Variability analysis of a low-cost paper dipstick nucleic acid extraction method for wastewater surveillance using gage repeatability and reproducibility

variability-analysis-of-a-low-cost-paper-dipstick-nucleic-acid-extraction-method-for-wastewater-surveillance-using-gage-repeatability-and-reproducibility
Variability analysis of a low-cost paper dipstick nucleic acid extraction method for wastewater surveillance using gage repeatability and reproducibility

References

  1. Zheng, X. et al. Wastewater surveillance provides spatiotemporal SARS-CoV-2 infection dynamics. Engineering 40, 70–77 (2024).

    Google Scholar 

  2. Grassly, N. C., Shaw, A. G. & Owusu, M. Global wastewater surveillance for pathogens with pandemic potential: Opportunities and challenges. Lancet Microbe 6, 100939 (2025).

    Google Scholar 

  3. Paul, J. R., Trask, J. D. & Culotta, C. Poliomyelitic virus in sewage. Science 90, 258–259 (1939).

    Google Scholar 

  4. Holm, R. H. et al. Surveillance of RNase P, PMMoV, and CrAssphage in wastewater as indicators of human fecal concentration across urban sewer neighborhoods, Kentucky. FEMS Microbes 3, xtac003 (2022).

    Google Scholar 

  5. Goetgeluck, C. et al. Using wastewater surveillance to investigate community-level differences in antibacterial resistance in a major urban center, USA. Appl. Environ. Microbiol. 91, e01684 (2025).

    Google Scholar 

  6. Egaña, I. et al. Wastewater based epidemiology for the surveillance of illicit drug and substance of abuse use in prison settings: A critical review. Wiley Interdiscip. Rev. Forensic Sci. 7, e70004 (2025).

    Google Scholar 

  7. Li, J. et al. In-sewer stability of 31 human health biomarkers and suitability for wastewater-based epidemiology. Water Res. 249, 120978 (2024).

    Google Scholar 

  8. Burnor, E. et al. Wastewater for public health: timely, sensitive, and reliable SARS-CoV-2 omicron variant monitoring in California. Environ. Sci. Water Res. Technol. 11, 876–890 (2025).

    Google Scholar 

  9. Girón-Guzmán, I. et al. Urban wastewater-based epidemiology for multi-viral pathogen surveillance in the Valencian region, Spain. Water Res. 255, 121463 (2024).

    Google Scholar 

  10. Perez-Zabaleta, M. et al. Wastewater surveillance of SARS-CoV-2 from aircraft to citywide monitoring. Nat. Commun. 16, 1–9 (2025).

    Google Scholar 

  11. Jarvie, M. M., Nguyen, T. N., Southwell, B. & Wright, D. Leveraging wastewater surveillance to actively monitor covid-19 community dynamics in rural areas with reduced reliance on clinical testing. Appl. Res. 3, e202400012 (2024).

    Google Scholar 

  12. Maree, G. et al. Wastewater surveillance overcomes socio-economic limitations of laboratory-based surveillance when monitoring disease transmission: The south African experience during the covid-19 pandemic. PLoS ONE 20, e0311332 (2025).

    Google Scholar 

  13. Karthikeyan, S. et al. Rapid, large-scale wastewater surveillance and automated reporting system enable early detection of nearly 85% of covid-19 cases on a university campus. Msystems 6, 10–1128 (2021).

    Google Scholar 

  14. Yang, Z. Low-cost and rapid sensors for wastewater surveillance at low-resource settings. Nat. Water 1, 405–407 (2023).

    Google Scholar 

  15. Pillay, L., Amoah, I. D., Kumari, S. & Bux, F. Potential and challenges encountered in the application of wastewater-based epidemiology as an early warning system for covid-19 infections in south Africa. Acs Es&T Water 2, 2105–2113 (2022).

    Google Scholar 

  16. Black, J. et al. Epidemiological evaluation of sewage surveillance as a tool to detect the presence of covid-19 cases in a low case load setting. Sci. Total Environ. 786, 147469 (2021).

    Google Scholar 

  17. Saththasivam, J. et al. Covid-19 (SARS-CoV-2) outbreak monitoring using wastewater-based epidemiology in Qatar. Sci. Total Environ. 774, 145608 (2021).

