Lab Matters Fall Winter 2021 - Page 20


Developing Advanced Molecular Assays for SARS-CoV-2 and Other Pathogens in Wastewater

By Madhuri Jasti , Thermo Fisher Scientific and Alexandre “ Sasha ” Vlassov , Thermo Fisher Scientific
Wastewater surveillance — tracing various deadly pathogens in wastewater and sewage — was ongoing for many years , however at only a handful of locations worldwide . Interest in wastewater molecular testing and its numerous applications started growing exponentially last year , with a primary goal to support COVID-19 pandemic response . Scientists in every major city of the United States and all key European countries are now monitoring the spread of SARS-CoV-2 in sewage , following the discovery that virus particles are shed in feces of infected individuals . 1 , 2 SARS-CoV-2

was also detected in wastewater samples collected from commercial passenger aircrafts and cruise ships , opening new applications for wastewater-based epidemiology beyond community monitoring . 3

The goals for various organizations performing testing and surveillance are very different , and include early-warning systems , prediction of surges , estimation of the overall numbers of infected people in cities or regions , and monitoring emerging virus strains . The newlydeveloped workflow solutions for robust detection of SARS-CoV-2 in wastewater samples using the MagMAX™ Wastewater Ultra Nucleic Acid Isolation Kit , automated on the KingFisher™ Purification System , with downstream analysis by qPCR , digital PCR or next generation sequencing ( NGS ) can enable superior wastewater surveillance .
Testing Protocols
1 . Parasa S , Desai M , Thoguluva Chandrasekar V , et al . Prevalence of gastrointestinal symptoms and fecal viral shedding in patients with coronavirus disease 2019 : a systematic review and meta-analysis . JAMA Netw Open 2020 ; 3 : e2011335 .
2 . Medema G , Heijnen L , Elsinga G , Italiaander R , Brouwer A . Presence of SARS-coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in The Netherlands . Environ Sci Technol Lett 2020 ; 7:511 – 6 .
3 . Ahmed W , Bertsch PM , Angel N et al . ( 2020 ) Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater : a surveillance tool for assessing the presence of COVID-19 infected travelers . J Trav Med 27 ( 5 ): taaa116 .
Madhuri Jasti processes wastewater samples with a MagMax™ kit on the KingFisher Apex instrument . Photo : Thermo Fisher Scientific
Prior to analysis , infectious agents in wastewater are typically concentrated to maximize the chances of successful detection . Once concentrated , nucleic acid is purified from the enveloped and non-enveloped viral particles and bacterial targets , and quantified using qRT-PCR or other molecular analysis techniques , which enable the accurate and sensitive detection and analysis . Thermo Fisher Scientific has developed cost-effective automation solutions that minimize hands-on time for molecular testing to support all diverse wastewater testing programs .
As a part of MagMax™ kit development , multiple workflows were developed to process different volumes of wastewater samples , using several upfront concentration methods .
Nucleic acid purification workflows were developed for :
• 200 µ l wastewater samples concentrated by centrifugation or ultrafiltration or precipitation
• 1mL pre-concentrated or direct sludge samples
• 10mL wastewater samples with pre-enrichment of viral targets using Dynabeads ® intact virus enrichment beads
• 50 – 500mL wastewater samples with pre-concentration of viral targets by filtration method .
All these workflows can be performed manually as well as in the fully automated format on the KingFisher™ Purification System ( Flex / Apex / Duo Prime ) to enable high-throughput sample processing . These workflows are fully compatible with downstream analysis of viral and bacterial pathogens by qPCR ( using target-specific TaqMan assays ), digital PCR and NGS .
This streamlined process of robust viral and bacterial nucleic acid purification , harnessing the power of automation and molecular analysis by a range of PCR and NGS platforms , is ensuring accuracy and efficiency to wastewater monitoring for

COVID-19 and other biological threats . n

Thermo Fisher Scientific is an APHL Diamond Level Sustaining Member .
18 LAB MATTERS Fall / Winter 2021
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