Introduction

Avian leukosis is a general term for avian neoplastic diseases of poultry caused by avian leukosis virus (ALV), a retrovirus belonging to the genus Alpharetrovirus of the family Retroviridae [3, 19]. There are several clinical types of avian leukosis, including erythroblastic, myeloblastic, myeloid, and lymphoblastic leukemias, with lymphoblastic leukemia being the most prevalent type [17, 33]. Based on virus interference tests, antigenic differences in their envelope glycoproteins, and genomic characteristics, ALV strains that naturally infect chickens are divided into seven subgroups: A, B, C, D, E, J, and K [14]. Among the subgroups, subgroups A, B, and J are the most common and are the main exogenous ALVs that cause tumors in chickens. Subgroup K is a new subgroup that has been detected in local chicken breeds in recent years [11]. In general, ALV-A, ALV-B, and ALV-J are often found coinfecting the same host. Endogenous ALV-E has low pathogenicity but may increase the pathogenicity of exogenous viruses of other subgroups [10, 21, 30]. The RNA genome of ALV contains gag/pro, pol, and env genes. The capsid protein p27 is encoded by the gag gene and is highly conserved in all ALV subgroups. It is the main group-specific antigen used for detection of ALV by immunological methods [4]. Avian leukosis can be transmitted vertically and horizontally, causing retardation of growth and development in infected chickens. This results in immunosuppression, vaccine failure, or coinfections with other pathogens, resulting in losses in poultry flocks [9, 12, 27]. The molecular mechanism by which ALV causes immune suppression or immune tolerance is not well understood, and there are no effective vaccines or drugs available to control ALV infection. Therefore, the primary measures to prevent and control the disease depend on the detection and eradication of ALV and the purification of breeder flocks [24, 28], and there is a need for a rapid, specific, and sensitive method for of this virus.

Since it was first reported in 1868, avian leukemia has occurred in almost all countries with large-scale poultry farming, causing immense economic losses [7, 23, 34]. The currently used methods for detection of ALV include virus isolation, indirect immunofluorescence, enzyme-linked immunosorbent assay, polymerase chain reaction (PCR), and virus neutralization tests. These detection methods are time-consuming, are sensitive to interference by contaminants, require expensive equipment, and require skilled operators, making them less than ideal for on-site testing and use by free-range poultry farmers. Although PCR-based analysis is the gold standard for molecular detection of viruses in clinical practice, it is expensive and time-consuming [1]. In 2000, Notomi et al. [16] described a loop-mediated isothermal amplification (LAMP) technique that employs four specific primers and a loop primer to target six regions of the viral genome, together with Bst DNA polymerase, which has high strand displacement activity, to efficiently amplify nucleic acids under isothermal conditions at 60-65 ℃. Using agarose gel electrophoresis or adding fluorescent dyes to the reaction system to allow direct readout of detection results, the complete process takes approximately 60 min [2, 15]. In contrast to other detection methods, LAMP does not require special equipment and has high sensitivity and specificity [31]. LAMP can also be modified to amplify RNA templates for detection of RNA viruses by replacing Bst DNA polymerase with reverse transcriptase, and this system is called reverse transcription loop-mediated isothermal amplification (RT-LAMP). RT-LAMP has high sensitivity and specificity and allows one-step amplification of RNA templates, simplifying the operation steps. This method has been used successfully for diagnosis of Newcastle disease, thrombocytopenia syndrome, COVID-19, and other viral diseases [6, 22, 32].

