Magnetic carbon bead-based concentration method for SARS-CoV-2 detection in wastewater

doi:10.21203/rs.3.rs-5299217/v1

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Magnetic carbon bead-based concentration method for SARS-CoV-2 detection in wastewater

https://doi.org/10.21203/rs.3.rs-5299217/v1

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Wastewater surveillance for pathogens is important to monitor disease trends within communities and maintain public health; thus, a quick and reliable protocol is needed to quantify pathogens present in wastewater. In this study, a method using a commercially available magnetic carbon bead-based kit, i.e., the Carbon Prep (C.prep) method (Life Magnetics), was employed to detect and quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bacteriophage Phi6 and pepper mild mottle virus (PMMoV) in wastewater samples. The performance of this method was evaluated by modifying several steps and comparing it with the polyethylene glycol (PEG) precipitation method to demonstrate its applicability to virus detection in wastewater. The protocol of the C.prep method, based on the manufacturer’s instructions, could not detect SARS-CoV-2 RNA, while the optimized protocol could detect it in the tested samples at concentrations that were not significantly different from those obtained using the PEG precipitation method. However, the optimized C.prep method performed more poorly in recovering Phi6 and detecting PMMoV than the PEG precipitation method. The results of this study indicated that the full workflow of the C.prep method was not sufficient to detect the target viruses and that an additional RNA extraction step was needed to remove inhibitors in wastewater.

Carbon bead

Carbon Prep

SARS-CoV-2

virus concentration

wastewater-based epidemiology

Wastewater-based epidemiology (WBE) is a potential approach for providing unbiased information on community-level incidents associated with several diseases, such as coronavirus disease 2019 (COVID-19) (Bibby et al., 2021; Kumar et al., 2021; Nemudryi et al., 2020; Róka et al., 2021) and gastroenteritis (Chacón et al., 2021; Hotta et al., 2023). Compared to clinical testing, WBE is more cost-effective because it requires fewer resources for the analysis of pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enteric viruses in the laboratory to describe the dynamics or fluctuation of disease incidence in sewer catchment areas (Jiang et al., 2023).

Many factors limit the application of WBE: one of them is pathogen analysis. As the first step of this analysis, concentrating the pathogens is necessary because of their low abundance in wastewater (Jiang et al., 2023). Depending on the concentration method applied, different virus detection and recovery values are obtained (Ahmed et al., 2021, 2020; Angga et al., 2023; Barril et al., 2021; Farkas et al., 2022; Kaya et al., 2022). Due to its simplicity and affordability, the polyethylene glycol (PEG) precipitation method is widely used to concentrate pathogens in wastewater. In addition, this method is more sensitive in detecting SARS-CoV-2 and enteric viruses than other methods (Angga et al., 2023).

A relatively new detection method based on carbon-coated magnetic nanoparticles is the Carbon Prep (C.prep) Wastewater RNA Isolation Kit (Life Magnetics, Plymouth, MI, USA). This protocol, known as the C.prep method, aims to isolate RNA in a single workflow by using magnetic carbon beads to adsorb the viruses contained in samples. Specifically, it has been proven successful in isolating RNA from cells (Jones et al., 2019; Li et al., 2020; Shah et al., 2018).

The performance of the C.prep method in concentrating viruses as well as isolating RNA from wastewater samples is still unknown compared with that of other detection methods. Therefore, this study aimed to evaluate and optimize the C.prep method and compare its performance with that of the PEG precipitation method and a second method that included an additional RNA extraction step. Indigenous SARS-CoV-2 and the spiked bacteriophage Phi6 were selected as performance indicators, along with pepper mild mottle virus (PMMoV) as a process control because of its abundance in wastewater (Kitajima et al., 2018).

2.1 Wastewater samples

Influent wastewater samples were collected from five wastewater treatment plants (WWTPs) located in Yamanashi Prefecture, Japan, via grab sampling between June and November 2023. Autoclaved polyethylene bottles were used to collect the samples, which were then transported to the laboratory on ice packs and processed within 48 h (concentration step).

2.2 Preparation of the spiked viral stock

The Pseudomonas phage Phi6 (NBRC 105899, National Institute of Technology and Evaluation (NITE), Tokyo, Japan), a surrogate of enveloped viruses (Aquino De Carvalho et al., 2017), was cultured using a host strain of Pseudomonas syringae (NBRC 14084, NITE), as previously described (Torii et al., 2021). The initial concentration of the Phi6 stock was approximately 10⁸ to 10¹⁰ plaque-forming units/mL. The Phi6 stock was diluted by 100–1,000 times, depending on the initial concentration, and then seeded into samples at a ratio of 1:1,000 to the processed volume. The spiked samples were slowly mixed using a rotator (Nichiryo, Koshigaya, Japan) at 20–30 rpm or a magnetic stirrer (As One, Osaka, Japan) for at least 10 min before being subjected to concentration methods. The recovery of spiked viruses was calculated from the copy number obtained from the spiked samples per copy number of diluted stocks.

