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. 2024 Jul;30(7):891–900. doi: 10.1261/rna.079891.123

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription

Pavana Khan 1, Lauren M Aufdembrink 1, Katarzyna P Adamala 1,2, Aaron E Engelhart 1,2,✉
PMCID: PMC11182012  PMID: 38637016

Abstract

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Keywords: fluorescent aptamer, RPA, T7 RNA polymerase, isothermal amplification, pathogen detection

INTRODUCTION

Early detection of emerging infectious diseases is the first step to containment and prevention of epidemics and pandemics. The SARS-CoV-2 pandemic underscored the need for disease surveillance mechanisms that enable rapid diagnosis (Peck 2020). Polymerase chain reaction (PCR) is the most widely used diagnostic method for the detection of infectious diseases, with high analytical sensitivity, specificity, and real-time monitoring of reaction progress (Garibyan and Avashia 2013). However, PCR reactions are energy-intensive and require expensive thermocyclers. Additionally, real-time readout in quantitative PCR (qPCR) requires further instrumentation and, typically, costly fluorescent probes (Arya et al. 2005).

Infectious diseases often emerge in regions of the world with limited access to laboratory detection equipment, such as qPCR instrumentation. Additionally, many viral diseases such as MERS, SARS-CoV-2, Ebola, and avian flu originate as zoonotic transmissions, highlighting the need for constant surveillance in farms and livestock markets (Magouras et al. 2020). Thus, according to the guidelines established by the World Health Organization, a need exists for diagnostic systems that (1) are cost-effective, (2) are energy-efficient, and (3) require minimal equipment and skilled personnel (Kosack et al. 2017).

Isothermal nucleic acid amplification methods are alternatives to thermal cycling-based PCR, and these allow amplification at constant temperatures with appropriate enzymes or enzyme cocktails (Fakruddin et al. 2013). Being more energy-efficient and cost-effective, isothermal diagnostics are highly applicable to resource-constrained settings, for use in field research or diagnostics, and for hospitals in remote regions where expensive, thermocycling equipment may be unavailable (Khan et al. 2020). A range of isothermal diagnostics have been developed, such as rolling circle amplification (RCA), loop-mediated amplification (LAMP), nucleic acid sequence–based amplification (NASBA), recombinase polymerase amplification (RPA), and signal-mediated amplification of RNA technology (SMART) (Notomi et al. 2000; Wharam et al. 2001; Deiman et al. 2002; Piepenburg et al. 2006; Mohsen and Kool 2016; Abdolahzadeh et al. 2019; Aufdembrink et al. 2020). These isothermal reactions use enzymes such as recombinases, strand displacement polymerases, or nucleases to recapitulate the exponential amplification of PCR without the attendant thermocycling requirements.

Of these isothermal amplification methods, RPA has been extensively used in isothermal detection systems. RPA uses recA recombinase proteins for the separation of double-stranded DNA duplexes (Armes and Stemple 2007; Piepenburg and Armes 2015). By using two primers to guide a recombinase, the technique enables recombinase-primer complexes to scan dsDNA for a homologous match to the primers, followed by strand invasion (Khan et al. 2020). The use of single-stranded-binding proteins (SSBs) helps stabilize the single-stranded DNA, whereas DNA polymerase performs stand synthesis in each direction. The output of RPA is double-stranded DNA that can undergo multiple rounds of amplification. Many techniques use RPA as a preliminary target amplification step and follow RPA with a second detection step, such as a combination of RPA and LAMP, or a combination of reverse transcription RPA and another technique, such as CRISPR-Cas12a detection (Song et al. 2021; Sun et al. 2021b) or XNAzymes (Yang and Chaput 2021, 2023). RPA amplification has also been coupled to fluorescence resonance energy transfer probes for fluorogenic detection postamplification.

CRISPR-RPA platforms are generally two-step and involve a preamplification step using RPA, followed by a Cas12 or Cas13 enzymatic assay to detect DNA targets such as in SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) (Gootenberg et al. 2017, 2018). An example that is closer to being a one-step reaction is the one-tube OR-DETECTR (Sun et al. 2021b), which keeps the CRISPR-Cas12a reagents separate from the RT-RPA reactions by having the CRISPR-Cas12a reagents on the lid of the tube, only to be mixed with the RPA reaction product following a 30-min preamplification incubation period. Additionally, a recent invention called ROSAR follows RPA with a fluorescent transcriptional output in a two-step system involving preamplification with RPA (Liang et al. 2024). Thus, many of these systems are multistep techniques, requiring additional time, adding complexity to the procedure, or using expensive probes for detection. These characteristics impede their application in point-of-care settings, where rapid diagnosis is key for immediate treatment opportunities, and in resource-constrained settings, where cost-effectiveness is essential. Our PACRAT (pathogen detection with aptamer-observed cascaded RPA–in vitro transcription) system improves upon these existing platforms by being a one-tube, one-step reaction, without any preamplification steps and multistep mixing protocols.

