Carya illinoinensis pustulata constant temperature amplification detection kit and application thereof

Abstract

This invention provides a method for detecting scab fungus in thin-shelled pecans ( Venturia effusa This invention relates to the field of biodetection technology, encompassing isothermal amplification primer and probe sets, rapid detection kits, methods, and applications. Addressing the limitations of current methods such as difficulty in identifying the pathogen causing thin-shelled pecan scab and over-reliance on expensive instruments and skilled personnel, this invention researches LFD-RPA detection technology for this pathogen. This invention provides a simple and efficient method for detecting the pathogen, exhibiting no cross-reactivity with other species in the *Neurospora* genus and closely related genera (such as *Cladosporium*), demonstrating high specificity, a minimum detection limit of 5 pg genomic DNA, and high sensitivity. This invention is suitable for real-time, rapid on-site detection of this disease by departments such as port quarantine, research institutions, agricultural and forestry management, and growers.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to isothermal amplification primers, probes, reagent kits, and methods for detecting *Venturia effusa*, the scab pathogen of pecans. It is suitable for on-site, real-time, and rapid detection of this disease by departments such as port quarantine, research institutions, agricultural and forestry management, and growers. Background Technology

[0002] Thin-shelled pecans (Carya illinoinensis), also known as American pecans, are increasingly affected by scab disease, a disease that has become the most serious health hazard in pecans due to the expanding planting area. Scab disease is prevalent in all planting regions, severely impacting yield and quality. Once it occurs in orchards, it can spread rapidly and cause significant damage under suitable temperature and humidity conditions. Therefore, rapid and accurate on-site detection of the disease in orchards and timely implementation of control measures are currently the most effective ways to control its outbreak. However, there is currently a lack of rapid on-site detection methods and kits for thin-shelled pecan scab both domestically and internationally.

[0003] The pathogen causing scab disease in pecans is *Venturia effusa*, which infects the leaves, young branches, and fruits of pecans. Currently, detection techniques for *Venturia effusa* are limited to laboratory testing, relying primarily on traditional pathogen detection techniques and ITS sequence analysis. In the laboratory, this requires extensive isolation, cultivation, and purification of suspected pathogen-carrying materials or diseased plant tissues. The purified isolates are then verified using Koch's postulates. This process is cumbersome, time-consuming, and labor-intensive, with a low detection rate. Furthermore, *Venturia effusa* grows slowly under artificial culture conditions, with a culture period of approximately 30 days, making it highly susceptible to contamination by other saprophytic fungi. Morphologically, it is also easily interfered with by *Cladosporium* sp., which share similar morphological characteristics. Therefore, detecting *Venturia effusa* using traditional techniques requires even longer testing times and is significantly more challenging. To overcome the detection problem of *Venturia effusa*, the inventors, based on the principle of quantitative real-time PCR (qPCR), used the ITS sequence of *Venturia effusa* as a target. By comparing the sequence differences between *Venturia effusa* and its closely related genera, a set of specific primers and TaqMan probes was designed and screened, developing a TaqMan quantitative real-time PCR detection technology for *Venturia effusa*. This detection technology has improved the detection efficiency of *Venturia effusa* to a certain extent and shortened the detection time. However, this detection technology relies entirely on expensive large-scale instruments and equipment in the laboratory, requires certain technical skills from the testing personnel, and cannot meet the needs of rapid on-site detection, thus greatly limiting its application scope. Therefore, existing rapid detection methods for *Venturia effusa* have certain shortcomings in terms of practicality.

