Introduction

Individual sex can provide important information in the fields of ecology and behavior1,2. The improvement in protocols for rapid and accurate sex identification of birds is of great importance in evolutionary biology, breeding, and conservation3,4. Many birds exhibit sexual size dimorphism, even at the nestling or subadult stage, enabling sex determination based on morphometry5. However, approximately 60% of bird species are monomorphic birds that are indistinguishable in their external morphology and behavior6. Therefore, accurately and quickly identifying the sex of birds is difficult. Currently, sex identification in birds is performed using morphological, surgical, cytological, or molecular methods7. Molecular sexing in birds is widely used because of its accuracy, minimal invasiveness, and convenience and was first initiated by Griffiths et al. in 1995 8,9. It is generally based on polymerase chain reaction (PCR) amplification of the duplication of the chromo-helicase-DNA-binding protein (CHD) sex-chromosome-specific gene located on the Z and W chromosomes8,9,10. With the development of this technology, multiple molecular methods have been applied for sex determination, including high-resolution melting analysis4, real-time PCR11, and loop-mediated isothermal amplification (LAMP)12. However, these methods either require expensive equipment or additional primers13,14.

Recombinase-based isothermal amplification assay, recombinase polymerase amplification (RPA), or recombinase-aided amplification (RAA) is a novel technique for nucleic acid isothermal amplification using a bacterial recombinase enzyme and isothermal polymerase in recent years15,16,17. The RPA/RAA technique could achieve exponential amplification of target DNA at 37–42 °C within 20–30 min. The RPA/RAA technique mainly utilized three crucial enzymes: a recombinase (anchor on a DNA single strand and search for homologous sequences), a strand displacing DNA polymerase (for amplification and extension), and a single-stranded DNA binding protein gp32 (stabilize the displaced strand DNA). The difference between RPA and RAA is the source of recombinant enzymes. The RPA recombinant enzyme is derived from bacteriophage T4, while the RAA recombinant enzyme is derived from bacteria or fungi. Furthermore, the amplification product of RPA/RAA can be monitored by gel electrophoresis, a fluorescent probe, gel electrophoresis, and lateral flow chromatography. Compared to conventional PCR, RPA/RAA offers a simpler and faster environment for gene amplification. Currently, it is widely used for pathogen detection18,19,20. Lateral flow chromatography strips (LFD) are suitable for visualizing RPA/RAA product21. Among the various visualization methods for RAA/RPA amplicons, the LFD assay is better suited for field applications because it allows rapid result visualization within 2–5 min without complex procedures22,23,24. Therefore, the RPA/RAA-LFD method could be applied to point-of-care testing (POCT) for sex identification of bird CHD genes.

The great white pelican (Pelecanus onocrotalus; order Pelecaniformes) is widely distributed throughout Eastern Europe, Asia, and Africa25. However, it appears that the population has severely declined and is hardly found in the wild as a result of urbanization, industrialization, and reduction in bird habitats. In 2021, the great white pelican was listed as a national priority wild animal protected by China. A recovery plan and reintroduction program are necessary for the conservation of the great white pelican. Determining the sex ratio of the population before reintroduction is critical. However, it is difficult to identify the sex of pelicans based on their external appearance alone, which is particularly evident in the nestlings26. Therefore, the aim of this study was to establish an RAA-LFD assay for sex determination of great white pelicans based on CHD located on the Z and W chromosomes (Fig. 1). To the best of our knowledge, this is the first study of pelican sex identification using isothermal amplification. Visualization of RAA-LFD detection will be extremely useful in bird conservation and captive breeding for the quick determination of sex.

Fig. 1
figure 1

The workflow of the overall process for sex of pelican rapid determination based RAA-LFD assay. Fragment of the target gene is isothermally amplified by recombinase polymerase amplification at 39°C for 30 min. After the formation of double-labeled amplicon containing biotin and carboxyfluorescein (FAM), the amplication product travel in a buffer stream containing antibody-coated gold particles and trapped at the test line by biotin streptavidin affinity, resulting in an appearance of red color indicative of a positive result. Non-captured gold particles move through the test line and are fixed to the control line by anti-mouse antibodies.

