Single-tube One-step Gel-based RT-RPA/PCR for Highly Sensitive Molecular Detection of HIV Virus

Abstract

We developed a single-tube one-step gel-based reverse transcription-recombinase polymerase amplification (RT-RPA)/polymerase chain reaction (PCR) (termed “SOG RT-RPA/PCR”) to detect human immunodeficiency virus (HIV). To improve the assay sensitivity, the RNA template is preamplified by RT-RPA prior to PCR. To simplify the detection process and shorten the assay time, we embedded PCR reagents into agarose gel, making it to physically separate the reagents from the RT-RPA reaction solution in a single tube. Due to the thermodynamic properties of agarose, the RT-RPA reaction first occurs independently on top of the PCR gel at a low temperature (e.g., 39°C) during the SOG RT-RPA/PCR assay. Then, the RPA amplicons directly serve as the template for the second PCR amplification reaction, which begins when the PCR agarose dissolves due to the elevated reaction temperature, eliminating the need for multiple manual operations and amplicon transferring. With our SOG RT-RPA/PCR assay, we could detect 6.3 copies of HIV RNA per test, which is a 10-fold higher sensitivity than that of standalone real-time RT-PCR and RT-RPA. In addition, due to the high amplification efficiency of RPA, the SOG RT-RPA/PCR assay shows stronger fluorescence detection signals and a shorter detection time compared to the standalone real-time RT-PCR assay. Further, we detected HIV viral RNA in clinical plasma samples and validated the superior performance of our assay. Thus, the SOG RPA/PCR assay offers a powerful method for simple, rapid, and highly sensitive nucleic acid-based molecular detection of infectious diseases.

Graphical Abstract

Single-tube one-step gel-based RT-RPA/PCR platform for simple, rapid and highly sensitive molecular detection of HIV virus.

Introduction

Human immunodeficiency virus (HIV) is a bloodborne virus that causes acquired immune deficiency syndrome (AIDS), which has become a global health problem.^1-4 Early and sensitive detection of HIV plays a crucial role in effectively preventing the spread of disease and timely treating and managing patients to control disease progression.^5-7 Polymerase chain reaction (PCR)/reverse transcription-PCR (RT-PCR) is the current standard method for molecular detection of many infectious diseases, including HIV/AIDS.^8-10

The detection performance of PCR/RT-PCR can be further improved by combining nucleic acid pre-amplification with PCR.^11-14 For instance, researchers have explored nested PCR, which involves two successive PCR reactions, to improve the sensitivity and specificity of nucleic acid detection.^{15, 16} To further simplify the amplification process and improve the detection efficiency, recombinase polymerase amplification (RPA) has recently been coupled to PCR for nucleic acid detection.¹⁷ Since RPA does not require temperature cycling and can produce many amplicons within a short period (less than 10 min), it significantly shortens the reaction time and improves the detection sensitivity.¹⁸ In addition, RPA isothermal amplification has a higher tolerance for potential inhibitors than PCR, ^{19, 20} potentially simplifying nucleic acid sample preparation. However, this approach typically requires manual operations to open the reaction tube and transfer the RPA amplicons to the second PCR reaction, which is a time-consuming and labour-intensive process and potentially causes cross-contamination.

Here, we propose a simple, rapid, highly sensitive single-tube one-step gel-based RT-RPA/PCR assay (termed “SOG RT-RPA/PCR”) by coupling RT-RPA pre-amplification with PCR using the PCR gel by taking advantage of the thermodynamic properties of agarose. In the SOG RT-RPA/PCR assay, the PCR reagents are embedded in agarose gel to enable physical separation from the RT-RPA reaction solution in a single tube. Initially, the first RT-RPA amplification occurs independently on the PCR gel while the gel remains in its gel state during the RPA incubation. Following the RT-RPA reaction on the PCR gel, the PCR gel dissolves completely at an elevated temperature and mixes with the RPA amplicons. Thus, the RPA amplicons directly serve as the template to initiate the second PCR amplification, which eliminates the need for manual operations and amplicon transferring and enables a simple single-tube assay. To demonstrate its use, we employed the SOG RT-RPA/PCR assay to detect HIV RNA and achieved a superior performance compared with the conventional standalone RT-PCR and RT-RPA approaches. Lastly, we further tested our assay using HIV clinical plasma samples and demonstrated great potential for rapid and highly sensitive molecular detection.

