The images in this article are from the internet and used for illustrative purposes. We respect intellectual property rights and comply with laws. If you own the copyright and believe an image infringes your rights, please contact us. Upon confirmation, we'll promptly remove or replace the image. Thank you for your understanding.

Since the advent of the Polymerase Chain Reaction (PCR) method, particularly with the continuous development of real-time Quantitative PCR (qPCR) technology, nucleic acid amplification techniques have permeated various fields of biological science. Recombinase Polymerase Amplification (RPA), developed by the UK-based TwistDx Inc. in 2006, is an isothermal amplification technology that has rapidly advanced in recent years. This technique boasts advantages such as rapidity, sensitivity, excellent specificity, and suitability for on-site rapid detection. It has been applied successfully in diverse areas, including the detection of genetically modified organisms, various pathogens, and food safety.

Reaction Principle

The RPA reaction primarily relies on the recombinase UvsX and the recombinase loading factor UvsY (auxiliary protein) derived from the T4 bacteriophage, along with the Single-Stranded DNA-Binding Protein (SSB) Gp32, and the strand-displacing DNA polymerase Bsu (Bacillus subtilis Pol) or Sau (Staphylococcus aureus Pol). The amplification of the template is achieved based on the T4 bacteriophage's nucleic acid replication mechanism.

Initially, in the presence of Adenosine Triphosphate (ATP), the primer forms a nucleoprotein filament complex with the recombinase UvsX. This complex bi-directionally scans the target double-stranded DNA for homologous sequences. Once a homologous sequence is found, the recombinase dissociates the double-stranded DNA at this position, forming a D-loop structure. One side of the D-loop remains double-stranded, undergoing strand displacement, while the other side becomes single-stranded, stabilized by the SSB protein Gp32. Subsequently, the nucleoprotein filament complex actively hydrolyzes ATP, causing a conformational change and exposing the 3' end of the primer. At this point, the DNA polymerase Bsu binds to the 3' end of the primer, initiating the DNA amplification reaction and forming a new complementary strand.

In this reaction, the processes mediated by forward and reverse primers occur simultaneously. The newly formed single strand pairs with the original strand, generating a complete amplicon. This amplification process repeats continuously, leading to exponential growth of the amplification product. The entire process is typically completed within 20 minutes. During the reaction, competition exists between the SSB protein and UvsX for primer binding sites. The auxiliary protein UvsY can invade the primer-covered binding sites of the SSB protein, preventing the recombination phenomenon that occurs when the SSB protein binds to the primer, and it can facilitate the binding of UvsX to the primer, as illustrated in Figure 1.

Figure 1: Schematic Diagram of Recombinase Polymerase Amplification (RPA)

Types of RPA Detection Methods

Basic RPA Detection

Basic RPA detection is a nucleic acid detection method that combines RPA technology with agarose gel electrophoresis. The main detection process involves performing RPA amplification reaction under constant temperature conditions (37-39°C) for 20-40 minutes. After the reaction is completed, agarose gel electrophoresis is used to detect the RPA amplification products. Components such as recombinase and single-stranded binding protein in the RPA reaction system can bind to the amplification products, inhibiting their migration in the gel. This can lead to smear patterns in gel electrophoresis results. To avoid this, phenol and chloroform are added to the amplification products in a 1:1 volume ratio. The products are then extracted, and the extracted amplification products are subjected to agarose gel electrophoresis. Similar to the gel electrophoresis process for PCR products, RPA amplification products are mixed with nucleic acid dyes like EB before gel electrophoresis. The presence or absence of bands and their sizes on the gel determine the detection results of the target nucleic acid.

Recombinase Polymerase Amplification with Lateral Flow Dipstick (RPA-LFD)

RPA-LFD is a nucleic acid detection method that combines RPA technology with lateral flow dipstick (LFD). In comparison to the basic RPA detection method, the RPA-LFD reaction system includes a DNA endonuclease (nfo), an nfo probe, and a downstream primer labeled with biotin. The nfo probe is approximately 50 nt long, with a FAM fluorescent group at the 5' end, a blocking group (typically C3spacer) at the 3' end, and a tetrahydrofuran (THF) molecule in the middle, as shown in Figure 2. The DNA endonuclease nfo recognizes the THF molecule in the nfo probe and cleaves it. After cleavage, the probe produces a free hydroxyl end, acting as an upstream primer. This new upstream primer, along with the biotin-labeled downstream primer, undergoes amplification to yield an amplification product with a FAM group at the 5' end and biotin at the 3' end. This amplification product can be detected using a lateral flow dipstick.

Figure 2: Schematic Diagram of the Structure of the nfo Probe

LFD, based on lateral flow chromatography, immunology, and colloidal gold technologies, is a type of test strip. The strip is mainly composed of three parts: the sample pad, detection line, and control line. The sample pad contains colloidal gold particles labeled with anti-FAM antibodies, the detection line has a ligand with biotin, and the control line has another antibody that can bind to anti-FAM antibodies. The specific detection principle of LFD is illustrated in Figure 3.

Firstly, the FAM groups on the amplification product bind to the anti-FAM antibodies on the sample pad, forming an immune complex. Then, all colloidal gold particles migrate to the detection line under the action of the buffer solution. When they reach the detection line, the biotin on the amplification product is captured by the biotin ligand on the detection line, causing the amplification product to aggregate. This aggregation results in the appearance of a red band on the detection line. Colloidal gold particles that fail to bind to the amplification product continue to migrate beyond the detection line. When they reach the control line, the anti-FAM antibodies on the colloidal gold particles bind to the antibodies on the control line, leading to the aggregation of colloidal gold particles and the appearance of a red band on the control line. The presence of a red band on the control line indicates the completion of LFD detection.

