scholarly journals SARS-CoV-2 RNA titers in wastewater anticipated COVID-19 occurrence in a low prevalence area

2020 ◽  
Author(s):  
Walter Randazzo ◽  
Pilar Truchado ◽  
Enric Cuevas-Ferrando ◽  
Pedro Simón ◽  
Ana Allende ◽  
...  
Keyword(s):  
Water Samples ◽  
N Gene ◽  
Environmental Surveillance ◽  
Effluent Water ◽  
Diarrhea Virus ◽  
Untreated Wastewater ◽  
Concentration Method ◽  
Diagnostic Panel ◽  
Influent Water ◽  
Low Prevalence

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), the area with the lowest COVID-19 prevalence within Iberian Peninsula. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was tested using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10.90 ± 3.54% and 10.85 ± 2.11% in influent water and 3.29 ± 1.58% and 6.19 ± 1.00% in effluent water samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater waters of 5.38 ± 0.21 log genomic copies/L on average. Two secondary water samples resulted positive (2 out of 18) and all tertiary water samples tested as negative (0 out 12). This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that SARS-CoV-2 was circulating among the population even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, this environmental surveillance could be implemented by municipalities right away as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

2021 ◽  
Author(s):  
Liyun Chang ◽  
Zhiyong Liu ◽  
Yuelan Zhao ◽  
Yan Li ◽  
Jianhua Qin
Keyword(s):  
Quantitative Pcr ◽  
Quantitative Methods ◽  
Diarrheal Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
N Gene ◽  
Established Method ◽  
Diarrhea Virus ◽  
Fluorescence Quantitative Pcr

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

scholarly journals One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

scholarly journals Pathogenicity and immunogenicity of a new strain of porcine epidemic diarrhea virus containing a novel deletion in the N gene

2020 ◽  
Vol 240 ◽  
pp. 108511 ◽  
Author(s):  
Xian-Wei Wang ◽  
Mi Wang ◽  
Jing Zhan ◽  
Qian-Yu Liu ◽  
Lin-lin Fang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
N Gene ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

An efficient virus concentration method and RT-nested PCR for detection of rotaviruses in environmental water samples

2005 ◽  
Vol 124 (1-2) ◽  
pp. 117-122 ◽  
Author(s):  
Leera Kittigul ◽  
Som Ekchaloemkiet ◽  
Fuangfa Utrarachkij ◽  
Kanokrat Siripanichgon ◽  
Dusit Sujirarat ◽  
...  
Keyword(s):  
Water Samples ◽  
Nested Pcr ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Virus Concentration ◽  
Concentration Method ◽  
Virus Concentration Method

scholarly journals Development of a Rapid Method for Simultaneous Recovery of Diverse Microbes in Drinking Water by Ultrafiltration with Sodium Polyphosphate and Surfactants

2005 ◽  
Vol 71 (11) ◽  
pp. 6878-6884 ◽  
Author(s):  
Vincent R. Hill ◽  
Amy L. Polaczyk ◽  
Donghyun Hahn ◽  
Jothikumar Narayanan ◽  
Theresa L. Cromeans ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Samples ◽  
Tap Water ◽  
Bacterial Species ◽  
Surfactant Solution ◽  
Tween 80 ◽  
Sample Collection ◽  
Sodium Polyphosphate ◽  
Concentration Method ◽  
Serovar Typhimurium

ABSTRACT The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.5-μm microspheres, Salmonella enterica serovar Typhimurium, Bacillus globigii endospores, and echovirus 1. Ten-liter tap water samples were concentrated to ∼250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2, C. parvum, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.

scholarly journals Self-Driven Pretreatment and Room-Temperature Storage of Water Samples for Virus Detection Using Enhanced Porous Superabsorbent Polymer (PSAP) Beads

2021 ◽  
Author(s):  
Wensi Chen ◽  
Ting Wang ◽  
Zeou Dou ◽  
Xing Xie
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Room Temperature ◽  
Low Cost ◽  
Virus Detection ◽  
Sample Pretreatment ◽  
Superabsorbent Polymer ◽  
Viral Pathogens ◽  
Untreated Wastewater ◽  
Degradation Rate Constant

Abstract The continuous emergence of infectious viral diseases has become a major threat to public health. To quantify viruses, proper handling of water samples is required to ensure the accuracy and reliability of the testing results. In this study, we develop enhanced porous superabsorbent polymer (PSAP) beads to pretreat and store water samples for virus detection. By applying PSAP beads to collect water samples, the viruses are captured and encapsulated inside the beads while undesired components are excluded. We have successfully demonstrated that the shelf life of the model virus can be effectively extended at room temperature (22°C) and elevated temperature (35°C). Both the infectivity level and genome abundance of the viruses are protected even in a complex medium like untreated wastewater. Under the tested conditions, the viral degradation rate constant can be reduced to more than 10 times using the PSAP beads. Therefore, the enhanced PSAP beads provide a low-cost and efficient sample pretreatment and storage method that is feasible and practicable for large-scale surveillance of viral pathogens in water samples.

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