Materials and methods

RNA templates.

HIV RNA was extracted from AcroMetrix HIV-1 High Control samples (Thermo Scientific) and clinical plasma samples using the QIAamp Viral RNA Mini kit (QIAGEN). The clinical plasma samples were de-identified and obtained by the University of Connecticut Health Center under the approval of its Institutional Review Board (protocol #21-014-2).

Oligonucleotides.

All oligonucleotides were purchased from Integrated DNA Technologies. The sequences of all oligonucleotides used are shown in Table S1. The RPA primers and RPA probe were selected from a previous study,²¹ whereas the PCR primers and PCR probe were designed in this study.

PCR gel preparation.

A PCR reaction mix containing 1 x SsoAdvanced Universal Probes Supermix (Bio-Rad), 0.5-0.9 μM PCR forward and reverse primers, and 0.2-0.25 μM PCR probe was prepared. The concentrations were calculated based on the final 50 μL one-pot system. The 2% agarose solution was prepared by resuspending agarose (ultra-low gelling temperature, molecular biology grade; Sigma, Cat No. A2576) in water and melting at 90°C. The melted agarose was added in the lid of the tube containing the PCR reaction mix. The melted agarose and PCR reagents were mixed well by inverting the tube several times immediately after the lid was closed and were promptly aliquoted into PCR tubes (42 μL in a tube). The PCR tubes were left on ice to generate the PCR gel. The agarose concentration of the gel was 0.2% unless otherwise specified. The PCR gels were used within 2 hours.

One-step RT-RPA/PCR.

RT-RPA reaction mix was prepared using the TwistAmp Basic kit (TwistDx). One freeze-dried RPA pellet was resuspended in 29.5 μL rehydration buffer, 1.2 μL 20 μM RPA forward primer, 1.2 μL 20 μM RPA reverse primer, varied volume RNA template, and 1 μL SuperScript IV reverse transcriptase (200 U/μL, ThermoFisher Scientific). For the assessment of the sensitivity and the PCR gel stability, 1.5 μL RNase H (5 U/μM, New England Biolabs) was also added. Finally, 2.5 μL of 280 mM magnesium acetate was added to the RT-RPA mixture. Immediately after the addition of magnesium acetate, 8 μL RT-RPA reaction mix was added on the 42 μL PCR gel. One-step RT-RPA/PCR was performed as follows: 39°C for 10 min, 95°C for 30 s, and 30–45 cycles at 95°C for 5 s followed by 60°C for 30 s, using the CFX96 Touch Real-time PCR System (Bio-Rad) or CFX Opus 96 Real-time PCR System (Bio-Rad).

Real-time RT-PCR.

Real-time RT-PCR reaction mix containing 1 x Reliance One-Step Multiplex RT-qPCR Supermix (BioRad), 0.9 μM PCR forward primer, 0.9 μM PCR reverse primer, 0.25 μM PCR probe, and RNA template was prepared. One-step RT-PCR was performed on a C1000 Touch Thermal Cycler (Bio-Rad) as follows: 50°C for 10 min, 96°C for 10 min, 45 cycles of 60°C for 2 min and 98°C for 30 s. Real-time readouts were acquired using the CFX96 Touch Real-time PCR System (Bio-Rad).

Real-time RT-RPA.

Real-time RT-RPA was performed using the TwistAmp exo kit (TwistDx). SuperScript IV reverse transcriptase was added to the rehydration solution. The final reaction mix contained 0.42 μM RPA forward primer, 0.42 μM RPA reverse primer, 0.15 μM RPA probe, 4 U/μL SuperScript IV reverse transcriptase, and 14 mM MgOAc in 50 μL. The reaction mix was incubated at 39°C for 45 min on a C1000 Touch Thermal Cycler (Bio-Rad). Real-time readouts were acquired using the CFX96 Touch Real-time PCR System (Bio-Rad).

