A Handy and Visual Detection Method for Potato Late Blight Utilizing Multi-Copy Specific Sequences and RPA-LFD Technology

Abstract

Potato late blight, caused by Phytophthora infestans, is one of the most destructive diseases affecting global potato production. Rapid and accurate detection of the pathogen is essential for effective disease prevention and control. In this study, a novel multi-copy and specific DNA sequence, designated SSPI1 (Specific Sequence of Phytophthora infestans 1), was identified through bioinformatic analysis. Using seven closely related Phytophthora species and two additional common potato pathogens as controls, PCR assays demonstrated that SSPI1 offers higher sensitivity than PiYPT1 and improved specificity over PiITS. Based on this novel multi-copy sequence, we developed a rapid, user-friendly, and highly sensitive detection method for potato late blight by combining recombinase polymerase amplification (RPA) with lateral flow dipstick (LFD) technology. The entire diagnostic process—including crude DNA extraction, RPA amplification, and visual detection—can be completed within 35 min without the need for specialized equipment. Optimization results indicated that among nine candidate RPA primer pairs, RPA-F1/RPA-R2 exhibited the highest amplification efficiency. The optimal RPA reaction temperature range was determined to be 27–42°C, with a detection limit of 11 copies/μl for SSPI1. This method could detect as little as 100 pg of genomic DNA, identify samples collected as early as 24 h post-inoculation, and even detect infections in asymptomatic leaves under field conditions, demonstrating superior sensitivity compared to the PiYPT1-targeted RPA-LFD assay. Overall, this study presents a highly sensitive, specific, practical, and efficient tool for the early and rapid detection of potato late blight.

Introduction

Potato late blight, caused by Phytophthora infestans (P. infestans), is a catastrophic soil-borne disease that instigated the Irish Potato Famine in the 19th century and continues to pose the most severe threat to global potato production [1-3]. Annually, global losses and control costs surpass $10 billion [1-3]. For example, as the world’s largest potato producer, China reports an average annual late blight incidence of 1.9747 million hectares, which constitutes 40.66% of its total potato planting area [4].

In the field environment, potato planting tubers with latent infections, leftover infected potato tubers in the soil, as well as chlamydospores and oospores, constitute the primary sources of initial infection for potato late blight [5-7]. Upon infection, P. infestans hyphae proliferate rapidly within plant tissues. Following a latent period, water-soaked lesions appear on leaves, accompanied by significant mycelial growth and sporangia formation [1, 8]. Under humid and cool (10–23°C) conditions, sporangia release zoospores, which spread rapidly through air or water [8]. After potatoes are infected by P. infestans, visible infection symptoms generally emerge within 3 to 6 days. However, under low-temperature and high-humidity conditions, the growth and reproduction of P. infestans accelerate dramatically, causing the rapid death of infected plants within 5 to 10 days [1, 8]. Early infection induces minimal damage to host cells, thereby delaying the manifestation of symptom. By the time symptoms become evident, tissues have already sustained significant damage. This underscores the critical importance of early detection to exploit the narrow therapeutic window before the onset of rapid pathogen dissemination [9].

Molecular biology-based detection technologies have provided accurate and efficient approaches for the identification of plant pathogen [10]. In the case of P. infestans, various molecular detection techniques have been developed, including conventional PCR [11], real-time PCR [12], nested PCR [13], loop-mediated isothermal amplification (LAMP) [14], and recombinase polymerase amplification (RPA) [15]. PCR-based amplification methods require specialized laboratory equipment and trained personnel, making them unsuitable for on-site applications. Although the earlier LAMP technology eliminates the need for thermal cycling instruments, its primer design is highly complex, and the reaction must be maintained at approximately 65°C, which presents challenges for field deployment [10, 16]. The RPA technique achieves isothermal amplification by mimicking the in vivo DNA replication process, allowing reactions to proceed at a constant temperature of 30–42°C. Furthermore, RPA reaction mixtures can be lyophilized into a dry powder format, facilitating storage and transport [17]. Owing to its advantages—such as rapid amplification, high specificity, low-temperature operation, and multiple detection modalities—RPA has been increasingly utilized for the detection of plant pathogens [17-20].

