Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is an airborne disease that endangers wheat during its entire growth period. In this study, the Pst134EA_003354 uncharacterized protein (GenBank: XM_047941824.1) of Pst was used as the target sequence, and the primers PS-RPA-F and PS-RPA-R, as well as the probe PS-LF-probe, were designed for recombinase polymerase amplification (RPA) technology. Flow chromatography was combined with the process to establish an RPA detection method for Pst. This method successfully established visual detection within 10 min under a constant temperature of 39 °C, and the detection results were consistent with those of ordinary PCR analysis. However, it only had high specificity for Pst, and the detection limit was 10 fg/μL. In addition, this rapid method successfully detected Pst from wheat leaves during the field incubation period, indicating substantial benefits for applied use. In summary, the RPA detection method established in this study has the favourable characteristics of high efficiency, simple functionality, and rapid and universal practicability, providing a theoretical basis for the early detection and prevention of Pst.

by the naked eye.Nucleic acid amplification can be completed by maintaining the activity of the enzyme within a suitable temperature range of 37-42 °C, which does not require high-temperature denaturation, annealing, or other steps.The mild reaction conditions and high amplification efficiency of RPA make it very suitable for rapid clinical diagnosis, food detection, epidemic prevention and control, industrial application, and on-site real-time detection 19 .RPA is gaining popularity because of its unique characteristics, including a low reaction temperature for amplification and a lack of sensitivity to plant inhibitors 20 .Because RPA does not require any advanced laboratory equipment, it is very suitable for on-site testing 21 .At present, RPA technology has not been widely used mainly because it is not a widely available technology and is only used for scientific research 15 .It is important to perform more assessments in the field on isothermal amplification technologies, including LAMP and RPA techniques 22 .Furthermore, RPA is more tolerant than PCR to inhibitors and background DNA 23 .Most components in RPA are supplied by the manufacturer in a freeze-dried pellet that allows components to be taken on site without refrigeration 24 .Hence, compared to previously reported methods, the advantages of the RPA method are obvious 25 .It has been used to detect citrus yellow pulse virus, broad bean virus, citrus leaf spot virus, wheat blight, and wheat sheath rot caused by various pathogens [26][27][28][29][30] .However, the detection of Pst with RPA has not yet been reported.
In this experiment, a wheat stripe rust Pst134EA_003354 uncharacterized protein was used as the target sequence, specific RPA primers and probes were designed and combined with lateral flow layer test strips, and an RPA-LFD rapid detection system for Pst was established.No specialized software has been developed for the design of RPA primers, and only PCR software can be used for design and screening 19 .This system was used to detect wheat stripe rust in the field and provides a theoretical basis for the early prevention of Pst.The abovementioned nucleic acid detection of Pst has achieved good results, which demonstrates that the detection efficiency of the RPA method is obviously better than that of the PCR method, and the reaction time is faster, which provides a rapid and accurate detection method for the disease prediction and field investigation of wheat seedlings in the later growth stage and provides a good basis for the prevention and control of Pst to reduce the decline in production and to mitigate economic losses.

Primer screening and probe design
The resulting eight primer sets were used for RPA detection and agarose gel electrophoresis.The results showed that RPA PS4 primers produced detection bands, while other primer pairs produced only quality control bands.The electrophoresis gel detection results also showed that the PS4 primer pair could amplify a 300 bp target band, primer PS1 produced multiple nontarget fragments, and primers PS2-3 and PS5-8 produced obvious primer dimers.Therefore, the primer PS4 set was selected as the primer for this study.Additionally, probes were designed using fragments with the lowest homology between the amplified product and the downstream sequence (Fig. 1A,B.Fig. S1 shows the original electrophoretic gels and blots.).The primer pairs and probe information used are shown in Table 1.

Specific detection of Puccinia striiformis f. sp. tritici
The RPA results showed that only the RPA amplification products of Pst CYR32, CYR33, and CYR34 DNA showed detection bands and quality control bands on the flow chromatography test strip.Of the five other pathogens tested, only wheat DNA and the blank control showed a quality control zone in ddH 2 O.In addition, PS-RPA-F and PS-RPA-R were primers for ordinary PCR detection, and the agarose gel electrophoresis results showed that Pst was amplified to a specific target of 300 bp with no other pathogen amplification occurring.Detection of Puccinia striiformis f. sp.tritici isolates Thirty-five samples from Datong, Huzhu, Ledu, Jianzha, Hualong and Gui-De counties were collected.Thirty samples were found to be infected with Pst using the RPA test, and five samples were not found to be infected with Pst, which was consistent with the results from ordinary PCR analysis.The test results are shown in Fig. 4A,B and Table 2. (Fig. S4 shows the original electrophoretic gels and blots.).