    Google Scholar 

  18. Rainey, A. L., Liang, S., Bisesi, J. H. Jr., Sabo-Attwood, T. & Maurelli, A. T. A multistate assessment of population normalization factors for wastewater-based epidemiology of covid-19. PLoS ONE 18, e0284370 (2023).

    Google Scholar 

  19. National Academies of Sciences, Engineering, and Medicine. Increasing the Utility of Wastewater-Based Disease Surveillance for Public Health Action: A Phase 2 Report (National Academies Press, 2024).

  20. Ahuja, S., Tallur, S. & Kondabagil, K. Simultaneous microbial capture and nucleic acid extraction from wastewater with minimal pre-processing and high recovery efficiency. Sci. Total Environ. 918, 170347 (2024).

    Google Scholar 

  21. Zou, Y. et al. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol. 15, e2003916 (2017).

    Google Scholar 

  22. Boese, B., Corbino, K. & Breaker, R. In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification. Nucleosides, Nucleotides Nucleic Acids 27, 949–966 (2008).

    Google Scholar 

  23. Illumina. Eco™ thermal and optical systems deliver high precision (Technical Note: Real-Time PCR). https://www.illumina.com/documents/products/technotes/technote_eco_uniformity.pdf (2024). Accessed December 2024.

  24. Docampo-Vázquez, C., Gragera-Alia, T., Fernández-Domínguez, M., Zubizarreta-Macho, Á. & Aragoneses-Lamas, J. M. Novel digital technique for measuring the volumetric healing process of free gingival grafts surrounding dental implants. Front. Dent. Med. 5, 1372312 (2024).

    Google Scholar 

  25. Betancourt-Rodríguez, J., Zamora-Gasga, V. M., Ragazzo-Sánchez, J. A., Zapata, J. A. N. & Calderón-Santoyo, M. A standardized method for genus colletotrichum characterization by isothermal microcalorimetry using thermokinetic parameters. J. Microbiol. Methods 204, 106651 (2023).

    Google Scholar 

  26. Thakkar, H. et al. Cell-engineered recombinant (alpha)-synuclein: A gage r&r validated protocol. J. Proteome Res. 23, 16–24 (2023).

    Google Scholar 

  27. Ross, R. A., Foley, C. M., Jones, H. M. & Osinski, M. A. A method for assessing and monitoring consistency of nonclinical ECG analysis. J. Pharmacol. Toxicol. Methods 116, 107189 (2022).

    Google Scholar 

  28. Vega, C. G. et al. ROTADIAL: The first nanobody-based immunoassay to detect Group A Rotavirus. J. Virol. Methods 298, 114279 (2021).

    Google Scholar 

  29. LLC, C. Ford motor company, general motors corporation. Potential Failure Mode and Effect Analysis (FMEA) 67–112 (2008).

  30. Saikaew, C. An implementation of measurement system analysis for assessment of machine and part variations in turning operation. Measurement 118, 246–252 (2018).

    Google Scholar 

  31. Canadas, N., Machado, J., Meireles, J., Delgado, P. & Portinha, A. Development of an automatic system for the measurement of force and stroke parameters of car radio keypads. Measurement 100, 84–92 (2017).

    Google Scholar 

  32. Bottani, E., Montanari, R., Volpi, A. & Tebaldi, L. Statistical process control of assembly lines in manufacturing. J. Ind. Inf. Integr. 32, 100435 (2023).

    Google Scholar 

  33. Amara, S. B., Dhahri, J., Samet, S. & Fredj, N. B. Method for improving the measurement system selection depending on part and process precisions. Measurement 98, 103–111 (2017).

    Google Scholar 

  34. Wheeler, D. J. & Lyday, R. W. Evaluating the Measurement Process (SPC Press, Incorporated, 1989).

    Google Scholar 

  35. Wheeler, D. J. EMP III Using Imperfect Data (SPC Press, 2006).

  36. Almeida, F.D et al. A multivariate GR&R approach to variability evaluation of measuring instruments in resistance spot welding process. J. Manuf. Process.36, 465–479 (2018).

  37. Marques, R. A. M. et al. Multivariate GR&R through factor analysis. Measurement 151, 107107 (2020).

    Google Scholar 

  38. Wheeler, D. J. EMP III (Evaluating the Measurement Process): Using Imperfect Data (SPC PRESS (Statistical Process Control), 2006).

