Wastewater-based epidemiology (WBE) is useful for the surveillance of severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) in communities, complementing clinical
diagnostic testing of individuals. In this Review, we summarize recent progress and
highlight remaining challenges in monitoring SARS-CoV-2 RNA in wastewater systems for
community and environmental surveillance. Very low concentrations of viral particles and
RNA present in the complicated wastewater and sewage sample matrix require efficient
sample processing and sensitive detection. We discuss advantages and limitations of
available methods for wastewater sample processing, including collection, separation,
enrichment, RNA extraction, and purification. Efficient extraction of the viral RNA and
removal of interfering sample matrices are critical to the subsequent reverse
transcription-quantitative polymerase chain reaction (RT-qPCR) for sensitive detection
of SARS-CoV-2 in wastewater. We emphasize the importance of implementing appropriate
controls and method validation, which include the use of surrogate viruses for assessing
extraction efficiency and normalization against measurable chemical and biological
components in wastewater. Critical analysis of the published studies reveals imperative
research needs for the development, validation, and standardization of robust and
sensitive methods for quantitative detection of viral RNA and proteins in wastewater for
WBE.
Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2
and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle
samples minimizes transmission to healthcare workers and relieves the pressure of
resources and healthcare personnel during the pandemic. This study aimed to develop an
enhanced method enabling simultaneous viral inactivation and RNA preservation during
on-site self-collection of saliva and gargle samples. Our method involves the addition
of saliva or gargle samples to a newly formulated viral inactivation and RNA
preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and
detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain
reaction directly from the magnetic beads. This method has a limit of detection of 25
RNA copies per 200 μL of gargle or saliva sample and 9–111 times higher
sensitivity than the viral RNA preparation kit recommended by the United States Centers
for Disease Control and Prevention. The integrated method was successfully used to
analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2
in 123 gargle and saliva samples collected daily from two NPS-confirmed positive
SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP
buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65
copies/200 μL sample) is stable in the VIP buffer at room temperature for at least
3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA
enables self-collection of samples, reduces risks associated with SARS-CoV-2
transmission, and maintains the stability of the target analyte.
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