APHL 2021 Poster Abstracts
A Direct Nucleic Acid Capture Method for Purification SARS-CoV-2 Genetic Material from Wastewater Provides a Convenient Tool for Epidemiological Surveillance
S . Mondal and N . Feirer , Promega Corporation , Madison , WI
Early in the COVID-19 pandemic , it was demonstrated that the genetic material of SARS-CoV-2 could be detected in the feces of infected individuals and thereafter in wastewater . This finding mobilized investigation if wastewater-based epidemiology ( WBE ) using the genetic signal of SARS-CoV-2 could be used to track the spread of COVID-19 in communities . The SARS-CoV-2 genetic signal is present at a low concentration in wastewater , making sample concentration a prerequisite for sensitive detection and utility in WBE . Most of the viral concentration methods were originally developed to concentrate non-enveloped viruses . We hypothesized that since the SARS-CoV-2 genetic material in the wastewater is primarily compromised virions present as nucleoprotein complex , a direct capture method that renders total nucleic acid conducive to binding to a silica-based affinity resin , may be able to overcome the cumbersome viral concentration steps . This led to development of a simple , rapid and modular alternative to existing purification methods from wastewater . Chaotropic agents are added to raw sewage allowing nucleic acid binding to a silica matrix , inhibitors that may have co-purified is washed by successive alcohol washes to and subsequently eluted in water . The eluted nucleic acid is further processed in a second step with either a spin column or using an automated nucleic acid purification system , like the Maxwell RSC ®. The process recovered 63.13 ± 4.16 %, 40.09 ± 10.89 %, 39.67 ± 10.66 %, of HCoV OC43 , 229E and bacteriophage MS2 respectively spiked into the wastewater samples . Assuming wastewater may contain RT-qPCR inhibitors , we formulated enzyme mixes containing MMLV-RT and Taq DNA polymerase with hot-start chemistry that demonstrated resistance to PCR inhibitors found in wastewater like humic acid , urea and bile salts . We developed a RTqPCR assay using this enzyme mix to detect N1 , N2 ( nucleocapsid ) and E ( envelope ) gene fragment of SARS-CoV-2 in a multiplexed assay . Pepper Mild Mottle Virus ( PMMoV ) a fecal indicator RNA virus present in wastewater and an exogenous inhibition control were included in all PCR reactions for quality control . The workflow had a limit of detection of 1.6 GC / ml with 40 ml of raw wastewater sample . Using this workflow , we monitored wastewater samples from three wastewater treatment plants in Dane county , in south-central Wisconsin , serving a population about 300,000 for four months covering the peak of community prevalence . Our analysis was compared to the COVID-19 cases declared by the municipalities , showed strong correlation between the viral loads and its trends in wastewater and the clinical cases . Data obtained here highlights the potential for WBE for surveillance of COVID-19 and other infectious agents in a community .
Presenter : Subhanjan Mondal , Promega , subhanjan . mondal @ promega . com
SARS-CoV-2 Next Generation Sequencing and Genomic Epidemiology in Florida
J . Motes , S . Schmedes , O . Tekin , J . Yglesias , J . Weiss , Schimenti , E . Kopp and J . Blanton , Florida Bureau of Public Health Laboratories , Jacksonville , FL
Introduction : Tiled-amplicon whole genome sequencing is currently the gold-standard for SARS-CoV-2 ( SC2 ) variant detection and molecular epidemiology , to aid in public health surveillance and outbreak investigations . In April 2020 , Florida Bureau of Public Health Laboratories ( BPHL ) implemented SC2 sequencing for state-wide surveillance and targeted sequencing for outbreak investigations . Here , we present our workflow for SC2 sequencing from primary clinical specimens at BPHL .
Methods : Wet lab : A representative subset of COVID-19-positive specimens , sent to BPHL for diagnostic testing , are selected for sequencing for state-wide surveillance . Additional samples are sequenced per request to aid in epidemiologic investigations . cDNA is generated from purified RNA , and tiled amplicons are generated using ARTIC v3 primer sets . Libraries are prepared using Illumina ’ s NexteraXT library prep kit and are sequenced on the Illumina MiSeq or NextSeq 550Dx . Bioinformatics : SC2 consensus assemblies and variant profiles are generated using a custom-built pipeline , FLAQ- SC2 , and lineages are assigned using Pangolin . Phylogenies used for cluster detection , outbreak investigation , and phylodynamics are constructed using NextStrain .
Results and Discussion : BPHL has sequenced 7,428 SC2 isolates that have passed internal quality control and have been uploaded to NCBI Genbank and GISAID . These isolates aid both state-wide and national SC2 surveillance efforts . Additionally , BPHL provides support to state epidemiologists for targeted investigations . Recently , BPHL aided a COVID-19 transmission investigation among high school athletes in Florida . BPHL sequenced 18 COVID-19- positive specimens from individuals with known attendance , or potential contact with individuals in attendance , at a high school wrestling tournament with a suspect index case . Three distinct clusters were observed in the Florida SC2 phylogeny among these 18 samples , with each cluster belonging to a different lineage ( B . 1.361 ( n = 14 ), B . 1.2 ( n = 3 ), B . 1.1.64 ( n = 1 )). In this investigation , SC2 sequencing provided genomic data to reject the hypothesis that COVID-19 cases with association to the wrestling tournament were the result of a super-spreader event from a single index patient . In addition , genomic data also was used to aid in contact tracing to exclude a potential exposure route among a household member of an individual with unknown attendance at the tournament .
Presenter : Jessy Motes , Florida Bureau of Public Health Laboratories , jessy . motes @ flhealth . gov
COVID-19
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Summer 2021 LAB MATTERS 45 |