Among nucleic acid amplification-based methods for detecting P. infestans, commonly targeted sequences include the single-copy sequences PiYpt1 [13, 15] and the multi-copy internal transcribed spacer PiITS region [14]. Multi-copy PiITS sequences generally offer higher sensitivity; however, their high degree of conservation across closely related species can compromise assay specificity [14]. Although PiYpt1 exhibits higher specificity, it is a single-copy sequence in the genome, which may lead to lower sensitivity compared to the multicopy sequence as previous similar report [21]. In addition to factors such as template quality, primer design, and reaction conditions, the genomic copy number of the target sequence plays a critical role in determining detection sensitivity [22]. Enhanced sensitivity in nucleic acid detection significantly improves the effectiveness of early pathogen screening.

Based on the aforementioned background, a novel multicopy-specific sequence, designated as SSPI1 (Specific Sequence of Phytophthora infestans 1), was identified through Self-BLAST analysis of the published P. infestans genome sequence. Using this sequence, we established a visual detection assay for potato late blight by combining RPA with LFD technology (RPA-LFD). The developed method exhibits simplicity in operation, rapid detection capability, high sensitivity, and excellent specificity, thereby offering a practical solution for on-site diagnosis of potato late blight by field personnel at the grassroots level.

Materials and Methods

Pathogens and DNA Extraction

The strain of P. infestans used in the experiments of this study was designated as 88069. Phytophthora capsici (P. capsici), Phytophthora sojae (P. sojae), Phytophthora parasitica (P. parasitica), Phytophthora palmivora (P. palmivora), Phytophthora cinnamomi (P. cinnamomi), Phytophthora cactorum (P. cactorum), and Phytophthora heveae (P. heveae), Ralstonia solanacearum (R. solanacearum) and Alternaria solani (A. solani), were selected as control pathogens. All aforementioned pathogens were isolated and maintained by the Guizhou Provincial Key Laboratory for Tobacco Quality Improvement and Efficiency Enhancement.

P. infestans strains were cultured on RSA (rye sucrose agar) medium plates at 20°C in the dark, whereas other Phytophthora species were cultivated on 10% V8 (V8 Juice Agar) medium at 25°C in the dark [18]. Ralstonia solanacearum was cultured on NA (nutrition agar) medium plates at 28°C [20]. Alternaria solani was cultivated on PDA (Potato Dextrose Agar) medium plates at 25°C [23].

Genomic DNA was extracted from the mycelia. Genomic DNA of Phytophthora species and Alternaria solani was extracted using the Fungal Genomic DNA Extraction Kit (Solarbio, China). The genomic DNA of Ralstonia solanacearum was isolated using the Bacterial Genomic DNA Extraction Kit (Sango, China).

The concentration and quality of the extracted DNA were determined by measuring the absorbance at 260, 230, and the ratio of OD260/280 and OD260/230 through the ND-1000 Spectrophotometer (Nanodrop, USA). The DNA samples were all stored at -20°C for further use.

Specific Sequence Screening and Sequencing Identification

To identify novel multi-copy sequences that were suitable for P. infestans detection, the P. infestans reference genome (GenBank Accession NO: GCA_000142945) was used for genome sequence alignment. We used BLASTN (v2.10.1) to identify potential multi-copy sequences using a self-blast strategy with the default parameters. Since PCR amplification and RPA amplification are compatible with individual mismatches in the intermediate sequences, the threshold for repetitive sequences is set as Query Coverage > 99% and Percent Identity > 95%. We screened the multi-copy sequence through two rounds of BLAST searches and manual proofreading. First, the sequences longer than 150 bp and with more than two high confidence hits (E value <0.01) were kept by “self-to-self ” comparisons of the genome. Then, Bedtools (v 2.30.0) was implemented to combine the overlapping multi-copy sequences, and based on these collapsed regions, the potential multi-copy sequences were collected with Samtools (V 1.9). Finally, the multi-copy sequences were used as query sequences in BLAST searches of the genome, and the number of hits and the mean of identity value for each multi-copy sequence were counted from the results. The operational procedure is illustrated in Fig. 1. The multi-copy sequence in reference genome was aligned with the genomes of 7 sequenced P. infestans strains. The 7 sequenced P. infestans strains genome information was downloaded from NCBI database. And then select conserved and high copy number sequences for blast to RefSeq Genome Database in NCBI database. This database included almost all known genomes, containing genomes of representing 203 Phytophthora species and representing 123 Pythium species. Finally, a novel multi-copy sequence was identified from P. infestans.