[0004] In recent years, with the rapid development of molecular biology techniques, scholars and companies worldwide have focused on the research and development of rapid molecular detection technologies and tools for pathogens, resulting in a continuous stream of novel rapid molecular detection technologies being researched and applied. Isothermal amplification techniques, due to their high sensitivity, strong specificity, ease of operation, and short processing time, have received widespread attention and favor. Recombinase polymerase amplification (RPA), a novel isothermal amplification technique developed by Piepenburg et al. in 2006, is considered to be the most promising isothermal amplification technique currently available. Under isothermal conditions (37–42°C), in this technical system, specific primers bind to recombinase to form a Rec / ssDNA complex. With the help of accessory proteins and single-stranded DNA-binding proteins (SSBs), this complex penetrates the template DNA double helix, forming a D-Loop region. The specific primers then recognize the template DNA sequence. At the target sequence, the Rec / ssDNA complex undergoes a conformational change, the recombinase dissociates, the 3' end of the primer is exposed, and it is recognized by DNA polymerase I (Pol I). Pol I performs base complementation based on the specific target sequence and initiates DNA amplification at the 3' end of the primer. Simultaneously, strand displacement DNA polymerase extends the primer and unwinds the double-stranded DNA structure, allowing the DNA sequence synthesis process to continue. Due to the different principles of RPA amplification product detection techniques, RPA detection technology is further divided into basic-RPA, fluorescent-RPA, and lateral flow dipstick RPA (LFD-RPA). Among them, LFD-RPA does not require complex and sophisticated instruments, is convenient, fast, sensitive, and efficient, and can meet the needs of rapid on-site detection. In the LFD-RPA system, endonuclease IV (nfo) and an nfo probe are added. The nfo probe has a fluorescent group at its 5' end and a blocking agent at its 3' end, with a debasement site (THF) labeled approximately 30 nt from the 5' end. During RPA-specific primer amplification, nfo specifically recognizes and cleaves the THF site on the probe, generating a free hydroxyl terminus. Simultaneously, the target sequence is elongated under the action of DNA polymerase. Because the reverse primer is labeled with biotin, the final RPA amplification product is a double-labeled product, i.e., the 5' end is labeled with a fluorescent group and the 3' end is labeled with biotin.When the RPA amplification product is dropped onto the lateral flow chromatography test strip, within 3–5 minutes, the biotin ligand binds to the 3' end of the biotin, appearing as a dark band on the test line. Uncaptured immune complexes continue to diffuse to the control line, where they are captured by specific antibodies, forming a dark band. Therefore, the presence of bands on both the test line and control line indicates a positive result; all other results are negative. LFD-RPA detection offers advantages such as high sensitivity, strong specificity, non-instrumental nature, visualization, and speed, making it highly practical and promising for fields requiring rapid on-site detection, such as medicine, law, and plant protection. However, LFD-RPA detection technology has not yet been developed or applied for the detection of *Carya carnosa* scab pathogen.

[0005] Based on the principle of LFD-RPA detection technology, the inventors previously established a TaqMan real-time PCR detection system for *Venturia* scab pathogen. Targeting the ITS gene sequence, they designed nine sets of LFD-RPA detection primers and two probes, including the specific primers VEF / VER and probe VEP, as well as upstream and downstream sequences. However, due to the high conservation of the ITS sequence among different species of *Venturia*, and the non-specific binding between the downstream primer and probe sequences, all candidate primers and probes failed to distinguish between different species of *Venturia* during extensive and repeated screening experiments, and false positives were observed. Therefore, further research and development are needed for LFD-RPA detection primers, probes, and detection systems for *Venturia* scab pathogen. Summary of the Invention

[0006] In view of this, the purpose of this invention is to solve the limitations of existing methods for detecting Venturia effusa, which rely excessively on expensive instruments, laboratory environments, and professional technicians. This invention provides a primer and probe set, reagent kit, and application method for isothermal amplification detection of Venturia effusa, enabling simple, rapid, and accurate detection without the need for special instruments.

[0007] This invention provides a set of isothermal amplification detection primer and probe combinations for Venturia effusa, characterized in that it includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.1, a downstream primer with the nucleotide sequence shown in SEQ ID NO.2, and a probe shown in SEQ ID NO.3; the probe has a fluorescent reporter gene at its 5' end and a quenching gene at its 3' end; the downstream primer has a biotin label at its 5' end.

[0008] The present invention also provides a kit for isothermal amplification detection of pecan scab pathogen, comprising the above-mentioned primer and probe combination, isothermal amplification detection reagent and flow chromatography test strip.