Digital RT-PCR.

Digital RT-PCR was carried out using the QuantStudio 3D Digital PCR system (ThermoFisher Scientific). The reaction mix containing 1x Reliance One-Step Multiplex Supermix (Bio-Rad), 0.9 μM PCR forward primer, 0.9 μM PCR reverse primer, 0.25 μM PCR probe, and RNA template was prepared and loaded on the QuantStudio 3D Digital PCR Chip v2 using a QuantStudio 3D Digital PCR Chip loader. PCR was carried out on the ProFlex 2x Flat PCR system (ThermoFisher Scientific) as follows; 50°C for 10 min, 96°C for 10 min, 44 cycles of 60°C for 2 min and 98°C for 30 s, 60°C for 2 min, and hold at 10°C. After PCR, the chips were imaged by a QuantStudio^™ 3D Digital PCR instrument. Imaging data were analyzed by QuantStudio 3D AnalysisSuite Cloud Software (ThermoFisher Scientific).

Fluorescence imaging.

Fluorescent images were acquired by the ChemiDoc MP Imaging System (Bio-Rad) and an LED transilluminator (Maestrogen). When using the ChemiDoc MP Imaging System, PCR tubes were placed on the blue sample tray and images were acquired using a blue transillumination excitation source and 590/110 emission filter.

Results and discussion

SOG RT-RPA/PCR assay

Figure 1A illustrates the workflow of the SOG RT-RPA/PCR assay for nucleic acid-based molecular detection. To prepare the PCR gel, we first melted agarose in water at 90°C and mixed it with PCR reaction reagents. Next, we promptly aliquoted the mixture into individual PCR tubes and placed the tubes on ice, enabling rapid gelation of the PCR gel. For the SOG RT-RPA/PCR assay, we directly added the RT-RPA reaction solution (with or without target nucleic acids) on the PCR gel in the reaction tube. Initially, the reaction tubes were incubated at 39°C for 10 min for the first RT-RPA pre-amplification. As the PCR gel does not dissolve at 39°C, and remains in its gel state (Figure S1), the RT-RPA reaction occurs independently on top of the PCR gel and generates many RPA amplicons. After the first RPA amplification, we increased the temperature of the reaction tube to 95°C for 30 s, which completely melts the PCR gel, activates the PCR polymerase, and denatures the RPA amplicons. Thus, the RPA amplicons generated from the RPA reaction mixes with the melted PCR reaction solution without the need for amplicon transferring or manual operation. During the PCR thermal cycling, the RPA amplicons directly serve as the template to initiate the PCR amplification reaction, because the PCR primers are designed within the RPA amplicons (Figure S2). Thus, the SOG RT-RPA/PCR assay provides a simple, direct, and robut approach for single-tube nucleic acid detection, potentially minimizing the risk of carry-over contamination.

Figure 1.

Operation and working principle of the SOG RT-RPA/PCR assay. (A) Experimental workflow of the SOG RT-RPA/PCR assay in the PCR gel. The PCR gel contains agarose gel and all PCR reaction agents except for the nucleic acid template. In the assay, the RT-RPA reaction solution is directly added on the PCR gel in the reaction tube. The RT-RPA pre-amplification is followed by PCR cycles. (B) Endpoint fluorescence after 30 cycles was visualized by an imaging system (top) and portable LED transilluminator (bottom). The RT-RPA reaction solution was mixed with the PCR reaction solution without agarose (samples 1–4) or added on the 0.2% PCR gel (samples 5–8). The concentration of HIV RNA template is 33 copies/uL. (C) Real-time fluorescence readouts of each sample.