The use of agarose gel electrophoresis to detect RT-LAMP amplification products has the disadvantages that it requires a dedicated gel imager, carries the risk of contact with the carcinogen ethidium bromide, and requires fluorescence detection, which can be costly. These factors have limited the widespread application of RT-LAMP for on-site testing of poultry [31]. In 2008, Kiatpathomchai et al. [5] described the use of RT-LAMP combined with an immunochromatographic strip in a lateral flow device (RT-LAMP-LFD) to detect the amplification products of Taura syndrome virus. In that system, biotin-labeled RT-LAMP amplification products in the reaction system hybridize specifically to a fluorescein isothiocyanate (FITC)-labeled probe, which binds to a gold-labeled anti-FITC antibody on the LFD binding pad. The complex is captured by streptavidin on the test line (T-line) of the nitrocellulose membrane to form a visible band. The remaining unhybridized labeled probe becomes enriched and is visible on the quality control line (C-line). RT-LAMP-LFD eliminates the need for electrophoresis and reduces false positive results caused by nonspecific amplification, making it useful in laboratory diagnosis and on-site detection [8]. This method has been used successfully for diagnosis of hepatitis C virus, white tail disease, West Nile encephalitis, and other diseases [13, 25, 29].

In this report, we describe an accurate, efficient, and sensitive RT-LAMP-LFD assay for detection of the p27 gene of ALV. The larger goal of this work was to develop a system for rapid assessment of the pathogenicity of common ALV strains in a large sample. The sensitivity and specificity of the newly established RT-LAMP-LFD assay were evaluated by comparison to conventional RT-PCR technology.

Materials and methods

Materials and reagents

Please refer to the Supplementary Information for details.

Design and synthesis of primers and probe

Based on the p27 gene sequence of ALV (GenBank accession number EU070902.1), a set of specific primers and probes was designed using PrimerExplorer V5 (http://primerexplorer.jp/e//). This included two external primers (F3 and B3), two internal primers (FIP and BIP), a loop primer (LP), and a probe (HP). The internal primer FIP was labeled with biotin, and the probe HP was labeled with FITC. A pair of PCR primers targeting the p27 gene was designed using Primer Premier 5.0 software, with the size of the amplified fragment predicted to be 717 bp. The primers and probes were synthesized and labeled by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) (Supplementary Fig. S1, Table 1).

Table 1 Specific primers and probe for detection of ALV genomic RNA in chicken samples
Full size table

Growth of avian leukemia virus and RNA extraction

Chicken embryo fibroblasts were grown to 60% confluency in a 10 cm2 cell culture plate. A virus suspension (0.8 ml, 500 TCID50/mL) was passed through a 0.22 μm filter membrane, diluted with 3 ml of DMEM medium containing 10% fetal bovine serum (FBS), and added to the cells. After 2 h of incubation, the supernatant was discarded, and DMEM containing 1% FBS was added. After 7 days, the virus was collected and stored at -80 °C.

RNA was extracted using a TaKaRa MiniBEST Viral RNA Extraction Kit Ver.5.0, following the manufacturer's instructions. The extracted viral RNA was analyzed using a NanoDrop 2000 ultraviolet spectrophotometer and stored at -80 °C.

Development of the RT-LAMP-LFD reaction system

The RT-LAMP reaction system was developed according to the recommendations included in the instructions of the manufacturer of the Bst 2.0 DNA polymerase large fragment (M0538). The amplification product was detected using 2.5% agarose gel electrophoresis and staining with calcein fluorescent dye. The RT-LAMP reaction was optimized as described in the Supplementary Information. After the reaction was completed, no termination step was performed, and 5 μL of ALV p27-HP probe was added to the reaction mixture for hybridization. Next, 5 μL of the hybridization solution was mixed with 90 μL of buffer and dropped onto the sample pad of the strip, and after 5-10 min, the strip test result was interpreted visually. If both the T and C lines on the strip were visible, the result was considered positive. If only the C-line was visible, the result was considered negative, and if only the T-line was visible or neither line was visible, the result was considered invalid.

Evaluation of the sensitivity and specificity of the RT-LAMP-LFD assay

The concentration of the extracted ALV RNA was determined by ultraviolet spectrophotometry, using a NanoDrop 2000 instrument, and the genomic RNA was serially diluted from 100 to 106 RNA copies/μL in DNase/RNase-free water. Using the optimized conditions, the RT-LAMP amplification was performed for each dilution, and the amplified products were analyzed by both 2.5% agarose gel electrophoresis and LFD. Simultaneously, a HiFiScript cDNA Synthesis Kit was used for reverse transcription of each dilution of RNA into cDNA, using the specific primers ALV-p27 F and ALV-p27 R, and the amplified product was visualized by 1.5% agarose gel electrophoresis in order to compare the sensitivity of the two methods.

