RECOMBINASE-AIDED AMPLIFICATION COMBINED WITH LATERAL FLOW DIPSTICK FOR THE RAPID AND SENSITIVE DETECTION OF TRICHOMONAS GALLINAE
ABSTRACT
Trichomonas gallinae is a protozoan parasite of the upper digestive tract of poultry and wild birds and poses a serious threat to the wild bird population and the pigeon industry globally. Therefore, rapid diagnosis and timely treatments are necessary to control T. gallinae. Although some conventional diagnostic methods have been described previously, these methods are hampered by omissions or high costs. In this study, the combination of biotin-labeled and FAM-labeled primers and probes for recombinase-aided amplification (RAA) with a lateral flow dipstick (LFD) was investigated to visualize the RAA results. The RAA-LFD assay was established for the on-site, rapid, and sensitive detection of T. gallinae. The RAA-LFD assay with Toxoplasma gondii, Neospora caninum, and Tetratrichomonas gallinarum did not produce a cross-reaction, which indicated that this RAA-LFD assay is specific for detecting T. gallinae. In addition, the RAA-LFD assay demonstrated high sensitivity with the lowest detection limit of 2.78 × 101 copies/μl of the Fe-HYD standard plasmid for T. gallinae. The complete detection process of the RAA-LFD assay was found to require 45 min. These results suggest that the RAA-LFD assay established in the present study may be promising for the rapid, specific, and sensitive detection of T. gallinae during surveillance efforts.
Trichomonas gallinae is a widely spread protozoan parasite that infects numerous avian species, primarily causing necrotic ulcerations in the upper digestive tract and occasionally the respiratory tract (Mehlhorn et al., 2009; Cai et al., 2024). Although many domestic and wild avian species can be infected, including Columbiformes, Falconiformes, and Strigiformes, pigeons are the primary hosts of T. gallinae (Stabler, 1954; Amin et al., 2014). Infection with T. gallinae has been considered the major cause of widespread mortality in pigeons, especially nestlings (Gerhold et al., 2007; Forrester and Foster, 2008; Lawson et al., 2011). The principal mode of transmission of T. gallinae is oral. Therefore, the disease is prone to break out among groups, which causes adverse economic effects in the pigeon breeding industry and might also lead to a significant decrease in wild bird populations. Hence, T. gallinae infection is regarded as one of the most significant parasitic diseases in current poultry and wildlife conservation policies (Kemp and Reid, 1965; Kietzmann, 1993). At present, there is no effective vaccine for T. gallinae (Xiang et al., 2023). Only when the disease conditions of poultry groups are monitored in the early stage can timely treatment and control be used to reduce risks to breeding enterprises and to stabilize the ecological structure.
The etiological diagnostic methods for T. gallinae comprise direct microscopic examination, cell culture, and staining. However, etiological diagnosis demands the definite detection of parasites for disease confirmation (Parker et al., 2003; Mostegl et al., 2010; Qiu et al., 2017; Arfin et al., 2019), which is prone to false negative results and is time-consuming and laborious. Serological diagnostic methods such as the enzyme-linked immunosorbent assay can facilitate diagnosis (Amin et al., 2011). However, this method is complex and has low diagnostic sensitivity. Therefore, these older methods are gradually being replaced by faster and more accurate molecular biology diagnostic methods, including polymerase chain reaction (PCR) assay and quantitative real-time PCR (qPCR) (Quillfeldt et al., 2018; Alejandro Mateo et al., 2022); however, these assays require costly equipment and professional staff. Thus, these diagnostic methods are manifestly not useful in resource-poor areas and for on-site detection. The emergence of isothermal amplification methods, including loop-mediated isothermal amplification and recombinase-aided amplification (RAA), is expected to solve challenges for on-site detection (Wang et al., 2020b).
The RAA is a novel nucleic acid isothermal amplification technique utilizing a single-strand DNA binding protein, recombinase UvsX (Escherichia coli), and DNA polymerase. Three crucial enzymes work together to complete the amplification within 20–30 min at 37–42 C (Gui et al., 2022; Zeng et al., 2024). Furthermore, methods such as a fluorescent probe, gel electrophoresis, and lateral flow chromatography can monitor the amplification product of RAA (Zeng et al, 2024). Lateral flow dipstick (LFD) can provide visualization of results within 2–5 min without complex procedures, and the LFD assay is better suited for visualizing RAA amplicons (Li et al., 2019; Bian et al., 2022). Compared with the PCR and qPCR assays, the RAA-LFD assay is more suitable for early on-site detection and for use in resource-poor areas.