[0009] The present invention also provides the application of the above-described primer-probe combination or the above-described kit in the preparation of reagents for detecting scab pathogens of pecan.

[0010] Preferably, the detection of scab pathogens in pecans includes the detection of Venturia effusa within the Venturia genus.

[0011] This invention also provides a method for detecting *Carex scabiei* of thin-shelled pecan using isothermal amplification and flow chromatography combined, comprising the following steps:

[0012] (1) Extract genomic DNA from the sample to be tested;

[0013] (2) Using the extracted DNA as a template, the template was amplified at an isothermal temperature using the primer and probe combination described above to obtain the isothermal amplification product.

[0014] (3) Drop the isothermal amplification product onto the flow chromatography test strip. If there are bands on the detection line and the control line, the sample to be tested contains pecan scab bacteria. If no detection band appears on the detection line, the sample to be tested does not contain pecan scab bacteria.

[0015] Preferably, the isothermal amplification procedure is as follows: reaction at 30℃~42℃ for 10~40 min.

[0016] Preferably, the isothermal amplification procedure is as follows: reaction at 38°C for 20–30 min.

[0017] Preferably, the isothermal amplification reaction system is 50 μL, comprising the following components: 2.5 μL upstream primer, 2.5 μL downstream primer, 0.5 μL probe, 29.4 μL AD buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 2.5 μL B buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 5 μL template, and 7.6 μL dd H2O.

[0018] In the reaction system, the concentrations of the upstream and downstream primers are both 0.5–0.6 μM; the concentrations of the probes are both 0.1–0.16 μM.

[0019] Beneficial effects

[0020] This invention focuses on common pathogens affecting pecans, including *Venturia effusa*, the causal agent of pecan scab, as well as other representative strains of the *Venturia* genus and representative strains of the closely related *Cladosporium* genus. A research system for the LFD-RPA detection of pecan scab disease was developed. This invention offers advantages such as high efficiency, sensitivity, and specificity, and does not rely on expensive instruments or specialized technicians. It enables rapid on-site detection of pecan scab disease and has promising applications in port quarantine, pecan scab disease monitoring and early warning, and disease prevention guidance. Verification has shown that the primers and probes provided by this invention can accurately detect *Venturia effusa*, with no cross-reactivity between species of the same or closely related genera, exhibiting high specificity and a minimum detection limit of 5 pg of total pathogen DNA, demonstrating high detection sensitivity. Attached Figure Description

[0021] Figure 1 Schematic diagram of primer and probe design;

[0022] Figure 2 For specific detection results, 1-3 are representative strains of *Venturia effusa* AH81, AH41, and AH82; 4-21 are, respectively, representative strains of *Diaporthe pseudophoenicicola* LSM1, *Colletotrichum siamense* JS2, *Colletotrichum aenigma* JSJT-1, *Colletotrichum fructicola* B-5, *Alternaria alternata*, *Pestalotiopsis microspora*, *Didymella sp.*, *Cladosporium cladosporioides*, *Pseudoveronaea ellipsoidea*, *Aureobacidium sp.*, *Trichoderma sp.*, *Penicillium sp.*, *V. pyrina*, *V. inaequalis*, *Cladosporium cucumerinum*, *Fusarium solan*, *Cercospora kikuchii*, and *Phomopsis*. Amygdali representative strain; 22 is the negative control (template is water).

[0023] Figure 3The results represent the sensitivity test results, where 1–8 represent template concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively; and 9 represents the negative control (template is water).

[0024] Figure 4 These are the results of field sample testing, where 1–6 are pecan leaf samples; 7–12 are pecan fruit samples; and 13 is a negative control (template is water). Detailed Implementation

[0025] This invention provides primers and probes for isothermal amplification of Venturia effusa, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO.1, a downstream primer with the nucleotide sequence shown in SEQ ID NO.2, and a probe with the nucleotide sequence shown in SEQ ID NO.3; the 5' end of the probe contains a fluorescent reporter gene; and the 3' end of the probe contains a quenching gene.

