This document describes evaluation of a simple post-use decontamination of Jumbosep™ centrifugal units following concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in wastewater samples. The method is effective in preventing carry-over of viral nucleic acids to successive samples, which could lead to false-positive test results.
Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the coronavirus disease 2019 (COVID-19). Detection of SARS-CoV-2 RNA in untreated wastewater has opened the window for its use as a tool to monitor the prevalence of SARS-CoV-2 in the community in an approach known as wastewater-based epidemiology (WBE) (1, 2).
Processing of wastewater samples includes a step to concentrate the virus prior to nucleic acid extraction. Concentration of viral particles can be carried out by adsorption to an electronegative MCE membrane (1, 3) or by ultrafiltration (1, 2). Subsequent detection and quantitation of viral sequences is then carried out via real-time reverse transcriptase (RT)-PCR.
Provided a simple reliable decontamination process is in place, use of Jumbosep centrifugal units for viral concentration offers economic and ecological benefits as all parts of the filter unit excluding the membrane insert can be reused.
This document describes evaluation of a simple post-use filter-unit decontamination process that prevents carry-over of viral nucleic acids to successive samples which could lead to false-positive test results.
Materials and methods
Sampling and detection methodology
We took a 24 h composite wastewater sample from the tributary of the wastewater plant with the aid of an autosampler. The resulting sample was kept at 4°C during transport to the laboratory and processed immediately upon arrival.
For the detection of the virus in the residual water sample, we performed viral concentration according to an adsorption-elution protocol using an electronegative MCE membrane (47 mm GN-6 Metricel™ MCE membrane disc filters) (1, 4). Briefly, prior to filtration the sample was acidified to pH 3.5 with 2N hydrochloric acid and magnesium chloride was added to a final concentration of 25 mM. After filtration, we transferred the filter with retained viral particles into a bead beating tube and viral RNA was extracted with the Quick-RNA viral kit (Zymo Research) with the aid of a bead mill. We performed detection/quantitation of the SARS-CoV-2 E gene sequences by real-time RT-PCR. Once confirmed positive, the sample was used for the decontamination experiment described below.
Viral concentration with Jumbosep centrifugal filters
We performed viral concentration by ultrafiltration using the Jumbosep according to the method described in reference 2. Briefly, large particulates (debris and bacteria) were first removed from the samples by centrifugation at 3000 × g for 30 min without brake. Subsequently, a 40 mL volume of the supernatant was filtered with a Jumbosep filter with a 100 kD molecular weight cut-off (MWCO) at 1500 × g for 15 min.
Viral extraction and quantitation
RNA extractions from 300 μL sample aliquots of the viral concentrate were carried out using the Quick-RNA viral extraction kit (Zymo Research) according to manufacturer’s instructions with sample elution volumes of 20 μL. We detected SARS-CoV-2 virus by real-time RT-PCR using the E gene primers from the Berlin-Charité protocol (4).
All RT-PCR tests were performed using the SuperScript III one-step RT-PCR system with Platinum Taq polymerase (Thermo Fisher Scientific). Each 25 μL reaction contained 12.5 μL of the reaction mix, 1 μL of enzyme mix, 0.5 μL of 5 μM probe, 0.5 μL of each primer of 20 μM, 3.5 μL of nuclease-free water and 5 μL of RNA. Amplification was performed on an Applied Biosystems 7500 real-time PCR instrument (Thermo Fisher Scientific). Reaction conditions consisted of 15 min at 50°C for reverse transcription, 2 min at 94°C for activation of Taq polymerase and 40 two-step amplification cycles of 3 s at 94°C and 30 s at 58°C, followed by a final extension of 3 min at 68°C. SARS-CoV-2 detection assays were conducted simultaneously together with a positive amplification control consisting of a plasmid containing SARS-CoV-2 E gene sequences (plasmid E) and a no template control (NTC).