2.3 C.prep method

The experimental design of this study is shown in Fig. 1. A total of 70 µL of magnetic carbon beads provided in the kit was added to 40 mL of spiked wastewater samples, followed by slow mixing in the rotator at 20–30 rpm for 20–30 min at room temperature. The magnetic carbon beads were separated using a DynaMag-15 Magnet rack (Thermo Fisher Scientific, Waltham, MA, USA), and the liquid portion was carefully removed using a transfer pipette and then discarded. The solid portion was resuspended with 750 µL of RPS Wash Buffer (provided in the kit) and transferred into a 1.5-mL tube. The workflow until this step was referred to as the “Half protocol” (Fig. 1a). The final product of this protocol was treated as the concentrated sample, which was then subjected to RNA extraction.

For the second workflow, steps similar to those in the Half protocol were followed, and the resuspended solid portion (with RPS Wash buffer) was placed in the DynaMag-2 Magnet rack (Thermo Fisher Scientific). Then, the liquid portion was carefully removed using a micropipette. The beads were resuspended again with 750 µL of Wash Buffer (provided in the kit), and the liquid portion was decanted again after bead separation. The washing step was repeated a second time using 500 µL of Wash Buffer. The remaining solid portion in the tube was dried by leaving the cap open for 10 min and then eluted with 40 µL of Elution Buffer (provided in the kit), following the manufacturer’s instructions. The eluted sample was heated at 65°C for 10 min and then centrifuged at 10,000 × g for 10 min at 4°C to separate the beads. Finally, the liquid portion was recovered as RNA. The protocol until this step was referred to as the “Full protocol 1” (Fig. 1a).

The third workflow was mainly like the second workflow, except that the heating in the last step was eliminated, and the amount of added Elution Buffer was increased to 140 µL to accommodate additional RNA. This protocol was referred to as the “Full protocol 2” (Fig. 1a).

Different volumes of magnetic carbon beads were added to the wastewater samples to optimize the C.prep method (Fig. 1b). The potential application of this method to an automated system was also investigated by reducing the processing volume to 10 mL (Fig. 1c). To reduce the overall processing time, the reaction time of the carbon beads and samples was also limited to 10 min (Fig. 1d).

2.4 PEG precipitation method

The PEG precipitation method was conducted by following the protocol of IDEXX Laboratories with slight modifications, as previously described (Malla et al., 2022). In brief, 4.0 g of PEG 8000 (Sigma-Aldrich, St. Louis, MO, USA) and 0.94 g of NaCl (Kanto Chemical, Tokyo, Japan) were added into a 40-mL sample without pre-treatment to obtain final concentrations of 10% (w/v) and 0.4 M, respectively. Then, the sample was centrifuged at 12,000 × g for 99 min at 4°C. The resulting supernatant was carefully removed until approximately 5 mL of the sample remained in the tube and then discarded. Then, the sample was centrifuged again at 12,000 × g for 5 min at 4°C and the supernatant was completely removed by carefully pouring it off. The pellet was resuspended in 800 µL of autoclaved ultrapure water (Merck, Darmstadt, Germany) to obtain a concentrated sample of ~ 1 mL.

2.5 RNA extraction

The extraction of RNA from the processed samples (Fig. 1) (including the RNA obtained from the Full protocol 2 of the C.prep method) was conducted using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The extraction was performed using 140-µL samples to obtain 60 µL of RNA extract and was done automatically using the QIAcube robotic system (QIAGEN).

2.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

SARS-CoV-2, PMMoV, and Phi6 were detected using a SARS-CoV-2 Detection RT-qPCR Kit for wastewater (Takara Bio, Kusatsu, Japan); then, the viral RNA extracts were subjected to one-step RT-qPCR based on Raya et al. (2024). In brief, each 25 µL of RT-qPCR mixture contained 5.0 µL of RNA, 2.5 µL of a mixture of primers and probe, 12.5 µL of One-Step RT-qPCR Mix, and 5.0 µL of RNase-free water. The primer and probe sequences of the target viruses used were based on previously published RT-qPCR assays (Centers for Disease Control and Prevention, 2020; Gendron et al., 2010; Haramoto et al., 2013; Zhang et al., 2006). RT-qPCR amplification was carried out using a Thermal Cycler Dice Real Time System III (Takara Bio) at the following thermal conditions for all assays: initial incubation at 25°C for 10 min, RT reaction at 52°C for 5 min and 95°C for 10 s, 45 cycles of denaturation at 95°C for 5 s, and annealing and extension at 60°C for 30 s.