Due to the presence of the TaqMan probe, the fluorescence signal of the PCR reaction can be visualized with a fluorescence imaging instrument at the endpoint (Figure 1B) or monitored in real-time by a PCR instrument (Figure 1C). When we directly mixed the RT-RPA solution with the PCR reaction solution without agarose, we observed no fluorescence signal (Figure 1B and C), which may be attributed to the poor compatibility of the RPA reaction and PCR reaction systems. However, we observed strong fluorescence signal when we placed the RT-RPA reaction solution, containing both reverse transcriptase and the RNA template, on the PCR gel (Figure 1B and C). This result indicates that the PCR gel not only improves the compatibility of the two reaction systems but also enables sequential RT-RPA followed by PCR. Unlike conventional real-time RT-PCR, the fluorescence signal of the SOG RT-RPA/PCR reaction rose with less than 5 cycles (Figure 1C), suggesting that the first RT-RPA pre-amplification generated sufficient template to initiate downstream PCR amplification, significantly shortening the detection time.

In general, fluorescence generated from TaqMan-based PCR is not intended for imaging because it is not strong enough to be visualized. However, in our SOG RT-RPA/PCR assay, we can directly distinguish the positive result from the negative one by using either an imaging system or a low-cost, portable LED transilluminator (Figure 1B). When using the LED transilluminator, the negative result appears red because the PCR supermix contains the ROX dye, whereas the positive result is indicated by a change to yellow following excitation of fluorescein. Thus, the SOG RT-RPA/PCR assay provides a visual approach for rapid detection of nucleic acids in a single reaction tube.

Optimization of the SOG RT-RPA/PCR assay

There is limited compatibility between the RPA reaction solution and PCR reaction solution. To this end, we embedded PCR reaction reagents into agarose gel to physically separate the PCR reaction solution from the RT-RPA reaction solution during the first RPA pre-amplification. To minimize the effect of RPA reaction solution on the second PCR reaction, we added 8 μL RT-RPA reaction solution into the 42 μL PCR gel reaction system. We selected SsoAdvanced Universal Probes Supermix for PCR, because it showed an excellent performance in the presence of the RPA reaction buffer.

We optimized the agarose concentration of the PCR gel in the assay. A previous study showed that the PCR efficiency does not decrease unless the agarose concentration is over 3%.²² Yet, a high agarose concentration solution makes it more difficult to accurately transfer and pipette the agarose solution due to its high viscosity. Thus, we prepared the PCR gel with an agarose concentration range of 0–1%. We evaluated the performance of the SOG RT-RPA/PCR assay by both real-time fluorescence monitoring and endpoint fluorescence detection. As shown in Figure 2A, the assay worked across a wide concentration range of 0.2–1%; however, poor amplification occurred in some cases when the PCR gel contained a high agarose concentration (Figure 2A). Thus, we used 0.2% agarose to prepare the PCR gel, which demonstrated consistent performance in the SOG RT-RPA/PCR assay (Figure 2A).

Figure 2.

Optimization of the SOG RT-RPA/PCR assay in the PCR gel. (A) We evaluated the performance of the SOG RT-RPA/PCR assay at various agarose concentrations and compared with real-time fluorescence PCR and endpoint fluorescence detection. The cycle threshold (Ct) values of the real-time PCR and endpoint fluorescence intensity after 30 cycles are shown with the median. (B) The performance of the SOG RT-RPA/PCR assay at various RPA reaction temperatures was determined. RT-RPA was first performed on the PCR gel at the indicated temperature for 10 min. The Ct values of the real-time PCR and endpoint fluorescence intensity at 30 cycles are shown with the mean. (n = 5).

Next, we assessed the effect of the RT-RPA reaction temperature on the assay. Generally, the optimal reaction temperature of RPA amplification ranges from 37–42°C.¹⁸ We incubated the RT-RPA reaction solution on the 0.2% PCR gel at different temperatures for 10 min. We found that the SOG RT-RPA/PCR assay worked across a wide temperature range of 37.3–41.7°C (Figure 2B). The incubation temperature of 39°C showed the lowest variation and strongest fluorescence signal (Figure 2B). Thus, we selected this temperature for the first RT-RPA pre-amplification on the PCR gel in our assay.

