key: cord-326911-va3x6au2
authors: Ramos-Mandujano, G.; Salunke, R.; Mfarrej, S.; Rachmadi, A.; Hala, S.; Xu, J.; Alofi, F. S.; Khogeer, A.; Hashem, A. M.; Almontashiri, N. A.; Alsomali, A.; Hamdan, S.; Hong, P.; Pain, A.; Li, M.
title: A Robust, Safe and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Environmental Samples
date: 2020-06-29
journal: nan
DOI: 10.1101/2020.06.28.20141945
sha: 
doc_id: 326911
cord_uid: va3x6au2

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magnetic- nanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

Testing for COVID-19 is vital for monitoring and mitigating the spread of SARS-CoV-2 and for safely restarting the normal economy. To date, molecular diagnosis of COVID-19 predominantly relies on detection of SARS-CoV-2 RNA using real-time reverse transcription polymerase chain reaction (rRT-PCR) assays, such as those approved by the US Centers for Disease Control and Prevention (US CDC) 1 . As SARS-CoV-2 spreads globally, it also accumulates approximately 1 to 2 single nucleotide variants (SNVs) in the 29,903 bp genome per month 2 . The emergence of new strains could have serious implications in the efficacy of diagnostic tests and success of vaccines. For example, 87 of 2816 genomes sampled between Jan and May 2020 have the T28688C SNV (GISAID, https://nextstrain.org/) that alters the sequence of the binding site of the forward primer of the CDC N3 rRT-PCR assay 1 , potentially compromising its effectiveness.

Thus, continued surveillance of the evolution and geographic distribution of viral strains by high-throughput sequencing 3, 4 is another pillar of public health measures to combat COVID-19.

Both rRT-PCR testing and high-throughput sequencing of SARS-CoV-2 require RNA extraction from nasopharyngeal swab samples. In the clinic, swabs are collected in viral transport media (VTM) and, if necessary, transported following specific cold-chain biological substances transport guidelines 1 for RNA extraction. The US CDC recommends several commercially available RNA extraction kits 1 . Fully automated diagnostic systems (e.g., Roche cobas® 6800 and 8800) that perform all steps from RNA extraction to rRT-PCR without human intervention are also popular among diagnostic laboratories. Commercial kits and procedures typically yield consistent quality RNA and are easy to use, but come with a high price tag. Moreover, the availability of commercial proprietary reagents is seriously affected by the disruption of the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint global supply chain caused by the COVID-19 pandemic. The high cost and low availability of proprietary reagents impose a bottleneck on testing capacities in rich and poor countries alike.

Additionally, monitoring pathogens in wastewater is an important public health measure, and it requires methods that satisfy the biosafety requirements of handling unknown infectious agents and can overcome the low virus concentration and PCR inhibitors that are ubiquitous in wastewater. Therefore, there is great incentive to develop alternative methods that only require locally available and inexpensive chemicals, are simple to perform, and rival the performance of commercial kits. Besides alleviating supply shortage, the alternative methods should ideally eliminate the risk of handling live viruses, thus lowering the strict biosafety and biosecurity requirements 5 on testing facilities. Any self-build RNA extraction method that satisfies the above-mentioned criteria can help increase testing capacity not only in clinical laboratories but also in rural healthcare facilities, university laboratories and field testing sites.

RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC) 6 (sold as TRIzol by Invitrogen or TRI Reagent by Sigma-Aldrich) has been successfully used in life sciences laboratories around the world for nearly four decades. It requires widely available chemicals at a low cost. The AGPC methods has been found to match the performance of commercial kits and automated systems in SARS-CoV-2 rRT-PCR detection 7, 8 . In these studies, swabs were first collected in VTM or cell culture media, which were then used in AGPC RNA isolation. This workflow necessitates handling of live viruses and requires Biosafety Level 2 (BSL2) facilities.

We hypothesized that it should be possible to collect swabs directly in AGPC, which achieve two goals: 1) completely inactivation of any infectious agent by AGPC so that the downstream . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint procedures (e.g., transportation, RNA isolation, rRT-PCR, and sequencing) can be carried without BSL2 requirements, and 2) preservation of RNA integrity by denaturing nucleases.