Fig. 1. Flowchart depicting the screening process for identifying multi-copy sequences.

To identify this sequence, DNA extracted from the standard P. infestans strain 88069 was used as a template. The full-length primers targeting the novel multi-copy sequence were designed using Primer 5 software. The full-length fragment was amplified using high-fidelity DNA polymerase, purified via gel electrophoresis, and subsequently ligated into a TOPO vector (Yeasen, China) for sequence confirmation through a commercial sequencing service.

RPA Primer Optimization and LF Probe Design

To reduce the false matching in the specific multi-copy sequences, the sequences of primers and probes were selected with the help of Primer5 software and NR Database of NCBI. All the primers and probes were compared with the GenBank of NCBI using Primer-BLAST to insure their species specificity. According to the principles of RPA primer design, the primer length should be within the range of 30–35 nucleotides, and the sequence should not contain palindromic sequences, consecutive single-base repeat sequences, or regions prone to forming internal secondary structures [24]. Based on these criteria, three forward primers and three reverse primers of the novel multi-copy sequence were designed, respectively (Table 1). According to the instructions provided in the Basic-DNA Isothermal Rapid Amplification Kit (Genenodel, China) manual, various primer combinations were introduced into the reaction system to perform isothermal amplification. The reaction was carried out at 37°C for 25 min. Upon completion of the amplification, an equal volume (50 μl) of chloroform solution was added to the reaction mixture, followed by gentle vortexing and centrifugation at 12,000 ×g for 2 min. After centrifugation, 15 μl of the supernatant was carefully collected and subjected to agarose gel electrophoresis. The primer combination yielding the most efficient amplification was selected based on the electrophoretic results.

Table 1.

List of primers and probes employed in the study.

The reverse primers (Table 1) used for RPA-LFD was modified with biotin labels at the 5' end. For the specific probe (Table 1), the 5' end was FAM labeled and the 3' end was blocked with a phosphate group, which was inserted between the 30th and 31st base. All primers and probe were synthesized by Sangon (China).

Establishment of the RPA-LFD and Optimization of the Detection Conditions

The RPA reaction was performed based on the nfo DNA Isothermal Rapid Amplification Kit (Genenodel). Each reaction volume was 50 μl and contained 2 μl of forward primer (10 mM), 2 μl of biotin-labeled reverse primer (10 mM), and 0.6 μl of multi-site modified probe (10 mM), 40.9 μl buffer A, 1 μl DNA template, 2.5 μl buffer B (Magnesium Acetate, 280 mM), and 1 μl ddH₂O. The dried reagent in the reaction tube supplied by the kit was suspended in buffer A and mixed with the other reagents, except for the buffer B. Then, the buffer B was added into each tube to start the RPA reaction, and the tubes were incubated at 30°C to 40°C for 10 min in water bath. Visualization was based on LFD. The RPA products were diluted at a ratio of 1:20. The LFD Single-Use Nucleic Acid Test Strip (Genenodel) were added to the dilutions, and the results were read within 3 to 10 min. The appearance of the control line confirmed that the strips functioned normally. Positive reactions occurred when two separate lines, including the control line and the test line, were generated in different locations.

The RPA-LFD detection system was optimized by systematically evaluating three key parameters: the RPA amplification duration (10, 15, 20, 25, and 30 min), the operational temperature range for RPA amplification (22°C, 27°C, 32°C, 37°C, 42°C, and 47°C), and the effective temperature range for LFD detection (10°C, 30°C, and 50°C).