Therefore, when the test result is positive, two red bands can be observed on the LFD – one on the detection line and one on the control line. When the result is negative, only a red band on the control line is visible on the LFD. Currently, LFD can be combined with various nucleic acid amplification technologies such as LAMP, RCA, and RPA for the detection of target nucleic acids. The advantage of RPA-LFD lies in its simple operation, absence of complex instruments, and the ability to directly observe the detection results with the naked eye, making it suitable for on-site rapid testing.

Figure 3: Schematic Diagram of LFD Detection Principle

Real-time Fluorescent RPA Detection

The real-time fluorescent RPA detection method is a nucleic acid detection approach that combines RPA technology with real-time fluorescent detection technology. In comparison to the basic RPA detection method's reaction system, the real-time fluorescent RPA reaction system incorporates the nucleic acid endonuclease exo and exo probes. The exo probe is a probe with a size of 46-52 nt, featuring a pair of thymine bases, each carrying a fluorescent group and a quenching group. These thymine bases are separated by a THF spacer, and the 3' end of the probe is connected to a blocking group. The specific structure of the probe is illustrated in Figure 4. The fluorescent group in the probe is generally FAM, the quenching group is typically BHQ, and the blocking group is commonly C3spacer.

The nucleic acid endonuclease exo can recognize THF molecules in the exo probe and, after hybridization of the exo probe with the amplification product, cleave the THF molecule. This cleavage separates the fluorescent and quenching groups in the probe, leading to the generation of fluorescence. Equipment capable of real-time monitoring of fluorescent signals can be used to detect the fluorescence intensity during the reaction process, allowing for the determination of the detection results for the target nucleic acid.

Figure 4: Schematic Diagram of the Structure of the exo Probe

Currently, real-time fluorescent RPA detection methods have found widespread application in pathogen detection, food safety, and other fields. In comparison to basic RPA detection methods and RPA-LFD methods, the real-time fluorescent RPA detection method offers notable advantages with a shorter detection time, completing within 20 minutes. Additionally, it exhibits high specificity and sensitivity.

Figure 5: Simplified Diagram of LFD-RPA

Applications of RPA

Since its inception, the RPA technology has been applied in various fields such as single-gene mutation detection, water sources, food safety, infectious disease detection, as well as the detection of common pathogens in agriculture and animal husbandry.

1. Bacterial Detection

Traditional bacterial cultivation is time-consuming, labor-intensive, and susceptible to contamination. RPA technology has shown significant advantages in bacterial detection. For instance, E. coli, a normal flora in the human intestinal tract, can cause severe enteritis and diarrhea when consumed through contaminated food. Liu Jingwen and others utilized RT-RPA with exo probes at 37°C to qualitatively detect E. coli within 20 minutes, achieving a significantly shorter detection time compared to traditional methods. The method also demonstrated a low detection limit of 0.01ng/μl, providing technical support for rapid clinical diagnosis.

Wang et al. developed a dual-detection biosensor based on RPA and a three-stage lateral flow strip. The biosensor performed dual-chain RPA reactions for Vibrio cholerae and Vibrio vulnificus. This method boasts high sensitivity, specificity, short reaction time, and simplicity, making it suitable for grassroots hospitals and real-time on-site detection.

2. Virus Detection

RPA technology is applicable for the immediate detection of various viruses without cross-contamination. Yehia combined RPA technology with reverse transcription to establish a detection method for the H5N1 virus, demonstrating high sensitivity capable of detecting 1 copy of viral RNA. Behrman integrated RPA technology with real-time fluorescence detection for a rapid detection method of novel coronaviruses, achieving results within 15-20 minutes for 7 copies of viral RNA. RPA technology is also applicable to the detection of Monkeypox virus, Newcastle disease virus, Spring viremia of carp virus, African swine fever virus, Canine parvovirus, and others.

3. Parasite Detection

In the realm of parasite detection, RPA methods have been established for protozoa, nematodes, trematodes, and other parasites. Kersting et al. targeted the amplification of the 18S rRNA gene fragment of the malaria parasite using RPA-LFD, demonstrating high specificity and sensitivity under mild reaction conditions. This method is suitable for on-site inspections in remote areas, contributing to global progress in malaria prevention and control.

Guo Qinghong developed an RPA-LFD detection method for diagnosing Japanese schistosomiasis in mice and sheep using the G01 fragment. The method exhibited high sensitivity (97.22% for mice, 93.75% for sheep) and 100% specificity for negative samples, showing no cross-reactivity with other flukes. The results indicate that the RPA-LFD method has high specificity and sensitivity for detecting Japanese schistosomiasis.

4. Food Safety Detection

RPA technology is widely applied in food safety detection. Choi established a detection method for Vibrio parahaemolyticus using RPA, achieving detection within 20 minutes. Tang developed a real-time fluorescent RPA detection method for Vibrio cholerae, capable of detecting 5 copies of bacterial DNA within 20 minutes. Additionally, RPA technology is utilized for the screening and detection of genetically modified crops, with Chandu developing real-time fluorescent RPA and RPA-LFD detection methods for genetically modified soybeans.

Conclusion

Compared to PCR and other isothermal amplification methods, RPA technology features isothermal amplification, rapidity, sensitivity, specificity, ease of result interpretation, and operational simplicity. It offers an economical solution that can be used under limited resource conditions, presenting extensive development prospects. However, there is currently no dedicated primer design software for RPA, and many primers used in experiments may still be those used in traditional PCR, potentially introducing experimental errors. With further research on RPA technology, it is expected to mature and better fulfill its role in the future.