To test the specificity of the RT-LAMP-LFD assay, RNA was extracted from Newcastle disease virus, pathogenic avian influenza virus, Salmonella pullorum (CVCC530), and Salmonella enteritidis (CVCC3949) and used as template for amplification using the optimized RT-LAMP reaction system. At least three replicates were performed for each amplification reaction, and the products were detected using both 2.5% agarose gel electrophoresis and the LFD.

Evaluation of the repeatability of the RT-LAMP-LFD assay

Genomic RNA was extracted from avian leukosis virus and serially diluted to the lowest concentration detectable by RT-LAMP-LFD, and three samples were prepared in parallel. RT-LAMP amplification was performed using the diluted samples, and the amplification products were detected using a strip to assess the repeatability of the assay.

Validation of the RT-LAMP-LFD assay using clinical samples

Viral nucleic acid extraction and reverse transcription were carried out, using the appropriate kits (TaKaRa), on 80 anal swabs from chickens suspected of avian leukemia. Each sample was then tested by both RT-LAMP and RT-PCR to evaluate the reliability of the RT-LAMP-LFD detection method.

Results

Establishment of RT-LAMP-LFD

RT-LAMP primers were designed using Explorer V5 and tested in an RT-LAMP reaction system. The biotin-labeled inner primer (ALV-p27 FIP) was amplified by RT-LAMP using ALV RNA as a template. The amplification products were detected by SYBR Green I staining and 2.5% agarose gel electrophoresis. As shown in Figure 1a, positive samples developed a green color, while the color of the negative control did not change. Analysis by 2.5% agarose gel electrophoresis showed that positive samples produced a series of bands of different sizes, whereas no bands were obtained with the negative control (Fig. 1b). This showed that the ALV p27 gene fragment had been amplified successfully. We then tested whether the amplicons could be detected by immunochromatography using an LFD (Fig. 2). In the LFD test, the ALV-p27 FIP amplification product and FITC-labeled ALV p27-HP probe hybridized, forming an FITC-labeled probe complex. This complex was then added dropwise to the sample pad and combined with anti-FITC antibody-labeled gold nanoparticles on the binding pad by capillary migration. When the complex migrated to the T-line of the NC membrane, it was captured by streptavidin to form an immune complex, which formed the first visible band. Unhybridized FITC-labeled probe and gold nanoparticle-labeled anti-FITC antibody complexes continued to migrate to the C-line, where they bound to goat anti-chicken IgY, producing a second visible band. If a sample was negative, the gold nanoparticles containing anti-FITC antibodies were not enriched on the T-line.

Fig. 1
figure 1

Detection of ALV amplification products. M, 2000 bp DNA marker; 1, RT-LAMP products. 2: negative control. (a) Calcein-based fluorometric detection. (b) Agarose gel electrophoresis detection. (c) LFD detection

Full size image
Fig. 2
figure 2

RT-LAMP-LFD diagram. The main components of the LFD include a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorption pad. The conjugate pad is sprayed with gold nanoparticles labeled with anti-FITC monoclonal antibody. On the NC membrane, the T-line is sprayed with streptavidin, and the C-line is sprayed with goat anti-chicken IgY. The C-line is 5 mm away from the T-line, and the strip size is 80.0 × 3.80 mm

Full size image

The standard reaction conditions for the RT-LAMP were optimized by testing different reaction temperatures and times. The results of gel electrophoresis showed that maximum amplification was achieved when the reaction time was found to be 60 min (Fig. 3a), and this time was selected for subsequent experiments. Similarly, the optimal reaction temperature was 64 °C (Fig. 3b).