[0026] In this invention, the upstream primer Ve-RPA-F3 with the nucleotide sequence shown in SEQ ID NO.1 is specifically: 5'-GAATGGAACGAATGGACCTATCTCAAAGAAG-3';

[0027] In this invention, the downstream primer Ve-RPA-R5 with the nucleotide sequence shown in SEQ ID NO.2 is specifically: [5'-biotin]-CCGAGATTCAACCCGAATTCTGTCCTCGCG-3';

[0028] In this invention, the nucleotide sequence of the probe Ve-RPA-P2, as shown in SEQ ID NO.3, is specifically: [5'FAM]-GACTACCGAATGACATCTTATCAAGATGGCTAATGCGAGAGCAG-[3'C3spacer].

[0029] In this invention, the fluorescent reporter gene preferably includes the FAM fluorescent reporter gene; the quenching gene does not fluoresce, and the quenching gene preferably includes the Spacer C3 quenching gene.

[0030] In this invention, the primers and probes are preferably isothermal amplification-specific primers and probes designed and screened based on the sequence of a unique, functionally unknown gene (FKW77_006955) on the Venturia effusa genome (GenBank accession number: GCA_007735645.1). Specifically, according to RPA primer design principles, a pair of specific primers is artificially designed and screened on this gene sequence, with the 5' end of the downstream primer containing biotin; simultaneously, an RPA probe is designed and screened on the upstream and downstream primers, with the 5' end labeled with a "FAM" group, the 3' end labeled with a "Spacer C3" group, and the base at position 30 from the 5' end replaced with a "THF" group.

[0031] In this invention, the primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0032] This invention provides a thermotropic amplification detection kit for Venturia effusa, the scab pathogen of pecans, comprising the primers and probes, RPA reaction reagents, and flow chromatography test strips described in the above-described scheme.

[0033] In this invention, the RPA reaction reagent is preferably purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., and in addition, positive control and negative control are also included.

[0034] In this invention, the flow chromatography test strip is preferably purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0035] The present invention also provides the application of the primers and probes described above or the kit described above in the detection of Venturia effusa, the scab pathogen of pecans.

[0036] In this invention, the detection of Venturia effusa, the scab pathogen of pecans, is preferably performed by detecting Venturia effusa from the genus Venturia.

[0037] In this invention, the preferred fungi detected from the genus *Venturia effusa* include *Venturia effusa*, other fungi in the genus *Venturia* (*Venturia sp.*), fungi in the genus *Cladosporium* (*Cladosporium sp.*), and other pathogenic fungi on pecans, including fungi in the genus *Colletotrichum* (*Colletotrichum* sp.), fungi in the genus *Diaporthe* (*Diaporthe* sp.), fungi in the genus *Alternaria* (*Alternaria* sp.), and fungi in the genus *Pestalotiopsis* (*Pestalotiopsis* sp.). Preferred fungi from the *Aureobasidium* genus include *V. pyrina* and *V. inaequalis*; preferred fungi from the *Cladosporium* genus include *Cladosporium cladosporioides* and *Cladosporium cucumerinum*; preferred fungi from the *Anthracis* genus include *Colletotrichum siamense*, *Colletotrichum aenigma*, and *Colletotrichum fructicola*; preferred fungi from the *Diaporthe pseudophoenicicola* genus; preferred fungi from the *Alternaria* genus include *Alternaria alternata*; and preferred fungi from the *Pestalotiopsis* genus include *Pestalotiopsis ellipsoidea*.

[0038] This invention also provides a method for detecting scab pathogens in thin-shelled pecans using a combination of RPA and flow chromatography, comprising the following steps:

[0039] (1) Extract genomic DNA from the sample to be tested;

[0040] (2) Using the genomic DNA of the sample to be tested as a template, the template is amplified at isothermal temperature using the primer and probe combination described in the above scheme to obtain the isothermal amplification product;

[0041] (3) Drop the isothermal amplification product onto the flow chromatography test strip. If there are bands on the detection line and the control line, the sample to be tested contains pecan scab bacteria. If no detection band appears on the detection line, the sample to be tested does not contain pecan scab bacteria.