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Results

Primer design and screening

In this study, each four RAA primers for the CHD-W and UCE genes were obtained (Table 1). An RAA-AGE assay was performed to identify the best primers. In Fig. S1a, the primer pairs W1, W2, and W3 were amplified in both male and female DNA. Primer pair W4 successfully amplified specific fragments of 171 bp based on CHD-W but not CHD-Z, and the amplified product was pure. Therefore, W4 was determined as the best primer pair for subsequent studies. As shown in Fig. S1b, primer pairs UCE1-4 both amplified the target region. The RAA of UCE2 produced a single, pure band. Therefore, we selected UCE2 for the RAA-LFD primer pair. Furthermore, to be visualized using the LFD system, the forward primer consisted of an antigenic label FAM group at the 5′ end, and the reverse primer was labeled at the 5′-end with biotin.

Table 1 Primers sequences used in this study.
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Establishment of the RAA-LFD assay

The RAA-LFD assay is that the amplified products can be visualized. We used genomic DNA (gDNA) from females as a template for a positive female identification and male gDNA to control for male identification at 39 °C within 30 min in the RAA-LFD assay using raaW4 primer pair. The RAA production was visualized using LFD strips for 5 min. Positive reactions showed two separate lines, including the control and test lines, whereas only the control line appeared in the negative assay (Fig. S2). For false-negative signals, we carried out the evaluation by raaUCE2-RAA-LFD assay to be used as a positive control for DNA quality. This experiment was considered valid when the raaUCE2-RAA-LFD assay was performed on both females and males. That is, the raaUCE2-RAA-LFD analysis showed that there were both control and test lines in the LFD strip.

Specificity and sensitivity of the RAA-LFD assay

RaaW4-RAA-LFD specificity was tested using 20 great white pelicans and 2 pink-backed pelicans of known sex and compared with conventional PCR. According to the conventional PCR using the 2550 F/2718R primer set, female samples were identified by a 450- and 960- bp amplification product or a single 450- bp product, and male samples were characterized by a single 960-bp product. The results showed that the 22 pelicans were identified as 16 females and 6 males; however, one was not detected using conventional PCR (Fig. 2a). The raaW2-RAA-LFD analysis revealed that all 22 pelicans could be detected, among which 16 of the 22 birds were female and 6 were male, and the detection rate and PCR were 100% accurate (Fig. 2b).

Fig. 2
figure 2

Comparative analysis of the specificity between conventional PCR (a) and raaW4-RAA-LFD assay (b). The PCR amplicon products were subjected to electrophoresis on a 2% agarose gel. M: DL2000 Marker. ♀: Female; ♂: Male.

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We used four-fold serial dilutions of female pelican gDNA (100 ng, 25 ng, 6.3 ng, 1.6 ng, 0.4 ng, 0.1 ng, 0.1 ng, 25 pg, and 6.3 pg) to assess the sensitivity exhibited by the RAA-LFD assays and compared it with conventional PCR. As shown in Fig. S3, a detection limit of 1.6 ng gDNA was identified for female pelican feathers by conventional PCR using the 2550 F/2718R and UCR-F/R primers. The RAA reaction products for each concentration of female gDNA were tested separately on the LFD, and the color of the test line became lighter with decreasing concentration. We note that the limit of detection with raaW4 primers was 0.1 ng (Fig. 3a) and that of raaUCE2 primers was 25 pg (Fig. 3b) in RAA-LFD assay, respectively.

Fig. 3
figure 3

Analysis of sensitivity RAA-LFD assay using four-fold serially diluted gDNA extracted from female pelican. The RAA amplified products using raaW4 (a) and raaUCE2 (b) primers were visible on the LFD at 5 min. NC: ddH2O.

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Comparative validation of the RAA-LFD assay using field samples

To evaluate the diagnostic validity and reliability of the RAA-LFD assay, gDNA was extracted from 15 white pelicans of unknown sex. Simultaneously, we used the 2550 F/2718R primers from conventional PCR for detection. All pelican feather gDNA samples were successfully detected, and the RAA-LFD identified 11 females and 4 males (Table 2). The results of the RAA-LFD assay were consistent with the conventional PCR results.

