Isothermal amplification detection kit for apocarya colletotrichum gloeosporioides and application thereof

Abstract

The invention provides an isothermal amplification primer probe set for detecting Colletotrichum fucicola of Carya illinoensis, a rapid detection kit, a method for detecting Colletotrichum fucicola and application of the isothermal amplification primer probe set and the rapid detection kit, and belongs to the technical field of biological detection. Aiming at the limitation problems that the current apocarya colletotrichum gloeosporioides is difficult to identify, excessively depends on expensive instruments and technicians and the like, the apocarya colletotrichum gloeosporioides LFD-RPA detection technology research is developed, the apocarya colletotrichum gloeosporioides can be simply, conveniently and efficiently detected, the apocarya colletotrichum gloeosporioides LFD-RPA has no cross reaction with 22 representative strains of the colletotrichum gloeosporioides, the specificity is high, the detection time is the lowest detection limit is 10 pg of 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 technology, and specifically relates to a constant temperature amplification primer, a probe, a kit and a method for detecting the anthracnose pathogen of pecan (Colletotrichum fructicola), which is 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] Thin-shell pecan (Carya illinoinensis), also known as American pecan, is native to the United States and northern Mexico. It belongs to the genus Carya of the Juglandaceae family. It is a deciduous tall tree and is a world-famous dried fruit. As a nutritious and health food, it is deeply loved by citizens. Because of its high economic value, it has been widely introduced to more than 20 countries around the world. my country began to introduce thin-shell pecans in the early 20th century. At present, the planting areas are mainly distributed in Zhejiang, Jiangsu, Anhui and Yunnan, and the planting area has reached more than 1.2 million mu. However, with the continuous expansion of the planting area of ​​thin-shell pecans in my country, anthracnose of thin-shell pecans has occurred in all planting areas, seriously affecting the yield and quality. It is currently one of the main diseases of thin-shell pecans. The disease has a long incubation period. The infected plants do not show necrosis symptoms for a long time. Only when they encounter high temperature and high humidity conditions will large-scale necrosis occur and an epidemic will break out. Therefore, rapid detection of the early infection in the orchard, mastering the infection situation of the disease and implementing prevention and control measures in time are the prerequisites for controlling the outbreak of the disease. At present, there is a lack of on-site rapid detection methods and kits for anthracnose of thin-shell pecans both at home and abroad.

[0003] The dominant pathogen of thin-shelled pecan anthracnose is Colletotrichum fructicola, which can infect the leaves and fruits of thin-shelled pecans. At present, the detection technology methods of fruit anthracnose bacteria only remain 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, not only time-consuming and labor-intensive, but also has a low detection rate. In addition, detection or identification is also interfered by other microorganisms, such as saprophytes, parasites and similar species, which also increases the difficulty of separation detection or identification. Therefore, the existing detection methods cannot meet the detection needs of on-site scenarios such as port quarantine, field quarantine and disease monitoring.

[0004] In order to overcome the problem of detecting anthracnose bacteria of thin-shelled pecans in the early stage, the inventor designed and screened the Padlock probe of thin-shelled pecan anthracnose bacteria and its detection method (patent number: ZL201910879229.X) according to the pectin lyase gene (pectin lyase, pl) sequence of anthracnose bacteria based on the principle of Padlock probe (PLPs) detection technology. The detection method can detect thin-shelled pecan anthracnose bacteria (Colletotrichum gloeosporioides) in the strain to be tested with high throughput and specificity, but it is not specific to the other 16 species of the genus Anthrax. In addition, the detection technology depends on the laboratory working environment, requires PCR instruments and molecular hybridization instruments, and cannot meet the needs of rapid on-site detection. Therefore, the existing rapid detection method of thin-shelled pecan anthracnose bacteria has certain defects in practicality.

[0005] 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, the test line and the quality control line have bands, 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 rapid on-site detection. However, the development and application of LFD-RPA detection technology has not yet been seen in the detection of thin-shell pecan anthracnose bacteria.

