Isothermal amplification detection kit for carya illinoensis scab and application of isothermal amplification detection kit

Abstract

The invention provides an isothermal amplification primer probe group, a rapid detection kit, a method and application for detecting Xanthomonas vesicans of Carya illinoensis, and belongs to the technical field of biological detection. Aiming at the limitation problems that the current carya illinoensis scab is difficult to identify, excessively depends on expensive instruments and technicians and the like, the research on the LFD-RPA detection technology of the carya illinoensis scab is developed, and the carya illinoensis scab can be simply, conveniently and efficiently detected; in addition, the kit has no cross reaction with 18 representative strains of other species and similar species (cladosporium) and other 14 genus in the genus of furazolospora, the specificity is strong, the lowest detection limit is 5 pg genome DNA, and the sensitivity is high. The method is suitable for port quarantine, scientific research institutions, agriculture and forestry management, farmer households and other departments to rapidly detect the diseases in real time on site.

Description

Technical Field

[0001] The invention belongs to the field of biological detection technology, and specifically relates to a constant temperature amplification primer, a probe, a kit and a method for detecting Venturia effusa, which are suitable for on-site real-time rapid detection of the disease by departments such as port quarantine, scientific research units, agricultural and forestry management and growers. Background Art

[0002] Carya illinoinensis is also known as American pecan. However, with the continuous expansion of the planting area of ​​Carya illinoinensis, Carya illinoinensis scab has occurred in all planting areas, seriously affecting the yield and quality, and has become the most serious disease on Carya illinoinensis. Once the disease occurs in an orchard, it will break out over a large area under suitable temperature and humidity conditions, causing serious damage. Therefore, the most effective way to control the outbreak of the disease is to quickly and accurately detect the occurrence of the disease on site in the orchard and take timely prevention and control measures. However, at present, there is a lack of on-site rapid detection methods and kits for Carya illinoinensis scab at home and abroad.

[0003] The pathogen of scab of thin-shelled pecan is Venturia effusa, which can infect the leaves, twigs and fruits of thin-shelled pecan. At present, the detection technology methods of Venturia effusa are only on the laboratory detection technology platform, mainly relying on traditional pathogen detection technology and ITS sequence analysis. In the laboratory, a large number of isolates need to be separated, cultured and purified from materials suspected of carrying pathogens or diseased plant tissues, and the purified isolates are verified by "Koch's postulates". The detection process is cumbersome, time-consuming and labor-intensive, and the detection rate is not high. In addition, Venturia effusa grows slowly under artificial culture conditions, with a culture period of about 30 days. It is very susceptible to contamination by other saprophytes and is easily interfered by Cladosporium sp. with similar morphological characteristics. Therefore, the detection time required for the detection of Venturia effusa using traditional technology is longer and the detection difficulty is higher. In order to overcome the problem of detecting Venturia effusa, the inventors, based on the principle of real-time fluorescence quantitative PCR (Quantitative Real-time polymerase chain reaction), took the ITS sequence of Venturia effusa as the target, and designed and screened a set of specific primers and TaqMan probes by comparing the sequence differences between Venturia effusa and its similar genera, and developed a set of TaqMan fluorescence quantitative PCR detection technology for thin-shelled pecan scab pathogens. This detection technology has improved the detection efficiency of Venturia effusa to a certain extent and shortened the detection time. However, this detection technology is completely dependent on expensive large instruments and equipment in the laboratory, has certain technical requirements for detection personnel, and cannot meet the needs of rapid on-site detection. Its application range is greatly limited, so the existing rapid detection method of Venturia effusa has certain defects in practicality.