Specificity and Sensitivity Test for RPA-LFD

To assess the specificity of the RPA-LFD detection system for P. infestans, genomic DNA samples from seven previously prepared Phytophthora species, along with those from R. solanacearum and A. solani, were analyzed using the RPA-LFD assay, and the resulting banding patterns on the lateral flow strips were visually interpreted.

To assess the sensitivity of the RPA-LFD assay, serial dilutions of P. infestans genomic DNA templates (100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg) and plasmid DNA templates containing the target sequence (1.1 × 10⁴, 1.1 × 10³, 1.1 × 10², 1.1 × 10¹, 1.1 × 10⁰, and 1.1 × 10 ^-1 copies/μl) were tested using the optimized RPA-LFD detection system, with the results interpreted through visual observation of colorimetric changes on the lateral flow strips.

The sensitivity of the optimized RPA primers was validated through real-time fluorescence detection [25]. The exo DNA isothermal rapid amplification kit (Genenodel) was employed to evaluate the sensitivity of the RPA reaction, with the reaction system strictly following the manufacturer's instructions. The fluorescence detection protocol was configured as follows: initial preheating at a constant temperature of 39°C for 1 min, followed by isothermal amplification at 39°C with 30 sec cycles repeated for a total of 40 cycles, during which FAM channel fluorescence values—selected based on the fluorescent probe design—were recorded at 30 sec intervals.

Practicability Test of RPA-LFD in Field Condition

For RPA-LFD detection in field condition, excise approximately 2–3 cm² of leaf tissue and place it into a 1.5 ml microcentrifuge tube containing 300 μl of PEG-NaOH solution [15]. Homogenize the sample for 2–3 min using a plastic pestle, then transfer 5 μl of the crude DNA extract as the template for RPA amplification. During the RPA reaction, maintain the required temperature by holding the reaction tube in the palm. Following completion of the reaction, add 100 μl of ultrapure water to dilute the product. Subsequently, apply 50–100 μl of the diluted amplification product onto the LFD test strip to enable equipment-free visual detection under field conditions (Fig. 2).

Fig. 2. Schematic diagram of the field-based RPA-LFD detection protocol.

Conventional PCR was performed to determine whether the collected samples were infected with P. infestans, with the widely accepted PiYpt1 selected as the detection gene [13, 15].

Results

Identification of Multi-Copy Specific Sequence SSPI1

The genomic data of 7 P. infestans strains were obtained from the NCBI Genome Database. Among these, the genome of strain T30-4 (GenBank: GCA_000142945) served as the reference genome for P. infestans. Through multi-copy sequence mining based on this reference genome, a conserved genomic sequence of 174 base pairs was identified (Fig. 3A). This sequence was present as 88 highly similar copies in the reference genome and ranged from 40 to 191 copies across the genomes of the other 6 P. infestans strains (Table 2). BLAST analysis against the NCBI database confirmed that this sequence is unique to P. infestans. The sequence was designated as SSPI1. Sanger sequencing show that the experimentally obtained SSPI1 sequence was entirely consistent with the corresponding region in the reference genome (Fig. 3B).

Fig. 3. Verification of nucleic acid amplification for the SSPI1 sequence.

(A) The sequence of SSPI1; (B) Sequencing peak diagram of plasmid Topo::SSPI1; (C-E). Electrophoretic diagrams of PCR detection sensitivity for SSPI1 (C), PiYPT1 (D), and PiITS (E) as target sequences under the same PCR amplification conditions, with lanes 1-5 representing the amounts of genomic DNA templates added as 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg respectively; (F-G). Specificity of PCR detection for PiITS (F) and SSPI1 (G), with lanes 1-10 representing the added DNA templates as P. infestans, P. capsici, P. sojae, P. parasitica, P. palmivora, P. cinnamomi, P. cactorum, P. heveae, R. solanacearum, and A. solani respectively.

Table 2.

The copy number of SSPI1 in each genome of P. infestans.