Decontamination of Jumbosep filter units
Following filtration of the SARS-CoV-2 E positive water sample as described in the sampling and detection methodology section, the decontamination process involved removal of the Jumbosep filter unit membrane insert. After this, the sample reservoir and filtrate receiver were first immersed for 1 h in a solution of 2% Extran MA 02 neutral detergent (EMD Millipore) and 0.05% sodium hypochlorite and subsequently rinsed three times with molecular biology-grade water (Condition A). A filter unit that did not undergo the detergent/sodium hypochlorite immersion step, but did undergo the molecular biology grade water rinses (Condition B), served as control for the decontamination process.
We evaluated the effectiveness of both procedures by passing molecular grade water through the treated Jumbosep units with new inserts in a 15 min centrifugation step at 1500 × g. The effect of the decontamination procedure on the detection assay was evaluated by filtering molecular grade water spiked with plasmid E using a filter unit that had undergone decontamination according to Condition A. We evaluated the resulting concentrates for the presence of SARS-CoV-2 E gene sequences as described in the viral extraction and quantitation section. Assay controls further consisted of a wastewater concentrate positive for SARS-CoV-2 (positive control) and no template control.
Results
Repeated use of Jumbosep filter units for concentration requires a simple and effective decontamination procedure to prevent carry-over contamination in subsequent samples.
The decontamination process we used consists of a 1 h immersion in a solution of a neutral detergent and 0.05% sodium hypochlorite followed by three washes with molecular grade water. The filters, which we first used for concentrating a known SARS-CoV-2 positive wastewater sample were then treated with the above-described decontamination process (Condition A) or a mock treatment (Condition B) consisting of three washes with molecular grade water and omitted the detergent/sodium hypochlorite immersion step.
Table 1 presents the results of a SARS-CoV-2 real-time RT-PCR assay carried out on the resulting sample concentrates. Samples obtained from filters that were decontaminated (Condition A) tested negative in the SARS-CoV-2 real-time RT-PCR test, whereas samples obtained from mock-treated filters tested positive.
The decontamination process did not affect the downstream detection as shown by the fact that the real-time RT-PCR assay was able to detect SARS-CoV-2 E gene sequences in a sample consisting of molecular biology grade water spiked with plasmid E after concentrating it in a decontaminated filter.
Together, these results indicate that the decontamination process is effective in preventing carry-over of viral nucleic acids to successive samples without impacting the detection assay.
Table 1. SARS-CoV-2 real-time RT-PCR test results of water sample concentrates from Jumbosep centrifugal filters
| Sample | Decontamination procedure | Real-time RT-PCR |
|
|---|---|---|---|
| Average Ct |
Score |
||
| Wastewater concentrate positive for SARs | N/A | 33.11 |
Positive |
| Molecular biology grade water | Condition A | > 38 |
Negative |
| Molecular biology grade water | Condition B | 36.68 |
Positive |
| Molecular biology grade water with plasmid E |
Condition A |
22.72 |
Positive |
| NTC control |
N/A |
— |
Negative |
Conclusions
Repeated use of Jumbosep centrifugal filters for concentration of viral particles in wastewater samples requires a simple and effective decontamination procedure to prevent carry-over contamination in subsequent samples to eliminate the possibility of carry-over contamination.
We show in this study that a decontamination process consisting of a 1 h immersion in a solution of a neutral detergent and 0.05% sodium hypochlorite followed by three washes with molecular grade water is effective in preventing carry-over of detectable viral nucleic acid sequences in subsequent samples.
The method described does not impact on the real-time RT-PCR detection assay and therefore is a viable way of treating the centrifugal filters between use.
References
- Ahmed W, Bertsch PM, Bivins A, Bibby K, Farkas K, Gathercole A, et al. Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater. Sci Total Environ. 2020;739 June:139960. doi:10.1016/j.scitotenv.2020.139960.
- 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. 2020. doi:10.1021/acs.estlett.0c00357.
- Bivins A, Ahmed W, North D, Bibby K. Wastewater concentration by adsorption and direct extraction for SARS-CoV-2 RNA detection and quantification using RT-ddPCR V.2. 2020. doi: 10.17504/protocols. io.bhiuj4ew
- Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25:1–8.
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