[0042] The present invention first extracts genomic DNA from the sample to be tested.

[0043] This invention utilizes a fungal DNA extraction kit or a rapid nucleic acid extraction method to extract genomic DNA from the sample to be tested.

[0044] In this invention, the fungal DNA extraction kit is preferably purchased from Omega; the rapid extraction method uses an ultra-rapid nucleic acid release agent, preferably purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0045] After obtaining the genomic DNA of the sample to be tested, the present invention uses the genomic DNA as a template and employs the primers and probes described in the above scheme to perform isothermal amplification of the template.

[0046] After obtaining the isothermal amplification product, the present invention drops the amplification product onto a flow chromatography test strip. If a positive detection band is detected, the sample to be tested contains Venturia effusa; if no positive detection band is observed, the sample to be tested does not contain Venturia effusa.

[0047] In this invention, the preferred isothermal amplification program is a reaction at 30℃~42℃ for 10~40 min.

[0048] In this invention, the isothermal amplification program is preferably performed at 38°C for 20–30 min.

[0049] In this invention, the isothermal amplification reaction system, in 50 μL, preferably includes the following components: 2.5 μL upstream primer, 2.5 μL downstream primer, 0.5 μL probe, 29.4 μL AD buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 2.5 μL B buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 5 μL template, and 7.6 μL dd H2O.

[0050] In this invention, the final concentrations of the upstream and downstream primers are preferably 0.5–0.6 μM, respectively; and the final concentration of the probe is preferably 0.1–0.16 μM.

[0051] The technical solution of the present invention will be described in detail below with reference to the embodiments thereof.

[0052] Test materials, reagents and instruments

[0053] Tested strains: Fifteen strains from the pecan host plant, including *Venturia effusa* strains AH81, AH41, AH82, *Diaporthe pseudophoenicicola* strain LSM1, *Colletotrichum siamense* strain JS2, *Colletotrichum aenigma* strain JSJT-1, *Colletotrichum fructicola* strain B-5, *Alternaria alternata*, *Pestalotiopsis microspora*, *Didymella sp.*, *Cladosporium cladosporioides* (*Cladosporium* strains), *Pseudoveronaea ellipsoidea*, *Aureobacidium* sp., *Trichoderma* sp., and *Penicillium* sp., were isolated and identified by the Jiangsu Institute of Botany, Chinese Academy of Sciences. Other strains included *V. pyrina* (pear) and *V. inaequalis* (apple), *Cladosporium cucumerinum* (melon), and *Fusarium*. The six strains, including solan (soybean), Cercosporakikuchii (soybean), and Phoopsis amygdali (peach), were all generously donated by Professor Hu Baishi of Nanjing Agricultural University.

[0054] Reagents and instruments used: Fungal DNA Kit (50) Fungal DNA Extraction Kit (Omega, catalog number: D3390-01), Ultra-fast Nucleic Acid Release Agent (Amp Future (Changzhou) Biotechnology Co., Ltd., DNA type); RPA Reaction Reagent (Amp Future (Changzhou) Biotechnology Co., Ltd., catalog number: WLN8203KIT); Fluorochromatographic Test Strips (Amp Future (Changzhou) Biotechnology Co., Ltd., catalog number: WLFS8204); DL2,000 DNA Marker (Baori Biotechnology Co., Ltd., catalog number: 3427Q); 2×Taq Master Mix (Novizan Biotechnology Co., Ltd., catalog number: P111-01); Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] Primer and probe design and screening

