Development of RT-RPA assays for rapid detection of mango-infecting viruses using high-throughput sequencing-derived genomic information

Abstract

Plant viral diseases pose a major threat to perennial fruit crops, where infections often remain latent and persist over long periods, facilitating unnoticed spread through planting material. Recent advances in high-throughput sequencing (HTS) have greatly expanded knowledge of plant viral diversity and plant-virus interactions, including the identification of emerging and re-emerging viruses. However, translating virome-level discoveries into practical disease management tools remains a significant challenge, particularly for field-level surveillance and sustainable crop protection. Mango (Mangifera indica), a globally important fruit crop, exemplifies this gap, as virus infections are poorly correlated with visible symptoms and reliable on-site diagnostic tools are limited. In this study, HTS was employed to reconfirm the presence of mangifera indica latent virus (MiLV) and mangifera virus 1 (MaV-1) in mango plants and to generate validated genomic information for diagnostic assay development. Building on these data, reverse transcription–recombinase polymerase amplification (RT-RPA) assays were developed and optimized for rapid virus detection. The assays operated efficiently under isothermal conditions (40 °C) within 25 min. The MiLV RT-RPA assay enabled direct detection from crude leaf extracts without the need of RNA purification, whereas MaV-1 detection required purified RNA as template. Sensitivity analysis showed that the MiLV RT-RPA assay detected viral RNA up to 0.01 fg µl⁻¹ (equivalent to a 10⁻¹⁰ dilution of 100 ng µl⁻¹ RNA) and MaV-1 up to 0.1 ng µl⁻¹ showed complete concordance with RT-PCR (Cohen’s K = 1.00). Validation using field-collected symptomatic and asymptomatic samples demonstrated complete concordance with conventional RT-PCR. The detection of both viruses in young grafted plants showed the potential role of vegetative propagation in virus dissemination and emphasizes the importance of screening both scion and rootstock materials. This study demonstrates how HTS-based insights into plant viral diversity can be effectively translated into rapid, field-deployable molecular diagnostics. The developed RT-RPA assays provide a practical tool for routine virus surveillance, certification of virus-free planting material, thereby contributing to improved stress resilience and sustainable mango production.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09448-z.

Introduction

Mango (Mangifera indica L.) is one of the economically most important fruit crops cultivated across tropical and subtropical regions of the world. India is the largest producer of mango, contributing nearly half of global production; however, overall productivity remains comparatively low due to susceptibility to multiple biotic and abiotic stresses [1–3]. Among biotic constraints, viral diseases represent a major challenge, particularly in perennial crops, where infections persist for years, often remaining undetected while continuously affecting plant vigor, fruit quality and yield [4].

The mango virome has long been considered limited, largely due to the absence of visible symptoms and the lack of systematic virus surveillance. To date, only a few viruses have been reported to infect mango, including mangifera indica latent virus (MiLV; family Benyviridae), mangifera indica totivirus 1 (MiTV1; family Totiviridae), and mangifera virus 1 (MaV-1; family Closteroviridae) [5–7]. MiLV, the first virus reported in mango, is considered latent and has not been consistently associated with distinct disease symptoms, which complicates its detection and epidemiological assessment [5, 8].

Recent advances in high-throughput sequencing (HTS) have transformed plant virus diagnostics by enabling unbiased detection of both known and novel viruses without prior sequence information [9–15]. Using HTS-based virome analysis, the presence of MiLV and a novel olivavirus, mangifera virus 1 (MaV-1), infecting mango plants in India was recently demonstrated, along with detailed genome characterization and phylogenetic placement of these viruses [7]. While HTS is vital for virus discovery and comprehensive virome profiling, its routine application for large-scale diagnostics is constrained by high cost, sophisticated infrastructure, and the need for specialized bioinformatics expertise.

Consequently, there is a serious necessity for rapid, sensitive and field-deployable diagnostic tools that can translate HTS-based virus discovery into practical disease management strategies. Conventional diagnostic methods such as enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) are widely used. However, ELISA often lacks sensitivity and requires virus-specific antibodies, whereas RT-PCR, although highly sensitive, depends on thermal cycling equipment and high-quality RNA, limiting its suitability for on-site applications [16–18].

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique that operates efficiently at low temperatures (37–42°C) and produces results within 20–30 minutes, without the need for complex instrumentation [19]. The assay involves a synergistic interaction between three primary enzymes viz. recombinase, a single strand binding protein (SSB) and a strand displacing DNA polymerase. RPA relies on the principle where an ATP-dependent recombinase forms a stable nucleoprotein filament with primers to scan and invade homologous double-stranded DNA (dsDNA) so as to initiate strand exchange. This structure is then stabilized by SSB proteins, allowing a strand-displacing DNA polymerase to initiate exponential template extension from the 3’ end of the invading primers [20, 21]. The amplification products of RPA can be detected using different methods like agarose gel electrophoresis, colorimetric visual assays, real-time fluorescence using exo or fpg probes, field-deployable lateral flow immunochromatographic assays (LFICA) and RPA-CRISPR/Cas12a integrated platforms [20–23].

Further, different RPA based assays have been successfully applied for the rapid detection of several pathogens in plants [20, 21, 24, 25], animals [26–28] and humans [29, 30]. In plants, these assays demonstrated high sensitivity, specificity, and compatibility with crude plant extracts, making it particularly suitable for field diagnostics and resource-limited settings [18, 31, 32].