To obtain a standard curve, the positive control DNA provided in the SARS-CoV-2 detection RT-qPCR Kit for wastewater was used in the SARS-CoV-2, PMMoV, and Phi6 assays. These standards were serially diluted by 10-fold using EASY Dilution solution (for Real Time PCR) (Takara Bio) to obtain concentrations from 5 × 10⁰ to 5 × 10⁵ copies/µL. A negative control was also included in every qPCR run. The reactions for all samples, including the standards and negative control, were performed in duplicate (qPCR technical replicates). Threshold cycle (Ct) values > 40 were considered negative.

2.7 Statistical analysis

The paired t-test was used to compare virus concentrations and recovery values between different protocols or methods. The statistical analysis was conducted using Microsoft Excel 2019 (Microsoft Corporation, Redmond, WA, USA).

3.1 Workflow of the C.prep method

Table 1 shows the performance of the C.prep method with the different tested workflows in detecting the target viruses in the wastewater samples. These viruses were not detected in any of the samples using the Full protocol 1, while the Full protocol 2 and Half protocol showed no significant differences (n = 4; paired t-test, P > 0.05) in SARS-CoV-2 (4.8 ± 0.3 log copies/L and 4.8 ± 0.4 log copies/L, respectively) and PMMoV (8.0 ± 0.1 log copies/L and 8.2 ± 0.3 log copies/L, respectively) concentrations. In addition, no significant differences (n = 4; paired t-test, P > 0.05) in the recovery of spiked Phi6 were detected between the Full protocol 2 and the Half protocol (0.5 ± 0.8% and 0.8 ± 0.9%, respectively). As these two protocols included an additional RNA extraction step, the Half protocol was selected as the optimized one due to the shorter processing time, considering that magnetic bead-based extraction and heat extraction were conducted in the Full protocol 2.

Table 1

Detection of target viruses using different workflows of the C.prep method
Workflow	No. of tested samples	SARS-CoV-2			Phi6	PMMoV
Workflow	No. of tested samples	No. of detected samples	No. of quantified samples	Concentration (mean ± SD^a; log copies/L)	Recovery (mean ± SD; %)	No. of detected samples	No. of quantified samples	Concentration (mean ± SD; log copies/L)
Full protocol 1	4	ND^b	ND	NA^c	ND	ND	ND	NA
Full protocol 2	4	4	4	4.8 ± 0.3	0.5 ± 0.8	4	4	8.0 ± 0.1
Half protocol	4	4	4	4.8 ± 0.4	0.8 ± 0.9	4	4	8.2 ± 0.3

^aSD, standard deviation; ^bND, not detected; ^cNA, not available.

3.2 Carbon bead concentration

As shown in Table 2, doubling the carbon bead volume from 70 µL to 140 µL resulted in no significant differences (n = 5; paired t-test, P > 0.05) in SARS-CoV-2 (4.5 ± 0.2 log copies/L and 4.6 ± 0.5 log copies/L, respectively) and PMMoV (7.3 ± 0.5 log copies/L and 7.0 ± 0.3 log copies/L, respectively) concentrations, or in the recovery of spiked Phi6 (4.4 ± 3.5% and 4.7 ± 3.1%, respectively). Thus, the carbon bead volume of 70 µL was used for the optimized C.prep protocol in this study.

Table 2

Effect of carbon bead addition on the performance of the C.prep method
Carbon beads (µL)	No. of tested samples	SARS-CoV-2			Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
Carbon beads (µL)	No. of tested samples	No. of detected samples	No. of quantified samples	Concentration (mean ± SD; log copies/L)	Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
70	5	5	5	4.5 ± 0.2	4.4 ± 3.5	7.3 ± 0.5
140	5	5	5	4.6 ± 0.5	4.7 ± 3.1	7.0 ± 0.3

3.3 Volume of processed samples

Processing volumes of 40 mL and 10 mL resulted in Phi6 recovery values of 3.8 ± 2.3% and 4.1 ± 0.8%, respectively, and PMMoV concentrations of 7.5 ± 0.2 log copies/L and 7.7 ± 0.2 log copies/L, respectively. While the two different processing volumes (Table 3) did not result in significantly different Phi6 recovery values and PMMoV concentrations (n = 5; paired t-test, P > 0.05), the use of 10-mL samples resulted in a less sensitive detection of SARS-CoV-2 (3 out of 5 samples detected) compared with the use of 40-mL samples (4 out of 5 samples detected). Therefore, the latter volume was still used in the optimized protocol.

