteriophages by plaque assay (e.g., Bacteroides fragilis bacteriophages), the detection of certain chemicals associated with human and nonhuman wastes (e.g., caffeine, laundry brighteners, fecal sterols, and stanols), and the detection of specific genetic markers by PCR. The molecular detection of human and animal viruses and bacteriophages falls into the latter category. Each method has advantages and disadvantages depending on the circumstances and the questions at hand. To date, none has been proposed as a standard method (9,34,40,45).The detection and genotyping of Fϩ RNA bacteriophages have been widely used as a tool for microbial source tracking (MST) (5,8,17,18,31,44). Fϩ RNA bacteriophage genogroup I (GI) and GIV have been associated with animal contamination, and genogroups II and III indicate predominantly human fecal contamination or domestic sewage-associated in-

First description of Adenovirus, Enterovirus, Rotavirus and Torque teno virus in water samples collected from the Arroio Dilúvio, Porto Alegre, Brazil

“…synthesis of cDNA, which was achieved with a High Capacity cDNA Reverse Primer sequences reported by Wolf et al (2010). b Primer sequences reported by Tsai et al (1993).…”

Adenoviruses of canine and human origins in stool samples from free-living pampas foxes (<italic>Lycalopex gymnocercus</italic>) and crab-eating foxes (<italic>Cerdocyon thous</italic>) in São Francisco de Paula, Rio dos Sinos basin

“…There is also serological evidence for infection by CAV on Brazilian maned wolves (de Almeida Curi et al, 2010). HAdV is ubiquitous in human populations (Eick et al, 2011;Ersching et al, 2010), as well as the virus often being found in environmental samples contaminated by human feces (Miagostovich et al, 2008;Silva et al, 2011;Wolf et al, 2010). HAdV-C has occurred in 14 of the 17 samples (82.35%) and 5 samples were co-infected by CAV (29.41%).…”

“…Real-time PCR (qPCR) was applied for the detection human adenovirus (HAdV) genomes using oligonucleotides described previously (VTB2-HAdVCf 5'-AGACGTACTTCAGCCTGAAT-3'; VTB2-HAdVCr 5'-GATGAACCGCAGCGTCAA-3') (Wolf et al, 2010). Nucleotides for the specific amplification of CAV-1 and -2 genomes were originally designed, the forward primer was CAV-F1: 5´-CACGATGTGACCACTGAGAG-3´; reverse: CAV-R1 5´-GGTAGGTATTGTTTGTGACAGC-3´), whose target is a fragment of 300 to 350 base pairs in the gene that encodes the hexon protein.…”

“…Not only adenoviruses. Those viruses are non-enveloped, being highly resistant on the environment; its genomes may be readily detectable on environmental samples (Miagostovich et al, 2008;Silva et al, 2011;Wolf et al, 2010). Other viruses which may be transmitted by the fecal-route are enteroviruses (EV, genus Enterovirus, family Picornaviridae) and rotaviruses (RV, genus Rotavirus, family Reoviridae), two non-enveloped RNA viruses.…”

“…Other viruses which may be transmitted by the fecal-route are enteroviruses (EV, genus Enterovirus, family Picornaviridae) and rotaviruses (RV, genus Rotavirus, family Reoviridae), two non-enveloped RNA viruses. Since they are host-specific viruses and excreted in high titers by the fecal route, its detection and identification may allow the tracking of sources involved on fecal contamination of water and other environmental matrices (Silva et al, 2011;Wolf et al, 2010).…”