Fig. 3
figure 3

Gel electrophoresis of amplified products under optimized RT-LAMP reaction conditions (a) Effect of reaction time. Lane M, 2000 bp DNA marker; lanes 1-4, 30, 45, 60, and 75 min; 5, negative control. (b) Effect of reaction temperature. Lane M, 2000 bp DNA marker, lane 1, negative control; lanes 2-9, amplification products obtained at 52, 54, 56, 58, 60, 62, 64, and 65 ℃, respectively; M, 2000 bp DNA marker

Full size image

The reaction system was further optimized by adjusting the concentrations of the internal and external primers, the volume of MgSO4 solution used, and the concentration of dNTPs. Gel electrophoresis of the RT-LAMP amplification products showed that optimal results were obtained when the concentration ratio of the internal and external primers in the RT-LAMP reaction system was 3:1 (Fig. 4a). When different volumes of MgSO4 were tested, the best results were obtained when 1.5 μL of 100 mM MgSO4 was used (Fig. 4b). Likewise, as shown in Fig. 4c, optimal results were obtained when the volume of 10 mM dNTP added to the reaction mixture was 3 μL. In summary, the optimal concentration ratio of primers for the RT-LAMP reaction system in this study was 3:1, and the optimal volume of MgSO4 and dNTPs was 1.5 and 3 μL, respectively.

Fig. 4
figure 4

Gel electrophoresis of amplified products under optimized RT-LAMP reaction conditions (M, 2000 bp DNA marker) (a) Analysis of RT-LAMP amplification products obtained using different ratios of internal and external primers. Lanes 1-4, 1:1, 2:1, 3:1, and 4:1; lane 5, negative control. (b) RT-LAMP amplification products obtained using different volumes of MgSO4. (c) RT-LAMP amplification products obtained using different dNTP concentrations. Lanes 1-6, 1, 1.5, 2, 2.5, 3, 3.5, and 4 μL of mM dNTP mixture; lane 7, negative control

Full size image

Evaluation of RT-LAMP-LFD performance

The sensitivity of the RT-LAMP-LFD was assessed by performing RT-LAMP-LFD and RT-PCR reactions on ALV genomic RNA serially diluted from 106 to 100 RNA copies/μL. The lowest detectable concentration of ALV RNA by RT-PCR was 104 RNA copies/μL (Fig. 5b). For RT-LAMP, the lowest detectable concentration was 102 RNA copies/μL when using either 2.5% agarose gel electrophoresis (Fig. 5a) or LFD (Fig. 5c) for detection. Thus, the sensitivity of the RT-LAMP assay was found to be 100 times that of RT-PCR.

Fig. 5
figure 5

Sensitivity of the RT-LAMP-LFD assay. M, 2000 bp DNA marker. In lanes 1-8, the ALV genome was serially diluted from 100 to 106 RNA copies/μL. (a) Agarose gel electrophoresis of RT-LAMP products. (b) Agarose gel electrophoresis of RT-PCR products. (c) LFD detection of RT-LAMP products

Full size image

The specificity of the RT-LAMP reaction was tested using RNA from five other pathogens as template. When ALV genomic RNA was tested, 2.5 % agarose gel electrophoresis showed the characteristic pattern of bands of different sizes, but when the other four pathogens were tested, no bands were observed (Fig. 6a). Likewise, analysis of the amplified products using LFD revealed that ALV genomic RNA yielded a positive result, but RNA from the other four pathogens did not, indicating the specificity of the RT-LAMP-LFD assay (Fig. 6b).

Fig. 6
figure 6

Specificity of the RT-LAMP-LFD assay. (M, 2000 bp DNA marker. Lane 1, negative control; lane 2, RT-LAMP products obtained using ALV as template. Lanes 3-8, RT-LAMP products obtained using five other pathogens as templates. (a) Detection of RT-LAMP products by agarose gel electrophoresis. (b) Detection of RT-LAMP products by LFD

Full size image

To test the reproducibility of the RT-LAMP, extracted genomic RNA of ALV was diluted to the lowest concentration at which it can be detected by RT-LAMP-LFD and used as a template for RT-LAMP amplification in three separate experiments. In each case, the amplified products were detected by both 2.5% agarose gel electrophoresis (Supplementary Fig. S2a) and LFD (Supplementary Fig. S2b).

