Search results for "RT-qPCR"

Recovering coronavirus from large volumes of water

2021

The need for monitoring tools to better control the ongoing coronavirus disease (COVID-19) pandemic is extremely urgent and the contamination of water resources by excreted viral particles poses alarming questions to be answered. As a first step to overcome technical limitations in monitoring SARS-CoV-2 along the water cycle, we assessed the analytical performance of a dead end hollow fiber ultrafiltration coupled to different options for secondary concentrations to concentrate viral particles from large volume of spiked tap water, seawater and surface water together with two quantitative RT-qPCR detection kits. Spiking the porcine epidemic diarrhea virus (PEDV), an enveloped virus surrogat…

Bias of library preparation for virome characterization in untreated and treated wastewaters

2020

The use of metagenomics for virome characterization and its implementation for wastewater analyses, including wastewater-based epidemiology, has increased in the last years. However, the lack of standardized methods can led to highly different results. The aim of this work was to analyze virome profiles in upstream and downstream wastewater samples collected from four wastewater treatment plants (WWTPs) using two different library preparation kits. Viral particles were enriched from wastewater concentrates using a filtration and nuclease digestion procedure prior to total nucleic acid (NA) extraction. Sequencing was performed using the ScriptSeq v2 RNA-Seq (LS) and the NEBNext Ultra II RNA …

Comparing analytical methods to detect SARS-CoV-2 in wastewater

2021

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene…

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.

2021

The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. T…

Monitoring Human Viral Pathogens Reveals Potential Hazard for Treated Wastewater Discharge or Reuse

2022

Wastewater discharge to the environment or its reuse after sanitization poses a concern for public health given the risk of transmission of human viral diseases. However, estimating the viral infectivity along the wastewater cycle presents technical challenges and still remains underexplored. Recently, human-associated crAssphage has been investigated to serve as viral pathogen indicator to monitor fecal impacted water bodies, even though its assessment as biomarker for infectious enteric viruses has not been explored yet. To this end, the occurrence of potentially infectious norovirus genogroup I (GI), norovirus GII, hepatitis A virus (HAV), rotavirus A (RV), and human astrovirus (HAstV) a…

HEV Occurrence in Waste and Drinking Water Treatment Plants

2020

Hepatitis E virus (HEV), particularly zoonotic genotype 3, is present in environmental waters worldwide, especially in industrialized countries. Thus, monitoring the presence of HEV in wastewater treatment plants (WWTPs) is an emerging topic due to the importance of reusing water on a global level. Given the limited data, this study aimed to monitor the occurrence of HEV in influent and effluent water in waste- and drinking-water treatment plants (WWTPs and DWTPs). To this end, different procedures to concentrate HEV in influent and effluent water from WWTPs and DWTPs were initially evaluated. The evaluated procedures resulted in average HEV recoveries of 15.2, 19.9, and 16.9% in influent, …

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR

2020

Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX™, EMA™, and PMAxx™ photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA…

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

2022

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested po…

Molecular analyses on HPV infections in semen

Introduction: Human Papillomaviruses (HPVs) are non-enveloped double stranded DNA viruses classified in different genera and several different genotypes. Depending on the genotypes, HPV infections can be asymptomatic or can cause from warts to malignant tumours. Some genotypes, such as HPV18 and HPV16, are considered high risk (HR) HPVs and they are the major cause of cervical cancer. HPV is highly tissue-tropic and infects epithelial cells, but it can also binds other cell types. It is known that HPV can be found in semen but its effects on spermatozoa and male reproductive system are not completely clarified. Materials and Methods: In order to study the HPV infection in semen, we develope…

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

2016

Background & Aims: A novel family of proton-sensing G-proteinâcoupled receptors, including ovarian cancer G-proteinâcoupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells. Methods: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and…