However, additional viruses such as mangifera indica totivirus 1 (MiTV-1) have been reported in mango through HTS-based studies, the present work focused on MiLV and MaV-1 because these viruses were consistently detected in the analyzed transcriptome datasets and provided sufficient genomic information for designing reliable diagnostic primers. Building upon earlier virome characterization, the primary objective of this work was not virus discovery but the translation of HTS-derived genomic information into a practical diagnostic application. We report the development, optimization and validation of RT-RPA assays for rapid detection of MiLV and MaV-1, with emphasis on assay sensitivity, specificity and field applicability. By integrating repeated HTS confirmation with simplified molecular diagnostics, this study aims to bridge the gap between virome research and routine virus indexing, supporting the production of certified virus-free mango planting material and sustainable orchard management.

Results

Preprocessing of raw HTS data

High-throughput sequencing of eleven mango samples (A1–A5 and L1–L6) generated large transcriptome datasets. The total number of raw reads obtained from these samples ranged from 49,243,894 to 69,959,678. After removal of adapter sequences and low-quality reads, 49,125,528 to 69,809,786 high-quality (HQ) reads were retained for further analysis. The HQ reads were mapped to the Mangifera indica reference genome to remove host-derived sequences. Following host read subtraction, 873,468 to 1,666,835 host-unaligned reads were obtained and subjected to de novo assembly. The assembly resulted in contigs with N50 values ranging from 310 to 391, while the assembled contig lengths varied between 3,808 and 8,415 nucleotides, indicating assembly quality. Homology-based analysis of the assembled contigs revealed sequences corresponding to mangifera virus 1 (MaV-1) and mangifera indica latent virus (MiLV) in several datasets. Viral reads corresponding to MaV-1 were detected in samples A1–A4, with mapped reads ranging from 16,438 to 102,811, resulting in genome coverage of 121.35× to 710.11× (Table 1). Similarly, MiLV-associated reads were identified in samples A1, A2, L2 and L5, with mapped reads ranging from 3,074 to 17,494, corresponding to genome coverage values ranging from 103.59× to 374.68× (Table 1). These results confirmed the presence of both viruses in the analyzed mango transcriptome datasets.

Table 1.

Summary statistic of mango transcriptomes

Samples	Statistics terms
	Total raw reads	Total filtered reads (HQ)	Host aligned reads	Host unaligned reads	De novo Assembly		MaV-1		MiLV
					N50	Contig length	Map read	Genome coverage (X)	Map read	Genome coverage (X)
A1	55,824,968	55,703,646	54,036,811	1,666,835	310	4,787	102811	710.11	5776(RNA 1)3074(RNA 2)	107.11(RNA 1)138.93(RNA 2)
A2	56,212,032	56,131,906	54,516,164	1,615,742	365	8,415	29252	198.21	17494(RNA 1)3392(RNA 2)	309.27(RNA 1)156.87(RNA 2)
A3	59,803,880	59,657,944	58,856,723	947,157	336	6,718	16438	121.35	-	-
A4	64,860,432	64,743,020	63,834,032	1,026,400	324	4,938	95233	694.96	-	-
A5	59,441,068	59,308,724	58,340,322	1,100,746	327	3,808	-	-	-	-
L1	63,184,310	63,052,532	61,976,907	1,207,403	325	3,947	-	-	-	-
L2	59,729,106	59,614,462	58,821,284	907,822	391	4,401	-	-	10763(RNA 1)7964(RNA 2)	204.50(RNA 1)374.68 (RNA 2)
L3	69,959,678	69,809,786	68,680,147	1,279,531	324	4,068	-	-	-	-
L4	49,243,894	49,125,528	48,370,426	873,468	326	4,701	-	-	-	-
L5	57,524,426	57,423,702	55,963,786	1,560,640	338	4,943	-	-	5299(RNA 1)4043 (RNA 2)	103.59(RNA 1)198.53 (RNA 2)
L6	57,365,534	57,252,260	56,358,147	1,007,387	337	4,278	-	-	-	-

Identification and genomic characterization of mango viruses using HTS data

Homology-based virus identification was performed using the BLASTx algorithm against the NCBI non-redundant database. The assembled contigs showed significant similarity to MiLV and MaV-1, belonging to the families Benyviridae and Closteroviridae, respectively. In A1, A2, A3 and A4 datasets, contigs ranging from 8,315 nt to 17,247 nt exhibited 99.03–99.92% nucleotide sequence identity with MaV-1. In the case of MiLV, BLASTx analysis identified seven contigs ranging from 2,594 nt to 6,758 nt, showing 98.45–99.66% nucleotide sequence identity with RNA1 and RNA2 of MiLV isolates available in GenBank. The complete genomic RNA sequences of the MaV-1 and MiLV were assembled from A1, A2, A3, A4 and A1, A2, L2, L5 datasets, respectively (Table 1). The genome organization of both viruses was consistent with previously reported virus genomes [7].

Amino acid sequence comparison of the taxonomically important proteins RdRp, HSP70h and CP of MaV-1 with previously reported isolates showed high sequence identity ranging from 99.4 to 99.9%. Phylogenetic analysis based on these proteins using the best-fit maximum-likelihood model placed the present MaV-1 isolate within the clade of previously reported Indian isolates (Fig. 1). Similarly, comparison of the RdRp and CP proteins of MiLV revealed amino acid sequence identities ranging from 98.2 to 99.8% and 98.3–99.5%, respectively, with previously reported MiLV isolates. Phylogenetic analysis grouped the present isolate with known MiLV isolates, including the previously reported Indian isolate (Fig. 2). These results confirm that the viral sequences identified in this study correspond to MiLV and MaV-1 and are closely related to previously reported isolates.