[0004] In recent years, with the rapid development of molecular biology technology, scholars and enterprises in various countries have focused on the research and development of molecular rapid detection technology and tools for pathogens, and there are continuous reports and applications of novel molecular rapid detection technology research. Isothermal amplification techniques have attracted widespread attention and favor due to their high sensitivity, strong specificity, convenient operation, and short time consumption. Recombinase Polymerase Amplification (RPA) is a new isothermal amplification technology developed by Piepenburg et al. in 2006 and is considered to be the isothermal amplification technology with the greatest application potential. Under constant temperature (37-42°C), the specific primers in this technology system bind to the recombinase to form the complex Rec / ssDNA; with the help of auxiliary proteins and single-stranded DNA binding protein (SSB), they invade the double-stranded DNA template to form a D-Loop region, and the specific primers perform sequence recognition on the template DNA; the conformation of the Rec / ssDNA complex changes on the target sequence, the recombinase dissociates, the 3' end of the primer is exposed, and is recognized by DNA polymerase I (Pol I). Pol I performs base complementation according to the specific target sequence and initiates DNA amplification response at the 3' end of the primer. At the same time, the strand displacement DNA polymerase extends the primer and unwinds the double-helix DNA structure, allowing the DNA sequence synthesis process to continue. Due to the different principles of RPA amplification product detection technology, RPA detection technology is divided into basic-RPA, fluorescence-RPA and lateral flow dipstick RPA (LFD-RPA). Among them, LFD-RPA does not require complex and sophisticated instruments and equipment, is convenient, fast, sensitive and efficient, and can meet the needs of on-site rapid detection technology. In the LFD-RPA system, exonuclease IV (nfo) and nfo probe are added. The 5' end of the nfo probe has a fluorescent group, the 3' end has a blocker, and the abasic site (THF) is marked at a sequence position of about 30 nt from the 5' end. During the RPA specific primer amplification process, nfo can specifically recognize the THF site on the probe and cut it off, thereby generating a free hydroxyl end, and at the same time, the target sequence is extended under the action of DNA polymerase. Because the reverse primer is labeled with biotin, the final RPA amplification product is a double-labeled product, that is, the 5' end is labeled with a fluorescent group and the 3' end is labeled with biotin.When the RPA amplification product is dripped onto the lateral flow chromatography test strip, within 3 to 5 minutes, the biotin ligand combines with the biotin at the 3' end, and a dark band appears on the test line. The uncaptured immune complex continues to diffuse to the quality control line and is captured by the specific antibody to form a dark band. That is, on the test strip, bands appear on the test line and the quality control line, and the test result is positive, and all other cases are negative. LFD-RPA detection has the advantages of high sensitivity, strong specificity, non-instrumentation, visualization, simplicity and speed. It has strong practicality and development potential in fields such as medicine, law and plant protection that require on-site rapid detection. However, the development and application of LFD-RPA detection technology has not yet been seen in the detection of thin-shell pecan scab pathogens.

[0005] Based on the principle of LFD-RPA detection technology, the inventors of the present invention, on the basis of the established TaqMan fluorescent quantitative PCR detection system for thin-shelled pecan scab pathogen, still used the ITS gene sequence as the target, and designed a total of 9 groups of LFD-RPA detection primers and 2 probes on the specific primers VEF / VER and probe VEP as well as the upstream and downstream sequences. Because the ITS sequence is relatively conservative between different species of the genus Venturia, and there is non-specific binding between the downstream primer and the probe sequence, in a large number of repeated test screening processes, all candidate primers and probes cannot distinguish between different species of pathogens in the genus Venturia, and there is interference from false positives. Therefore, the LFD-RPA detection primers, probes and detection systems for thin-shelled pecan scab pathogens are still to be further developed. Summary of the invention

[0006] In view of this, the purpose of the present invention is to solve the limitation problems of the prior art detection method of Venturia effusa, such as excessive reliance on expensive instruments and equipment, laboratory environment and professional technicians, and to provide a primer probe set, a kit and an application method for isothermal amplification detection of Venturia effusa, so as to achieve simple, fast and accurate detection of Venturia effusa without special instruments.

[0007] The invention provides a set of Venturia effusa isothermal amplification detection primer-probe combinations, which are characterized in that they comprise an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, and a probe as shown in SEQ ID NO.3; the 5' end of the probe has a fluorescent reporter gene, and the 3' end contains a quencher gene; the 5' end of the downstream primer has a biotin label.

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

[0009] The present invention also provides the use of the primer-probe combination or the kit in preparing a reagent for detecting the scab pathogen of Hickory nut.

[0010] Preferably, the detection of the pathogen of pecan scab includes detecting Venturia effusa in the genus Venturia.

[0011] The present invention also provides a method for detecting the pathogen of walnut scab by combining isothermal amplification and lateral flow chromatography, which comprises the following steps:

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

[0013] (2) using the extracted DNA as a template, and using the primer probe combination as described above to perform isothermal amplification on the template to obtain an isothermal amplification product;

[0014] (3) dropping the isothermal amplification product onto a flow chromatographic test strip; if the detection line and the quality control line have bands, the sample to be tested contains the thin-shelled hickory scab pathogen; if the detection line does not have a detection band, the sample to be tested does not contain the thin-shelled hickory scab pathogen.

