“…The sodium nitrate added increases conductivity and hence assists in the detachment of virus particles from organic matter ( 50 ). Studies also showed that precipitation-based methods outperform IP concentration for the recovery of poliovirus, crAssphage, and SARS-CoV-2, suggesting that this process is robust and more resilient to organic matter than ultrafiltration ( 12 , 51 ), whereas others found that PEG precipitation performs similarly to Amicon ultrafiltration, with both methods resulting in approximately 40% SARS-CoV02 recoveries ( 29 ). The precipitation-based methods are very easy to perform, and the associated reagents are inexpensive and usually available in bulk quantities.…”
Section: Discussionmentioning
confidence: 99%
“…The precipitation-based methods are very easy to perform, and the associated reagents are inexpensive and usually available in bulk quantities. The bench time required for these methods is short, and many samples can be handled at once; however, the incubation times, especially for the PEG methods, significantly increase the overall processing time ( 29 ). Another disadvantage of precipitation-based methods is the potential resuspension of the pellet concentrate after centrifugation, and hence this step should be performed without delays.…”
Section: Discussionmentioning
confidence: 99%
“…Furthermore, the filter membranes are subject to clogging if the samples have a high turbidity. Nonetheless, precipitation- and ultrafiltration-based approaches are more robust and versatile and hence may be more suitable for many WBE applications ( 27 – 29 ).…”
As wastewater-based epidemiology is utilized for the surveillance of COVID-19 at the community level in many countries, it is crucial to develop and validate reliable methods for virus detection in sewage. The most important step in viral detection is the efficient concentration of the virus particles and/or their genome for subsequent analysis.
“…The sodium nitrate added increases conductivity and hence assists in the detachment of virus particles from organic matter ( 50 ). Studies also showed that precipitation-based methods outperform IP concentration for the recovery of poliovirus, crAssphage, and SARS-CoV-2, suggesting that this process is robust and more resilient to organic matter than ultrafiltration ( 12 , 51 ), whereas others found that PEG precipitation performs similarly to Amicon ultrafiltration, with both methods resulting in approximately 40% SARS-CoV02 recoveries ( 29 ). The precipitation-based methods are very easy to perform, and the associated reagents are inexpensive and usually available in bulk quantities.…”
Section: Discussionmentioning
confidence: 99%
“…The precipitation-based methods are very easy to perform, and the associated reagents are inexpensive and usually available in bulk quantities. The bench time required for these methods is short, and many samples can be handled at once; however, the incubation times, especially for the PEG methods, significantly increase the overall processing time ( 29 ). Another disadvantage of precipitation-based methods is the potential resuspension of the pellet concentrate after centrifugation, and hence this step should be performed without delays.…”
Section: Discussionmentioning
confidence: 99%
“…Furthermore, the filter membranes are subject to clogging if the samples have a high turbidity. Nonetheless, precipitation- and ultrafiltration-based approaches are more robust and versatile and hence may be more suitable for many WBE applications ( 27 – 29 ).…”
As wastewater-based epidemiology is utilized for the surveillance of COVID-19 at the community level in many countries, it is crucial to develop and validate reliable methods for virus detection in sewage. The most important step in viral detection is the efficient concentration of the virus particles and/or their genome for subsequent analysis.
“…This explains the low absorbance signal (OD 450 ) for the viral titer tested from 10 1 to 10 4 TCID 50 /mL in the direct ELONA and also could be due to the presence of fetal bovine serum and other substances like antibiotics present in the DMEM culture medium ( Choi et al., 2014 ), where the variants of SARS-CoV-2 were propagated, and can potentially affect the detection signal. A study also reported that high centrifugal speeds generated by fixed angle centrifuge rotors allow the precipitate to concentrate along the vertical axis of the sample tube, making it less visible and difficult to reconstitute, potentially resulting in viral precipitate loss and reduced yields ( Engler and Selepak, 1994 ; Mailepessov et al., 2022 ). As a result of that, centrifugation of the samples could also be a reason for low absorbance signal due to loss of viral precipitate or reduced viral loads.…”
The exponential spread of COVID-19 has prompted the need to develop a simple and sensitive diagnostic tool. Aptamer-based detection assays like ELONA are promising since they are inexpensive and sensitive. Aptamers have advantages over antibodies in wide modification, small size, in vitro selection, and stability under stringent conditions, which aid in scalable and reliable detection. In this work, we used aptamers against SARS-CoV-2 RBD S protein to design a simple and sensitive ELONA detection tool. Screening CoV2-RBD-1C and CoV2-RBD-4C aptamers and optimizing assay conditions led to the development of a direct ELONA that can detect SARS-CoV-2 RBD S glycoprotein in buffer solution and 0.1 % human nasal fluid with a detection limit of 2.16 ng/mL and 1.02 ng/mL, respectively. We detected inactivated Alpha, Wuhan, and Delta variants of SARS-CoV-2 with the detection limit of 3.73, 5.72, and 6.02 TCID50/mL, respectively. Using the two aptamers as capture and reporter elements, we designed a more sensitive sandwich assay to identify the three SARS-CoV-2 variants employed in this research. As predicted, a lower detection limit was obtained. Sandwich assay LOD was 2.31 TCID50/mL for Alpha, 1.15 TCID50/mL for Wuhan, and 2.96 TCID50/mL for Delta. The sensitivity of sandwich ELONA was validated using Alpha and Wuhan variants spiked in 0.1% human nasal fluid sample condition and were detected in 1.41 and 1.79 TCID50/mL LOD, respectively. SEM was used to visualize the presence of viral particles in the Delta variant sample. The effective detection of SARS-CoV-2 in this study confirms the potential of our aptamer-based technique as a screening tool.
“…Even the absence of wastewater treatment plant could not stop researchers to implement WBE studies in the remote location against all the odds. Figure-1 depicts a simple word cloud of the most used word in the publications pertaining to the domain 1 , 2 , 3∗ , 4∗ , 5 , 6∗ , 7 , 8∗ , 9∗ , 10 , 11∗ , 12∗ , 20 , 21 , 22 , 23 , 24 , 25 , 26∗ , 27∗ , 28∗ , 29 , 30 , 31∗∗ , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40∗ , 41 , 42∗∗ , 43∗∗ , 44 , 45 , 46 , 47 , 48∗ , 49 , 50∗∗ , 51 . Fig 1 A word cloud depicting the most frequently used keywords in the publications pertaining to the domain of WBE.…”
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