Isothermal amplification reactions with RNA as an output are highly desirable, and RNA aptamers have been used successfully in multiple biosensing technologies (Dunn et al. 2017; Kitto et al. 2021). The unique folding properties, catalytic capacity, and the ability to bind fluorogenic dyes enable a range of real-time readouts using RNA (Abdolahzadeh et al. 2019; Aufdembrink et al. 2020). Here, we show that careful tuning of solution conditions allows for a one-pot, isothermal, aptamer-based pathogen detection technique, that combines reverse-transcriptase RPA with in vitro transcription of the produced dsDNA, enabling an aptamer-based output (Fig. 1). We previously reported Apta-NASBA (Aufdembrink et al. 2020) for detection of transcripts, which uses the NASBA replication cycle. Here, we report an amplification system that uses RPA, which in turn cascades to an in vitro transcription reaction. Using this reaction, we produce fluorescent aptamers, which bind to inexpensive, small-molecule fluorogenic dyes, thus enabling real-time detection (Ouellet 2016). We have termed this aptamer-observed cascaded amplification technique PACRAT. PACRAT enables rapid, highly sensitive, fluorometric detection of pathogenic RNA, allowing quantitative analysis of PACRAT reactions by fluorescence. PACRAT involves three steps: (1) reverse transcription of pathogenic RNA target, (2) RPA on the cDNA product, and, finally, (3) transcription of RPA DNA product. All these steps occur in one pot, in <10 min, requiring no intermediate reagent addition steps and exhibiting single-copy detection limits of the pathogenic target. PACRAT can also be multiplexed, enabling simultaneous detection of two different pathogenic targets—Escherichia coli and SARS-CoV-2—and two different SARS-CoV-2 strains—wild-type and Delta variants in a one-pot reaction with real-time, multicolor fluorescence detection. Additionally, we show that PACRAT can directly detect E. coli, with single-cell detection limits, without the need for sonication or for extensive lysis steps. We also demonstrate the robustness of our system to saliva matrix, which is a common PCR inhibitor (Morais et al. 2022). PACRAT thus improves upon prior infectious disease diagnostics by increasing cost and energy efficiency and by reducing reliance on complex equipment and skilled on-site personnel. The system, therefore, promises to be an attractive platform for use in point-of-care settings.

FIGURE 1.

FIGURE 1.

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(A) Schematic of PACRAT. Reactions begin with an RNA target and reverse transcriptase reverse transcribes the single-stranded RNA to cDNA. DNA polymerase completes synthesis of a complementary strand, making a double-stranded DNA template. RPA begins with recombinases binding single-stranded primers, which then invade dsDNA. SSBs bind single-stranded DNA to stabilize it and DNA polymerase synthesizes a new DNA strand in each direction. DNA synthesis continues until two new DNA duplexes are formed. The dsDNA products have a T7 RNA polymerase promoter region (blue) and an aptamer coding sequence (green). T7 RNA polymerase can then transcribe the dsDNA product, resulting in an RNA amplicon conjugated to a fluorescent aptamer. The aptamer can bind its small-molecule cognate dye, generating a specific, real-time fluorescence signal. (B) Initial validation of PACRAT with urea-PAGE gels. SYBR Gold stained urea-PAGE shows the size of the DNA amplicon produced by RPA amplification at 39°C. The lane containing the reaction with 4.5 × 1010 copies of SARS-CoV-2 nucleocapsid N3 gene target shows the correct band size for the product at 155 bp. (C) Urea-PAGE gels of RNA products from PACRAT reactions with SARS-CoV-2 versus reactions with no target. (Left) SYBR-stained gel shows the correct RNA product size of 127 nt. (Right) DFHBI-stained gel, in which DFHBI is the cognate dye for the Broccoli aptamer, shows the band that contains the Broccoli aptamer.

