key: cord-0949904-raftx1iu
authors: Hammerling, Michael J.; Warfel, Katherine F.; Jewett, Michael C.
title: Lyophilization of premixed COVID‐19 diagnostic RT‐qPCR reactions enables stable long‐term storage at elevated temperature
date: 2021-06-10
journal: Biotechnol J
DOI: 10.1002/biot.202000572
sha: d9ca345660f5263606d80f180d8ab3a1e14a42e3
doc_id: 949904
cord_uid: raftx1iu

Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) diagnostic tests for SARS‐CoV‐2 are the cornerstone of the global testing infrastructure. However, these tests require cold‐chain shipping to distribute, and the labor of skilled technicians to assemble reactions and interpret the results. Strategies to reduce shipping and labor costs at the point‐of‐care could aid in diagnostic testing scale‐up and response to the COVID‐19 outbreak, as well as in future outbreaks. In this study we test both lab‐developed and commercial SARS‐CoV‐2 diagnostic RT‐qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization. Fully assembled reactions were lyophilized and stored for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS‐CoV‐2 RNA. Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization. We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze‐dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days. We anticipate that the incorporation of these methods into SARS‐CoV‐2 diagnostic testing will improve testing pipelines by reducing labor at the testing facility and eliminating the need for cold‐chain shipping.

RT-qPCR remains the gold standard to deliver highly accurate diagnoses of ongoing viral infection. [5] In order to combat the ongoing pandemic and ensure that we have adequate diagnostic responses prepared for future threats, we must improve the quality, ease-of-use, and distribution of established RT-qPCR-based diagnostics.

One strategy to enable distribution of preassembled RT-qPCR diagnostic reactions without the need for the cold-chain storage is lyophilization (i.e., freeze drying), which would reduce distribution and storage costs and labor in the diagnostic lab. Lyophilization is a common strategy to confer stability to biological samples and biochemical reactions, enabling the storage of samples as a dry powder at ambient temperature for later rehydration. [6] In recent years, lyophilization has been used by synthetic biologists to enable cell-free systems for ondemand biomanufacturing, biosensing, and educational kits. [7] [8] [9] [10] [11] [12] [13] [14] Further, lyophilized in vitro transcription and PCR-based detection mixtures have demonstrated superior qualities for providing diagnostics in resource-limited settings. [13, 15, 16] To prevent the loss of activity during lyophilization and storage, additives referred to here as lyoprotectants can be implemented and optimized to stabilize biological molecules in freeze-dried mixes. The most commonly used lyoprotectants are sugars, ranging from nonreducing disaccharides to larger polymeric saccharides, but can also include molecules such as osmolytes and sugar alcohols. [10, 17] Established mechanisms of protein stabilization are water replacement, in which lyoprotectants replace water by hydrogen bonding with proteins to maintain native conformation, [18, 19] and vitrification, in which lyoprotectants trap the protein in a glassy matrix, therefore reducing mobility and improving stability. [20] Combinations of various lyoprotectants have also been found to have synergistic properties. [18, 21, 22] Many factors play a role in choosing an effective formulation for lyophilization, requiring optimization of lyoprotectant identity and concentration for each system of interest. [23] In this work, we explore the use of lyophilization and lyoprotectants for stabilization and long-term storage of fully assembled SARS-CoV-2 RT-qPCR diagnostic reactions. We test the tolerance to lyophilization of several commercially available kits and a recently developed noncommercial mix using the novel synthetic thermostable reverse transcriptase, RTX. [24] We also explore stabilization of these lyophilized mixtures with a variety of lyoprotectant formulations and concentrations which help preserve fidelity at ambient and elevated temperatures. We find that a single RT-qPCR kit validated for COVID-19 diagnostics is highly robust to lyophilization, and can be formulated for storage for at least 30 days at up to 37 • C while retaining the ability to detect down to 50 copies of SARS-CoV-2 RNA. In addition to eliminating the need for expensive and logistically challenging coldchain storage, the pre-mixed reactions can improve result turn-around times and reduce the opportunity for reaction assembly error by minimizing operator handling, holding promise for improving result quality and consistency. [25, 26] Our lyoprotectant optimizations show how currently available diagnostic tools can be adapted in order to prepare for pandemic response by enabling ease of use and reducing distribution challenges while maintaining reaction quality.

In this study, we aimed to use lyophilization to improve the ease of use and potential distribution of RT-qPCR-based diagnostics. We first benchmark a set of commercial kits used for SARS-CoV-2 detection against a recently developed synthetic reverse transcriptase mix.

We then evaluate the tolerance of each of these mixes to lyophilization with a variety of lyoprotectant formulations and storage at ambient temperature. Finally, we test the most promising kit under our defined conditions with higher lyoprotectant concentrations and expose these mixes to a more rigorous regime of elevated temperatures and extended incubation times to demonstrate the viability of this method for shipping and long-term storage of these reactions outside the cold-chain.