[0056] Download the genomes of eight typical strains of the genus *Venturia effusa* (GenBank: GCA_007735645.1), *V. pyrina* (GenBank: GCA_023079195.1), *V. inaequalis* (GenBank: GCA_003689225.1), *V. carpophila* (GenBank: GCA_014858625.1), *V. nashicola* (GenBank: GCA_004522665.1), *V. asperata* (GenBank: GCA_003689335.1), *V. oleaginea* (GenBank: GCA_013176395.1), and *V. aucupariae* (GenBank: GCA_003693225.1) from the Genomic database of NCBI, as well as the genomes of *Cladosporium*. cucumerinum (GenBank: GCA_023634325.1), C. cladosporioides (GenBank: GCA_018408255.1), C. sphaerospermum (GenBank: GCA_000261425.2), C. halotolerans (GenBank: GCA_011745625.2), C. oxysporum (GenBank The genomes of nine typical strains of the genus *C. effusa*, including *C. phlei* (GenBank: GCA_003614995.1), *C. rectoides* (GenBank: GCA_046128805.1), *C. endophyticum* (GenBank: GCA_037044255.1), and *C. tenuissimum* (GenBank: GCA_046128905.1), were analyzed. Comparative genomic analysis and screening of the coding genes in the genomes of these 17 typical strains were performed using Orthofinder software. Single-copy genes present only in the *Venturia effusa* genome and absent in other strains were screened, resulting in the identification of 10 conserved genes unique to *Venturia effusa*. Using the unique conserved gene (FKW77_006955) as a candidate target, three candidate upstream primers and three candidate downstream primers were designed on the candidate target gene according to the RPA primer design principle. Three candidate probes were designed in the middle of the upstream and downstream primers. The 5' end of the downstream primer contains a biotin label, the 5' end of the probe contains a "FAM" group, and the 3' end contains a "Spacer C3" group. Any base at a position about 30 nt from the 5' end was replaced with dSpacer.Through orthogonal experimental design, 27 combinations of upstream and downstream primers and probes were explored. The optimal combination of candidate target primers and probes was screened through specificity and sensitivity tests. Finally, isothermal amplification primers and probes for thin-shelled pecan scab disease (Venturia effusa) with high specificity and sensitivity were obtained, namely upstream primer Ve-RPA-F3: 5'-GAATGGAACGAATGGACCTATCTCAAAGAAG-3'; downstream primer Ve-RPA-R5: [5'-biotin]-CCGAGATTCAACCCGAATTCTGTCCTCGCG-3'; probe Ve-RPA-P2: [5'FAM]-GACTACCGAATGACATCTTATCAAGATGG[THF]CTAATGCGAGAGCAG-[3'C3spa cer].

[0057] Experimental Example 1: Primer and Probe Specificity Determination

[0058] Genomic DNA of the tested strains was extracted using a fungal genomic DNA miniaturization kit (OMEGA), following the kit's instructions. The extracted genomic DNA was stored at -20°C for later use. The concentration of genomic DNA from the strain Venturia effusa was adjusted to 1 ng / μL for specific detection.

[0059] The primer and probe powder synthesized by the commissioned company were dissolved in sterile deionized water to a concentration of 10 μM as a working solution, according to the specific parameters on the synthesis report.

[0060] Following the reaction system (50 μL reaction system) instructions of the RPA kit (Anpu Future (Changzhou) Biotechnology Co., Ltd.), add the following to the dry powder tube in sequence: 29.4 μL AD buffer, 2 μL each of 10 μM forward and reverse primers, 0.6 μL each of 10 μM probe, 5 μL of 1 ng / μL DNA template, 8.5 μL ddH2O, and finally 2.5 μL B buffer. After thorough vortexing and centrifugation for 5 seconds, place the reaction tube in a 39℃ metal bath for 30 min. After the reaction, dilute the amplification product 10-fold with sterile deionized water, mix well, and then add 80 μL of the reaction solution to the sample well of the flow chromatography strip. Observe the detection results after 15 min. The results are as follows: Figure 2As shown, clear detection bands were observed for Venturia effusa strains AH81, AH41, and AH82, while no detection bands were observed for other representative strains of the genus *V. pyrina* and *V. inaequalis*, representative strains of the genus *Cladosporium* *cucumerinum* and *Cladosporium cladosporioides*, and representative strains of other genera such as *Diaporthe pseudophoenicicola*, *Colletotrichum siamense*, *Colletotrichum aenigma*, *Colletotrichum fructicola*, *Alternaria alternata*, *Pestalotiopsis microspora*, *Didaemella sp.*, *Pseudoveronaea ellipsoidea*, *Aureobacidium sp.*, *Trichoderma sp.*, *Penicillium sp.*, *Fusarium solan*, *Cercospora kikuchii*, and *Phomopsis amygdali*. These results indicate that the primers and probes designed and screened for Venturia effusa in this invention have strong specificity.