In this study, we combined RAA and LFD to establish a method for detecting T. gallinae. The combination method facilitates the analysis of results with the naked eye and can be completed within 45 min, with high specificity and sensitivity. The rapid, convenient, and sensitive characteristics of RAA-LFD make it a promising approach for early on-site detection of T. gallinae.
MATERIALS AND METHODS
Sample collection
Oropharyngeal swab samples were collected from pigeons infected with T. gallinae. Other samples of pathogen DNA, including that from Toxoplasma gondii, Neospora caninum, and Tetratrichomonas gallinarum were provided by the Animal Parasites and Parasitic Epidemiology Laboratory, Anhui Agricultural University, Hefei, Anhui Province, China. DNA of T. gallinae was extracted with the TIANamp Genomic DNA kit (TIANGEN Biotechnology, Beijing, China) following the instructions with the kit.
Design of RAA primers and RAA assay
The specific conserved sequences of T. gallinae (Fe-HYD) (GenBank accession number AF446077.1) were chosen as target regions for this study. The Fe-HYD gene sequence was analyzed using Clustal Omega to identify regions containing TTTN or AAAN. Following the methods reported in previous articles, 4 pairs of primers and a probe were synthesized by Tsingke Biotechnology (Beijing, China). According to the instructions of the RAA basic kit (Qitian Gene Biotechnology, Jiangsu, China), the RAA assay and analysis of the products were carried out using the methods described previously (Zhou et al., 2025).
RAA-LFD assay
The RAA technique was used to pre-amplify the target of T. gallinae detection by labeling biotin and fluorescent group FAM at the 5′ end of the downstream primers and probes of the optimal RAA assay. The LFD assay was used to identify the product generated by RAA. A 10 µl sample of the amplified product was combined with 100 µl of phosphate buffered saline, and an LFD strip was placed into the mixture at room temperature. When the presence of distinct and clear test line and control line on the LFD strip indicated that nucleic acid fragments were present in the sample being tested, the RAA-LFD assay was configured for the detection of T. gallinae and to allow visualization of the results of the assay.
Sensitivity and specificity of the RAA-LFD assay for detecting T. gallinae
RAA-LFD assay was carried out with the recombinant plasmid of various copies (0, 2.78 × 100, 101, 102, 103, 104, 105, and 106 copies/μl) to reflect the sensitivity. Under optimal reaction conditions, the RAA-LFD assay was performed to detect pathogens such as T. gondii, N. caninum, T. gallinarum, and T. gallinae to evaluate the specificity of the RAA-LFD assay.
Utility of the RAA-LFD assay for detecting T. gallinae
Genomic DNA was extracted from 8 pigeon oropharyngeal swab samples collected from a pigeon farm in Anhui Province. The RAA-LFD assay was used for detection of T. gallinae.
Ethical approval
All procedures involving animals were reviewed and approved by the Research Ethics Committee of the Anhui Agricultural University (number AHAUB 2022014).
RESULTS
Isolation and culture of T. gallinae
After the oral and pharyngeal swab samples of pigeons were collected and transported to the laboratory, they were transferred into new culture media and left overnight. Direct microscopic observation was used to determine the state of the parasites. T. gallinae were translucent and oval with flagella, the cell surface undulated, and the cells moved irregularly and rapidly. After isolation and purification, T. gallinae with stable passage was obtained.
Primer screening and optimization of RAA reaction time
Under the same conditions and utilizing the previously outlined RAA primer screening method (Zhou et al., 2025), the primers exhibited strong band specificity for the F4/R4 primer pair as determined by the naked eye. The F4/R4 primer pair was then selected for optimization of the RAA reaction time, and 40 min was chosen as the optimal reaction time for RAA based on previously reported results.
Sensitivity test of RAA-LFD assay
The plasmid pUC57-FeHYD harboring T. gallinae was diluted to concentrations of 0, 2.78 × 10°, 101, 102, 103, 104, 105, and 106 copies/μl to determine the sensitivity of the RAA-LFD assay, and the results indicated that the limit of detection was 2.78 × 101 copies/μl (Fig. 1).
Citation: The Journal of Parasitology 111, 5; 10.1645/25-34
Specificity test of RAA-LFD assay
Pure water and the genomic DNA of T. gondii, N. caninum, T. gallinarum, and T. gallinae were used as templates, and the products were amplified by RAA-LFD assay. The results showed that the RAA-LFD assay could specifically amplify the target sequence of the Fe-HYD gene without cross-reaction with nucleic acids of other parasites (Fig. 2).
Citation: The Journal of Parasitology 111, 5; 10.1645/25-34
Practicability test of RAA-LFD assay
When the RAA-LFD assay was used to detect the extracted genomic DNA, the results show that this assay could be used to successfully distinguish positive samples, demonstrating the practicality of this RAA-LFD assay (Fig. 3).