However, the AGPC method as is commonly practiced has several drawbacks that make it unsuitable for high-volume testing. It requires extensive manual pipetting of hazardous chemicals and multiple centrifugation steps, which increase the risk of human errors and personnel injury especially when the sample number is large. Solid-phase reversible immobilization (SPRI) of nucleic acid on magnetic nanoparticles (MNPs) offers a simple and elegant alternative to centrifuge-or column-based methods 9 . Nucleic acid (e.g. RNA) reversibly binds to functionalized MNPs under dehydrating conditions and can be separated from contaminants in solution by a strong magnet. This allows fast and thorough washes to eliminate inhibitors of downstream molecular biology reactions and yields high quality RNA for PCR and high-throughput sequencing. Because it requires no centrifugation and only low-cost materials, the MNP-based RNA extraction is inherently scalable and amenable to automation. Although the combination of the AGPC and SPRI technologies would be obviously advantageous in consideration of reagent availability, cost, biosafety and ease-of-use, development of AGPC compatible MNP-based RNA extraction protocols has been limited.

Here we developed the Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) workflow (Fig. 1A) . MAVRICS only requires widely available and low-cost materials and can be self-assembled in a basic laboratory setting. It is compatible with AGPC inactivated . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint samples to alleviate the shortage of commercial kits, lower biosafety risks, and enable sample and scalable sample preparation. MAVRICS performed on par or better than commercial RNA extraction kits in rRT-PCR detection of SARS-CoV-2, influenza viruses and respiratory syncytial virus in various clinical and environmental samples.

MNPs can be functionalized with either a carboxyl or silica coating to bind nucleic acids 10 .

Carboxylated MNPs (cMNPs) are available commercially (e.g., RNAClean XP from Beckman Coulter) and widely used in molecular biology workflows such as PCR cleanup and sequencing library preparation. Unfortunately, cMNPs (in the form of RNAClean XP) failed to recover detectable RNA from AGPC solutions (in the form of TRIzol) spiked with high quality total RNA from human cells, while the conventional AGPC method based on organic phase separation and centrifugation recovered ~45% of input RNA. On the other hand, cMNPs were capable of 84-96% recovery when the same RNA was spiked in water, suggesting that AGPC interferes with RNA binding onto cMNPs. Silica magnetic nanoparticles (SiMNP) have been used to extract total nucleic acid from samples lysed and inactivated in AGPC without centrifugation and phase separation 10 . Since commercial SiMNPs are expensive and difficult to procure during the COVID-19 crisis, we synthesized SiMNP from scratch using a published open-source protocol 10 . The synthesis took ~14 hours with 3 hours hands-on time and required only base chemicals, a strong magnet, and standard lab equipment (Fig. 1A, Supplementary Fig. 1A-D) . In our case, all . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint materials were locally available ( Table 1) . One synthesis yielded enough SiMNPs for 5,000-10,000 extractions, and the material cost was miniscule. Another benefit of SiMNP is its chemical inertness. Our SiMNPs have been stored at room temperature for 6 weeks at the time of writing without noticeable change in performance.

We first tested if SiMNPs could isolate RNA from contrived SARS-CoV-2 saliva samples (see methods) inactivated in AGPC (in the form of TRIzol). As previously reported, SiMNPs were able to isolate RNA directly from TRIzol using the total nucleic acid extraction protocol (hereafter referred to as TNA protocol) described in 10 . We used the US CDC 2019-nCoV_N3 rRT-PCR assay to quantitate the recovery of SARS-CoV-2 sequences. SiMNPs coupled with the TNA protocol resulted an increase of 3.1 in Ct value compared to the official TRIzol Reagent protocol, which means a 11.1% yield of viral RNA relative to the AGPC method ( Fig. 1B-C) . In contrast, RNA received by the cMNP (RNAClean XP) methods was negligible ( Fig. 1B-C) . Interestingly, the yield of SiMNPs improved when the sample in TRIzol was first phase separated by chloroform and the aqueous phase was used in combination with an enzymatic reaction cleanup protocol described in 10 (cleanup CHCl3 protocol, Fig. 1B-C) . However, this modification defeated the purpose of using SiMNPs to simplify the workflow. Together, these results showed that SiMNPs could isolate viral RNA directly from AGPC inactivated samples, but existing SiMNP protocols significantly underperformed compared to the AGPC method, thus reducing the sensitivity of diagnostic tests.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint Next, we aimed to develop an efficient SiMNP-based RNA extraction protocol using the contrived SARS-CoV-2 samples and US CDC 2019-nCoV_N1 and N3 rRT-PCR assays. Increasing the amount of SiMNPs 2.5 times significantly improved the recovery of both the TNA and cleanup CHCl3 protocols. We also noticed an improvement by washing the SiMNPs once with TRIzol and RNA binding buffer (1:1), presumably further removing RNases. Nonetheless, none of these protocols could better the TRIzol Reagent protocol ( Fig. 2A-B, Supplementary Fig. 2B -C). Since the cleanup CHCl3 protocol had consistently outperformed the TNA protocol, we suspected that the RNA binding buffer 10 in the TNA protocol might not be optimal. Indeed, after adding buffering agents (Tris-HCl or Bis-Tris, pH6.5) to the RNA binding buffer and increasing its guanidinium chloride concentration to 3M, the yield of RNA doubled ( Fig. 2A-B 