  39. Torii, S., Furumai, H. & Katayama, H. Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater. Sci. Total Environ. 756, 143067 (2021).

    Google Scholar 

  40. Hsu, S.-Y. et al. Biomarkers selection for population normalization in SARS-CoV-2 wastewater-based epidemiology. Water Res. 223, 118985 (2022).

    Google Scholar 

  41. Centers for Disease Control and Prevention. Developing a wastewater surveillance sampling strategy. https://archive.cdc.gov/www_cdc_gov/nwss/sampling.html (2020). Accessed 28 Jan 2026.

  42. Lau, M. et al. Selection of surrogate pathogens and process indicator organisms for pasteurisation of municipal wastewater-a survey of literature data on heat inactivation of pathogens. Process Saf. Environ. Prot. 133, 301–314 (2020).

    Google Scholar 

  43. Babler, K. M. et al. Comparison of electronegative filtration to magnetic bead-based concentration and V2G-qPCR to RT-qPCR for quantifying viral SARS-CoV-2 RNA from wastewater. ACS ES&T Water 2, 2004–2013 (2022).

    Google Scholar 

  44. Ahmed, W. et al. Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Sci. Total Environ. 805, 149877 (2022).

    Google Scholar 

  45. D’Aoust, P. M. et al. Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low covid-19 incidence and prevalence. Water Res. 188, 116560 (2021).

    Google Scholar 

  46. Mason, M. G. & Botella, J. R. Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks. Nat. Protoc. 15, 3663–3677 (2020).

    Google Scholar 

  47. Rucki, M., Barisic, B. & Szalay, T. Analysis of air gage inaccuracy caused by flow instability. Measurement 41, 655–661 (2008).

    Google Scholar 

  48. Rameez, R. et al. Evaluation of alternative methods for estimating the precision of REML-based estimates of variance components and heritability. Heredity 128, 197–208 (2022).

    Google Scholar 

  49. Kenett, R. S. & Shmueli, G. Clarifying the terminology that describes scientific reproducibility. Nat. Methods 12, 699–699 (2015).

    Google Scholar 

  50. Al-Refaie, A. & Bata, N. Evaluating measurement and process capabilities by GR&R with four quality measures. Measurement 43, 842–851 (2010).

    Google Scholar 

  51. Rosengart, A. L., Bidwell, A. L., Wolfe, M. K., Boehm, A. B. & Townes, F. W. Spatiotemporal variability of the pepper mild mottle virus biomarker in wastewater. ACS ES&T Water 5, 341–350 (2024).

    Google Scholar 

  52. Goitom, E. et al. Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto. Sci. Total Environ. 932, 172917 (2024).

    Google Scholar 

  53. Sreejith, S. et al. A comprehensive review on graphene FET bio-sensors and their emerging application in DNA/RNA sensing & rapid covid-19 detection. Measurement 206, 112202 (2023).

    Google Scholar 

  54. Alatraktchi, F. A. Rapid measurement of the waterborne pathogen pseudomonas aeruginosa in different spiked water sources using electrochemical sensing: Towards on-site applications. Measurement 195, 111124 (2022).

    Google Scholar 

  55. Ahuja, S., Kumar, M. S., Nandeshwar, R., Kondabagil, K. & Tallur, S. Longer amplicons provide better sensitivity for electrochemical sensing of viral nucleic acid in water samples using PCB electrodes. Sci. Rep. 12, 8814 (2022).

    Google Scholar 

  56. Kumar, M. et al. Electrochemical sensing of SARS-CoV-2 amplicons with PCB electrodes. Sens. Actuators, B Chem. 343, 130169 (2021).

    Google Scholar 

  57. Nandeshwar, R. et al. Portable absorbance platform for sensing of viral and bacterial nucleic acid leveraging intercalation with methylene blue: Application for wastewater-based epidemiology. Biosens. Bioelectron.: X 14, 100373 (2023).

    Google Scholar 

  58. Chu, D. et al. Simple, direct amplification of RNA-containing paper discs for diagnosing the hepatitis C virus. J. Med. Virol. 96, e29919 (2024).

    Google Scholar 

  59. Strohmeier, O. et al. Automated nucleic acid extraction from whole blood, B. subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk. RSC Adv. 5, 32144–32150 (2015).

    Google Scholar 

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