Citation: The Journal of Parasitology 111, 5; 10.1645/25-34
DISCUSSION
Although T. gallinae is one of the most important avian pathogens in poultry husbandry, there has been no effective vaccine against this parasite until now. A rapid and accurate detection method is essential for timely clinical diagnosis and disease control to prevent T. gallinae epidemics. With the rapid development of isothermal nucleic acid amplification technologies, RAA and recombinase polymerase amplification (RPA) have emerged, and both methods are similar in principle and process. Even though both RAA and RPA have advantages regarding simplicity, sensitivity, and specificity, RAA is more easily obtained and is less costly (Wu et al., 2022). Therefore, we established the RAA-LFD assay, and the results were visualized using lateral chromatography test strips, which can detect T. gallinae rapidly, conveniently, and sensitively.
RAA alone has some disadvantages, such as the possibility that interactions between RAA primers may generate secondary structures and that the high sensitivity of RAA could result in false positive results (Wang et al., 2020a; Wu et al., 2020). The specific amplification efficiency of RAA primers is key to the sensitivity and specificity of the RAA-LFD assay. Based on Fe-HYD, T. gallinae is divided into 3 subtypes: A1, A2, and C1. Our sequence belonged to subtype C1 (Ahmadabad et al., 2024). However, we selected the conserved region of Fe-HYD following sequence comparison when designing RAA primers, thus resulting in the RAA primer covering all 3 gene subtypes. Multiple pairs of primers should be designed for the RAA-LFD assay to screen for the optimal primer pair. In the present study, F4/R4 was selected as the optimal primer pair for RAA based on previous reports (Zhou et al., 2025), which avoided the formation of secondary structures and false positive results. The RAA-LFD assay can be completed within 45 min at 37 C. However, the RAA-LFD assay requires multiple open-cap operations, which can result in the accumulation of amplification product aerosols in the laboratory environment and may lead to cross-contamination of products. Therefore, upon completion of RAA amplification, it is necessary to move to a well-ventilated area for dilution and detection to prevent false-positive results.
Currently, the conventional method of PCR and the “gold standard” qPCR are the most accurate methods for detecting T. gallinae (Sigrist et al., 2022). In this study, the performance of the RAA-LFD assay was compared with that of PCR and qPCR to determine the feasibility of the RAA-LFD assay. According to the available data on the detection of T. gallinae (Zhou et al, 2025), the lowest detection limit recorded for the qPCR assay was 2.78 × 101 copies/μl and that of the conventional PCR assay was 2.78 × 103 copies/μl, which is consistent with findings in other studies (Rentería-Solís et al., 2020; Sigrist et al., 2021). The lowest detection limit of the RAA-LFD assay was 2.78 × 101 copies/μl, indicating that a lower concentration of parasites could be detected and revealing that the RAA-LFD assay was more sensitive than the conventional PCR method for detecting T. gallinae, supporting the potential use of the RAA-LFD assay as an early diagnostic method. In addition, the RAA-LFD assay had sensitivity comparable to that of the gold standard qPCR assay for detecting T. gallinae but also had a shorter detection time, a simplified operational process, and no requirement for sophisticated instrumentation. Although the RAA-LFD assay cannot be used to distinguish between the 3 T. gallinae subtypes, as can conventional culture and PCR followed by DNA sequencing, this drawback does not impact the capacity of this RAA-LFD assay to facilitate the expeditious detection of T. gallinae in outdoor settings.
CONCLUSIONS
In this study, the RAA-LFD assay for T. gallinae detection was used at an isothermal temperature of 37 C with high specificity, and the whole process required no complicated technical and operational procedures or complex instruments. The lowest detection limit of the RAA-LFD assay was 2.78 × 101 copies/μl of the Fe-HYD standard plasmid for T. gallinae, a result comparable to the sensitivity of the qPCR assay and 100 times more sensitive than the conventional PCR assay. This RAA-LFD assay is expected to become a new solution for detecting T. gallinae, with high specificity, simplicity, rapidity, and sensitivity.
Sensitivity of recombinase-aided amplification assay with lateral flow dipstick for the detection of Trichomonas gallinae. H2O: DNA template is was double-distilled water; 1e0–1e6: plasmid standards with DNA templates of 2.78 × 100–106 copies/μl.
Specificity of recombinase-aided amplification assay with lateral flow dipstick for the detection of Trichomonas gallinae. Lanes 1–5: assay results for pure water, Toxoplasma gondii, Neospora caninum, Tetratrichomonas gallinarum, and Trichomonas gallinae.
Practicability of recombinase-aided amplification assay with lateral flow dipstick. Lanes 1–8 contain 8 different DNA samples.
Contributor Notes