Strain name	GeneBank	Assembly	Copy number
T30-4	GCA_000142945	ASM14294v1 (reference genome)	88
1306	GCA_026225685	Pinf1306_UCR_1	41
VZR-VIR21a	GCA_044509515	PHIN_VZR-VIR21a	40
KR_1_A1	GCA_012295175	ASM1229517v1	138
KR_2_A2	GCA_012552325	ASM1255232v1	152
RC1-10	GCA_011316315	UAnd_PInf_RC1-10.1	191
HP-10-31	GCA_001661535	ASM166153v1	126

Amplification and detection were performed using the full-length primers targeting the SSPI1 sequence, along with the previously reported detection primers for the PiYPT1 sequence [13, 15] and PiITS sequence [14]. The results demonstrated that the sensitivity of PCR amplification targeting SSPI1 was superior to that of the PiYPT1, achieving a detection limit at the 100 fg level, which was comparable to the sensitivity observed for the PiITS (Fig. 3C–3E). Using 7 other closely related Phytophthora species and two common potato pathogens as controls, PCR amplification results confirmed that the assay targeting SSPI1 exhibited higher specificity than that targeting PiITS (Fig. 3F and 3G). These results collectively demonstrate that SSPI1 is a high-copy, species-specific sequence, and that PCR amplification targeting SSPI1 could offer higher sensitivity and specificity in the detection of P. infestans than previous reported targets.

Screening of Optimal RPA Primers for SSPI1

To obtain an optimal primer combination for RPA amplification targeting the acquired multicopy-specific sequence SSPI1, a set of primers was designed following standard RPA primer design principles, including three forward primers and three reverse primers (Table 1). And then, RPA amplification reactions were performed by using 1 ng of P. infestans genomic DNA as the template under identical conditions using the following 9 primer combinations: RPA-F1 / RPA-R1, RPA-F1 / RPA-R2, RPA-F1 / RPA-R3, RPA-F2 / RPA-R1, RPA-F2 / RPA-R2, RPA-F2 / RPA-R3, RPA-F3 / RPA-R1, RPA-F3 / RPA-R2, RPA-F3 / RPA-R3. The resulting amplicons were analyzed by agarose gel electrophoresis. As shown in Fig. 4A, the primer pair RPA-F1 / RPA-R2 produced the most abundant and specific amplification products, indicating superior performance in both yield and specificity. Therefore, RPA-F1 / RPA-R2 was selected as the optimal primer pair targeting SSPI1 for subsequent RPA-LFD and real-time RPA assays conducted in this study.

Fig. 4. Evaluation of RPA-LFD operational conditions.

(A) Electrophoretic detection diagrams of RPA amplification of SSPI1 under different primer combinations, with lanes 1-9 representing the primer combinations in the RPA amplification system as RPA-F1/RPA-R1, RPA-F1/RPA-R2, RPA-F1/RPA-R3, RPAF2/ RPA-R1, RPA-F2/RPA-R2, RPA-F2/RPA-R3, RPA-F3/RPA-R1, RPA-F3/RPA-R2, and RPA-F3/RPA-R3; (B). The LFD test results after RPA amplification at 37°C, respectively conducted under conditions of 10°C, 30°C and 50°C; (C) RPA-LFD Test Results for RPA at 22°C, 27°C, 32°C, 37°C, 42°C, and 47°C; (D) The RPA-LFD test results under conditions of RPA amplification time of 10 min, 15 min, 20 min, 25 min and 30 min. All experiments were independently repeated three times, with consistent results obtained each time.

Establishment of RPA-LFD Detection System for SSPI1

Using 1 ng of P. infestans genomic DNA as the template, we established an RPA-LFD detection system with primer RPA-F1, biotin-labeled primer RPA-R2-Biotin, and nfo probe RPA-probe (Table 1). To optimize the assay conditions, we systematically evaluated three key parameters: the operational temperature range for LFD detection (Fig. 4B), the optimal temperature range for RPA amplification (Fig. 4C), and the minimum duration required for RPA amplification (Fig. 4D). In this study, experimental evaluations were performed on the operational temperature range for LFD detection (Fig. 4B), the optimal temperature range for RPA amplification (Fig. 4C) and the required duration of RPA amplification (Fig. 4D). As illustrated in Fig. 4B, LFD detection remains functional across ambient temperatures ranging from 10°C to 50°C. As shown in Fig. 4C, the effective RPA amplification temperature range is 27°C to 42°C. As depicted in Fig. 4D, at an amplification temperature of 37°C, a reaction time of 20 min in RPA is sufficient to meet the requirements for subsequent LFD detection.

