Passive Sampler

What is passive sampling of SARS-CoV-2 in wastewater?

Passive sampling involves the deployment of a device in a waterbody for a known time period, allowing for pollutants in the water to interact with the device. This interaction could include the association of a pollutant with a particular medium or substance or induces a chemical reaction within the device. At the end of the deployment, the passive sampler is analysed through visual inspection or via advanced laboratory analytical methods. A notable advantage of passive sampling in water systems is that the deployment is easy (i.e. no specialised skills required), rapid and usually does not require confined space entry permits. Furthermore, the continuous exposure of the passive sampler to the water column reduces the sampling errors that exist when taking discrete water samples. Consequently, passive sampling has had a significant uptake in freshwater resource settings, especially in the field of water chemistry, where both time- and flow-based passive sampling techniques have been validated.

The application of passive sampling in water and wastewater microbiology has not received much research attention. Some studies have used glass bead passive samplers, one to characterise colonising biofilms in groundwater and the other to monitor for pathogens in wastewater. Some have trialled several passive samplers, including Zetapor membranes, nylon materials, low-density polyethylene and polyvinylidene difluoride for the detection of herpesviruses and noroviruses in seawater. But most studies monitored pathogens in wastewater systems using the Moore’s swab, which is a piece of medical gauze that is placed in the wastewater for 1 to 7 days and is attached to a string for retrieval. Slight modifications to the Moore’s swab have been adopted to monitor for polioviruses in wastewater. Recently, researchers have revived the use of passive samplers for the detection of SARS-CoV-2 in wastewater. If you are interested in these publications, please navigate to https://www.passives4covid.org/publications.php.

Publication on our passive samplers

We now have our paper published online in Environmental Science and Technology: https://pubs.acs.org/doi/10.1021/acs.est.1c01530

Torpedo Passive Sampler Housing Units

We have developed some neat little units that can house many types of passive sampling materials - they are designed in a torpedo shape to reduce clogging and fouling during deployment in wastewater sewers. You can either place an order for some to be delivered to your work or you can download the 3D print files - for free!

ORDER YOURS TODAY: Please use this form to order some 3D printed torpedoes - these will be delivered to your door, fully assembled ready to deploy: https://forms.gle/ntPJj4iQSzG7cryX7

DOWNLOAD DESIGN FOR FREE: If you have access to your own 3D printer, you can download and use as you wish! To download, please fill out the following google form (we do not collect any personal information here, so please never enter passwords or anything like this): https://forms.gle/31XgRkGvfVc5fKvW7

Smaller Torpedos - PENSIVES: We have developed some nice little new passive sampling housings/units, small enough to fit into individual allotment connections. Want to download these little beauties? Check out this link: https://doi.org/10.17632/566rkkxcf9.1 You can also order these to your door - just use the same form as above and place "PENSIVE" in the comment box.

Assembly Procedure

This video demonstrates the appropriate method of putting together the passive sampler (for those who are printing their own). Please note, in this case we are using three different types of passive materials; a gauze, a membrane and a Qtip. We also make passive samplers with just membranes. The bolt that you see going into the lid of the passive sampler is to help reduce the buoyancy of the device and ensure it remains inundated during the deployment period.

Deployment Procedure

This video demonstrates the appropriate method of deploying a passive sampler into the sewer network. Please note, in this case the field worker checks for dangerous gases first and also collects a grab/spot sample.

Dismantling Procedure

This video demonstrates the appropriate process for dismantling of the passives sampler.

Extraction Procedure

Pre-processing and storage. All samples were transported to the laboratory on ice and pre-processed on the day of collection. Immediately after retrieval, passive sampling units were cleared of all obvious ragging materials. Passive sampling units were dismantled on the day of retrieval. Electronegative membranes and cotton buds were either used immediately for RNA extraction or directly frozen at -80oC until extraction was possible.

RNA Extraction. The electronegative membranes were directly placed into 2 mL garnet-type bead-beating tubes and then processed using a Qiagen RNeasy PowerMicrobiome kit (Qiagen, Germany), with the following modifications: (1) 100µL of phenol:chloroform:isoamyl (25:24:1, pH 6.5-8.0) was added to the bead beating tube before adding the sample, (2) beat-beating for 30sec at 4m/s (MP-Bio, USA), (3) DNase treatment was conducted for 15 minutes, and (4) final elution done using 50 µL of DEPC water (Sigma-Aldrich, Germany), passed through twice to ensure maximum yield. At least once on every day extraction was conducted, we also processed a method extraction blank. On some occasions, the Qiagen RNeasy PowerMicrobiome kits were not available so we used the Macherey-Nagel NucleoSpin RNA Stool kit (Macherey-Nagel, Germany) as per manufacturer’s instructions.

RNA transcription and qPCR

The SARS-CoV-2 Real-time RT-PCR Assay (PerkinElmer, USA; hereafter referred to as the PE assay), which is a combined reverse transcription and TaqMan based qPCR, was used to detect both the nucleocapsid N (via the FAM fluorophore) and the ORF-1ab (via the ROX fluorophore) genes of the SARS-CoV-2 virus. After significant testing, our process included slight variations from that of the PE manufacturer’s recommendation: 5µL of template was used in each reaction together with 10µL of the PE mastermix and 15µL of ultrapure DNase/RNAase free water (Invitrogen, USA). During the early stages of the experiment, between three and five technical replicates were used to help explore the between-replicate variability, after which we used duplicates. We always ran duplicate no template controls, which were always negative, and standard curves using five dilutions of the Twist synthetic SARS-CoV-2 RNA control 1 (GenBank ID: MT007544.1, Cat No: 102019), resulting in very high coefficients of determination (R2>0.99) and consistent intercepts (mean for N gene: 43.6; ORF-1ab gene: 42.5) and slopes (mean for N gene: -3.47; ORF-1ab gene: -3.38) resulting in acceptable qPCR efficiencies (E=94% and 98%, respectively). The manufacturer suggests their MS2 phage internal control (detected via the VIC fluorophore) is added to samples prior to bead-beating. However, the bead-beating appeared to shear the MS2 RNA, limiting its use as a full extraction control, and hence we instead added the MS2 RNA after the bead beating step. According to the manufacturers specifications, we re-ran samples that were inhibited according to this MS2 control at 1:10 dilutions (and if still inhibited at 1:33); this resulted in 34% of our samples being run at least 1:10 dilutions of the template. All assays were run with 45 cycles on a Bio-Rad Laboratories CFX-96 qPCR machine (Bio-Rad, USA). Each amplification curve was manually inspected by the same individual and cross-checked by another. Thresholds to determine Cq values were estimated using the “auto threshold” option in the Bio-Rad CFR Maestro 1.1 program (Bio-Rad, USA, 2017) and were compared between runs for consistency.