We first chose a set of RT-qPCR kits for use in COVID-19 diagnostic mixes from different manufacturers, including the Invitrogen Super-Script III One-step RT-PCR (SuperScript), the Promega GoTaq Probe 1-step RT-qPCR (GoTaq), and the Takara One Step PrimeScript RT-PCR (PrimeScript) kits for comparison. These kits were benchmarked against reaction mix containing the thermostable synthetic reverse transcriptase RTX, which can perform single-enzyme RT-PCR and was previously shown to function as the RT component of TaqMan based COVID-19 RT-qPCR diagnostic reactions. [24, 27] Given the thermostability and general robustness of this enzyme, we hypothesized that it may be especially amenable to stabilization by lyophilization and longterm storage at ambient or elevated temperatures. Indeed, Escherichia coli cells expressing RTX have previously been lyophilized into "cellular reagents" as ready-to-use PCR reagents which require no enzyme purification. [28] However, RTX has not been lyophilized in a fully premixed diagnostic reaction mix to our knowledge.

RT-qPCR was performed using these kits and an RTX/Taq reaction mixture (see Materials and Methods) on a dilution series of synthetic SARS-CoV-2 RNA (Twist Biosciences, MT007544.1) and a no-template control (NTC). Reaction series were performed using both the N1 and N2 probe mixes, which target different regions of the N gene of the SARS-CoV-2 genome (Integrated DNA Technologies), to assess performance of these diagnostic setups on various concentrations of synthetic target RNA ( Figure 1 ). We found that all reaction mixes performed well using the N1 probe, generating a log-linear relationship between target concentration and the cycle in which fluorescence can be detected, or the quantitation cycle (Cq), of the diagnostic reaction ( Figure 1A , C). The Cq value is the critical metric for determining viral RNA concentration in a sample, and thus a loglinear relationship between synthetic SARS-CoV-2 concentration and Cq value is an essential outcome for a successful testing regime. In contrast, when using the N2 probe mix, the RTX reaction mix failed to detect the target RNA except at high concentrations of target RNA F I G U R E 1 Benchmarking of RTX SARS-CoV-2 diagnostic reactions against commercial reactions. Each column represents the results from SARS-CoV-2 reaction mixes featuring a different RT-qPCR mix, including RTX, GoTaq, SuperScript, and PrimeScript from left to right. (A) Amplification curves for each kit with a dilution series of SARS-CoV-2 synthetic genomes using the N1 probe mix. All reaction mixes detect SARS-CoV-2 RNA at all concentrations without false positives in the absence of target RNA. (B) Amplification curves for each kit with a dilution series of SARS-CoV-2 synthetic genomes using the N2 probe mix. The RTX mix fails to detect SARS-CoV-2 below 5,000 copies of the target RNA. Each data point represents the average of n = 6 experiments, with errors bars representing standard deviation. (C) Standard curve of Cq values measured across a 10-fold serial dilution of SARS-CoV-2 synthetic genomes from 5 through 50,000 copies for N1 (black) and N2 (blue) probe mixes. All commercial mixes perform comparably, generating a log-linear relationship for both the N1 and N2 probes of template concentration versus Cq value. In contrast, the RTX custom mix performs well for N1 but not N2 probe mixes.

( Figure 1B) , and thus did not yield a log-linear relationship between Cq value and synthetic SARS-CoV-2 concentration ( Figure 1C ). Commercial kits performed well using both N1 and N2 probes. These results show that each RT-qPCR formulation using the N1 probe can detect synthetic SARS-CoV-2 RNA at the attomolar level, but the N2 probe failed to adequately detect SARS-CoV-2 RNA in the RTX-based mix.

Based on these results, we proceeded with lyophilization tests using only the N1 probe mix for testing and optimizing lyophilization of premixed diagnostic reactions.

We next tested the amenability of fully-assembled SARS-CoV-2 diagnostic reactions to lyophilization using the commercial kits and the homemade RTX mix. To attempt to identify lyophilization conditions which stabilized premixed diagnostic reactions, we tested concentra-tion gradients of the commonly used nonreducing disaccharide lyoprotectants sucrose and trehalose, and the large polymeric saccharide dextran 70. [10, 21, 29] Each of the previously assayed RT-qPCR mixes Finally, lyoprotectants are known to be especially important in preserving reaction mixtures exposed to elevated temperatures. [10] Therefore, we hypothesized that lyoprotection of diagnostic reactions may improve the stability of Cq values when reactions are exposed to elevated temperatures and longer incubation times.

To test this hypothesis, we proceeded with a second lyophilization experiment with longer incubation times and higher temperatures. Due to their comparable performance and low reagent cost in the GoTaq reactions, and to reduce the overall number of formulations to be tested, sucrose and sucrose/trehalose mixes were chosen as the lyoprotectants in these experiments. Since final RFU of GoTaq reactions incubated for 14 days in the prior experiment continued to increase up to 50 mg mL -1 of lyoprotectant ( Figure 2C ), higher concentrations were tested in this experiment. GoTaq reaction mix was assembled with N1 primer/probe and lyophilized with no lyoprotectant, sucrose concentrations of 50, 75, or 100 mg mL -1 , or with sucrose/trehalose mixes of 20, 30, and 40 mg mL -1 each. A set of non-lyophilized control reactions was also included. These reactions were incubated at 23 • C, 37 • C, or 50 • C for 30 days. After 30 days, reactions were tested with a dilution series of SARS-CoV-2 RNA at concentrations ranging from 5 to 50,000 copies.