[0015] Preferably, the isothermal amplification procedure is: reacting at 30°C to 42°C for 10 to 40 minutes.

[0016] Preferably, the isothermal amplification procedure is: react at 38°C for 20 to 30 minutes.

[0017] Preferably, the reaction system of the isothermal amplification is 50 μL, including the following components: 2.5 μL of upstream primer, 2.5 μL of downstream primer, 0.5 μL of probe, 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 template, and 7.6 μL of dd H2O.

[0018] In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.5-0.6 μM; the concentration of the probe is both 0.1-0.16 μM.

[0019] Beneficial Effects

[0020] The present invention takes common pathogens on thin-shelled pecans, including the pathogen of thin-shelled pecan scab (Venturiaeffusa), as well as other representative strains of the genus Venturia and representative strains of the similar genus Cladosporium as research objects, and carries out research on the LFD-RPA detection technology system for thin-shelled pecan scab. The present invention has the advantages of high efficiency, sensitivity and strong specificity, and does not rely on expensive instruments and equipment and professional and technical personnel. It can quickly realize the on-site detection of thin-shelled pecan scab, and has good application prospects in port quarantine, thin-shelled pecan scab monitoring and early warning, and disease prevention measures guidance. It has been verified that the primer probe provided by the present invention can accurately detect scattered Venturia (Venturia effusa), there is no cross-reaction between the same genus and similar genus species, the specificity is strong, and the minimum detection limit is 5pg of pathogenic bacteria total DNA, and the detection sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram for primer-probe design;

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

[0023] Figure 3are the sensitivity test results, where 1 to 8 are 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; 9 is the negative control (the template is water).

[0024] Figure 4 These are the test results of field samples, where 1 to 6 are thin-shelled pecan leaf samples; 7 to 12 are thin-shelled pecan fruit samples; and 13 is a negative control (the template is water). DETAILED DESCRIPTION

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

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

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

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

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

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

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

[0032] The invention provides a constant temperature amplification detection kit for Venturia effusa, comprising the primers and probes described in the above scheme, an RPA reaction reagent and a lateral flow chromatography test strip.

[0033] In the present 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 the present invention, the lateral flow chromatography test strip is preferably purchased from Amp Future (Changzhou) Biotechnology Co., Ltd.

[0035] The present invention also provides the use of the primer probe or the kit described in the above scheme in detecting Venturia effusa.

[0036] In the present invention, the detection of Venturia effusa is preferably the detection of Venturia effusa from the genus Venturia.

[0037] In the present invention, the detection of scattered Venturia (Venturia effusa) from the Venturia genus preferably includes scattered Venturia (Venturia effusa), other Venturia sp., Cladosporium sp. and other pathogens on thin-shelled pecans, including Colletotrichum sp., Diaporthe sp., Alternaria sp., and Pestalotiopsis sp. The fungi in the other V. genus preferably include V. pyrina and V. inaequalis; the fungi in the genus Cladosporium preferably include Cladosporium cladosporioides and Cladosporium cucumerinum; the fungi in the genus Colletotrichum preferably include Colletotrichum siamense, Colletotrichum aenigma, and Colletotrichum fructicola; the fungi in the genus Diaporthe pseudophoenicicola preferably include Alternaria alternata; the fungi in the genus Pseudodiscoidea preferably include Pestalotiopsis ellipsoidea.

[0038] The present invention also provides a method for detecting the pathogen of walnut scab by combining RPA and lateral flow chromatography, comprising the following steps:

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

[0040] (2) using the genomic DNA of the sample to be tested as a template, and using the primer-probe combination described in the above scheme to perform isothermal amplification on the template to obtain an isothermal amplification product;

[0041] (3) dropping the isothermal amplification product onto a flow chromatographic test strip; if the detection line and the quality control line have bands, the sample to be tested contains the thin-shelled hickory scab pathogen; if the detection line does not have a detection band, the sample to be tested does not contain the thin-shelled hickory scab pathogen.

[0042] The present invention first extracts the genomic DNA of the sample to be detected.

[0043] The present invention utilizes a fungal DNA extraction kit or a rapid nucleic acid extraction method to extract the genomic DNA of the sample to be detected.

[0044] In the present invention, the fungal DNA extraction kit is preferably purchased from Omega Corporation; the rapid extraction method using an ultra-fast nucleic acid releaser is 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 primer probes described in the above scheme to perform isothermal amplification on the template.