We combined the modifications, i.e., the additional wash step and new binding buffers, that improved the recovery of viral RNA by SiMNPs and showed that they outperformed the TRIzol Reagent protocol as judged by both the N1 and N3 rRT-PCR assays (TNA 2X Bis-Tris or Tris, Fig.   2C -D). The number of SARS-CoV-2 RNA molecules captured by the SiMNP-TNA 2X Bis-Tris or SiMNP-TNA 2X Tris protocol was estimated by the standard curve method to be very close to the input value (Fig. 2E) . Similar results were obtained using an independent synthesis of SiMNPs, proving the robustness of the protocols (Supplementary Fig. 2E . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We next compared MAVRICS with commercial kits using real-world COVID-19 swab samples 

Since the first reports of SARS-CoV-2 shedding in stool 11, 12 , the presence of the virus has been confirmed in municipal wastewater, sometimes even before the first confirmed cases in the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint community 13 . This suggests that wastewater surveillance could be effective for monitoring the total COVID-19 case load (including asymptomatic cases) in the population. Detecting pathogens by rRT-PCR in wastewater requires methods that satisfy the biosafety requirements of handling unknown infectious agents and can overcome the low virus concentration and PCR inhibitors that are ubiquitous in wastewater. MAVRICS could be a safe and easy-to-implement workflow to extract viral RNA in wastewater. We first tested the recovery of known quantities of SARS-CoV-2 RNA and intact murine noroviruses (MNVs) spiked in wastewater concentrate, in which viral particles in 250 ml raw sewage were concentrated on electronegative membranes followed by ultrafiltration with Centripep YM-50 to a final volume of 700 ul 14 . The wastewater concentrate was first inactivated by 10X volume of TRIzol and extracted using MAVRICS. The result showed an 88% recovery of the input SARS-CoV-2 RNA (Fig. 4A) . The amount of norovirus RNA captured by the SiMNPs was almost identical to that by the conventional Qiagen RNA purification kit (Fig. 4B) .

We further simplified the preparation of wastewater by using TRIzol to inactivate and lyse the sewage biomass (including viral particles) immobilized on the electronegative membranes, followed by RNA extraction by MAVRICS. Again, the spike-in SARS-CoV-2 was efficiently recovered (Fig. 4C) , and the amount of pepper mild mottle virus (PPMoV, ubiquitous in wastewater) captured by the SiMNPs was almost identical to that by the conventional QIAamp viral RNA mini kit (Fig. 4D) .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Lastly, we validated the MAVRICS method for detection of other human pathogenic viruses than SARS-CoV-2. A commercial human respiratory pathogens control panel that contains influenza A and B viruses, and respiratory syncytial virus (RSV) was lysed in TRIzol and used for RNA extraction by MAVRICS. We then used a clinical diagnostic rRT-PCR panel to quantitate the viruses. Interestingly, influenza A, influenza B and RSV were readily detectable in samples extracted using SiMNPs, but the Ct value of the same pathogens lagged by 4.08, 4.24 and 5.57, respectively, for samples extracted using the TRIzol Reagent protocol (Fig. 4E) . No virus was detected in blank controls extracted either by SiMNPs or TRIzol (Fig. 4E) .

We described an SiMNP-based RNA extraction workflow, MAVRICS, that is compatible with pathogen detection in clinical and environmental samples. All reagents used in MAVRICS are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. The longest preparation step is the synthesis and silica coating of MNPs, which can be done overnight with ~ 3-hour hands-on time. The material cost for one synthesis is inconsequential yet can support thousands of RNA extractions. Because MAVRICS works for samples inactivated and preserved in AGPC (e.g., TRIzol), it allows potentially infectious samples to be handled safely without special biocontainment facilities. Importantly, MAVRICS matches, and exceeds in many cases, the performance of commercial proprietary reagents using established molecular diagnostic tests of SARS-CoV-2, influenza viruses, and RSV. These tests entail molecular biology reactions that require high quality input RNA. This suggests that the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint RNA produced by MAVRICS is free of contaminants and maintains good integrity. It will be of interest to study if MAVRICS is compatible with other molecular biology techniques, such as next-generation sequencing (NGS), in the future. Since NGS library preparation uses similar reactions, including reverse transcription and PCR, one would expect the answer is affirmative.