RPA practicality test
Twenty-one wheat leaf samples were collected from seven areas in Qinghai Province.The leaves were tested, and the results from the Dashijia, Ahetan, Shangduoba, and Sheren villages were positive.However, Pst was not detected in samples from Heerjia, Xiaduoba, and Baiwujia villages.The results from the ordinary PCR tests were comparable.Pst was detected on the first day after inoculation.However, the RPA and ordinary PCR analysis showed weak bands, indicating that the amount of Pst was relatively low on the first day after infection.Puccinia striiformis f. sp.tritici was detected from Days 2-8, and observation stopped on the ninth day when sporadic urediospores appeared on the surface of the leaves.This shows that the established RPA system can detect Pst at the seedling stage (Fig. 5A-D, and Table 3. Fig. S5 shows the original electrophoretic gels and blots.).

Discussion
In this study, we designed and screened specific RPA primers and probes based on the Pst134EA_003354 uncharacterized protein and established a rapid detection method for Pst using RPA technology in combination with lateral flow chromatography test strips to detect Pst visually during the incubation period.
Primer and probe design are key factors in the success of RPA detection.The length of RPA primers is generally between 30 and 35 bp; however, TwistDx has recently announced that PCR primers (18 bases and higher)  can successfully be used 31 .The target band of the amplified product in this study was between 100 and 500 bp.
The primers PS-RPA-F and PS-RPA-R and the probe PS-LF-Probe used in this study specifically detected the physiological races CYR32, CYR33, and CYR34 and can be used to detect the pathogens of other wheat leaf diseases.This illustrates that the specificity of the test was strong.It has been reported that the detection limit of the LAMP detection system for Pst is 1 pg/μL 9  LAMP detection of Pst requires the design of four primers and a loop primer for amplification 36 , while RPA technology requires only forwards and reverse primers to detect the target band.Additionally, the complexity of RPA primer design is relatively low compared to LAMP detection technology, and the RPA technique can specifically detect Pst within 20 min at a constant temperature of 39 °C.The method is simple, fast, and can be conducted at room temperature, and the detection buffer can be divided into aliquots to avoid false-positive results.The practicality of RPA technology is an important factor warranting the promotion of this method.
In this study, thirty mixed isolates of Pst and five uninfected samples were collected from the Datong, Huzhu, Ledu, Jianzha, Hualong and Gui-DeGui-De counties of Qinghai Province, and 21 self-inoculated leaves of spring www.nature.com/scientificreports/wheat seedlings were collected from areas with a high incidence of Pst that were infected for 1-8 days.A small amount of Pst was found in the mixed isolates on the first day after infection.In addition, 12 samples were detected in areas with a high incidence of Pst.This shows that the application of this system can rapidly and efficiently detect Pst in leaves during the incubation period.This study provides a theoretical basis for the early prevention and control of wheat stripe rust.
The technique has been applied to detect wheat take-all 32 and wheat sheath blight 33 .RPA detection is of great significance for the early diagnosis, prediction, and comprehensive control of various wheat diseases.In the future, we will use this technique to distinguish the physiological races of wheat stripe rust.The traditional method is to use differential hosts.This process is long and complex.However, if RPA detection technology is  www.nature.com/scientificreports/used, the physiological race can be identified quickly, saving time, material resources, and workforce.This will be the direction of our subsequent efforts.

Test materials and reagents
The test pathogens in this study were Puccinia striiformis f. sp.tritici races CYR32, CYR33, CYR34; isolates of Pst; Puccinia graminis Pers tritici Eriks and E Henn; Puccinia recondita, Blumeria graminis f. sp.tritici, Puccinia striiformis West.f. sp., Puccinia recondita.f. sp.agropyri.The pathogens were provided by the Key Laboratory of Comprehensive Management of Agricultural Pests of Qinghai Province and the College of Plant Protection, Northwest A&F University.
The test materials in this study were Mingxian 169 wheat seedling leaves collected from the Dashijia, Heerjia, and Chada villages in Gui-De County; from the Shangduoba, Xiaduoba, Xingfu, and Longshang villages in Hualong County; from Ahetan village in Xunhua County; from Baiwujia village in Minhe County; from the Kangjia, Hanjia, Baojia, Maojiazhai and Benkang villages in Datong County; from Gelong village in Huzhu County; from Xiakou village in Ledu County; and from Jianzha County in Yangjiacun (Fig. 6).There was a distance of at least 50 m between samples in a single wheat field to avoid duplication of any original isolate during the season.
The reagents used in this study were as follows: RAA-nfo nucleic acid amplification reagent (type test strip) was acquired from Qinghai Baisai Trading Co., Ltd.; HybriDetect was purchased from Milenia Biotec Versailler Table 3. Application of recombinase polymerase amplification (RPA) in detection isolators of Puccinia striiformis f. sp.tritici in field."+"experimentally verified positive; "−"experimentally verified negative.