[0061] Experiment Example 2: Optimization of the Isothermal Amplification Reaction System

[0062] 1) Primer concentration optimization

[0063] In the reaction system, 5 μL of template DNA (1 ng / μL) and 0.6 μL of probe (10 μM) were added. The amounts of upstream and downstream primers (10 μM) added were set to 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, and 3.5 μL, respectively, resulting in final primer concentrations of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, and 0.7 μM. The reaction system was incubated at 39℃ for 30 min. The reaction solution was then diluted 10-fold, and 80 μL of the diluted solution was added to the sample well of the flow chromatography test strip. The detection results were observed after 15 min, and the optimal primer concentration in the reaction system was determined based on the clarity of the detection band. The primer concentration optimization experiment showed that the clarity of the positive detection band gradually improved with the increase of primer concentration. When the final concentration reached 0.5 μM and 0.6 μM, there was no significant difference in the clarity of the band. Therefore, the optimal reaction concentration of primers in the reaction system was 0.5 μM, that is, the amount of upstream and downstream primers (10 μM) added in the reaction system was 2.5 μL.

[0064] 2) Probe concentration optimization

[0065] In the reaction system, 5 μL of template DNA (1 ng / μL) and 2.5 μL of forward and reverse primers (10 μM) were added. The probe addition volumes were set to 0.2 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, and 0.8 μL, respectively, resulting in final probe concentrations of 0.04 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.14 μM, and 0.16 μM. The reaction was carried out at 39℃ for 30 min, and LFD-RPA was used for detection. The optimal probe concentration in the reaction system was selected based on the clarity and appearance time of the detection band. The results showed that the clarity of the positive detection bands improved significantly with increasing probe concentration. When the concentrations reached 0.1 μM, 0.12 μM, 0.14 μM, and 0.16 μM, there was no significant difference in the clarity of the detection bands. Therefore, the optimal reaction concentration of the primers in the reaction system was 0.1 μM, which means that the amount of probe (10 μM) added to the reaction system was 0.5 μL.

[0066] 3) Optimization of reaction temperature

[0067] Based on the optimization of primer and probe concentrations, the reaction system was set up at 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃ for 30 min. The reaction results were detected using LFD-RPA. The results showed that detection bands appeared at temperatures ranging from 30℃ to 42℃. However, with increasing temperature, the clarity of the detection bands first increased and then decreased. The clarity of the detection bands was most pronounced at 38℃; therefore, the optimal reaction temperature was 38℃.

[0068] 4) Optimization of reaction time

[0069] Based on optimization of primers, probes, and temperature, reaction times were set at 38℃ for 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min. The reaction results were detected using LFD-RPA. The results showed that the clarity of the detection bands gradually increased with increasing reaction time, and after 20 min, there was no significant difference in the bands. Therefore, the optimal reaction time was 20 min.

[0070] Through optimization experiments of the LFD-RPA detection system, the final LFD-RPA detection system for Venturia effusa was determined as follows: 2.5 μL each of upstream and downstream primers (10 μM), 0.5 μL of probe (10 μM), 29.4 μL of AD buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 2.5 μL of B buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 5 μL of DNA template, 7.6 μL of dd H2O, and a total reaction volume of 50 μL. The total reaction volume was reacted at a constant temperature of 38℃ for 20 min.