We initially chose the ultra-low gelling temperature agarose for the SOG RT-RPA/PCR assay because the gelling point of the agarose is lower than the annealing and extension temperature for PCR, thereby keeping the PCR reagent melted during PCR cycles. Also, we found that the SOG RT-RPA/PCR could work with Certified Molecular Biology Agarose (Bio-Rad #1613102). However, it is a challenge to hand its liquid agarose solution due to its high viscosity. Moreover, its PCR gel may not completely dissolve during PCR cycles due to its high melting and gelling temperatures. Therefore, the ultra-low gelling temperature agarose was used in our SOG RT-RPA/PCR assay.

We also assessed the stability of the PCR gel. We found that the PCR gel was stable at 4°C for at least 1 week (Figure S3). It may be stable for a longer time, because, according to the manufacture’s instruction, SsoAdvanced Universal Probes Supermix can be stored at 4°C for up to 3 months. The excellent stability of the PCR gel facilitates to conveniently perform the assay because it is not necessary to freshly prepare the PCR gel.

The SOG RT-RPA/PCR assay is more sensitive than standalone RT-PCR and RT-RPA

To accurately evaluate the detection sensitivity, we first quantified the copy number of HIV viral RNA sample using digital RT-PCR. Next, we prepared tenfold serial dilutions of the HIV RNA template and performed the SOG RT-RPA/PCR assay. For comparison purpose, we also ran standalone real-time RT-PCR and real-time RT-RPA using the same RNA template samples. To make a rigorous comparison, we used the same PCR primers and RPA primers as the SOG RT-RPA/PCR assay for the standalone real-time RT-PCR and RT-RPA assays, respectively (Figure S2). We chose Reliance One-Step Multiplex RT-qPCR Supermix for real-time RT-PCR because it contained the same DNA polymerase used for the SOG RT-RPA/PCR assay.

As shown in Figure 3, we could detect 6.3 copies of HIV viral RNA by using the SOG RT-RPA/PCR assay, showing more than 10 times higher sensitivity than standalone real-time RT-PCR or real-time RT-RPA. In addition, the SOG RT-RPA/PCR assay demonstrated faster amplification than that of conventional real-time RT-PCR (Figure 3A and D). For instance, the mean cycle threshold (Ct) values of the SOG RT-RPA/PCR were 4.7 and 5.6 for 63 and 630 copies of RNA per test, respectively (Figure S4A), which is almost 7 times faster than that of real-time RT-PCR (typically above 35).

Figure 3.

Sensitivity evaluation and comparison of the SOG RT-RPA/PCR assay with conventional real-time RT-PCR and real-time RT-RPA. (A and B) Real-time fluorescence detection and endpoint fluorescence measurement of tenfold serial dilutions of HIV RNA samples by the SOG RT-RPA/PCR assay (n = 3). (C) Endpoint fluorescence images of the SOG RT-RPA/PCR products by an imaging system (top) and LED transilluminator (bottom). Individual images of three replicates are shown. NTC, non-template control. (D) Real-time fluorescence detection of tenfold serial dilutions of HIV RNA samples by real-time RT-PCR (n = 3). (E) Real-time fluorescence detection of tenfold serial dilutions of HIV RNA samples by real-time RT-RPA (n = 2).

The overall reaction time of the SOG RT-RPA/PCR is similar to one-step real-time RT-PCR if PCR runs until 40 cycles (Figure S5). However, the SOG RT-RPA/PCR assay may be terminated at an early PCR cycle because of its fast amplification, indicating that the SOG RT-RPA/PCR assay is faster than one-step real-time RT-PCR (Figure S4B). The SOG RT-RPA/PCR assay may be as rapid as RT-RPA if PCR is terminated earlier (e.g., 20 or 30 cycles). Thus, the SOG RT-RPA/PCR assay offers highly sensitive and rapid detection of HIV viral RNA.