Fig. 1.

Phylogenetic analysis of MaV-1 genomic segments. Maximum-likelihood trees were constructed using deduced amino acid sequences of (a) RdRP and (b) HSP70 and (c) CP. The best-fit substitution models were LG + G+I for RdRP, LG + G+F for HSP70 and WAG + G+I for RNA2. Bootstrap support values (1,000 replicates) ≥ 50% are shown at the corresponding nodes. Branch lengths represent substitutions per site, as shown by the scale bar

Fig. 2.

Phylogenetic analysis of MiLV genomic segments. Maximum-likelihood trees were constructed using deduced amino acid sequences of (a) RNA1 and (b) RNA2. The best-fit substitution models were LG + G for RNA1 and WAG for RNA2. Bootstrap support values (1,000 replicates) ≥ 50% are shown at the corresponding nodes. Branch lengths represent substitutions per site, as shown by the scale bar

Comparison of the amino acid sequences of two taxonomically important proteins RdRp and CP from MiLV with homologous proteins from other isolate of MaV-1, revealed amino acid sequence identities ranging from 98.2% to 99.8% and 98.3% to 99.5%. Present isolate of MiLV shared highest identity with previously reported MiLV from India. Phylogenetic analysis of the RdRp and CP sequences from benyviruses, conducted using the best fit maximum-likelihood model, showed that RdRp and CP sequence of MiLV clustered with Indian isolate of MiLV (Fig. 2). Currently, only two complete genomic RNA1 and RNA2 sequences of MiLV isolate from Israel and India are available in public domain. This is the another near-complete genome/ complete coding genome sequence of MiLV from India.

Screening and optimization of primer pairs for RT-PCR/RT-RPA detection of mango viruses

Based on the viral sequences obtained from HTS, specific primer pairs were designed for the detection of MaV-1 and MiLV. Screening of the primer pairs using purified RNA as a template in RT-PCR resulted in amplification products of approximately 210 bp, 128 bp and 184 bp for MaV-1 with primer pairs M1F/1R, M2F/2R and M3F/3R, respectively. Similarly, evaluation of three primer pairs designed for MiLV (IL1F/1R, IL2F/2R and IL3F/3R) produced amplification products of approximately 272 bp, 169 bp and 243 bp, respectively. Among the primer pairs tested, M1F/1R for MaV-1 and IL3F/3R for MiLV produced the strongest and most consistent amplification and were therefore selected for further assay optimization (data not shown). No amplification was observed with RNA extracted from healthy mango leaf samples. Similar results were obtained in the RT-RPA evaluation using RNA as a template, producing specific and bright amplifications of ~ 210 bp (MaV-1) and ~ 243 bp (MiLV) with M1F/1R and IL3F/3R respectively (Fig. 3). Thus, these primer pairs were selected for all subsequent assay optimizations and validations. All amplicons were confirmed by Sanger sequencing, showed 100% identity to the corresponding viral sequences obtained through HTS.

Fig. 3.

Optimization of RT-RPA assay using three set of primer pair each for mangifera virus 1 (MaV-1) (M1F/1R, M2F/2R, M3F/3R) and mangifera indica latent virus (MiLV) (IL1F/1R, IL2F/2R, IL3F/3R). a Lanes 1, 3, 5: purified RNA template of MaV-1; 2, 4, 6: Healthy control for MaV 1. b 7, 9, 11: purified RNA template of MiLV; 8, 10, 12: Healthy control for MiLV, L: 100 bp DNA ladder (GeneDireX, India)

Optimization of the RT-RPA assay

Eight different extraction buffers were tested for their efficacy in preparing crude leaf extracts suitable for the RT-RPA assay. The NaOH: EDTA (1:2) buffer showed the most consistent and bright amplification in case of MiLV, while with MaV-1 none of the buffers showed amplification. Thus, NaOH: EDTA (1:2) buffer was used for all subsequent experiments involving crude sap for the detection of MiLV (Supplementary Fig. 1). Whereas, only RNA was used as a template in case of detection of MaV-1.

After the selection of the best primer pair, the RT-RPA assay was optimized using different incubation temperatures and time periods. Results revealed 40 °C for 25 min was found to be the optimal isothermal conditions, which provided the strongest and most specific amplification for both MiLV (using RNA/crude sap as a template) and MaV-1 (purified RNA as a template) (Figs. 4 and 5; Supplementary Fig. 2and 3). Amplification was detectable at earlier time points such as 15 min, the reactions incubated for 25 min consistently produced stronger and more reproducible amplification across independent replicates. Therefore, 25 min was selected as the optimal reaction time for routine assay performance. However, no amplification was observed in the negative controls containing healthy plant samples, indicating a high degree of specificity under these conditions.

Fig. 4.

Standardization of different incubation temperatures for the detection of (a) MaV-1 (~ 210 bp) and (b) MiLV (~ 243 bp) using RNA (as template) in RT-RPA assay. Lanes 1, 5: 34 °C; Lanes 2, 6: 37 °C; Lanes 3, 7: 40 °C; Lanes 4, 8: 43 °C; Lanes N: Negative control, M: 100 bp DNA ladder (GeneDireX, India)

Fig. 5.