Reactions that were not lyophilized could not survive prolonged incubation at ambient or elevated temperatures, and after 30 days none of these reactions retained any activity (Figure 3, lower table) .

In contrast, lyophilization of GoTaq reactions imparted robust thermostability to the reaction mix. After 30 days, reactions incubated at 23 • C and 37 • C were still capable of detecting SARS-CoV-2 RNA at concentrations as low as five total copies ( Figure 3A, 3B ). However, if the limit of detection (LOD) is defined as the concentration of template at which > 95% of samples containing SARS-CoV-2 RNA are identified as positive, [30] then the optimal LOD is five total copies (1,000 copies mL -1 ) for non-lyoprotected reactions incubated at 23 • C and 50 total copies (10,000 copies mL -1 ) for lyoprotected reactions incubated at 37 • C ( Figure 3A-B, lower table) . While this value for samples incubated at 37 • C is higher than the LOD of the non-lyophilized CDC assay at 1,000 copies mL -1 , [31] the sensitivity of the lyophilized assay could theoretically be brought up to this value by increasing the scale of the reaction from 5 to 50 µL. In addition, reactions incubated at 23 • C or 37 • C displayed no significant difference in Cq value between the unprotected reactions and the lyoprotected reactions.

In contrast, the reactions incubated at 50 • C did not generally reach the threshold of 95% detection to define a limit of detection [30] except in the 5,000 and 50,000 copy test reactions for the non-lyoprotected cases ( Figure 3C ). Those reactions that did have a definable LOD had significantly higher Cq values in the absence of lyoprotectant (black dots) compared to samples with lyoprotectant (purple and green dots) (Welch's two-sided t-test, p = 0.01 and 0.0004 for 5,000 and 50,000 genome copies, respectively), and also retained a higher rate of positive test results at low template concentration ( Figure 3C ). Combined with the results for the 23 • C and 37 • C samples, these results show that lyoprotectants preserved freeze-dried reaction mixes, improving the likelihood of SARS-CoV-2 RNA detection and preserving Cq value in samples exposed to higher temperatures.

We demonstrated that lyophilization of pre-mixed COVID-19 RT- Finally, we found that lyoprotectants are not necessary for preserving these diagnostic reactions at room temperature, but that lyoprotectants are associated with improved performance when the reactions are exposed to elevated temperatures. We hope that these results will spur the development and distribution of preassembled, lyophilized diagnostic reactions to reduce distribution costs and enable streamlined workflows, especially in resource-limited settings.

For commercial reaction mixes, reactions were assembled per the manufacturer's recommendations as laid out in the Promega GoTaq, Invitrogen SuperScript III, or Takara One Step PrimeScript (RR064A) manuals and including 0.5 µL of N1 or N2 probe mix (IDT: 10006713) in a 5 µL reaction. For diagnostic reactions using RTX, reactions were F I G U R E 3 Lyoprotectants are effective at stabilizing lyophilized GoTaq SARS-CoV-2 diagnostic reactions exposed to elevated temperatures. GoTaq diagnostic reactions were premixed with no lyoprotectant (None), 50, 75, or 100 mg mL -1 sucrose (Suc50, Suc75, Suc100) or a mixture of 20, 30, or 40 mg mL -1 each of sucrose and trehalose (ST20, ST30, ST40) and lyophilized. Reactions were incubated for 30 days at (A) 23 • C, (B) 37 • C, or (C) 50 • C to assess stability of reactions exposed to elevated temperatures for long periods. Diagnostic reactions were performed on a dilution series from 5 to 50,000 copies of synthetic SARS-CoV-2 RNA. Each dot of the dotplot is a Cq value of a single reaction, and the columns of the Samples were considered to be positive if they reported a Cq value of < 40.

Reactions were assembled as described above and mixed thoroughly with lyoprotectant, briefly spun down, and flash frozen. An SP Scientific

Benchtop Pro with Omnitronics lyophilizer was prepared by bringing pressure down to < 100 mTorr and condenser temperature to < -80 • C.

Reactions were transferred to dry ice to keep them frozen and caps were removed. Reactions were transferred to the lyophilization chamber and the chamber was immediately brought to < 100 mTorr. Reactions were lyophilized overnight and inspected the next day to ensure complete drying. They were then capped and incubated at the appropriate test temperature. Reactions were reconstituted using 5 µL of the appropriate dilution of SARS CoV2 RNA (Twist: 102019) by pipetting up and down exactly 10 times using an Integra Voyager pipette and read in a Bio-Rad CFX96 qPCR instrument as described above.

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CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel. Revision, 3, 30. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article

Lyophilization of premixed COVID-19 diagnostic RT-qPCR reactions enables stable long-term storage at elevated temperature