However, we found that the SOG RT-RPA/PCR assay was not quantitative, because the amount of the RNA template did not always correlate with the fluorescence intensity. This may be attributed to inconstant RT-RPA amplification (Figure 3E).

HIV viral RNA detection in clinical plasma samples by the SOG RT-RPA/PCR assay

To validate the clinical utility of the SOG RT-RPA/PCR assay, we applied it to detect HIV viral RNA using clinical plasma samples. We first extracted HIV viral RNA from clinical plasma samples by using the QIAamp Viral RNA Mini kit. For comparison, we detected the HIV viral RNA samples by both real-time SOG RT-RPA/PCR and real-time RT-PCR, which has been considered as the gold standard. As shown in Figure 4A, the fluorescence of two positive samples (samples 6 and 7) rose within 10 cycles and showed strong fluorescence signals in our real-time SOG RT-RPA/PCR, which we attribute to the excellent amplification efficiency of RT-RPA on the PCR gel. By contrast, the HIV RNA samples in the positive clinical samples (6 and 7) were difficult to detect by standalone real-time RT-PCR until 35 PCR cycles were completed (Figure 4B). In addition, the endpoint fluorescence signals of the standalone real-time RT-PCR were much lower than that of the SOG RT-RPA/PCR assay. We compared the clinical detection of 14 clinical plasma samples by using the SOG RT-RPA/PCR and real-time RT-PCR, which demonstrated 100% agreement (Figure 4C). Thus, the SOG RT-RPA/PCR assay is suitable for rapid and highly sensitive detection of HIV viral RNA in clinical plasma samples.

Figure 4.

HIV viral RNA detection in clinical plasma samples by using the SOG RT-RPA/PCR assay and standalone real-time RT-PCR. (A) Real-time fluorescence readouts of the SOG RT-RPA/PCR assay. (B) Real-time fluorescence readouts of the standalone real-time RT-PCR assay. (C) Table comparing the clinical detection of 14 clinical plasma samples by using SOG RT-RPA/PCR and real-time RT-PCR.

Conclusions

In the present study, we developed a simple, rapid, and highly sensitive SOG RT-RPA/PCR assay by coupling RT-RPA pre-amplification with PCR on a PCR gel. The PCR gel-embedded PCR reagents allow us to perform RT-RPA pre-amplification prior to the PCR reaction in the same tube, as the PCR gel only dissolves once the temperature is raised following completion of the RT-RPA reaction. The SOG RT-RPA/PCR assay can detect 6.3 copies of HIV RNA target per test. Further, we validated its clinical utility by detecting HIV clinical plasma samples, demonstrating superior performance compared with the standalone real-time RT-PCR method.

The SOG RT-RPA/PCR assay offers several advantages over existing approaches: i) It is simple and fast. By embedding PCR reagents into agarose gel, the RT-RPA reaction solution can be directly added on the PCR gel, enabling simple, one-step nucleic acid detection in a single tube without the need for manual operation or liquid transferring. ii) It shows high sensitivity. By combining RT-RPA pre-amplification with PCR in the PCR gel, the SOG RT-RPA/PCR achieved 10 times higher sensitivity compared with conventional real-time PCR and RPA amplification. iii) It enables visual detection. Due to the production of many amplicons during the first RPA pre-amplification, the fluorescence signals of the SOG RT-RPA/PCR significantly increase, making it possible to directly visualize the testing results under a low-cost, portable fluorescence reader. To achieve a reliable and objective visual detection of the SOG RT-RPA/PCR assay, a smartphone app can be developed to record and analyse the images. In the future, the SOG RPA/PCR assay can be further integrated into a microfluidic chip to develop a multiplex detection platform for simultaneous detection of HIV co-infections with other pathogens (e.g., hepatitis C virus, hepatitis B virus, human papillomavirus).^{23, 24} Therefore, the SOG RPA/PCR assay has great potential for providing a simple, rapid, and highly sensitive approach for nucleic acid-based molecular detection of infectious diseases.