Standardization of different time periods (15, 20, 25, 30, 35 & 40 min) for the detection of (a) MaV-1 (~ 210 bp) and (b) MiLV (~ 243 bp) using RNA (as template) in RT-RPA assay. Lanes 1–12, 1, 7: 15 min, 2, 8: 20 min, 3, 9: 25 min, 4, 10: 30 min, 5, 11: 35 min, 6, 12: 40 min; Lanes N: Negative control, M: 100 bp DNA ladder (GeneDireX, India)

Determination of sensitivity and specificity for the detection of MiLV and MaV-1

The sensitivity of the optimized RT-RPA assay was compared with conventional RT-PCR using 10-fold serial dilutions of purified viral RNA and infected crude leaf sap. For MiLV, the RT-RPA assay showed higher sensitivity, with detection possible upto a 10^− 10 dilution using either RNA or crude sap as the template, which was found to be equivalent to the sensitivity of conventional RT-PCR (Fig. 6, Supplementary Fig. 4). Whereas, for MaV-1, the RT-RPA assay showed a detection limit of 10^− 3 when using RNA as a template, which was slightly less sensitive than RT-PCR, which could detect the virus upto a 10^− 4 dilution (Fig. 6).

Fig. 6.

Comparison of sensitivity for the detection of (a) & (b) MaV-1 (~ 210 bp) and (c) & (d) MiLV (~ 243 bp) using RNA (as template, ten-fold serial dilutions) in RT-PCR and RT-RPA assays. Lanes L: 100 bp DNA ladder (GeneDireX, India); Lane 1: positives MaV-1 and MiLV stock, Lanes 2–11: ten-fold serial dilutions, Lanes 12: healthy control

Specificity of the developed primers was evaluated using MiLV-infected, MaV-1-infected and healthy mango plant samples. No cross-amplification was observed when MiLV primers were tested on MaV-1-infected samples or when MaV-1 primers were tested on MiLV-infected samples, confirming the specificity of the assay. Minor bands observed below 100 bp in some reactions likely correspond to primer-dimer, which are occasionally observed during amplification assays and were not considered during interpretation of results because the expected target amplicons (~ 210 bp for MaV-1 and ~ 243 bp for MiLV) were clearly detected. Further, the specific amplicons were also confirmed through Sanger sequencing and submitted to NCBI (Accession numbers: PX915425 and PX915426).

Validation of the developed assay for the detection of viruses infecting mango

The developed RT-RPA assay was validated by testing 25 leaf samples of mango from two different locations. The results were compared to those obtained by conventional RT-PCR using the same samples. The RT-RPA assay successfully detected MiLV in seven (07) out of 25 samples (using RNA/crude sap as template) and MaV-1 in four (04) mango samples (using RNA as template). Both symptomatic and asymptomatic samples were tested positive for both viruses with RT-PCR and RT-RPA. The results showed a 100% concordance between the RT-RPA and RT-PCR methods for both virus detections (Table 2). The diagnostic agreement between RT-RPA and conventional RT-PCR was further evaluated using Cohen’s kappa coefficient. The analysis showed perfect agreement between the two assays for detection of both MiLV and MaV-1, with a kappa value of 1.000 and p < 0.001 (Table 3). These results confirm that the developed RT-RPA assay performs comparably to conventional RT-PCR for detection of mango viruses. This validation confirms the reliability and reproducibility of the developed RT-RPA assay for the rapid and specific detection of MiLV and MaV-1 in mango plants. However, the number of positive samples available for validation was limited due to the low incidence of these viruses in the surveyed plant material, the developed RT-RPA assay showed complete agreement with conventional RT-PCR results, indicating its reliability for rapid detection of MiLV and MaV-1 in mango plants.

Table 2.

Detection of mangifera indica latent virus (MiLV) (using RNA/crude sap as template) and mangifera virus 1 (MaV-1) (using RNA as template) in mango leaf samples in reverse-transcription recombinase polymerase amplification (RT-RPA) and reverse-transcription polymerase chain reaction (RT-PCR)

S.No	Code for mango plants	Symptoms	Variety	MiLV			MaV-1
				RT-PCR (RNA)	RT-RPA (RNA)	RT-RPA (Crude)	RT-PCR (RNA)	RT-RPA (RNA)
1	MG1	Leaf deformation	Ambika	+	+	+	+	+
2	MG3	Mosaic, undulated leaf margins	Ambika	+	+	+	+	+
3	MG4	Leaf deformation	Ambika	-	-	-	+	+
4	MG5	Mosaic, undulated leaf margins	Ambika	-	-	-	+	+
5	MA1	upward leaf curling of leaves	Arunika	-	-	-	-	-
6	MA2	upward leaf curling of leaves	Arunika	-	-	-	-	-
7	MA3	Asymptomatic	Arunika	+	+	+	-	-
8	MA4	Asymptomatic	Arunika	-	-	-	-	-
9	MA5	Asymptomatic	Arunika	-	-	-	-	-
10	MA6	Leaf deformation	Arunika	+	+	+	-	-
11	MA7	upward leaf curling of leaves	Arunika	-	-	-	-	-
12	MA8	upward leaf curling of leaves	Arunika	-	-	-	-	-
13	MA9	Mosaic, undulated leaf margins	Arunika	-	-	-	-	-
14	MA10	Mosaic, undulated leaf margins	Arunika	-	-	-	-	-
15	ML1	upward leaf curling of leaves	Local	-	-	-	-	-
16	ML2	Asymptomatic	Local	+	+	+	-	-
17	ML3	Asymptomatic	Local	-	-	-	-	-
18	ML4	upward leaf curling of leaves	Local	-	-	-	-	-
19	ML5	upward leaf curling of leaves	Local	-	-	-	-	-
20	ML6	Asymptomatic	Local	-	-	-	-	-
21	ML7	Leaf deformation	Local	+	+	+	-	-
22	ML8	Leaf deformation	Local	-	-	-	-	-
23	ML9	Leaf deformation	Local	-	-	-	-	-
24	ML10	Asymptomatic	Local	-	-	-	-	-
25	ML11	Asymptomatic	Local	+	+	+	-	-
Total positive samples				07	07	07	04	04