RT-LAMP-LFD and RT-PCR were then used to analyze 80 anal swab samples from chickens with clinically suspected ALV infection. The results obtained by RT-PCR and RT-LAMP-LFD were consistent, with seven samples testing positive and 73 testing negative (positive rate, 8.7%; Table 2). This demonstrates that the detection rate of the RT-LAMP-LFD assay when using clinical samples is equal to that of the gold standard RT-PCR method.

Table 2 Clinical application of the RT-LAMP-LFD detection method
Full size table

Discussion

The widespread prevalence of avian leukemia in the chicken population has seriously affected chicken production and caused substantial economic losses to the chicken industry. Because ALV is a retrovirus that can be transmitted vertically and cause early congenital infection, it is difficult to prevent and control the disease through vaccination. Therefore, the primary measure for controlling the disease is to make a timely and accurate diagnosis of ALV infection and eliminate all infected birds to purify the population. The ALV-infected chickens can have multiple serotypes based on antigen and antibody tests: no viremia, antibody (V-A+); viremia, no antibody (V+A-); viremia, antibody (V+A+); no viremia, or no antibody (V-A-) [18]. However, detection of ALV antigens and antibodies alone is not sufficient to identify all ALV-infected chickens.

The application of various nucleic acid amplification techniques to detect pathogenic bacteria and viruses has improved the efficiency of testing, but some of these techniques require sophisticated instruments and methods for amplification and detection of the amplification products because of the low selectivity of the target sequences [26]. The current molecular biology detection techniques include RT-PCR, nested PCR, and PCR-gene chips, all of which have high sensitivity and accuracy but require special equipment and trained personnel, limiting their usefulness for on-site testing. The newly developed RT-LAMP-LFD technology has the advantages of other amplification techniques but also has the advantages of simple operation, the lack of a need for special equipment, and rapid real-time visualization of detection results. Previously, Peng et al. [20] developed a simple and rapid LAMP assay for rapid detection of the common ALV subgroups in 35 min under isothermal conditions at 63 °C. That assay had a detection limit of 20 copies of ALV DNA, making it 10 times more sensitive than the conventional PCR assay. However, the LAMP method is prone to false-positive results, and the detection accuracy needs to be improved. Xiang et al. [30] employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection technique for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR), and real-time PCR (RT-PCR) were compared. The results showed that when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, comparable with RT-PCR. This method only identified ALV-J, but the common subgroups of ALV often had co-infection. The visual RT-LAMP-LFD assay developed in the present study improves the amplification efficiency of this method and is convenient and fast. The amplification reaction can be completed within 1 h, with good sensitivity and specificity, without requiring special equipment. The results can be judged based on the color bands on the test strips, and the problem of false positivity caused by nonspecific amplification products is largely avoided. The assay’s detection limit is 102 RNA copies/μL, which is 100 times lower than that of the traditional RT-PCR method.

In this study, we used the highly conserved and specific p27 gene of ALV virus as the detection target. The RT-LAMP-LFD for visual detection of avian leukemia was established by optimizing the LAMP reaction system. The assay is highly sensitive, specific, convenient, and practical and compares favorably to the gold-standard RT-PCR method. Experiments using clinical samples showed that this technique has practical application value and can potentially be used for on-site detection of ALV.

In summary, we developed an RT-LAMP-LFD for convenient and sensitive detection of ALV from RNA extracted from diseased chicken samples. This method includes one-step RT-LAMP amplification of the target RNA and user-friendly visual readout of the amplification product detection results on the LFD. This assay has the advantage of enabling real-time detection of ALV in settings with limited resources.