Sensitivity of RPA-LFD and Real-Time Fluorescent RPA

To assess the sensitivity of the SSPI1-targeted RPA assay, serial dilutions of P. infestans genomic DNA and plasmid DNA harboring the SSPI1 sequence were used as templates for RPA-LFD detection. The results showed that the minimum detectable concentration of plasmid DNA was 11 copies/μl (Fig. 5A), while the limit of detection (LOD) of the RPA-LFD assay for P. infestans genomic DNA was determined to be 100 fg (Fig. 5B). Except RPA-LFD detection, real-time fluorescent RPA was also conducted on a quantitative PCR (qPCR) instrument using P. infestans genomic DNA of graded concentrations as templates. The data revealed that the LOD of real-time fluorescent RPA was as low as 10 fg of genomic DNA (Fig. 5C and 5D). To further validate the sensitivity of this assay in P. infestans-infected plant tissues, we collected samples from inoculation sites at different time points (Fig. 5E), extracted genomic DNA from these samples, and subjected the extracts to RPA-LFD testing. RPA-LFD detection yielded positive results for potato leaves inoculated with P. infestans spores as early as 24 h post-inoculation (hpi) (Fig. 5F).

Fig. 5. RPA test results obtained with varying template quantities.

(A) RPA-LFD detection results of the 50 μl RPA reaction system supplemented with Topo::SSPI1 plasmid at concentrations of 1.1×10⁴, 1.1×10³, 1.1×10², 1.1×10¹, 1.1×10⁰, and 1.1×10^-1 copies/μl, respectively; (B) Sensitivity evaluation of the RPA-LFD assay: detection results of the 50 μl RPA reaction system supplemented with different amounts ofPhytophthora infestans genomic DNA (100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg); (C) Real-time fluorescent RPA amplification curves based on the SSPI1 sequence: curves were generated from 50 μl RPA reaction systems containing P. infestans genomic DNA at concentrations of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg, respectively; (D) Standard curve for real-time fluorescent RPA detection based on the SSPI1 sequence. The linear regression equation was established between the cycle threshold (Ct) value (X) and the logarithm of the initial DNA quantity (Y), expressed as Y = –3.508X + 36.337. Error bars represent the standard error from three independent real-time fluorescent RPA experiments; (E) Phenotypic photographs of potato leaves at 0, 6, 12, 24, 36, 48, 60, and 72 hours post-inoculation (hpi) with P. infestans. (F). RPA-LFD detection results of P. infestansinfected potato leaves at 0, 6, 12, 24, 36, 48, 60, and 72 hpi. All experiments were independently repeated three times, with consistent results obtained each time.

Specificity of RPA-LFD

To further evaluate the specificity of the P. infestans RPA-LFD detection system targeting the SSPI1 sequence, genomic DNA from 7 closely related Phytophthora species (P. capsici, P. sojae, P. parasitica, P. palmivora, P. cinnamomi, P. cactorum, P. hevea) and two other common potato pathogens (R. solanacearum and A. solani) was used as controls. As shown in Fig. 6A, a distinct test band was observed exclusively in the P. infestans genomic DNA sample. No amplification signals were detected in the genomic DNA of P. capsici, P. sojae, P. parasitica, P. palmivora, P. cinnamomi, P. cactorum, P. heveae, R. solanacearum, or A. solani. These results confirm that the RPA-LFD assay targeting SSPI1 possesses high specificity for P. infestans, with no cross-reactivity to closely related oomycetes or other common potato pathogens. To further evaluate the applicability of the established P. infestans detection method to different P. infestans strains, we performed RPA-LFD assays on 4 P. infestans strains (GS-1, SX-1, CQ-1, and GZ-1) isolated from 4 distinct provinces in China, as well as 2 commonly used reference strains in scientific research (88069 and T30-4). The results demonstrated that all tested P. infestans strains were successfully detected by the RPA-LFD assay (Fig. 6B). Furthermore, all RPA-LFD results were consistent with those obtained from PCR validation (Fig. 6C).