Table 3.

Diagnostic agreement between RT-RPA and conventional RT-PCR for detection of mango viruses in field samples

Virus	Assay comparison	RT-PCR (+) / RT-RPA (+)	RT-PCR (+) / RT-RPA (−)	RT-PCR (−) / RT-RPA (+)	RT-PCR (−) / RT-RPA (−)	Total samples	Kappa (K)	Std. Error	95% CI	p-value
MiLV	RT-PCR vs. RT-RPA (RNA)	7	0	0	18	25	1.000	0.000	1.000–1.000	< 0.001
MiLV	RT-PCR vs. RT-RPA (Crude sap)	7	0	0	18	25	1.000	0.000	1.000–1.000	< 0.001
MaV-1	RT-PCR vs. RT-RPA (RNA)	4	0	0	21	25	1.000	0.000	1.000–1.000	< 0.001

Agreement between RT-RPA and RT-PCR was evaluated using Cohen’s kappa coefficient (K). A K value of 1.00 indicates perfect agreement between the two diagnostic assays

TP true positive, FN false negative, FP false positive, TN true negative

Discussion

Viral diseases represent a persistent and often underestimated constraint to mango (Mangifera indica) productivity, largely because infections in perennial crops frequently remain latent or asymptomatic for long periods. Recent HTS-based virome studies have substantially expanded knowledge of mango-associated viruses, revealing a more complex viral landscape than previously recognized [9–15]. In our earlier study, HTS-based virome analysis led to the identification and genomic characterization of mangifera indica latent virus (MiLV) and a novel olivavirus, mangifera virus 1 (MaV-1) infecting mango plants in India [7]. While HTS has become an crucial tool for virus discovery and characterization, translating these findings into practical diagnostic tools remains a major challenge for routine disease surveillance and management.

In the present study, HTS was employed to reconfirm the presence of MiLV and MaV-1 and to obtain reliable genomic information required for diagnostic assay development. The primary objective of this work was not further virus discovery but the development of a rapid and deployable diagnostic method based on HTS-derived sequence data. By developing RT-RPA assays for MiLV and MaV-1, this study bridges the gap between virome research and routine virus indexing in mango production systems.

HTS provides comprehensive insights into plant viral diversity, its routine application in large-scale diagnostics is limited by high cost, sophisticated instrumentation, and the requirement for specialized bioinformatics expertise. Therefore, there is a growing need for rapid, sensitive and field-deployable diagnostic methods that can complement HTS-based discovery pipelines. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique that operates efficiently at low temperatures (37–42 °C) and produces results within 20–30 min without the need for thermal cycling equipment [18, 19]. Due to its simplicity and rapid reaction kinetics, RPA has been increasingly applied for the detection of plant viruses affecting several crops including grapevine, potato and other horticultural species [12, 18, 19, 31–33]. Recent studies have further demonstrated the versatility of RT-RPA assays for rapid detection of diverse plant viruses such as rice stripe virus, tobacco streak virus and cassava brown streak viruses [12, 34–36]. These advances highlight the growing importance of RT-RPA as a practical molecular diagnostic platform for plant health monitoring.

In this study, the RT-RPA assays developed for both MiLV and MaV-1 were optimized for temperature and time, with the optimal conditions of 40 °C for 25 min providing specific and robust amplification. Similar temperature ranges and reaction times have been reported for RT-RPA assays developed for other plant viruses [19, 31], confirming the robustness of this amplification system for plant virus diagnostics.

A key advantage of the RT-RPA assay for MiLV was its ability to use crude leaf sap as a template, prepared with the optimized NaOH: EDTA (1:2) buffer. This eliminates the need for time-consuming and costly RNA extraction kits, making the assay rapid and cost-effective diagnostic for on-site detection. Although agarose gel electrophoresis was used in the present study to visualize amplification products under laboratory conditions, RT-RPA assays can be adapted to portable detection techniques such as lateral flow dipsticks or fluorescence-based devices. These approaches eliminate the requirement for gel electrophoresis and enable practical point-of-care or field-level diagnostics. However, the inability to detect MaV-1 using crude sap, while still successful with purified RNA, may be attributed to relatively lower viral RNA abundance of MaV-1 in the tested samples, which could reduce amplification efficiency when crude plant extracts are used as the template. To address this limitation and enable crude sap-based detection for MaV-1, future research needs to explore the alternative extraction buffers or additives.