Table 3

Effect of the volume of processed samples on the performance of the C.prep method
Processing volume (mL)	No. of tested samples	SARS-CoV-2			Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
Processing volume (mL)	No. of tested samples	No. of detected samples	No. of quantified samples	Concentration (mean ± SD; log copies/L)	Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
40	5	4	4	4.9 ± 0.2	3.8 ± 2.3	7.5 ± 0.2
10	5	3	3	4.8 ± 0.1	4.1 ± 0.8	7.7 ± 0.2

3.4 Time reaction of the carbon beads

A lower detection of SARS-CoV-2 in wastewater samples (Table 4) was observed when the reaction time was 10 min (4 out of 5 samples detected) than when it was 25 min (5 out of 5 samples detected), while no significant differences (n = 5; paired t-test, P > 0.05) were observed between those two reaction times for Phi6 recovery (0.9 ± 0.6% and 0.8 ± 0.5% for reaction times of 25 min and 10 min, respectively) and PMMoV concentration (7.6 ± 0.3 log copies/L and 7.6 ± 0.3 log copies/L for reaction times of 25 min and 10 min, respectively). Hence, the 25-min reaction time was maintained in the optimized protocol as per the manufacturer’s instructions.

Table 4

Effect of different reaction times of the carbon beads on the performance of the C.prep method
Reaction time (min)	No. of tested samples	SARS-CoV-2			Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
Reaction time (min)	No. of tested samples	No. of detected samples	No. of quantified samples	Concentration (mean ± SD; log copies/L)	Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
10	5	4	4	5.1 ± 0.2	0.8 ± 0.5	7.6 ± 0.3
25	5	5	4	5.0 ± 0.2	0.9 ± 0.6	7.6 ± 0.3

3.5 Performance comparison: optimized C.prep method vs. PEG precipitation method

Table 5 shows a comparison between the optimized C.prep method and the PEG precipitation method in terms of virus detection and recovery. Both methods successfully detected SARS-CoV-2 in all tested samples (4.8 ± 0.4 log copies/L and 5.0 ± 0.1 log copies/L, respectively) with no significant differences in the observed concentrations (n = 4; paired t-test, P > 0.05). However, lower Phi6 recovery values (0.8 ± 0.9% and 17.7 ± 1.6%, respectively) and PMMoV concentrations (8.2 ± 0.3 log copies/L and 8.8 ± 0.2 log copies/L, respectively) were obtained using the optimized C.prep method (n = 4; paired t-test, P < 0.05).

Table 5

Comparison of the optimized C.prep method with the PEG precipitation method in terms of detection, recovery, and measured concentration of target viruses
Method	No. of tested samples	SARS-CoV-2			Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
Method	No. of tested samples	No. of detected samples	No. of quantified samples	Concentration (mean ± SD; log copies/L)	Phi6 recovery (mean ± SD; %)	PMMoV concentration (mean ± SD; log copies/L)
Optimized C.prep	4	4	4	4.8 ± 0.4	0.8 ± 0.9	8.2 ± 0.3
PEG precipitation	4	4	4	5.0 ± 0.1	17.7 ± 1.6	8.8 ± 0.2

Various concentration methods based on different mechanisms are available to detect nucleic acids in samples, which can then be quantified via molecular methods such as qPCR or digital PCR. Numerous studies conducted so far have shown that each concentration method has its benefits and drawbacks.

Regardless of its simplicity and low initial cost (no need for centrifugation), the C.prep method with the full single workflow has some drawbacks, especially with respect to virus recovery. Considering that PMMoV is normally abundant in wastewater (Kitajima et al., 2018), the inability to detect it in such samples using the full workflow (Full protocol 1) indicated that RNA could not be successfully extracted. This fact is supported by a fair virus recovery achieved using the Full protocol 2, in which an additional RNA extraction step was conducted after obtaining RNA from the full workflow, showing that RNA extraction or purification using magnetic carbon beads and heat extraction was not sufficient to obtain a purified RNA extract. While many studies have reported that magnetic bead-based extraction resulted in higher nucleic acid concentrations than spin-column-based extraction for clinical samples (Ribeiro-Silva et al., 2007; Triant and Whitehead, 2009), no direct comparisons between these two extraction methods have been yet made for wastewater samples. The present study could provide an insight into whether spin column-based extraction works better than magnetic bead-based extraction for wastewater samples. Such superiority may be due to inhibitors present in wastewater (Ahmed et al., 2022; Schrader et al., 2012; Wilson, 1997). In addition, this study supports another previously reported finding, i.e., that heat extraction was ineffective for wastewater samples because of inhibitors (Bivins et al., 2022), despite the good performance of heat extraction in the detection of SARS-CoV-2 in saliva samples (Barza et al., 2020; Mahmoud et al., 2021).