RESULTS

PACRAT reaction design

In PACRAT, all the reagents are added to one tube and all three steps of the system (reverse transcription, amplification, and transcription) occur simultaneously in the reaction (Fig. 1A). The enzymes used include reverse transcriptase, DNA polymerase, and T7 RNA polymerase, along with recombinases and SSBs (Stringer et al. 2018). As in PCR and conventional RPA, the two oligonucleotide primers base-pair to specific regions of the DNA template; however, in PACRAT an additional sequence is concatenated to each primer. The addition of a 5′-T7 RNA polymerase promoter to one primer enables the DNA produced in RPA to undergo transcription, and the inclusion of a 3′-aptamer coding sequence in the other primer installs the template required for the production of quantitative readout. Thus, using these two primers and the RPA reaction, a dsDNA is assembled containing a 5′-T7 RNAP promoter and a 3′-aptamer coding sequence. The dsDNA is then transcribed, resulting in an RNA consisting of the pathogen target amplicon with a 3′-tail containing a fluorescent RNA aptamer, which forms a complex with a dye that is added to the reaction. This enables real-time, quantitative tracking of fluorescence intensity. Qualitative detection can also be performed by observing the amplicons under a transilluminator. The RNA produced can then participate in additional rounds of reverse transcription and RPA amplification, further feeding into the exponential RPA cycle.

Validation of PACRAT with SARS-CoV-2 target

We first sought to detect SARS-CoV-2, the causative pathogen of the COVID-19 pandemic, using PACRAT. With many regions of the world lacking adequate testing infrastructure, low-cost, isothermal detection systems are of high value in meeting the need for rapid testing (Khan et al. 2020; Budd et al. 2023). Specifically, we targeted two genes in SARS-CoV-2: the surface glycoprotein gene, commonly termed spike (S), and the nucleocapsid gene (N), which have both been widely targeted in available nucleic acid amplification methods (Naqvi et al. 2020; Zhou et al. 2022). We initially used primers targeting the N3 region of the SARS-CoV-2 nucleocapsid gene (Supplemental Table S1). This region is of high interest for viral detection because it is subject to less genetic drift than the well-known spike protein (Dutta et al. 2020), and it is used as a target in many “generalist” SARS-CoV-2 detection platforms, such as the antigen-based lateral flow immunoassays that are widely available (Peto and UK COVID-19 Lateral Flow Oversight Team 2021). The aptamer coding primer contained the sequence for the Broccoli aptamer, which binds the dye DFHBI-1T and produces GFP-like fluorescence in PACRAT (Filonov et al. 2014). We analyzed both the DNA and RNA products of PACRAT reactions targeting this sequence. We first analyzed the DNA to confirm RPA produced the correct size DNA product that matches the expected amplicon size (Fig. 1B). The RNA gel confirmed the presence of the correct RNA product. Furthermore, the RNA product bound DFHBI and activated its fluorescence, confirming a correctly folded aptamer was present in the amplicon–aptamer fusion sequence produced (Fig. 1B).

Optimization of the PACRAT system

We next aimed to optimize PACRAT conditions to achieve highly sensitive detection. We tested a range of different temperatures (37°C–42°C) and observed that 39°C was the optimum temperature (Supplemental Fig. S1).

The commercial RPA kit contains three kit reagent mixes (Armes and Stemple 2007). The patent for this proprietary kit describes the components of this reaction (Armes and Stemple 2007; Piepenburg et al. 2010). The 2× Reaction Buffer is described as containing MgCl2, KCl, DTT, Tris-HCl buffer, dNTPs, crowding agents, and bovine serum albumin. The 20× Core Reaction Mix contains polymerase, helicase, and resolvase, whereas the 10× E-Mix contains the D-loop formation and resolution components (RecA, RecF, RecO, RecR, ATP, SSBs, and DNA polymerase V). Additionally, our PACRAT reactions contained added buffer components to support transcription and aptamer folding (Heili et al. 2018). We speculated that the optimal protein concentrations for “pure” RPA might differ from that required for PACRAT, which requires “handoff” from the RPA complex to T7 RNA polymerase. For this reason, we varied the concentrations of the components of the commercial RPA kit. We found that the optimum for the commercial kit reagents in PACRAT was substantially lower than that for conventional RPA, with a final reaction concentration of 0.3× of RPA reagents yielding the best results in PACRAT (Fig. 2A). While investigating which component of the RPA kit may be detrimental to PACRAT, we found that omitting the kit 2× Reaction Buffer gave the best results (Fig. 2B). This is presumably because our added transcription buffer components were compatible with RPA, but the addition of further salts and other buffer components from the 2× Reaction Buffer was detrimental to the transcription reaction in PACRAT.

FIGURE 2.