[0071] Experimental Example 3: Determination of Primer and Probe Sensitivity

[0072] The DNA of strain *Venturia effusa* was serially diluted to concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively. Sensitivity was then assessed using the optimized LFD-RPA detection system of this invention. The results are as follows: Figure 3 As shown, when the template DNA concentration is 1 pg / μL, i.e., the template DNA content is 5 pg, a detection band is present, but the band clarity is not obvious; however, when the template DNA concentration is 100 fg / μL, i.e., the template DNA content is 500 fg, no detection band is observed. Sensitivity test results indicate that the detection limit of this invention is 5 pg, approximately the number of 4 copies of the *Venturia effusa* genome.

[0073] Test Example 4: Actual Sample Testing

[0074] This experiment selected 12 samples from different thin-shelled pecan growing areas in China for actual sample testing. A suitable amount of tissue (1-2g) was selected, and genomic DNA was extracted according to the instructions of the ultra-rapid nucleic acid release agent (DNA type) (Anpu Future (Changzhou) Biotechnology Co., Ltd.), or according to the method of Wang et al. (1993). The specific method was as follows: 100μL of 0.5M NaOH was added to each gram of tissue, and the mixture was thoroughly ground in a mortar and transferred to a 1.5ml EP tube. The tube was centrifuged at 12000rpm for 5min, and 5μL of the supernatant was added to 495μL of 0.1M Tris (pH 8.0). After mixing, 5μL was used as the detection template. The results are as follows: Figure 4As shown, the detection system of this invention can detect four positive samples, namely samples 1, 4, 5, and 8. To verify the accuracy of the detection system of this invention in detecting actual samples, 12 collected samples were subjected to traditional pathogen isolation and purification, and analyzed by TaqMan quantitative PCR and ITS sequence sequencing. The results showed that the results of the isothermal rapid detection system established in this invention were all correct.

[0075] In summary, this invention focuses on common pathogens affecting pecans, including *Venturia effusa*, the causal agent of pecan scab, as well as other representative strains of the *Venturia* genus and representative strains of the closely related *Cladosporium* genus. A research system for the LFD-RPA detection of pecan scab disease was developed. This invention offers advantages such as high efficiency, sensitivity, and specificity, and does not rely on expensive instruments or specialized technicians. It enables rapid on-site detection of pecan scab disease and shows promising application prospects in port quarantine, monitoring and early warning of pecan scab disease, and guidance on disease prevention measures.

[0076] Although the above embodiments provide a detailed description of the present invention, they are only a part of the embodiments and not all of the embodiments. Researchers can obtain other embodiments based on the embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of primer-probe combination in the preparation of reagents for detecting scab pathogens of pecan, characterized in that, The primer-probe combination consists of an upstream primer with the nucleotide sequence shown in SEQ ID NO.1, a downstream primer with the nucleotide sequence shown in SEQ ID NO.2, and a probe shown in SEQ ID NO.3; the probe has a fluorescent reporter gene at its 5' end and a quenching gene at its 3' end; the downstream primer has a biotin label at its 5' end, and the scab pathogen of pecan is Venturia effusa.

2. A method for detecting scab pathogens of thin-shelled pecans using a combination of RPA and flow chromatography, characterized in that, The pathogen causing scabies in thin-shelled pecans is *Venturia effusa*. The method includes the following steps: (1) extracting genomic DNA from the sample to be tested; (2) Using the extracted DNA as a template, the template is amplified at an isothermal temperature using the primer and probe combination described in claim 1 to obtain the isothermal amplification product; (3) Drop the isothermal amplification product onto the flow chromatography test strip. If there are bands on the detection line and the control line, the sample to be tested contains pecan scab bacteria. If no detection band appears on the detection line, the sample to be tested does not contain pecan scab bacteria.

3. The method according to claim 2, characterized in that, The isothermal amplification procedure is as follows: react at 30℃~42℃ for 10~40 min.

4. The method according to claim 2, characterized in that, The isothermal amplification procedure is as follows: react at 38℃ for 20-30 minutes.

5. The method according to claim 2, characterized in that, In the reaction system, the concentrations of the upstream and downstream primers are both 0.5–0.6 μM; the concentrations of the probes are both 0.1–0.16 μM.

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