Advances in virus detection methods for wastewater-based epidemiological applications.

Wastewater-based epidemiology (WBE) is a powerful tool that has the potential to reveal the extent of an ongoing disease outbreak or to predict an emerging one. Recent studies have shown that SARS-CoV-2 concentration in wastewater may be correlated with the number of COVID-19 cases in the corresponding population. Most of the recent studies and applications of wastewater-based surveillance of SARS-CoV-2 applied the "gold standard" real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) detection method. However, this method also has its limitations. The paper aimed to present recent improvements and applications of the PCR-based methods for SARS-CoV-2 monitoring in wastewater. Furthermore, it aimed to review alternative methods utilized and/or proposed for the detection of the virus in wastewater matrices. From the review, it was found that several studies have investigated the use of reverse-transcription digital polymerase reaction (RT-dPCR), which was generally shown to have a lower limit of detection (LOD) over the RT-qPCR. Aside from this, non-PCR-based and non-RNA based methods have also been explored for the detection of SARS-CoV-2 in wastewater, with detailed attention given to the detection of SARS-CoV-2 proteins. The potential methods for protein detection include mass spectrometry, the use of immunosensors, and nanotechnological applications. In addition, the review of recent studies also revealed two types of emerging methods related to the detection of SARS-CoV-2 in wastewater: capsid-integrity assays to infer about the infectivity of SARS-CoV-2 present in wastewater, and alternative methods for detection of SARS-CoV-2 variants of concern (VOCs) in wastewater. The recent studies on proposed methods of SARS-CoV-2 detection in wastewater have considered improving this approach in one or more of the following aspects: rapidity, simplicity, cost, sensitivity, and specificity. However, further studies are needed in order to realize the full application of these methods for WBE in the field.