Detection of adenoviruses in stools from healthy persons and patients with diarrhea by two‐step polymerase chain reaction

Allard, Annika; Albinsson, Bo; Wadell, Göran

doi:10.1002/jmv.1890370214

Cited by 162 publications

(125 citation statements)

References 32 publications

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“…The amplicons from HAdV-40 and HAdV-41 present two restriction fragments of 191bp and 110bp after the digestion, while the adenoviruses from other species (HAdV-A to E) do not (3,29). Based on this assay, the presence of human adenovirus from species F was detected in 41 (82%) samples and only 1 (2%) sample did not show human adenovirus F. Eight (16%) samples were not characterized due to low DNA concentration for the restriction assay.…”

Section: Resultsmentioning

confidence: 99%

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Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo

Barrella¹,

Garrafa²,

Monezi³

et al. 2009

Braz. J. Microbiol.

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The aim of this study was to verify the presence and annual distribution of adenoviruses and hepatitis A virus in domestic sewage in the city of Limeira, São Paulo. Fifty samples with a volume of 8 liters each were collected weekly from December 2004 to December 2005. The viruses were concentrated by filtration through positively charged ZP60S filter membranes, followed by ultracentrifugation. Human adenoviruses (HAdV) were detected by PCR followed by nested-PCR and screening for species F was done by restriction of the PCR product with TaqI endonuclease. Virus infectivity assays were performed by inoculation of concentrates onto HEp-2 cell monolayers. RT-PCR was used for the detection of hepatitis A virus. HAdV were detected in all samples, and 64% of samples were positive for infectious virus. Species F was present in 82% of the samples. Hepatitis A virus was detected in 48% of the samples. These results demonstrate that HAdV and HAV were present in the domestic sewage of Limeira throughout the period of study, demonstrating the importance of an adequate treatment before the disposal in the environment.

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Section: Resultsmentioning

confidence: 99%

“…At the present, the enteric adenoviruses of specie F, HAdV-40 and HAdV-41, are the third cause of non-bacterial diarrhea among young children (3,9). HAdV-41 is fatal in 50% of immunosupressed individuals infected (11).…”

mentioning

confidence: 99%

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo

Barrella¹,

Garrafa²,

Monezi³

et al. 2009

Braz. J. Microbiol.

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“…Molecular techniques are based on the detection of highly conserved portions of the viral genome (Allard et al 1992). The most widely used method, the Polymerase Chain Reaction (PCR), offers advantages over cell culture methods to detecting viruses in environmental samples.…”

Section: Molecular Detectionmentioning

confidence: 99%

Methods of Virus Detection in Soils and Sediments

Staggemeier¹,

Almeida²,

Spilki³

2011

VR&R

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Anthropogenic impacts on soils have become one of the major environmental concerns nowadays. Soils and sediments under water bodies may contain viruses and bacteria on higher loads than those identified in contaminated waters. Thesecontaminants may interfere with the whole environment of the affected area, not only damaging the soil and the water but also affecting wildlife. Viruses can travel across the ground contaminating groundwater. The focus of research concerning viruses in soil matrices has been the destination, transport, and detection of pathogenic viruses exogenous to soils. Many methods have been suggested in the last few years for viral detection in soil and sediments. Viruses can be extracted from soil by elution, concentrated and finally detected by molecular techniques cell culture. There are many elution methods described in the literature and 11 techniques are cited in this review. Molecular detection can be performed by Polymerase Chain Reaction (PCR) and its modifications. New assays for viral quantification are emerging, such as adaptation of plaque assays (PAs), most-probable number assays (MPNs), transmission electron microscopy (TEM), epifluorescence microscopy (EfM), and flow cytometry (FC).

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“…The nucleic acids were recovered in 45 μL and stored at -80°C until PCR analysis. AdV DNA was detected in sewage sludge samples by using the oligonucleotide primer pair hexAA 1885 and hexAA 1913, which amplifies a 300 bp fragment from the AdV hexon gene (Allard et al 1992). HAV RNA was detected in sewage sludge samples by RT nested-PCR using the oligonucleotide primer pair F6 (+) and F7 (-), which amplifies a 392 bp fragment suitable for detecting all HAV genotypes (universal primers).…”

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confidence: 99%

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

Schlindwein¹,

Simões

Barardi³

2009

Mem. Inst. Oswaldo Cruz

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Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. They contain numerous pathogenic microorganisms, mostly of faecal origin.Wastewater treatment processes are likely to reduce the number of pathogens in the water prior to its return to the environment. The high incidence of pathogenic viruses in sewage sludge is due to the preferential adsorption of viruses to sludge solids. Depending upon the method used to treat domestic sewage, between 50-99.99% of the viruses can be inactivated. Therefore, infectious viruses potentially remain after treatment. With enteric viruses, a low infectious dose can be enough to cause illness (Moe 1991).Studies that compared the persistence of enteric viruses indicated that enteroviruses such as poliovirus (PV) are not reliable indicators of other human enteric viruses of major health significance such as rotavirus (RV), astrovirus, hepatitis A virus (HAV) (Abad et al. 1994) and adenovirus (AdV). For this reason, we selected four enteric viruses (AdV, HAV, PV and RV) for use in a model of virus recovery in sewage sludge.Nucleic acids can be extracted from contaminated environmental samples by many different protocols. These include organic-based extraction by phenol (in the case of DNA viruses) or Trizol ® (in the case of RNA viruses), silica methods and commercial kits. These methods aim to extract and purify nucleic acids by removing cell debris and inhibitors. This extraction step is crucial to environmental virology because the main challenge in molecular detection techniques [such as polymerase chain reaction (PCR)] is inhibition by substances such as humic acids, polysaccharides and other chemicals (Kingsley & Richards 2001). Non-kit-based methods are preferred for routine laboratory procedures all over the world because they are less expensive than kit-based protocols. The reagent Trizol LS is a mono-phasic solution of phenol and guanidine isothiocyanate that is preferred for total RNA isolation from cells and tissues because it has the ability to lyse cells and inactivate nucleases (Boom et al. 1990). When this method is used, total RNA segregates into the aqueous phase, while DNA and proteins remain in the phenol phase and interface. For this reason, the traditional phenol/chloroform protocol is more suitable for DNA isolation. Silica methods are rapid, easy to use and efficient at removing inhibitors (Jiang et al. 2001).In this study, we compared organic-based and silica nucleic acid extraction methods on the basis of their ability to isolate viruses seeded into sewage sludge samples. The efficiency of viral elution was evaluated by PCR, reverse transcription (RT) PCR and RT-nested-PCR. The use of samples contaminated with known amounts of the viruses facilitated direct comparisons between the different extraction methods. MATERIALS AND METHODSHAV-cytophatic strain HM 175 and human AdV (genogroup C, serotype 5) (AdV5) were propagated in a continuous line of foetal FRHk-4 cells (rhesus kidneyderived cells) and Hep-2 cells (human la...

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Detection of adenoviruses in stools from healthy persons and patients with diarrhea by two‐step polymerase chain reaction

Cited by 162 publications

References 32 publications

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo

Methods of Virus Detection in Soils and Sediments

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

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