Fig. 6. Specificity evaluation of the RPA-LFD assay.

(A) The RPA-LFD detection results by adding different phytopathogen genomic DNA. The genomic DNA from left to right are from P. infestans, P. capsici, P. sojae, P. parasitica, P. palmivora, P. cinnamomi, P. cactorum, P. hevea, R. solanacearum and A. solani. All experiments were independently repeated three times, with consistent results obtained each time. (B) Detection results of the RPA-LFD assay for six distinct P. infestans strains, with sterile water as the negative control. The six P. infestans strains from left to right were GS-1, SX-3, GZ-1, CQ-1, 88069 and T30-4. All experiments were independently repeated three times, with consistent results obtained each time. (C) PCR amplification results analyzed by agarose gel electrophoresis, using primer pair SSPI1-FL-F/SSPI1-FL-R. Lanes 1–6: PCR templates were genomic DNA from the six P. infestans strains described in (B); Lanes 7– 15: PCR templates were genomic DNA from P. capsici, P. sojae, P. parasitica, P. palmivora, P. cinnamomi, P. cactorum, P. heveae, R. solanacearum and A. solani; Lane 16: Sterile water as the negative control.

The Application Using RPA-LFD in Field Condition

To validate the applicability of the RPA-LFD detection system for potato late blight developed in this study, five field samples suspected of potato late blight were collected from Qijiang, Chongqing, China, along with one artificially inoculated sample serving as a positive control and one healthy potato sample serving as a negative control. All the samples were subjected to analysis using both the RPA-LFD assay and conventional PCR. The genomic DNA of the suspected pathogenic tissue was extracted using the PEG-NaOH solution as described in the previous method. The required maintenance temperature for the RPA reaction is provided by hand holding. As shown in Fig. 7A and 7B, all the results of the RPA-LFD assay (Fig. 7A) in field condition were consistent with those obtained through PCR detection in the laboratory (Fig. 7A). These results strongly indicated that the RPA-LFD-based method for detecting potato late blight, developed in this study, demonstrated high accuracy and reliability, thereby facilitating efficient and convenient field diagnosis of P. infestans infections.

Fig. 7. The application using RPA-LFD in field condition.

(A) The electrophoresis results of PCR amplification using the PiYpt1 gene as the detection target are as follows: Lanes 1–5 correspond to the collected potato diseased samples, Lane 6 corresponds to the artificially inoculated diseased sample, and Lane 7 represents the healthy control sample; (B) The results of the rapid and portable RPA-LFD detection of the samples conducted in the outdoor environment are as follows: Lanes 1–5 correspond to the collected potato diseased samples, Lane 6 corresponds to the artificially inoculated diseased sample, and Lane 7 represents the healthy control sample; (C) Schematic diagram illustrating the sampling of different leaf sections from potato late blight-affected plants following manual inoculation; (D) RPA-LFD detection outcomes obtained from sampling various leaf sections and performing the assay without laboratory equipment.

To further assess the feasibility of applying this RPA-LFD system for early detection in field condition, potato leaves were sampled 48 hours post-inoculation and divided into four sections for DNA extraction and RPA-LFD testing as previous description in field condition. The results demonstrated that the SSPI1-based RPA assay successfully detected P. infestans not only in visibly symptomatic areas (Section I), but also in regions adjacent to lesions (Section II) and even in areas distant from visible symptoms (Section III) (Fig. 7C and 7D). These results highlighted that the RPA-LFD assay can detect P. infestans in both symptomatic tissues and asymptomatic leaf regions during the early stages of infection. This early detection capacity underscores the assay’s practical utility in field settings for timely and precise disease management interventions.