FIGURE 2.

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Optimization of PACRAT reactions. (A) Bar plot showing the normalized end point fluorescence values for PACRAT reactions targeting the SARS-CoV-2 nucleocapsid N3 gene, with varying concentrations of reagents from the TwistDx RPA Kit. TwistDx reagents include the 2× Reaction Buffer, 20× Core Reaction Mix, and 10× Probe E-Mix. (B) Bar plot showing the normalized end point fluorescence values for PACRAT reactions with varying concentrations of the 2× Reaction Buffer from the TwistDx RPA Kit. Error bars represent SEM, where n = 3. Individual data points for each triplicate are shown as black circles.

Additionally, crowding agents are known to play an important role in RPA (Piepenburg et al. 2006; Li et al. 2019). The 2× Reaction Buffer contains 1%–12% by volume of polyethylene glycol (PEG)—a crowding agent that has been shown to be essential for RPA (Piepenburg et al. 2010). PEGs have been found to affect RecA and gp32(N) mediated DNA invasion and extension, along with aiding polymerase processivity and increasing amplification efficiency (Piepenburg et al. 2010). Thus, we hypothesized that omitting 2× Reaction Buffer from the reactions may be decreasing the efficiency. To test this, we conducted reactions with titrations of PEG 20,000, going up to a final PEG concentration of 40% in the reactions. We saw that PACRAT reactions containing 20% PEG 20,000 by volume had higher fluorescence signals (Supplemental Fig. S2). This may be due to better transcription rates or more efficient aptamer folding due to more crowding.

Another beneficial reagent for in vitro transcription reactions is inorganic pyrophosphatase (IPP), which enhances RNA yield because of its ability to catalyze the hydrolysis of pyrophosphate and prevent magnesium from precipitating out of reactions (Cunningham and Ofengand 1990; Wang et al. 2015). We found that a concentration of 1.1 μM IPP gave an optimum signal in PACRAT (Supplemental Fig. S3).

Following validation and optimization of one-pot, one-step PACRAT reactions, we sought to investigate the limit of detection using real-time fluorescence results in reactions with the N3 region of the Nucleocapsid gene of SARS-CoV-2 as a target. PACRAT showed high sensitivity with this amplicon, readily distinguishing a negative reaction from a single-digit-copy number (4.5 copies) reaction, with the distinguishable difference in as little as 1 h (Fig. 3A). This was validated by both real-time fluorescence analysis and gel analysis (Fig. 3B). Additional incubation time allows for better distinction between the reaction containing the target and the negative control.

FIGURE 3.

FIGURE 3.

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Limit of detection of PACRAT with SARS-CoV-2 N gene. (A) The limit of detection for PACRAT reactions with SARS-CoV-2 RNA is shown to be 4.5 copies/reaction. Shaded area represents SEM, where n = 3. Transilluminator image shows fluorescence of reactions. (B) (Left) Urea-PAGE gel is stained with SYBR Gold to show RNA product from the end point of reactions with 4.5 copies of SARS-CoV-2 RNA target versus reactions with no target. The gel is staining the expected RNA product at a size of 127 nt. (Right) DFHBI-stained gel, where DFHBI is the cognate dye for the Broccoli aptamer, shows the band that contains the Broccoli aptamer, also at the expected size.

PACRAT exhibits single-copy detection and <10-min time to detection

Finally, we proceeded to investigate the time to detection for PACRAT reactions and, here, we switched to the SARS-CoV-2 B.1.167 spike gene as a target. The spike gene is prone to multiple mutations as seen in variants of SARS-CoV-2 such as the Alpha (B.1.17), Delta (B.1.167), and Omicron (BA.1/BA.2) variants (Ou et al. 2022). Thus, we hoped to show the versatility of our system by investigating its ability to detect the SARS-CoV-2 Delta (B.1.167) spike gene. We observed distinguishable differences in Pepper aptamer fluorescence between reactions with and without target, within only 10 min of starting the reaction (Fig. 4A). We also conducted a dilution series to ascertain our limit of detection, establishing that PACRAT was highly sensitive. Our results show the detection of a single copy of the SARS-CoV-2 B.1.167 spike gene (Fig. 4B), which is comparable to the sensitivity of PCR (Arnaout et al. 2020).

FIGURE 4.

FIGURE 4.