Sensitivity analysis showed that the developed RT-RPA assays for MiLV, the assay was found to be highly sensitive, capable of detecting the virus at a 10⁻¹⁰ dilution of RNA (100 ng/µl) /crude sap, which was similar to the sensitivity of conventional RT-PCR. This high sensitivity makes the assay suitable for detecting low viral loads, which is particularly important for latent viruses like MiLV [5]. For MaV-1, the RT-RPA assay was slightly less sensitive than RT-PCR, with a detection limit of 10⁻³ (equivalent to a concentration of 0.1 ng/µl) compared to 10⁻⁴ (0.01 ng/µl). In this study, virus presence was determined based on the amplification of the expected target fragment followed by sequencing confirmation. Sensitivity analysis showed that the developed RT-RPA assay could detect MiLV up to a 10⁻¹⁰ dilution and MaV-1 up to a 10⁻³ dilution of RNA. While this is a minor difference, the speed and field-deployable nature of RPA often outweigh the marginal difference in sensitivity for routine diagnostic purposes. The high specificity of the assay for both the above-mentioned viruses was confirmed by the lack of cross-reactivity between the MiLV and MaV-1 primers and the non-amplification in healthy plant samples. However, real-time PCR provides quantitative estimation of viral copy numbers, the primary objective of the present study was to develop a rapid and field-deployable diagnostic assay. Therefore, the performance of the developed RT-RPA assay was compared with conventional RT-PCR, which remains a widely used reference method for plant virus detection. Future studies may incorporate quantitative real-time PCR to further evaluate the detection limits and viral copy numbers associated with these viruses.

Validation of the RT-RPA assays using 25 field samples demonstrated a 100% concordance with the conventional RT-PCR method. The successful detection of both MiLV and MaV-1 in symptomatic and asymptomatic samples using the developed assays confirms their reliability and robustness. Statistical evaluation further confirmed the reliability of the developed assay. Cohen’s kappa analysis demonstrated perfect agreement between RT-RPA and conventional RT-PCR (K = 1.000), indicating that the RT-RPA assay provides diagnostic performance equivalent to the reference RT-PCR method. This high level of agreement supports the applicability of RT-RPA as a rapid and reliable molecular diagnostic tool for detection of mango-infecting viruses. Further, no correlation was found between the symptoms and the presence of virus as some of the asymptomatic samples were also found to be positive. The co-detection of both viruses in some samples reinforces the HTS findings and highlights the potential for complex viral infections in mango. This is consistent with findings in other crops, where mixed viral infections are common and can lead to more severe disease symptoms than single infections [12, 37]. Although isothermal amplification techniques such as RT-RPA may be prone to contamination if strict laboratory practices are not maintained, the use of appropriate negative controls, sequencing confirmation of amplicons, and validation against conventional RT-PCR ensured the reliability of the assay in the present study. The complete concordance observed between RT-RPA and RT-PCR during validation indicates that RT-RPA can serve as a reliable rapid screening tool for field-level detection of mango viruses.

The occurrence of MiLV and MaV-1 in both grafted Arunika plants and local rootstock propagated through stones indicates the potential role of grafting practices in virus dissemination within nursery systems. In the present study, all sampled plants were grafted onto locally raised mango seedlings used as rootstocks, indicating that infected rootstock material may act as a silent reservoir for virus transmission. Vegetative propagation through grafting is a well-recognized pathway for the spread of latent plant viruses, particularly in perennial fruit crops where asymptomatic infections frequently escape visual inspection [8, 38–42].

The detection of viruses in six-month-old nursery plants suggests early-stage infection, most likely introduced through infected rootstocks or contaminated nursery environments. These observations reinforce the importance of strict nursery sanitation and routine screening of propagation material. Similar results have been reported in other perennial cropping systems, where infected rootstocks contribute to sustained virus circulation [40, 43–45]. The detection of viruses in young nursery plants therefore highlights the importance of early screening of propagation material to prevent dissemination of infected planting stock.

These findings highlight the importance of routine molecular indexing of both scion and rootstock materials prior to grafting. Implementation of rapid RT-RPA-based screening at nursery level would enable early identification of infected propagation material, thereby preventing large-scale dissemination of viruses through planting stock. Integration of such diagnostics into certification programs is essential for producing virus-free planting material and for protecting long-term orchard productivity.

From a broader perspective, the combined HTS RT-RPA framework demonstrated in this study represents a scalable and practical strategy for managing viral diseases in perennial fruit crops. HTS enables comprehensive identification and characterization of known and emerging viruses, while RT-RPA provides a rapid, cost-effective, and field-deployable method for routine surveillance. Such integration is particularly relevant for certification programs aimed at producing virus-tested planting material, which remains the most effective long-term strategy for mitigating virus-associated yield losses in mango.

This study complements previous mango virome characterization by delivering a robust diagnostic application that is directly relevant to field conditions. The RT-RPA assays developed here provide a valuable tool for rapid virus detection and surveillance and establish a model for translating virome discoveries into actionable plant health management solutions.

Materials and methods

Sample collection and symptom observation

In 2024, a survey was conducted in Lucknow, India and mango plants displaying virus-like symptoms such as mosaic, undulated leaf margins, upward leaf curling and leaf deformation were observed in one-year-old nursery plants (Fig. 7). Eleven mango (Mangifera indica) nursery plants showing growth retardation and leaf deformities were collected from Uttar Pradesh, India. The plants were approximately six months old at the time of sampling. Four samples (A1-A4) belonged to the commercial cultivar Arunika and the remaining seven samples (A5 and L1-L6) represented local rootstock of mango variety multiplied through stones. All plants had been grafted onto locally raised mango seedlings used as rootstocks.