[0046] After obtaining the isothermal amplification product, the present invention drops the amplification product on a lateral 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 the present invention, the isothermal amplification procedure is preferably: reacting at 30°C to 42°C for 10 to 40 minutes.

[0048] In the present invention, the isothermal amplification procedure is preferably: reacting at 38° C. for 20 to 30 minutes.

[0049] In the present invention, the reaction system of the isothermal amplification is 50 μL, and preferably includes the following components: 2.5 μL of upstream primer, 2.5 μL of downstream primer, 0.5 μL of probe, 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 template, and 7.6 μL of dd H2O.

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

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

[0052] Test materials, reagents and instruments

[0053] Test strains: 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 strain), Pseudoveronaea ellipsoidea, Aureobacidium sp., Trichoderma sp., Penicillium sp., etc., all of which were isolated and identified by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province; other strains of Venturia, V. pyrina (pear tree) and V. inaequalis (apple), Cladosporium cucumerinum (melon), and other strains of Fusarium solan (soybean), Cercosporakikuchii (soybean), and Phomopsis amygdali (peach) were kindly donated by Professor Hu Baishi of Nanjing Agricultural University;

[0054] Reagents and instruments: Fungal DNA Kit (50) fungal DNA extraction kit (Omega, catalog number: D3390-01), ultra-fast nucleic acid release agent (Anpu Future (Changzhou) Biotechnology Co., Ltd., DNA type); RPA reaction reagent (Anpu Future (Changzhou) Biotechnology Co., Ltd., catalog number: WLN8203KIT); flow chromatography test strips (Anpu Future (Changzhou) Biotechnology Co., Ltd., catalog number: WLFS8204); DL2,000DNA Marker (Baori Medical Biological Technology Co., Ltd., catalog number: 3427Q); 2×Taq Master Mix (Novozyme Biotechnology Co., Ltd., catalog number: P111-01); primers and probes were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] Primer probe design and screening

[0056] The genomes of eight typical strains of 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) as well as the genome of Cladosporium were downloaded from the Genomic database of NCBI. 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 Cladosporium, including C. effusa (GenBank: GCA_035771495.1), 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 by comparative genomics analysis and screening of the coding genes in the genomes of 17 typical strains using Orthofinder software. Single-copy genes that only exist in the genome of Venturia effusa but not in the genomes of other strains were screened, and a total of 10 conserved genes unique to Venturia effusa were screened. Taking the unique conserved gene (FKW77_006955) as the candidate target, according to the RPA primer design principles, 3 candidate upstream primers and 3 candidate downstream primers were designed on the candidate target gene, and 3 candidate probes were designed between the upstream and downstream primers, wherein the 5' end of the downstream primer contained a biotin label, the 5' end of the probe contained a "FAM" group, the 3' end contained a "Spacer C3" group, and any base at a position approximately 30 nt away from the 5' end was replaced with dSpacer.Through orthogonal experimental design, 27 combinations of upstream and downstream primers and probes were carried out, and the best combination of candidate target primers and probes was screened through specificity tests and sensitivity tests. Finally, the isothermal amplification primers and probes with strong specificity and high sensitivity for Venturia effusa 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] The genomic DNA of the test strains was extracted using a fungal genomic DNA miniprep kit (OMEGA). The specific method was carried out according to the kit instructions. The extracted genomic DNA was stored in a -20°C refrigerator for future use. The concentration of the Venturia effusa genomic DNA was adjusted to 1 ng / μL for specific detection.

[0059] The dry powder of primers and probes synthesized by the entrusted company was dissolved into 10 μM using sterile deionized water as working solution according to the specific parameter indicators on the synthesis report.

[0060] Referring to the reaction system (50μL reaction system) in the instruction manual of the RPA kit (Anpu Future (Changzhou) Biotechnology Co., Ltd.), add 29.4μL of AD buffer, 2μL of upstream and downstream primers at a concentration of 10μM, 0.6μL of each probe at a concentration of 10μM, 5μL of DNA template at a concentration of 1ng / μL, 8.5μL of ddH2O, and finally add 2.5μL of B buffer to the dry powder tube in the kit. After thorough oscillation and mixing, centrifuge for 5s, and place the reaction tube in a 39°C metal bath for reaction for 30min. After the reaction is completed, dilute the amplified product 10 times with sterile deionized water, mix well, take 80μL of the reaction solution and drop it into the sample well of the flow chromatography test strip, and observe the test results after 15min. 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 Cladosporium cucumerinum and Cladosporiumcladosporioides, and representative strains of other genera Diaporthe pseudophoenicicola, Colletotrichum siamense, Colletotrichum aenigma, Colletotrichum fructicola, Alternaria alternata, Pestalotiopsis microspora, Didymella sp., Pseudoveronaeaellipsoidea, Aureobacidium sp., Trichoderma sp., Penicillium sp., Fusarium solan, Cercospora kikuchii, and Phomopsis amygdali. The results showed that the primers and probes designed and screened for Venturia effusa of the present invention had strong specificity.