We noticed that the correlation between SiMNP and DIRECT-zol was lower than that between SiMNP and TRIzol (compare Fig. 3A and 3C) . In the case of SiMNP vs. TRIzol, each sample was divided equally between SiMNP and TRIzol protocols and processed in parallel. On the other hand, the samples used in the SiMNP and DIRECT-zol comparison was extracted at different times. This was due to clinical reasons. Priority was given to extract enough RNA for NGS using the DIRECT-zol kits. As a results, samples were not equally divided between the SiMNP and DIRECT-zol extractions, and the swab might be present in one but not the other extraction method. These reasons could contributed to the lower correlation between the two methods.

Nonetheless, evidence from 36 clinical samples, 2 wastewater samples and 1 pathogens control sample showed that MAVRICS ravels performance of commercial reagents.

We noticed an interesting lack of correlation between the amount of total RNA and viral RNA (Supplementary Fig. 2A-C, 2D-G, Supplementary Fig. 3D vs. Fig. 3A-B) . For example, RNA concentration of S667 was below the detection range of Qubit fluorometer, and yet the copy number of SARS-CoV-2 was higher than S659, which had the one of the highest RNA concentration (Supplementary Fig. 3B-C) . SiMNP tends to have lower total RNA yield, but has lower Ct values when compared to other methods (Supplementary Fig. 3C ). There could be at . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint least two possibilities. First, SiMNPs may favor binding of RNA similar to the viral RNA. This could be due to the surface chemistry or high surface area to mass ratio of nanoparticles.

Second, SiMNPs may be more efficient in removing contaminants that inhibit reverse transcription and PCR. The exact reasons for this phenomenon need to be further studied.

In summary, we developed MAVRICS to enable safe, economical and effective extraction of RNA from clinical and environmental samples. The performance of MAVRICS rivals commercial RNA extraction kits in validated diagnostic tests of SARS-CoV-2, influenza viruses and respiratory syncytial virus. MAVRICS has the potential to become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint and the tubes were vortexed and settled on a magnetic stand and the cleared supernatant was removed. 90% Ethanol (400 µl) was added and the tubes were vortexed and settled on a magnetic stand. The supernatant was then removed. Ethanol washing was repeat three more times for a total of four washes. The beads were dried on a heat block at 50 °C for ~20 min. To elute the RNA 40 μl of nuclease-free water was added and mixed at 1300 rpm at RT for 5 min.

Finally, the tube was settled on a magnetic stand and the eluted RNA transferred to a new tube.

Total RNA extraction of the samples was performed following instructions as described in the is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint epT.I.P.S.® LoRetention series) and centrifuge tubes (Eppendorf DNA LoBind Tubes, Cat. No 0030108051) used in this study were PCR-clean grade. All of the operations were performed carefully following standard laboratory operating procedures. For Influenza and RSV assays, the following program was used: 50°C for 2 min, 55°C for 120 sec, 60°C for 360 sec, 65°C for 240 sec, followed by 5 cycles 95°C for 5 sec and 55°C for 30 sec, and then 45 cycles of 91°C for 5 sec and 58°C for 25 sec. MNV and PMMoV real-time PCR assay was conducted using the primer and probes which described previously 15, 16 . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. S659  S660  S661  S662  S663  S664  S665  S666  S667  S668  S669  S670  S742  S743  S744  S745  S746  S747  S748  S749  S750  S751  S752  S753  S504  S505  S506  S479  S482  S483  S468  S486  S488 S487 S150 S151 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint

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We thank KAUST Rapid Research Response Team (R3T) for supporting our research during the COVID-19 crisis. We thank members of the KAUST R3T for generously sharing materials and advices. We thank Professor Imed Gallouzi of McGill University for the useful discussion. We thank members of the Li laboratory, Chongwei BI, Baolei Yuan, Xuan Zhou, Samhan Alsolami, Yingzi Zhang, and Yeteng Tian, for helpful discussions; Marie Krenz Y. Sicat for administrative support. We thank members of the Pain lab for technical assistance.

ML and GRM performed majority of the molecular biology experiments. ML and GRM analyzed the data and wrote the manuscript. RS, SM, and JX performed experiments. AR and PH