Another disadvantage of the C.prep workflow is the high potential for human error since decantation of the liquid portion must be done carefully to prevent losing viral material, given that the attached solid portion containing complexes of the magnetic carbon beads and viruses are not strongly attached to the magnetic bar. Some studies have used PEG as an aggregation agent (Angga et al., 2022; Dai et al., 2021) to improve the attachment of the magnetic carbon–virus complexes to the magnetic bar.

As many pathogens are found at very low concentrations in wastewater, one of the simplest ways to increase the sensitivity of detection is to increase the sample volume. However, this could also increase the concentration of inhibitors; thus, choosing the initial sample volume is important, considering the trade-off between virus recovery and the effect of inhibitors. Many researchers have already used the automated workflow in the concentration step (Hayase et al., 2023; Karthikeyan et al., 2021) to minimize human errors, and most automated methods require a small starting volume (≤ 10 mL). In this study, a lower sensitivity was observed in the detection of SARS-CoV-2 after reducing the processing volume to 10 mL.

Despite several drawbacks, high throughput is expected from the C.prep method if multiple magnetic bars are available. The sensitivity in detecting indigenous SARS-CoV-2 shown by the optimized C.prep method was comparable with that obtained using the PEG precipitation method, which points to the applicability of the former method to recover enveloped viruses in wastewater. However, a lower spiked Phi6 recovery was observed when using the optimized C.prep method. It is plausible that these bacteriophages were not properly attached to the suspended solids present in the wastewater samples and were discarded along with the liquid portion. Further experiments need to be conducted to draw firmer conclusions in regard to this method’s performance in detecting enveloped viruses. Furthermore, the lower concentration of indigenous PMMoV detected using the C.prep method suggested that this technique is not so effective in recovering non-enveloped viruses.

This study evaluated the C.prep method, a relatively new concentration-extraction kit for the detection of viruses in wastewater samples. The full workflow of this method could not detect PMMoV, which is normally abundant in wastewater, while a modified version of the method with an additional RNA extraction step could detect the target viruses, suggesting that the full workflow could not remove the inhibitors present in the wastewater samples. Modifications of the protocol such as 1) increasing the carbon bead volume, 2) decreasing the volume of processed samples, and 3) increasing the carbon bead reaction time did not improve the sensitivity of the C.prep method; thus, when using it, the manufacturer’s recommendations should be followed. When compared with the PEG precipitation method, the optimized C.prep method was almost equally sensitive in detecting SARS-CoV-2 but performed more poorly in recovering Phi6 and detecting PMMoV. Based on these results, the optimized C.prep method did not perform as well as the another tested method and needs to be improved through further modifications.

CRediT authorship contribution statement

Made Sandhyana Angga: Conceptualization; Formal analysis; Investigation; Methodology; Writing - original draft. Sunayana Raya: Investigation. Soichiro Hirai: Investigation. Eiji Haramoto: Conceptualization; Funding acquisition; Methodology; Resources; Supervision; Writing - review & editing.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by the Japan Science and Technology Agency (JST) through e-ASIA Joint Research Program (grant number JPMJSC20E2) and the Japan Society for the Promotion of Science (JSPS) through Grant-in-Aid for Scientific Research (B) (grant number JP23H01536 and JP23K26230). The authors acknowledge M&S Instruments Inc. (Osaka, Japan) for supporting the experiment using the Carbon Prep Wastewater RNA Isolation Kit and the staff of the WWTP for their support and permission on wastewater sampling.

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Magnetic carbon bead-based concentration method for SARS-CoV-2 detection in wastewater

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Wastewater samples

2.2 Preparation of the spiked viral stock

2.3 C.prep method

2.4 PEG precipitation method

2.5 RNA extraction

2.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

2.7 Statistical analysis

3. Results

3.1 Workflow of the C.prep method

3.2 Carbon bead concentration

3.3 Volume of processed samples

3.4 Time reaction of the carbon beads

3.5 Performance comparison: optimized C.prep method vs. PEG precipitation method

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1