[0061] Experimental Example 2 Optimization of Constant Temperature Amplification Reaction System

[0062] 1) Primer concentration optimization

[0063] In the reaction system, the template DNA (1ng / μL) was added in an amount of 5μL, the probe (10μM) was added in an amount of 0.6μL, and the upstream and downstream primers (10μM) were added in amounts of 1μL, 1.5μL, 2μL, 2.5μL, 3μL, and 3.5μL, respectively, that is, the final concentrations of the primers were 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.6μM, and 0.7μM, respectively. The reaction system was placed in a constant temperature of 39°C for 30 minutes; the reaction solution was diluted 10 times, and 80μL of the dilution solution was dripped into the sample well of the flow chromatography test strip. After 15 minutes, the test results were observed, and the optimal reaction concentration of the primers in the reaction system was screened according to the clarity of the detection band. The results of the primer concentration optimization test showed that with the increase of primer concentration, the clarity of the positive detection band gradually improved. When the final concentration reached 0.5μM and 0.6μM, there was no obvious difference in the clarity of the band. Therefore, the optimal reaction concentration of the primers in the reaction system is 0.5μM, that is, the amount of upstream and downstream primers (10μM) added to the reaction system is 2.5μL.

[0064] 2) Optimization of probe concentration

[0065] In the reaction system, the template DNA (1ng / μL) was added in an amount of 5μL, the upstream and downstream primers (10μM) were added in an amount of 2.5μL, and the probe addition amounts were set to 0.2μL, 0.4μL, 0.5μL, 0.6μL, 0.7μL, and 0.8μL, respectively, that is, the final concentrations of the probes were 0.04μM, 0.08μM, 0.1μM, 0.12μM, 0.14μM, and 0.16μM. The reaction was carried out at 39°C for 30min, and the detection was performed by LFD-RPA. The optimal reaction concentration of the probe in the reaction system was screened according to the clarity and appearance time of the detection band. The results showed that with the increase of probe concentration, the clarity of the positive detection band was significantly improved. When the concentration reached 0.1μM, 0.12μM, 0.14μM, and 0.16μM, there was no significant difference in the clarity of the detection band. Therefore, the optimal reaction concentration of the primer in the reaction system is 0.1μM, that is, the amount of probe (10μM) added to the reaction system is 0.5μL.

[0066] 3) Reaction temperature optimization

[0067] Based on the optimization of primer and probe concentrations, the reaction system was set to react for 30 min at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C. The reaction results were detected by LFD-RPA. The results showed that the detection bands appeared at 30°C to 42°C, but with the increase of temperature, the clarity of the detection bands increased first and then decreased. When the temperature was 38°C, the clarity of the detection bands was most obvious, so the optimal reaction temperature was 38°C.

[0068] 4) Optimize response time

[0069] Based on the optimization of factors such as primers, probes, and temperature, the reaction time was set at 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min at 38 °C. The reaction results were detected by LFD-RPA. The results showed that with the increase of reaction time, the clarity of the detection band gradually increased. After 20 min, there was no obvious difference in the detection band, so the optimal reaction time was 20 min.

[0070] Through the LFD-RPA detection system optimization experiment, the LFD-RPA detection system for Venturiaeffusa was finally determined as: 2.5 μL of upstream and downstream primers (10 μM) each, 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 system of 50 μL; the total reaction system was reacted at a constant temperature of 38°C for 20 minutes.