Fig. 7.

Virus-like symptoms on Mangifera indica (a): mosaic and undulated leaf margins, (b) upward leaf curling of leaves, (c) leaf deformation

RNA extraction and quality assessment

Total RNA was extracted from 100 mg of leaf tissue using Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA) with little modifications. Briefly, after grinding the leaf samples in liquid nitrogen, the powder was transferred to RNA extraction buffer modified by adding 2% PVP-40 followed by 2% β-mercaptoethanol. Then the extract was incubated for 5 min at 56 °C followed by manufacturer’s instructions for remaining steps of RNA isolation. The concentration and purity of extracted RNA were determined using a NanoDrop™ UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), The quality was checked on 0.8% agarose gel and assessed using an Agilent 4150 TapeStation (Agilent Technologies, USA) and Qubit™ 4 Fluorometer (Invitrogen, USA). The Plant riboPOOL kit (siTOOLs Biotech, Germany) was employed for ribosomal RNA depletion according to the manufacturer’s guidelines.

Library preparation and high-throughput sequencing

RNA libraries were prepared using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, USA) as per manufacturer’s instructions. Library quality and quantity were assessed using an Agilent 4150 TapeStation, followed by sequencing on an Illumina NovaSeq 6000 platform generating paired-end reads of 2 × 150 bp.

Bioinformatics analysis

Raw reads were imported into the CLC Genomics Workbench v20.0.4 (Qiagen, Germany). Adapter sequences and low-quality reads were trimmed and high-quality reads were mapped against the M. indica reference genome (GCA_011075055.1). Unmapped reads were extracted and assembled de novo using CLC Genomics Workbench [41]. The assembled contigs were analyzed with BLASTx search against the NCBI non-redundant protein database and viral genome sequences. Conserved domains were predicted using the NCBI Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Pfam (http://pfam.xfam.org). Multiple sequence alignments were performed using ClustalW and maximum-likelihood phylogenetic trees were constructed in MEGA X with 1000 bootstrap replicates [46].

RT-PCR validation and Sanger sequencing

The assembled viral contigs were used for the primer design (Supplementary Table 1). First-strand cDNA was synthesized using the FIREScript^® RT cDNA Synthesis Kit (Solis BioDyne, Estonia) with random primers according to the manufacturer’s instructions. PCR amplification was carried out in a Thermal Cycler under standard conditions as mentioned by Prajapati et al., [7]. Amplicons were visualized on 1.6% agarose gel, purified, cloned and confirmed by Sanger sequencing.

Development of Reverse transcription-recombinase polymerase amplification (RT-RPA) assay for the detection of viruses infecting mango

Primer designing, RNA isolation and their use in RT-PCR and RT-RPA

The mango viruses [mangifera indica latent virus (MiLV) and mangifera virus 1 (MaV-1)] confirmed through HTS were targeted for the development of RT-RPA assay for their rapid detection. Three set of primer pairs (each) were designed from the conserved regions of the RNA-dependent RNA polymerase (RdRp) gene for MaV-1 and the coat protein (CP) gene for MiLV using Primer3 software (Supplementary Table 1) and as mentioned in manual (TwistDx, Cambridge, UK). In order to find out the most appropriate and specific primer pair, RT-PCR was used for the detection of the respective viruses.

The RNA was extracted as mentioned above from the symptomatic and asymptomatic (healthy) mango leaf samples and stored at -80 °C till further use. Further, first-strand cDNA was synthesis was done using random primers and the FIREScript^® RT cDNA Synthesis Kit (Solis BioDyne, Estonia) as per manufacturer’s instructions followed by PCR in a Thermal Cycler. The reaction mixture for PCR comprised of 1 µl of dNTP mix (2.5 mM each), 1 µl of cDNA, 2 µl of 10X reaction buffer, 10 pmol each of upstream and downstream primers for MiLV and MaV-1 respectively (Supplementary Table 1), 0.2 µl of Takara Taq™ DNA Polymerase enzyme (5U/µl) (Takara, Japan). The volume was raised to 20 µl with nuclease-free water. The conditions used for PCR were as follows: Initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56–61 °C for 30 s, extension at 72 °C for 30 s and final extension at 72 ◦C for 10 min were used for the detection of the respective viruses mentioned above.

Simultaneously, using the extracted RNA as a template RT-RPA was carried out using the TwistAmp^® Basic kit (TwistDx, Cambridge, UK) according to manufacturer’s instructions with little modifications and the different set of primer pair for the respective viruses. Firstly, a master mix was prepared by reconstituting freeze-dried pellets with 43.5 µl of reaction mixture prepared by using 29.5 µL rehydration buffer, 2.1 µL of each forward and reverse primer pair (10 µM), 9.8 µl of nuclease free water. Further, the reaction mixture was divided equally into two tubes. In each tube add 1 µl RNA (500 ng) followed by 1 µl of reverse transcriptase and the reaction was initiated by adding 280 mM of magnesium acetate (1.25 µl) followed by brief centrifugation and incubated at test conditions. Then around 10 µl of the RT-RPA products were incubated for 10 min at 65 °C and visualized on 2% agarose gel. The amplified products were then eluted from the gel using GeneJET Gel Extraction kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and the amplicons were confirmed through sequencing. The suitable primer pair were selected based on the results obtained and further used for standardization of the RT-RPA assay.

