Nejvíce citovaný článek - PubMed ID 22207081
Optimisation of a triplex real time RT-PCR for detection of hepatitis E virus RNA and validation on biological samples
This study presents the results of systematic wastewater monitoring of SARS-CoV-2 RNA and basic wastewater parameters from four different wastewater treatment plants (WWTPs) in the Czech Republic over the 2020-2022 epidemic. Two-step reverse-transcription quantitative PCR targeting genes encoding the N and Nsp12 proteins was employed to detect SARS-CoV-2 RNA loading in 420 wastewater samples. The results obtained were used to evaluate the potential of wastewater analysis for describing the epidemiological situation in cities of different sizes and determining temporal differences based on the prevailing SARS-CoV-2 variant. Strong correlations between the number of active and hospitalised COVID-19 cases in each WWTP catchment area and the concentration of SARS-CoV-2 RNA detected in the wastewater clearly demonstrated the suitability of this wastewater-based epidemiological approach for WWTPs of different sizes and characteristics, despite differences in SARS-CoV-2 variant waves, with some WWTPs showing high predictive potential. This study demonstrated on the data from the Czech Republic that targeted systematic monitoring of wastewater provides sufficiently robust data for surveillance of viral loads in sample populations, and thus contributes to preventing the spread of infection and subsequent introduction of appropriate measures.
- Klíčová slova
- COVID-19, PCR, Virus, Wastewater treatment plant, Wastewater-based epidemiology,
- Publikační typ
- časopisecké články MeSH
Hepatitis E virus (HEV) is the etiological agent behind hepatitis E infection. Domestic pigs and wild boars are the main animal reservoirs of HEV. Very few papers describe HEV infection in goats and sheep. As the data pertaining to the presence of HEV virus in the milk of small ruminants in Europe are lacking, the aim of this paper was to examine a representative number of milk samples from these animals. The detection of HEV genome (HEV RNA) was performed using reverse transcriptase real-time polymerase chain reaction (RT-qPCR). HEV RNA was found in 2.8% of the examined samples. Positivity ranged from 101 to 103 genome equivalents/mL (GE/mL) with a median of 9.99 × 102 GE/mL. On the basis of these results, the milk of small ruminants could represent a source of HEV infection to consumers.
- Klíčová slova
- HEV, RT-qPCR, milk, small ruminant,
- MeSH
- hepatitida E veterinární MeSH
- kozy virologie MeSH
- mléko virologie MeSH
- nemoci zvířat diagnóza virologie MeSH
- ovce virologie MeSH
- RNA virová MeSH
- virus hepatitidy E genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- RNA virová MeSH
The virus SARS-CoV-2, which has caused the recent COVID-19 pandemic, may be present in the stools of COVID-19 patients. Therefore, we aimed to detect SARS-CoV-2 in wastewater for surveillance of SARS-CoV-2 in the population. Samples of untreated wastewater were collected from 33 wastewater treatment plants (WWTPs) of different sizes within the Czech Republic. SARS-CoV-2 RNA was concentrated from wastewater and viral RNA was determined using real-time reverse transcription polymerase chain reaction (RT-qPCR). SARS-CoV-2 RNA was detected in 11.6% of samples and more than 27.3% of WWTPs; in some of them, SARS-CoV-2 was detected repeatedly. Our preliminary results indicate that an epidemiology approach that focuses on the determination of SARS-CoV-2 in wastewater could be suitable for SARS-CoV-2 surveillance in the population.
- Klíčová slova
- RT-qPCR, SARS-CoV-2, coronavirus, early warning system, epidemic, virus, wastewater, wastewater-based epidemiology,
- MeSH
- Betacoronavirus izolace a purifikace MeSH
- COVID-19 MeSH
- feces virologie MeSH
- koronavirové infekce epidemiologie virologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- odpadní voda virologie MeSH
- pandemie MeSH
- RNA virová MeSH
- SARS-CoV-2 MeSH
- virová pneumonie epidemiologie virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Názvy látek
- odpadní voda MeSH
- RNA virová MeSH
The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV- MS2 phage-like particles (MS2 PLP) - in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV - they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.
- Klíčová slova
- MS2 phage-like particle, RNA virus, RT-qPCR, detection, extraction efficiency calculation, isolation, process control virus, quantification,
- Publikační typ
- časopisecké články MeSH
The monitoring of wastewater treatment plants is important for their proper functioning as well as for re-use of water and also to avoid possible circulation of human or animal pathogens in our environment. The samples in this study originated from a full-scale wastewater treatment plant where the structure of the bacterial community was monitored using 454-pyrosequencing. The composition differed in different parts of the plant. In the effluent, bacteria belonging to phyla Proteobacteria, Actinobacteria, TM7 and Bacteroidetes were most frequently detected. The presence of Mycobacterium sp., Mycobacterium avium, Norovirus, Hepatitis A and E viruses was examined using quantitative real-time PCR. Mycobacterium sp. was detected in the effluent in quantities of up to 10(4) cells/ml. Mycobacterium avium subsp. paratuberculosis and subsp. hominissuis were detected in amounts of up to 10(3) cells/ml, and Norovirus group 1 and 2 were also detected. Our findings show the importance of monitoring and controlling the occurrence of specific pathogens in effluent, mainly because of the negative impact on human health when the water is reused.
Hepatitis E virus (HEV) is now accepted as a zoonotic virus, and domestic pigs, wild boars and deer are recognised as natural reservoirs of the pathogen. In this study, 762 animals (wild boars, fallow deer, red deer, sika deer, roe deer and mouflons) originating from the wild and from game enclosures were tested for the presence of HEV RNA by qRT-PCR. HEV RNA was detected in wild boars (96/450), red deer (2/169), roe deer (1/30) and mouflons (5/39). The sequence relationship between HEV isolates from wild boars and domestic pigs or humans indicate a circulation of HEV in the Czech Republic.
- Publikační typ
- časopisecké články MeSH