[0071] Experimental Example 3 Primer and probe sensitivity determination

[0072] The strain Venturia effusa DNA was diluted in series to make its concentration 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL and 1fg / μL, respectively, and the sensitivity detection was performed using the LFD-RPA detection system optimized by the present invention. The results are shown in FIG. Figure 3 As shown in the figure, when the concentration of template DNA is 1pg / μL, that is, the content of template DNA is 5pg, there is a detection band, but the clarity of the detection band is not obvious; but when the concentration of template DNA is 100fg / μL, that is, the content of template DNA is 500fg, there is no detection band. The sensitivity test results show that the minimum detection limit of the detection system of the present invention is 5pg, which is about 4 copies of the genome of the strain Venturia effusa.

[0073] Test Example 4: Actual Sample Testing

[0074] This experiment selected 12 samples from different thin-shell pecan planting areas in China for actual sample testing. Select a portion of tissue (1-2g) and extract genomic DNA according to the instructions of the ultra-fast nucleic acid releaser (DNA type) (Anpu Future (Changzhou) Biotechnology Co., Ltd.), or refer to the method of Wang et al. (1993) to quickly extract genomic DNA. The specific method is: add 100μL 0.5M NaOH per gram of tissue, grind it thoroughly in a mortar and transfer it to a 1.5ml EP tube, centrifuge at 12000rpm for 5min, take 5μL of supernatant and add 495μL 0.1M Tris (pH 8.0), mix well and take 5μL as the detection template. The results are as follows: Figure 4As shown, the detection system of the present invention can detect 4 positive samples, namely samples 1, 4, 5 and 8. In order to verify the accuracy of the detection system of the present invention in detecting actual samples, the 12 collected samples were separated and purified by traditional pathogens, and TaqMan fluorescent quantitative PCR detection and ITS sequence sequencing analysis were performed. The results showed that the results of the constant temperature rapid detection system established by the present invention were all correct.

[0075] In summary, the present invention uses common pathogens on thin-shelled pecans, including the pathogen of thin-shelled pecan scab (Venturia effusa), as well as other representative strains of the genus Venturia and representative strains of the similar genus Cladosporium as research objects, and conducts research on the LFD-RPA detection technology system for thin-shelled pecan scab. The present invention has the advantages of high efficiency, sensitivity and strong specificity, and does not rely on expensive instruments and professional technicians. It can quickly realize the on-site detection of thin-shelled pecan scab, and has good application prospects in port quarantine, thin-shelled pecan scab monitoring and early warning, and disease prevention measures guidance.

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

Claims

1. A method for detecting the pathogen of scab of walnut Venturia effusa ) A primer-probe combination, characterized in that, It comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a probe as shown in SEQ ID NO.3; the 5' end of the probe has a fluorescent reporter gene and the 3' end contains a quencher gene; the 5' end of the downstream primer has a biotin label.

2. A constant temperature amplification detection kit for scab pathogen of walnut, characterized in that: The invention comprises the primer-probe combination as claimed in claim 1, a constant temperature amplification detection reagent and a flow chromatography test strip.

3. Use of the primer-probe combination of claim 1 or the kit of claim 2 in preparing a reagent for detecting the pathogen of scab of Hickory nutsedge or a reagent for diagnosing scab of Hickory nutsedge.

4. The use according to claim 3, characterized in that: The detection of the thin-shelled pecan scab pathogen includes detecting scattered black star fungi in the genus Black Star Fungi ( Venturia effusa ).

5. A method for detecting the pathogen of Hickory nut scab by combining isothermal amplification and lateral flow chromatography, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the sample to be tested; (2) using the extracted DNA as a template, and using the primer-probe combination of claim 1 to perform isothermal amplification on the template to obtain an isothermal amplification product; (3) Dropping the isothermal amplification product onto a flow chromatographic test strip, if there are bands on the detection line and the quality control line, the sample to be tested contains the thin-shelled hickory scab pathogen; if no detection band appears on the detection line, the sample to be tested does not contain the thin-shelled hickory scab pathogen.

6. The method according to claim 5, characterized in that The procedure of the isothermal amplification is: reacting at 30°C to 42°C for 10 to 40 minutes.

7. The method according to claim 6, characterized in that The procedure of the isothermal amplification is: react at 38°C for 20-30 minutes.

8. The method according to claim 5, characterized in that The reaction system of the isothermal amplification (RPA) is 50 μL, including the following components: 2.5 μL of upstream primer, 2.5 μL of downstream primer, 0.5 μL of probe, 29.4 μL of AD buffer, 2.5 μL of Bbuffer, 5 μL of template, and 7.6 μL of dd H2O.

9. The method according to claim 5, characterized in that In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.5-0.6 μM; the concentration of the probe is both 0.1-0.16 μM.

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