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Time to detection and limit of detection of PACRAT with SARS-CoV-2 spike gene. (A) Testing the time to detection for PACRAT. Real-time fluorescence graph shows Pepper aptamer fluorescence curves for reactions with SARS-CoV-2 B.1.167 spike gene and reactions with no target. Time to detection (distinguishable difference between reactions with target and reactions without target) is 10 min for optimized reactions. Shaded area represents SEM, where n = 3. (B) Testing the sensitivity of PACRAT. Bar plot shows the normalized end point fluorescence values for PACRAT reactions with different target copies of SARS-CoV-2 B.1.167 spike gene. Individual data points for each triplicate are shown as black circles. Error bars represent SEM, where n = 3.

PACRAT can detect multiple pathogen RNAs in multiplexed reactions

We next sought to demonstrate the detection of two different pathogens in the same one-pot reaction. E. coli is a well-established diarrhea-causing agent, and one gene associated with diarrheagenic E. coli is a transcriptional activator of aggregative adherence fimbriae I (AggR). Patients with confirmed SARS-CoV-2 pulmonary infection have experienced digestive symptoms similar to that of diarrheagenic E. coli, additionally including nausea and vomiting, underscoring the need for simultaneous detection of these pathogens with overlapping symptomology (Carvalho et al. 2020; Megyeri et al. 2021).

We made two different aptamer coding primers for AggR detection coding for either the Broccoli aptamer (Filonov et al. 2014) or the Pepper aptamer (Chen et al. 2019), showing the easy adaptability of the system for the detection of any pathogenic agent. We used the Pepper aptamer with the HBC620 fluorophore, which has a fluorescent emission maximum in the red region of the visible spectrum, because this provides high spectral separation from the green-emitting Broccoli/DFHBI-1T complex (Filonov et al. 2014; Chen et al. 2019). We found that PACRAT can achieve sensitive detection of the AggR target (Supplemental Fig. S4).

For multiplexed target detection, we then used our designed primer sets—one set (Broccoli) for AggR in E. coli and one set (Pepper) for the spike protein gene in B.1.167 SARS-CoV-2—and tested their efficiency in simultaneous detection of these two different RNA targets (Fig. 5A). Consistent with our expectations, reactions containing the E. coli AggR gene (Broccoli/DFHBI-1T/green) and/or SARS-CoV-2 B.1.167 spike gene (Pepper/HBC620/red) each yielded signals in the expected channel depending on the presence of the appropriate amplicon (Fig. 5B). That is, reactions with only AggR RNA gave green fluorescence, whereas reactions with SARS-CoV-2 B.1.1.167 spike RNA gave red fluorescence. Reactions with both amplicons showed high fluorescence in both channels.

FIGURE 5.

FIGURE 5.

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Multiplexed detection of pathogens by PACRAT. (A) Schematic of multiplexed detection of two RNA targets: AggR gene in E. coli and the spike gene in B.1.167 SARS-CoV-2. Reactions with only E. coli target should only exhibit Broccoli fluorescence, whereas reactions with only the SARS-CoV-2 target should exhibit only Pepper fluorescence. Reactions containing both targets should show dual fluorescence and reactions with no target should show no fluorescence in any channel. (B) Bar graph showing end point fluorescence values for Broccoli and Pepper aptamers in reactions with SARS-CoV-2 RNA target or E. coli RNA target, or both targets or neither. Individual data points for each triplicate are shown as black circles. Error bars represent SEM, where n = 3.

These results indicate that the PACRAT system can provide multiplexed detection by using multiple gene-specific primer sets, thus enabling successful one-pot, dual-detection of two different pathogenic targets. As proof of concept, we performed another multiplexed reaction (Supplemental Fig. S5), in which we detected two variants of SARS-CoV-2: the Delta variant (B.1.167) and wild-type SARS-CoV-2 (WT) by designing primers targeting the spike gene of these variants (Supplemental Table S1).

Saliva sample matrix does not interfere with PACRAT

To determine the versatility of our system with animal samples, we examined PACRAT reactions in the presence of saliva. Saliva has been widely used as a testing specimen for the detection of SARS-CoV-2 because of the facility of sample collection (Sun et al. 2021a), but it is well-known as an interfering matrix component in PCR (Morais et al. 2022). We sought to demonstrate the resilience of PACRAT to saliva matrix by performing PACRAT with the SARS-CoV-2 spike gene as a target, in the presence of pre-COVID-19 saliva. Saliva did not interfere with these reactions (Fig. 6), and target-containing reactions without saliva had similar fluorescence values to target-containing reactions with saliva. Thus, when using our PACRAT system, point-of-care technicians working with minimal training in suboptimal laboratory conditions can potentially avoid complex and costly sample preparation and nucleic acid extraction methods required for analysis via PCR (Morais et al. 2022).