Table 2 Comparative performances of RAA-LFD and conventional PCR assays for pelican sex identification.
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Discussion

Morphological sex identification in birds can be difficult if there is no significant sexual dimorphism. Many bird species exhibit one of the highest population declines owing to hunting and habitat fragmentation. Therefore, conservation has become a priority. Sex determination is difficult because of sexual monomorphism, and traditional sexing methods are traumatic. Molecular sexing is a minimally invasive, effective, and rapid technique to determine the sex of individuals1,27. Sex identification using molecular methods can be applied to monomorphic birds and is highly accurate because it targets the sex chromosomes directly28. Sexing methods for molecular DNA amplification using PCR have been widely used8. PCR detection of amplified nucleic acid products relies primarily on electrophoretic equipment and gel imaging systems29. Since the early 1990s, isothermal amplification methods for nucleic acids have been developed, some of which have been successfully commercialized for convenience30. Many isothermal amplification methods have also been used for sex identification of birds14,31,32. Compared to other isothermal amplification methods, the RPA/RAA technique has the advantages of short reaction time, simple method design, and low reaction temperature33,34,35. In this study, we confirmed the application of RAA-LFD to the diagnosis of sex using random samples. An RPA/RAA-LFD assay targeting the CHD gene was examined and demonstrated the ability to detect the sexing of pelicans with a high degree of specificity and sensitivity in 30 min. The sensitivity assay indicated that the detection limit of the RAA-LFD method was 100 times higher than that of the PCR. Furthermore, the analytical specificity assay showed that the assay could detect female pelicans but did not react with males. Fifteen clinical sample sets were evaluated using the RAA-LFD and PCR assays, and the results of the RAA-LFD assay were consistent with those of conventional PCR.

The CHD gene seems to have certain advantages in that it has significant sequence divergence between the W and Z chromosomes as a target for sex identification and has been used for sex identification in numerous avian species9,27,36. Classically established CHD-based 2550 F/2718R primer pairs are powerful tools for sex determination in most bird species. However, a preliminary test showed that they did not work as well as our established RAA-LFD because it performed worse at low DNA concentrations. Compared with traditional PCR, the RAA-LFD assay is generally more sensitive23,37. In this study, we designed specific primers using a differential sequence based on the CHD-W/CHD-Z genes and successfully amplified all female samples. In addition, to avoid false-negative scoring, the present method of identifying DNA quality by establishing the RAA-LFD of UCE was successfully applied to monitor RAA reactions. UCE genes have 99.9% identity in avian species, and we suggest using UCE as a positive control when applying isothermal amplification based bird sex identification4,38,39,40.

In the wild, bird sexing is a difficult task; therefore, the RAA-LFD method can be very useful in field research for fast and easy visualization of amplification products41,42,43. One advantage of the RAA-LFD assay is that the amplified products can be visualized using colloidal gold particles on the strip. The RAA-LFD method established in this study allows for RAA amplification and product visualization without any large laboratory equipment. Therefore, this method is suitable for rapid and simple DNA amplification in field tests. However, for field research further studies on simplified DNA extraction methods are required. In the present study, we used a DNA extraction kit that required approximately 2 h of laboratory work, which is unrealistic for field research. Therefore, it is necessary to develop a simple DNA extraction method that can be combined with RAA-LFD to allow easy, reliable, and quick sex determination in birds in the field using only basic laboratory tools12,44.

In summary, the CHD-W/CHD-Z primer sets of RAA-LFD were successfully applied to pelican sexing. For the amplification reaction, only at 39 °C was needed and without sophisticated equipment. The RAA amplicon analysis was performed on the LFD. Thus, the findings of this study provide a basis for RAA-LFD as a tool for simple, rapid, and sensitive sex identification in great white pelicans (Pelecanus onocrotalus), which could potentially be applied in the sexing and captive breeding management of pelicans.

Materials and methods

Sample collection

The 22 feather samples from two bird species, including 20 great white pelicans (Pelecanus onocrotalus) and 2 pink-backed pelicans (Pelecanus rufescens), were plucked during vaccination and obtained from the Guangzhou Zoo in Guangdong Province, China. Feathers were stored in a plastic bag labeled with a numerical code for a specific individual and frozen at − 20 °C until use. All experimental animal protocols were reviewed and approved by the Guangzhou Zoo Animal Use and Care Committee. All methods were performed in accordance with the relevant regulations and followed the recommendations outlined in the ARRIVE guidelines for conducting research.

