Approach for Aim 2
Wastewater Characterization
WBT Sample Collection Strategy
To optimize the sampling strategy, two sets of experiments will be conducted to evaluate the utility of grab versus composite samples, one under controlled conditions in the laboratory (experiment #1A) and the other under field conditions (experiment #1B). Laboratory experiments (#1A) will focus on evaluating hold times of sewage samples in the laboratory. Field experiments (#1B) will focus on evaluating both hold times and hour-to-hour variability of sewage samples in the field. These samples will be collected from locations with contributions from watersheds characterized by the county, community, and building scales. The primary difference between experiments #1A and #1B is that experiments #1A will be conducted on one large 24 L grab sample to evaluate hold times in the laboratory. This process eliminates the effects of natural variability of sewage quality in the field. Experiments #1B will be conducted on separate 1 L grab samples as they vary naturally in time at the chosen sampling point. This set of experiments will be influenced by changes in water quality due to hold times, and also by the natural time fluctuations in water quality at a specific sampling point.
For the laboratory experiments (#1A; Fig. 1, below, left panel), a total of 7 samples will be evaluated at 24, 1-hour intervals, one collected from a county-scale network (CDWWTP), three from the community-scale (at University-owned pump stations servicing building clusters) and three from the building scale (University dormitory, hospital, academic building). Upon collection, these samples will be brought to the Sylvester Biospecimen Shared Resource (BSSR), and upon receipt, the sample will be split into 24 sub-samples. Each sub-sample will be analyzed in one-hour intervals. Each sub-sample will be split three ways, into A, B, and C.
Split A (100 mL) Ultrafiltration. We will use standard protocols for ultrafiltration (29), including separating the sediments followed by ultrafiltration (Centricon-70). The pellet from the first centrifugation step is then recombined with the concentrate. The target volume at the end of the ultrafiltration step is 1500 µL, thus providing a concentration factor of 47 (70/1.5). The concentrate will be preserved in an equal volume of DNA/RNA preservative (Zymo Shield) and stored at -80°C, with PCR/LAMP/RNA-seq testing for SARS-CoV-2, and E. coli by culture and qPCR, and PCR for fecal indicator virus human polyomavirus (HPyVs) (41).
Split B (800 mL) Normalization. Normalization parameters will include fecal indicator bacteria, FIB, (E. coli) by culture using standard spread plate procedures. As FIB also degrade in sewage over time, the degradation of the FIB may be an indicator of the degradation of viral RNAs. Once the FIB samples are processed, the remaining portion of Split B will be used for measuring physical chemical parameters using a calibrated water quality sonde (YSI) fitted with sensors for pH, conductivity, turbidity, temperature, and dissolved oxygen.
Split C (100 mL) Autosampler controls. Autosamplers composite samples will be held and then processed in a cumulative fashion. Samples will be composited in a cumulative fashion in 1-hour intervals for a total of 2.4 liters of sample at the end of the process. The composite sample generated from the “C” splits will then be split into a composite “A” split and a composite “B” split for the analysis of the same samples as listed above.
For the field experiments, a set of 7 autosamplers will be used. Six will be new autosamplers, the 7th will be an existing autosampler available at the CDWWTP (county-scale sewage). Of the new autosamplers, 3 will collect community-scale sewage and 3 will collect building-scale sewage. The 6 community-scale autosamplers will be set up as follows: 2 at the Gables campus (both servicing dormitories and academic buildings); 1 at the RSMAS campus (academic buildings without dormitories); and 3 at the MSoM campus (with one servicing the hospital system and two servicing the academic clusters of buildings). At each of the 6 sites, autosamplers will be set up to collect 24, 1-hour samples. The autosamplers will collect separate bottles for each grab sample. At each hour, the 1-L grab sample collected will be sent to the BSSR to be split into sub-samples A, B, and C, as described above. In a similar fashion, as for experiment #1A, at the end of the 24 hours, a composite will be prepared from the 24 individual grab samples and will be subsequently processed as per procedures for split A and B.
WBT Sample Concentration Comparison
Sample concentration techniques will be compared for a subset of the samples collected from Aim 1. The concentration techniques to be compared include ultrafiltration (UF) and flat filter electronegative concentration (EC). Samples to be processed will include the 7 samples from the holding time experiment. In addition to the 24 splits for the 1-hour holding experiment (analyzed by UF), these samples will be also analyzed by EC.
WBT Viral Detection Technology Development and Application
Three technologies will be utilized to detect the RNA of SARS-CoV-2 in wastewater and in biobanked samples at UM. These technologies include qRT-PCR, qLAMP, and a novel FA system. Once the samples are concentrated, they will be diluted in DNA/RNA shield until extraction. Nucleic acid extraction will be conducted using an Applied Biosystems Nucleic Acid Isolation Kit (MagMAX for Viruses and Pathogens) according to manufacturer instructions, which includes processing 200 µL through a binding bead kit using positive and negative controls. Nucleic acid is eluted from the beads using 50 µL of elution solution. The nucleic acid will undergo QC/QA, including RNA fragment analyzer and Qubit RNA quantification. The qRT-PCR will be conducted as per standard protocol for wastewater (29) and using the Perkin Elmer New Coronavirus Nucleic Acid Detection Kit to detect N gene and ORF1ab RNA. Standard curves will be prepared with inactivated virus and synthetic controls (BEI Resources NR-52350, heat-inactivated SARS-CoV-2 viral particles; Twist Bioscience, Synthetic SARS-CoV-2 RNA controls 1 & 2). If the qRT-PCR is positive, the sample will be sequenced for strain identification. cDNA will be synthesized from DNAse treated (Ambion DNA-fee) total nucleic acids using the NEBNext Ultra II first and second-strand synthesis. This will be used as a template for Swift Biosciences SNAP SARS-CoV-2 Panel sequencing.
The concentrate split from UF that is sent to WCM will be analyzed with qLAMP and also with RNA sequencing for metatranscriptomic analysis. Specifically, each aliquot will be measured with a qBit and split into three tubes (RNA-seq, gLAMP, and storage), including at least 100ng for the total RNA-seq (metatranscriptomics) and 10ng for qLAMP. If post-centrifugation samples are too low to split with those minimum values, then the total sample amount will be proportionally split into the two main assays (RNA-seq and qLAMP). qLAMP will be run on the TINY device and also on our qRT-PCR machine at WCM.
A subset of samples (n=100) will be analyzed by FA, a novel technology developed at UM by Dr. Mark Sharkey (see letter of support), combining standard PCR with a novel polymerase that can efficiently use both RNA and DNA as templates and retains polymerase activity in unprocessed biological fluids. This approach eliminates RNA extraction and cDNA synthesis steps. Positive viral RNA amplification is detected via sequence-specific fluorescent hydrolysis probes visualized at PCR endpoint by blue light excitation (Fig. 5). This approach has been used to test unprocessed clinical samples, but has not been validated for use with environmental samples.