FIGURE 6.

FIGURE 6.

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PACRAT is robust to saliva sample matrix. Pre-COVID-19 saliva was added to PACRAT reactions. Bars show end point fluorescence for reactions with SARS-CoV-2 B.1.167 Spike RNA target (left two bars) or without target (right two bars). Individual data points for each triplicate are shown as black circles. Error bars represent SEM, where n = 3.

PACRAT can detect as little as one E. coli cell without sonication steps

Finally, we applied PACRAT to detect nonpathogenic E. coli. We targeted a 204-nt region of the conserved 16S rRNA of the E. coli bacterial genome as shown previously in a classical RPA system (Biyani et al. 2021). We first used a short sonication lysis protocol followed by PACRAT on the E. coli lysate to discover that PACRAT can detect as little as one E. coli cell (Fig. 7A; Supplemental Fig. S6). Furthermore, we found that we could eliminate the sonication step and replicate the same trend (Fig. 7B; Supplemental Fig. S6) by adding lysis buffer to bacteria-containing samples and incubating for 5 min. This further demonstrates the versatility of our system in point-of-care settings with limited instrumentation.

FIGURE 7.

FIGURE 7.

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PACRAT can directly detect E. coli. E. coli cell lysate was added to PACRAT reactions either after sonication and spin-down (A) or without sonication and spin-down (B). Error bars or shaded areas represent SEM, where n = 3.

DISCUSSION

Here, we have shown that the RPA reaction can be coupled for the first time to in vitro transcription in a one-pot reaction, enabling readout of pathogen detection reactions with PACRAT, which possesses single-copy sensitivity or single-cell sensitivity, along with sub-10-min speed. PACRAT circumvents the requirement for expensive, bespoke labeled probes and enables straightforward primer design, while the one-pot nature of the reaction and the real-time fluorescent output mitigates the need for highly skilled technicians, making it ideal for point-of-care and field use. The robustness of PACRAT to saliva matrix and its ability to detect E. coli without sonication procedures underscore the effectiveness of the system in biological samples. PACRAT also allows for multiplexed, multicolor simultaneous detection of pathogens, exhibiting its usefulness in multiplexed detection without the need for expensive and time-consuming sequencing methods. The use of the RPA cycle, which exhibits PCR-like sensitivity and a high degree of resistance to sample inhibitors, coupled with in vitro transcription–based detection, represents a significant advance over other isothermal techniques, which exhibit lower sensitivity and require longer time or multiple-step amplification.

PACRAT is a robust tool and proof of concept of a potential diagnostic technology. It is suitable for rapid, early detection of pathogens and their variants. It promises to be a significant invention in combating SARS-CoV-2 and other pathogens. With the swift emergence of multiple SARS-CoV-2 variants, rapid, sensitive detection of SARS-CoV-2 and its variants is becoming increasingly important. Although real-time-PCR methods are the most well-established tests for SARS-CoV-2, the requirements for expensive reagents and equipment, skilled technicians, and more complicated workflows impede simple and fast turnaround of diagnostic results at point-of-care locations and in resource-poor settings (Khan et al. 2020). PACRAT offers the advantage of amplification at a constant low temperature of 39°C, a temperature easily achievable by using a water bath and hence avoiding the use of expensive thermocyclers (Liu et al. 2017). For use in these settings, PACRAT could be complemented by other enabling technologies for rapid sample preparation, such as HUDSON (heating unextracted diagnostic samples to obliterate nucleases) and rapid, low-cost silica-based purification schemes (Myhrvold et al. 2018; Rabe and Cepko 2020; Capriotti et al. 2024).

PACRAT proceeds in one pot while using multiple replication enzymes—recombinases, strand-displacing polymerases, and SSBs—for isothermal replication of dsDNA. This is further followed in the same reaction by in vitro transcription–based detection with T7 RNA polymerase. We have shown that multiple optimizations of buffer and reagent concentrations enabled the one-pot, one-step, multienzyme cascade that underlies PACRAT. This cascade is reminiscent of biological systems. The multienzyme cascade used in PACRAT that allows reverse transcription, amplification and transcription to occur in a one-pot reaction demonstrates possibilities for other cascaded enzymatic reactions, with potential promise for enhanced future cell-free biocomputational systems (Sharon et al. 2023), enzymatic circuits using T7 RNA polymerase aptamers (Kim et al. 2022), and engineered RNA-based systems, such as small transcription activating RNAs (STARs) (Chappell et al. 2015).