DNA extraction

Approximately 2–10 mm segments from two to three individual feather calami were cut off and placed in a 1.5 mL Eppendorf tube. Genomic DNA (gDNA) was extracted using a HiPure Tissue & Blood DNA Kit (D3018; Megan, Guangzhou, China) according to the manufacturer’s protocol. After the extraction, DNAs were measured using a Nano-Drop ND-2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and stored at − 20 ℃ until needed.

Primer design for sex determination

According to the guidelines of the RAA reaction kit manual, RAA primers [30–35 base pairs (bp), GC content 30–70%] specific for the ChromoHelicase-DNA-binding (CHD) gene sequences found on the Z and W chromosomes were self-designed by Primer Premier 5.0 software using the sequences derived from our Lab sequencing of pelicans (Table 1) and the primers were selected using the RAA agarose gel electrophoresis (RAA-AGE) assay. An optimal RAA primer pair was selected for the RAA-LFD assay. The RAA-LFD forward primer was labeled at the 5′-end with Carboxyfluorescein (FAM), reverse primer was labeled at the 5′ end with biotin.

A positive control for DNA quality and/or monitoring of RAA reactions consisted of highly conserved DNA in the so-called ultra-conserved element (UCE; GenBank: JQ331584.1) located on chromosome 6 of birds to design a primer set to develop the RAA-LFD assay, as previously described38. RAA primers based on the UCE gene were designed as described above. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and are listed in Table 1.

Conventional PCR amplifications

PCR amplicons were prepared using 2550 F/2718R primers for the molecular sex identification of the pelican45. The PCR reaction was performed using 2 µL DNA solution obtained from feathers in a final volume of 20 µL containing 10 µL of 2× DreamTaq PCR Master Mix polymerase (K1081, ThermoFisher Scientific), 4.0 µL of ddH2O, and 1 µL (10 µM) of each 2550 F/2718R primer. The amplification protocol was composed of the following steps: initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C denaturation for 30 s, annealing at 49 ℃ for 45 s, and extension at 72 ℃ for 1 min, followed by a final extension at 72 ℃ for 5 min. Amplification products were separated using electrophoresis on a 2% agarose gel and visualized using an ultraviolet transilluminator (Bio-Rad). Distilled water was used as the negative control.

RAA-LFD reaction for sex identification

The RAA assay was performed using an RAA nucleic acid amplification kit (Zongche Bio-Sci&Tech Co., Ltd., Hangzhou, China) according to the manufacturer’s protocol. Each reaction system contained one RAA lyophilized powder, 29.4 µL of buffer A, 2 µL of forward primer (raaW4-F/raaUCE2-F; 10 µM), 2 µL of reverse primer (raaW4-R/raaUCE2-R; 10 µM), 1 µL DNA template, 13.1 µL of ddH2O, and 2.5 µL of buffer B. After reaction at 39 °C for 30 min, 1 µL of amplified product was immediately mixed with 99 µL PBS buffer and tested using for HybriDetect LFD (Milenia Biotech, Giessen, Germany) at room temperature (RT). The dipsticks were dipped in the diluted solution for 5 min prior to observation. For the raaW4-RAA-LFD assay, the dipstick indicated a female result when the control and test lines were both visible; if only the control line was visible, the result was considered to indicate male sex. It is worth noting that the assay were only valid when the test line of raaUCE2-RAA-LFD was visible. A positive control was constructed using female pelican gDNA. Distilled water was used as the negative control.

Evaluation of the RAA-LFD assay

To determine the specificity of the RAA-LFD assay, DNA extracted from feather samples of 22 pelicans of known sex, including six females and sixteen males, was used as the DNA template to perform the RAA-LFD assay, as described above. In addition, to detect the sensitivity of the RAA-LFD assay, a four-fold serial dilution of female pelican gDNA (100 ng, 25 ng; 6.3 ng; 1.6 ng; 0.4 ng; 0.1 ng; 25 pg; 6.3 pg; total eight dilutions) was used to amplify the RAA reaction and compared with that of conventional PCR. Finally, we collected 15 feathers of unidentified white pelicans from the Nansha Waterfowl Ecological Park, Guangzhou, and verified the field test of sex identification using the RAA-LFD assay. The effectiveness of the RAA-LFD assay was compared to that of conventional PCR using the 2550 F/2718R primer pair described above. Distilled water was used as the negative control.