Standardization of extraction buffers for the preparation of crude leaf extract

Eight different extraction buffers: CTAB (10%), 0.1 M phosphate buffer pH 7.4, Mackenzie buffer (Mackenzie et al., 1997), various ratio (1:1, 1:2 and 1:3) of 50 mM NaOH: 2.5 mM EDTA pH 8.0, NaOH solution (0.5 M) and general extraction buffer-3 (GEB3, Agdia, USA) containing 2% Tween-20 were standardized (Sharma et al., 2023) for the preparation of crude leaf extract. The extract was prepared by grinding ~ 100 mg of fresh mango leaves in extraction buffer (1 ml) using mortar and pestle. Then the extract was transferred to micro-centrifuge tubes and centrifuged for 5 min at 10,000 g. Then the supernatant was transferred to fresh micro-centrifuge tube and used for further standardization of the assay as a template.

Optimization of RT-RPA assay using different incubation temperatures and time for the detection of MiLV and MaV-1

Using the best primer pair for MiLV and MaV-1 and keeping the time constant (30 min) different incubation temperatures i.e. 34, 37, 40 and 43 °C were optimized using RNA/crude sap as template. Further, different incubation time viz. 15, 20, 25, 30, 35 and 40 min were also standardized after optimization of the isothermal temperature for the two respective viruses. Healthy controls were used in the assay so as to check the non-specific amplification.

Determination of sensitivity and specificity of RT-RPA assay using RNA and crude extract as a template

The sensitivity of the RT-RPA assays developed for the detection of MiLV and MaV-1 was evaluated using 10-fold serial dilutions of purified RNA (~ 100 ng/µl)/ infected crude leaf sap (100 mg of leaf sample/1 ml lysis buffer) from infected mango plants. The performance of RT-RPA was compared with conventional RT-PCR. Further, healthy mango leaf samples were used as a negative control and the developed assay was repeated thrice in order to see the reproducibility of the results. After the completion of the reaction, results were analysed on agarose gel as mentioned above. The specificity of the developed assay was determined by testing mango samples for the presence of MiLV in MaV-1 infected samples and vice-versa and healthy plants were used as a negative control.

Validation of the developed RT-RPA assay in specific detection of MiLV and MaV-1 viruses

Total 25 symptomatic and asymptomatic mango leaf samples were collected from the plants growing in the campus of ICAR-IARI, New Delhi and from Gomtinagar, Lucknow, UP. The samples were subjected to RT-RPA assay using purified RNA/ crude leaf sap (freshly extracted) as a template and tested for the presence of MiLV (RNA/crude sap as template) and MaV-1 (RNA as template) viruses respectively (Table 1). Simultaneously, the same samples were also tested using RT-PCR and primers as mentioned above, along with positive and negative controls and the results of were analysed on agarose gel as mentioned previously, while the RT-PCR products were directly loaded on the gel.

Statistical analysis of diagnostic agreement

Diagnostic agreement between the developed RT-RPA assay and conventional RT-PCR was evaluated using Cohen’s kappa coefficient (K). The analysis was based on the classification of true positive (TP), false positive (FP), true negative (TN) and false negative (FN) results obtained from the 25 mango samples tested. The kappa statistic, standard error, 95% confidence interval, and p-value were calculated using the MedCalc statistical calculator (MedCalc Software Ltd., Belgium) [47]. A kappa value of 1.0 indicates perfect agreement between the two diagnostic methods.

Conclusion

The present study provides a valuable contribution to the understanding of the mango virome by identifying MaV-1 and MiLV. More significantly, we have established a rapid, sensitive, and reliable RT-RPA based diagnostic assays for the detection of these viruses. This combined HTS-RPA approach offers a powerful strategy for comprehensive disease management in perennial crops. The HTS component enables the discovery of new and emerging viral threats, while the RT-RPA component provides a practical, field-deployable tool for routine surveillance, certification programs and rapid response to disease outbreaks. This is particularly crucial for the mango industry, as it will facilitate the production of certified virus-tested planting material, which is the most effective long-term strategy for safeguarding orchard productivity and mitigating economic losses.

Abbreviations

HTS

High–throughput sequencing

RPA

Recombinase polymerase amplification

RT-PCR

Reverse transcription polymerase chain reaction

MiLV

Mangifera indica latent virus

MaV-1

Mangifera virus 1

RdRp

RNA–dependent RNA polymerase

CP

Coat protein

SRA

Sequence Read Archive

NCBI

National Center for Biotechnology Information

Authors’ contributions

M.R.P.: Data curation, Formal analysis, investigation, validation, methodology, writing—original draft preparation. P.B: Data curation, formal analysis, investigation, validation writing—original draft preparation. J.G.: Resource. P.S.: Writing—review and editing. D.S.: Resource. N.G.: Supervision, Writing—review and editing. V.K.B.: Supervision, writing—review and editing. S.K.S.: Conceptualization, supervision, resources, methodology, writing—review and editing.

Funding

The research work was supported by the Indian Council of Agricultural Research, Department of Agricultural Research and Education, Government of India under All India Network Project on Emerging Pests (AINP-EP) – Plant Virus Sub-component (CS 8–13/2024-PP).

Data availability

The datasets generated and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) and GenBank under accession numbers: SRR36746814, SRR36746815, SRR36746813, SRR36746823, SRR36746822, SRR36746821, SRR36746820, SRR36746819, SRR36746818, SRR36746817, SRR36746816, PX898077-PX898088.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.