MATERIALS AND METHODS

The primer design for RPA involves two primers, one of which is an aptamer coding primer that, in the presence of the target RNA, allows the amplicon to be conjugated to a fluorescent aptamer. Supplemental Table S1 contains the sequences of all the oligonucleotides used. Aptamer coding primers were designed using secondary structure prediction of the amplicon–aptamer fusion sequence produced, ensuring correct aptamer folding (Supplemental Fig. S7), and we observed that predictions of incorrect aptamer folding can lead to unsuccessful PACRAT reactions (Supplemental Fig. S8). Oligonucleotides were obtained as desalted grade from Integrated DNA Technologies (Coralville) and were used after resuspension. The TwistAmp Liquid Basic RPA kit (TwistDX) was used for all the PACRAT reactions. T7 RNAP and M-MuLV reverse transcriptase were overexpressed in-house as described previously (Aufdembrink et al. 2020). Target RNA templates were also prepared in-house as described previously (Aufdembrink et al. 2020). A 10× transcription buffer stock was prepared with 400 mM Tris-HCl, 240 mM MgCl2, 20 mM Spermidine-HCl, 10 mM DTT, and 1 M KCl (pH 7.9). A 20 mM stock of dNTP mix (Thomas Scientific) was prepared and a 20 mM stock of NTP mix (Larova GmbH) was made with 2× the amount of GTP as other NTPs.

PACRAT reactions

PACRAT reactions were run in a total volume of 50 µL. They contained reagents from the TwistAmp Liquid Basic RPA kit, with final concentrations of 0.3× Basic E-Mix and 0.3× of 20× Core Reaction Mix (as optimized in Fig. 2). 10× transcription buffer was added to a final concentration of 1×. Amplification reactions contained a final concentration of 0.34 μM T7 primer, 0.34 μM aptamer coding primer, 1.8 mM dNTP mix (0.45 mM each of dATP, dTTP, dGTP, and dCTP), 4 mM NTP mix (0.8 mM ATP, 1.6 mM GTP, 0.8 mM UTP, 0.8 mM CTP), 10 μM cognate aptamer-binding dye, 1.1 μM IPP (Bayou Biolabs), 1 μM T7 RNA polymerase, 0.25 μM M-MuLV reverse transcriptase, and 20% w/v PEG 20000 (Santa Cruz Biotechnology). Finally, 8.4 mM Mg(OAc)2 was added to the caps of the reaction tubes and the reactions were mixed with six inversions, followed by a quick spin-down. Immediately after mixing, the reactions were loaded into plates and monitored on a Bio-Rad CFX96 for 2 h at 39°C or until fluorescence plateaued. For PACRAT reactions using pooled human saliva, pre-COVID-19 saliva was used (Lee BioSolutions), and saliva was added to 2(v/v)% in the reaction mixture. Real-time fluorescence values and end point fluorescence values in each graph were normalized based on the lowest and highest average values in the system. Except where otherwise stated, all reactions were done with 4.5 × 1010 copies of SARS-CoV-2 B.1.167 spike gene target.

Gel staining

Urea-PAGE gels were made at 10% acrylamide with 1× TBE and 8 M urea. Gels stained for RNA product visualization were stained in 1× SYBR Gold dye for 15 min and imaged on an Omega Lum G (Aplegen) using the SYBR Safe filter. Gels stained with cognate dye for aptamer visualization were incubated in a folding buffer containing 10 mM Tris-Acetate pH 8, 1 mM MgCl2, and 50 mM KCl for two intervals of 30 min, changing the buffer between the intervals. This was followed by incubation in a folding buffer containing 10 μM aptamer-binding dye for 15 min and imaged on an Omega Lum G using a suitable filter for the fluorogen used.

E. coli cell culture and lysis

BL21 E. coli cells were grown in LB media at 37°C shaking at 250 RPM until reaching OD600 ≈ 1. For experiments involving sonication, cultures were kept on ice and subjected to sonication with two 2.7-kJ steps. Five hundred microliters of lysis buffer containing 1% Nonidet P-40 solubilizing agent (Sigma N-6507, Lot # 62H2508) was pipetted into the culture and incubated for 5 min on ice. This was followed by a spin-down at 4000 RPM, 4°C. The supernatant was used directly for PACRAT reactions. For experiments without any sonication, 500 μL of lysis buffer was pipetted into the E. coli cell cultures, followed by a 5 min incubation period. The lysate was directly used for PACRAT reactions.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

ACKNOWLEDGMENTS

We thank Peter Unrau, Andrej Lupták, and members of the Engelhart laboratory for helpful discussions. Several figures in this manuscript were created with BioRender.com. This work was supported by NASA Contract 80NSSC18K1139 under the Center for Origin of Life (to A.E.E. and K.P.A.).