Extraction of pathogenic fungus DNA
Wheat stripe rust samples were stored at 4 °C in a desiccator.There were 10 blades of Mingxian 169 and 30 blades of each sample at each sampling point.The genomic DNA of Pst and other control pathogens was extracted using the (Cetyltrimethylammonium Bromide)CTAB method 37 .The concentration of the obtained DNA was measured using a NanoDrop™ One ultramicro ultraviolet spectrophotometer, and the DNA integrity was checked using a 2% agarose gel.It was stored at − 80 °C until use.

RPA reaction system establishment
Preparation began by adding 40.9 μL of buffer A, 2 μL of forwards and reverse primers (10 μmol/L), and 0.6 μL of probe (10 μmol/L) to the detection tube containing the test dry enzyme preparation.Then, 2.0 μL of the test sample DNA and 2.5 μL of buffer B were added.This was mixed thoroughly by inverting the tube 5-6 times; then, the solution was centrifuged at a low speed for 10 s.The PE tube was then placed in a water bath at a constant temperature of 39 °C and incubated for 10 min.After the reaction, 50 μL DNA extract (volume ratio = 24:25:1) was added for extraction, the tube was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected.Finally, 20-40 μL of the supernatant was diluted 20-50 times with sterile water or PBS, resulting in a dilution volume that was not less than 100 μL.RPA-LFD technology is based on the principle of RPA amplification.The primers with the biotin marker and the probe with the carboxyl fluorescein (FAM) marker are used to amplify the target nucleic acid so that the final amplification product carries both FAM and biotin marker LFD.The front end is coated with gold nanoparticles with FAM antibody, and the detection line is covered with a biotin antibody.
When the reaction solution enters the test strip, the amplification product with FAM and biotin forms a biotin antibody-nucleic acid-gold nanoparticle complex on the detection line through antigen-antibody binding and colour development.There is also a quality control line on the LFD, which is coated with a fixed antibody, that can be directly combined with gold nanoparticles with FAM antibody and coloured on the quality control line to ensure the effectiveness of the test strip.

The ordinary PCR system
A specific primer of Pst, PS4F/PS4R, was used for ordinary PCR detection.The reaction system was as follows: MasterMix 12.5 μL, primers (10 μmol/L) 1 μL each, template 1 μL, and ddH 2 O 9.5 μL.The amplification program was as follows: 95 °C predenaturation for 3 min; 35 cycles of 95 °C denaturation for 1 min, 67.2 °C annealing for 30 s, and a 72 °C extension for 1 min; extension at 72 °C for 5 min; and storage at 4 °C.A sample of 4 μL was taken for electrophoresis.

Design of the RPA primers and probes
In this study, the Pst134EA_003354 uncharacterized protein was used as the target sequence (GenBank: XM_047941824.1) in conjunction with the characteristics of RPA primers.The primers and probe were designed using the Twist Amp nfo assay design manual guidelines (http:// www.twist dx.co.uk).Eight sets of specific RPA
, and the dual real-time quantitative fluorescent system established by Pan et al. can detect Pst at a minimum of 0.4 pg/μL 32 .When using RPA technology to detect other diseases, the detection sensitivities have been reported as follows: Wei et al. reported a sensitivity of 75 fg/μL for the detection of the causal agent of tomato leaf bacterial spot pathogen 33 , the RPA detection sensitivity of Pythium aphanidermatum established by Zhao et al. was 3.75 fg/μL34 , and Shen constructed an RPA for the detection of tobacco and found the sensitivity of bacterial wilt to be 1 pg/μL35 .Relatively speaking, the PS-RPA-F, PS-RPA-R, and PS-LF-Probe established in this study had a lower detection limit of 10 fg/μL for Pst genomic DNA, and the sensitivity was relatively high.

Table 1 .
Primer and probe sequences and amplicon.
Straße, Gießen Milenia; Premix Ex TaqTM II was purchased from TaKaRa; Shanghai Sangon Biotech Co., Ltd.synthesized all the primers that were used.Ordinary PCR is in vitro enzymatic synthesis of specific DNA fragments, which is based on the level of DNA concentration.In this experiment, the detection band began to weaken when the DNA concentration was lower than 1 pg/μL.The reaction mode of the RPA technology is different from that of PCR in that it does not require thermal cycle steps such as denaturation and annealing.This technology mainly relies on recombinase, polymerase, and single-stranded binding protein (SSB).
Figure 6.The sampling site marker map of the Puccinia striiformis f. sp.tritici sample isolates.