Search results for " RT-qPCR"
showing 9 items of 9 documents
A sensitive real-time RT-PCR reveals a high incidence of Southern tomato virus (STV) in Spanish tomato crops
2018
[EN] Southern tomato virus (STV) is a double-stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae). STV has been detected in tomato plants showing different symptoms although it has not been demonstrated that STV is the causal agent. To study the STV incidence and its pathogenic role, a sensitive and quantitative real-time reverse transcription-polymerase chain reaction assay (RT-qPCR) was developed. The standard curve perfonned with viral RNA transcripts allowed a wide dynamic range for STV quantitation from 10(4) to 10(11) copies/ng of total RNA. STV detection by RT-qPCR was 10(2)-fold more sensitive than conventional RT-PCR or RT-LAMP and 10(4)-fold more sens…
Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids
2018
The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To thi…
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 …
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…
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…
Detection of Parietaria Mottle Virus by RT-qPCR: An Emerging Virus Native of Mediterranean Area That Undermine Tomato and Pepper Production in Southe…
2021
Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin, especially on tomato and pepper crops. Symptoms on tomato leaves and fruits can be easily confused with those induced by cucumber mosaic virus (CMV) with necrogenic satellite RNA (CMV-satRNA), tomato spotted wilt virus (TSWV) or tomato mosaic virus (ToMV). Mixed infection of these viruses has been also reported in some tomato cultivars, with an increase in the complexity of the symptoms and severity of the disease. Although a specific serum and riboprobes have been produced, nowadays no sensitive diagnostic methods are available for the rapid PMoV detection. Here, we have developed a…