Author contributions: The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

Footnotes

MEET THE FIRST AUTHOR

Pavana Khan.

Pavana Khan

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Meet the First Author(s) is an editorial feature within RNA, in which the first author(s) of research-based papers in each issue have the opportunity to introduce themselves and their work to readers of RNA and the RNA research community. Pavana Khan is the first author of this paper, “PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription.” Pavana is a PhD candidate in Dr. Aaron Engelhart's laboratory, in the Molecular, Cellular, Developmental Biology, and Genetics Graduate Program at the University of Minnesota. The laboratory investigates the role of DNA and RNA as functional nucleic acids in the form of aptamers, DNAzymes, and RNAzymes. They integrate these functional nucleic acids as tools for detecting and tracking disease or as models for understanding evolutionary biology. A longstanding interest of the laboratory has been to engineer and use RNA aptamers in isothermal, pathogen detection systems.

What are the major results described in your paper and how do hey impact this branch of the field?

We developed a unique, fluorescent aptamer-based isothermal detection system called PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription). PACRAT can detect SARS-CoV-2 with single-copy detection limits, E. coli with single-cell detection limits, and 10-min detection times. For the first time, our PACRAT technology shows that with optimization of reagent and buffer conditions, recombinase polymerase amplification (RPA) can be cascaded into an in vitro transcription reaction with subsequent fluorescence detection in a one-pot reaction. Previous systems have usually incorporated a two-step method, with an amplification step followed by a detection step, adding complication, and increasing test cost and time. PACRAT circumvents this by being a one-step, one-pot detection system, thus enabling the use of this system in resource-poor regions and at-home settings.

What led you to study RNA or this aspect of RNA science?

I was fascinated by the potential of RNA beyond its canonical role as an information carrier and especially interested in all the light-up RNA aptamers that span the fluorescence spectrum. I was first drawn to the project because of the potential of isothermal nucleic acid amplification tests to facilitate disease detection in resource-poor regions around the world. Coming from Bangladesh, where there are no medical laboratories in remote villages, technology that enables rapid, easy-to-use, point-of-care detection kits for infectious diseases could help save millions of lives. PACRAT is thus a product of my fascination for fluorescent RNA aptamers and my motivation for facilitating access to sensitive and user-friendly pathogen detection tests.

During the course of these experiments, were there any surprising results or particular difficulties that altered your thinking and subsequent focus?

I started working on isothermal detection before COVID-19 hit. Since that time, the field has changed a lot. Once the pandemic started, we pivoted to focusing on SARS-CoV-2 detection but faced many challenges in optimizing the test. It was a difficult couple of years, as suddenly, many people were working and publishing successful results for new isothermal systems, and it was hard not to become demotivated and give up on PACRAT. Although challenging, I tried multiple different combinations of reagent and buffer concentrations, different temperatures, and different aptamer–primer designs and finally optimized the system to have the same sensitivity as a commercially available PCR test.

If you were able to give one piece of advice to your younger self, what would that be?

I would encourage my younger self to embrace failure in experiments because it is an inevitable part of the scientific process. A failed project is not a waste of time, because you still learned many invaluable skills, you learned how to troubleshoot, and you increased your knowledge in the field. Throughout graduate school, I put deadlines on when I should finish a project or when I should have a publication ready, and while reasonable expectations are great for productivity, there is no race, and it is okay to take things slow. Setbacks are not signs of defeat but rather opportunities for growth, and I would tell my younger self to take solace in the small victories. Even if nine gels fail and one succeeds, that is still a win.

What are your subsequent near- or long-term career plans?

In the short term, I aim to complete my graduate studies and then pursue a postdoctoral position, where I can further expand my skills and knowledge in RNA biology. Ultimately, my aspiration is to transition into academia and assume a professorial role within a primarily undergraduate institution. Drawing inspiration from my own transformative undergraduate experience at Carleton College, where the close-knit community and dedicated faculty ignited my passion for science, I am eager to replicate that environment, guiding and inspiring the next generation of scientists. I hope to establish a small teaching laboratory where I can engage students in hands-on research exploring the roles of functional nucleic acids in living organisms.

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