Bovine enterovirus (BEV), a single-stranded RNA virus belonging to the genus Enterovirus in the Picornaviridae family, has increasingly drawn attention due to its wide host range and potential implications for animal health and public safety (Goens et al., 2004). The BEV genome is approximately 7500 base pairs (bp) in length, comprising a 5′-untranslated region (5′UTR), a large open reading frame (ORF), and a 3′-untranslated region (3′UTR). BEV is classified into two primary serotypes: Enterovirus E and Enterovirus F, both characterized by significant genetic variability and adaptability (Zell et al., 2017).

Although BEV infections are commonly subclinical, specific stress conditions can induce severe symptoms such as diarrhea, dehydration, and, in severe cases, death, particularly among calves with immature immune systems (Ley et al., 2002; Boros et al., 2012; Ji et al., 2022; Zhang et al., 2014; Luo et al., 2023). These clinical manifestations pose a direct threat to livestock productivity and the economic viability of cattle farming.

Since its initial isolation by Moll and Davis in 1959, BEV has been detected globally (Ji et al., 2022, Zhang et al., 2014, Luo et al., 2023, Moll and Davis, 1961, Jiménez-Clavero et al., 2005). It has been identified not only in cattle but also in goats, sheep, deer, and environmental samples, including water and soil. Recent studies suggest potential zoonotic risks associated with BEV, with antibodies detected in human serum samples; however, its capacity to cause clinical symptoms in humans remains uncertain. BEV's ability to contaminate soil, water, and food products through fecal shedding highlights its significant potential for widespread environmental and foodborne transmission. Furthermore, inadequate farm hygiene and poor waste management practices exacerbate these risks, potentially facilitating viral entry into the food chain and endangering public health.

Accurate and timely detection of BEV is critical for controlling outbreaks and mitigating economic and public health impacts. While conventional diagnostic methods such as reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RT-qPCR), and droplet digital PCR (ddPCR) offer sensitivity, they are hampered by lengthy workflows, reliance on specialized infrastructure, and technical expertise. These limitations underscore the urgent need for rapid, cost-effective, and field-deployable diagnostic tools.

Recombinase-aided amplification (RAA) is an emerging isothermal nucleic acid amplification technology offering significant advantages for molecular diagnostics (Ceruti et al., 2023, Wu et al., 2022). The RT-RAA method integrates reverse transcription and recombinase-mediated amplification. Initially, reverse transcriptase converts target RNA into complementary DNA (cDNA). Subsequently, cDNA amplification occurs using specific primers and enzyme systems—including recombinases, single-stranded DNA-binding proteins, and heat-resistant DNA polymerases—that effectively replace the phages commonly employed in recombinase-polymerase amplification (RPA) techniques (Wu et al., 2022). The recombinase, aided by single-stranded binding proteins, facilitates the opening of double-stranded DNA, creating a stable single-stranded structure. DNA polymerase then initiates synthesis from the primer's 3′ end, resulting in exponential amplification (Fig. 1).

Probes utilized in this method are specifically designed to bind amplified cDNA sequences. Each probe ranges from 46 to 52 nucleotides, carefully avoiding palindromic sequences, internal secondary structures, and repetitive bases. There are four modification sites, with the tetrahydrofuran (THF) residue positioned at least 28 nucleotides from the 5′ end to act as an exonuclease recognition site. Fluorophores are labeled upstream of the THF site, while quenching groups are placed downstream, separated by a 1–4 nucleotide distance. The THF site itself is located at least 15 nucleotides away from the 3′ end, which is modified with a C3-spacer group (Fig. 2).

The RT-RAA enzyme system demonstrates high amplification efficiency and specificity, making it suitable for detecting low-level target RNA within complex sample matrices. RT-RAA does not require complex laboratory equipment or highly trained personnel. Its isothermal reaction conditions allow portable equipment use, making it particularly beneficial for field testing in remote areas or during outbreaks with limited laboratory access. Compared to traditional RT-PCR methods, RT-RAA is typically more cost-effective due to its operational simplicity and reduced requirement for specialized instrumentation (Li et al., 2024).

Unlike PCR-based methods, RAA operates at a constant low temperature (39–42°C), eliminating the need for intricate thermal cycling equipment. This characteristic, combined with its simplicity, rapidity, and robustness, renders RAA particularly suitable for on-site testing. Integration of reverse transcription in RT-RAA further enables sensitive and specific amplification of RNA target sequences. Additionally, employing fluorescence-based probes in real-time RT-RAA assays facilitates continuous amplification monitoring, enhancing accuracy and reliability while minimizing interpretation errors.

In this study, we developed a rapid and reliable real-time fluorescence RT-RAA method for detecting BEV. By targeting the conserved 5′UTR region of the BEV genome, we designed specific primers and probes that ensure high sensitivity and specificity. This innovative approach overcomes the limitations inherent in conventional diagnostic techniques, offering a practical solution for rapid BEV detection in clinical and field settings. Besides enabling early diagnosis and effective monitoring of BEV infections, this method significantly contributes to enhanced disease management within the cattle industry, ensuring beef safety and reducing potential zoonotic threats.

Firstly, RNA is reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase. Subsequently, recombinase forms a complex with primers, enabling the recombinase-primer complex to bind and invade the double-stranded DNA template. This process unwinds the DNA helix, exposing single-stranded regions stabilized by single-stranded DNA-binding proteins. Once the primer identifies its complementary sequence on the template, the recombinase dissociates, allowing DNA polymerase to initiate the synthesis of a new double-stranded DNA fragment.