Discussion

Late blight in potato is characterized by inconspicuous symptoms during its early infection stage, followed by a rapid outbreak and swift spread at later stages [1]. Early detection of this disease provides a crucial window for timely intervention, enabling growers to apply fungicides more precisely and effectively. In this study, we targeted a newly identified multi-copy specific sequence, SSPI1, and combined it with the RPA-LFD technique to establish a convenient visual detection system for potato late blight. This system offers notable advantages including high sensitivity, strong specificity, field applicability without the need for sophisticated equipment, visual readout of results, and simple operation, making it particularly suitable for on-site diagnosis in resource-limited field conditions.

In molecular amplification-based detection systems such as PCR, LAMP, and RPA, the selection of target sequences and primers is critical [21, 26]. Homan et al. [21] demonstrated that PCR detection targeting high-copy-number genomic sequences significantly improves sensitivity compared to low-copy-number targets. Apart from Homan et al. [21], Jiang et al. [27] identified a 171-bp sequence within multiple paralogous copies of the msp2 gene in Anaplasma phagocytophilum and used it to develop a recombinase polymerase amplification assay with improved sensitivity and specificity than previous target. Additionally, Chng et al. [28] also achieved higher diagnostic sensitivity for Sarcoptes scabiei by targeting a high-copy repetitive DNA sequence via PCR. Thus, mining multi-copy sequences is an effective strategy to enhance the sensitivity of nucleic acid amplification assays. In previous studies on P. infestans detection, commonly used targets included the single-copy PiYpt1 gene [15] and the multi-copy PiITS sequence [14]. Through genomic sequence analysis and NCBI BLAST comparisons, we identified a novel candidate target sequence, SSPI1, with a length of 174 bp and a copy number ranging from 40 to 191. BLAST analysis confirmed that SSPI1 is unique to the P. infestans genome. These characteristics make SSPI1 an ideal target for RPA-based detection, combining high specificity with enhanced sensitivity.

Previous studies [15, 17, 19, 24, 29] have reported that, during RPA amplification, different primer pairs targeting the same sequence can vary significantly in amplification efficiency, which in turn impacts detection sensitivity. In this study, we screened nine primer combinations and ultimately identified one pair that exhibited both high amplification efficiency and strong specificity, thereby improving the sensitivity of our RPA-LFD assay. Additionally, incorporating a nfo probe into the RPA-LFD system effectively minimized false positives caused by non-specific amplification products or primer dimers on the LFD, further enhancing assay specificity. Using this SSPI1-targeted RPA-LFD assay, we achieved a detection sensitivity of 100 fg for P. infestans genomic DNA and successfully detected SSPI1 in samples with template concentrations as low as 11 copies/μl. Our RPA-LFD assay demonstrated improved detection sensitivity for P. infestans compared to previous reports, with a detection limit of 100 fg of genomic DNA under optimized conditions [15]. Furthermore, real-time fluorescence RPA assays performed with a qPCR instrument demonstrated an even higher sensitivity of 10 fg genomic DNA.

The RPA-LFD method developed in this study is simple to operate and does not require additional temperature control equipment, making it highly suitable for field deployment in the absence of laboratory infrastructure. Our results demonstrated that the SSPI1-targeted RPA reaction can be efficiently performed within a temperature range of 27–42°C, which is compatible with body-heat incubation in field conditions [30]. Moreover, the LFD operates effectively within a broad temperature range of 10–50°C, supporting its use in potato-growing fields. Notably, RPA-LFD results obtained in field settings without temperature-controlled devices were consistent with laboratory PCR detection results targeting the PiYpt1 gene, underscoring the robustness and reliability of the assay. Importantly, our RPA-LFD assay could detect P. infestans not only in symptomatic tissues but also in asymptomatic tissues during the early infection stage. This indicates that the SSPI1-based RPA-LFD assay is a promising tool for rapid, accurate, and early field diagnosis of potato late blight by extension technicians, providing a valuable basis for precise and timely disease management.