[0006] Based on the principle of LFD-RPA detection technology, the inventors, on the basis of the invention of the Padlock probe and detection method of thin-shelled pecan anthracnose bacteria in the early stage, took the pectin lyase gene sequence as the target, designed the detection primers and probes of fruit anthracnose bacteria, and performed sequence comparison analysis, and found that the pectin lyase gene is relatively conservative in the genus Anthracnose, and theoretically does not meet the requirements of RPA detection primer and probe design. Although the inventors designed 3 groups of LFD-RPA detection primers and 2 probes for fruit anthracnose bacteria, specificity tests found that the candidate primers and probes are not specific to fruit anthracnose bacteria. Therefore, the LFD-RPA detection primers, probes and detection systems for thin-shelled pecan anthracnose bacteria have yet to be developed. Summary of the invention

[0007] In view of this, the purpose of the present invention is to solve the limitation problem that the detection method of anthracnose bacteria in thin-shelled pecan fruit in the prior art is overly dependent 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 anthracnose bacteria in thin-shelled pecan fruit, so as to achieve simple, fast and accurate detection of Colletotrichum fructicola without special instruments.

[0008] The invention provides a set of isothermal amplification detection primer-probe combinations for thin-shelled pecan anthracnose pathogen (Colletotrichum fructicola), which are characterized by 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 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.

[0009] The invention also provides a constant temperature amplification detection kit for anthracnose bacteria of pecan nuts, comprising the above-mentioned primer-probe combination, constant temperature amplification detection reagent and flow chromatography test strip.

[0010] The present invention also provides the use of the primer-probe combination or the kit in preparing a reagent for detecting anthracnose bacteria of pecan nuts.

[0011] Preferably, the detection of thin-shelled pecan anthracnose bacteria includes detecting Colletotrichum fructicola in the genus Colletotrichum.

[0012] The present invention also provides a method for detecting anthracnose bacteria of thin-shelled pecan fruit by combining isothermal amplification and lateral flow chromatography, comprising the following steps:

[0013] (1) extracting genomic DNA from the sample to be tested;

[0014] (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;

[0015] (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 fruit anthracnose bacteria; if no detection band appears on the detection line, the sample to be tested does not contain fruit anthracnose bacteria.

[0016] Preferably, the isothermal amplification procedure is: reacting at 32°C to 42°C for 10 to 40 minutes.

[0017] Preferably, the isothermal amplification procedure is: react at 37° C. for 25 to 40 minutes.

[0018] Preferably, the reaction system of the isothermal amplification is 50 μL, including the following components: 2 μL of upstream primer, 2 μL of downstream primer, 0.7 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 8.4 μL of dd H2O.

[0019] In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.4-0.6 μM; the concentration of the probe is both 0.14-0.16 μM.

[0020] Beneficial Effects

[0021] The 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-shell pecan anthracnose, and has good application prospects in port quarantine, thin-shell pecan anthracnose monitoring and early warning, and disease prevention measures guidance. It has been verified that the primer probe provided by the invention can accurately detect fruit anthracnose fungi (Colletotrichum fructicola), there is no cross reaction between different species of the same genus, the specificity is strong, and the minimum detection limit is 10pg of pathogen total DNA, and the detection sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram for designing target genes and primer probes;

[0023] Figure 2 are specific detection results, among which 1 to 4 are Colletotrichum fructicola strains B-5, W5-1, LHG-3901-9-3, and S3-5; 5 to 26 are C. gloeosporioides strain W5-2; C. siamense strain JS2; C. karst strain HB-3726-15-8; C. sojae strain CZ-2916-7-2-3; C. fioriniae strain JR-3802-B5-1-2; C. higginsianum strain cx101; C. destructivum strain c-5; C. li nicola strain mg02; C.caudatum strain bm; C.sublineolum strain gl; C.falcatum strain gz110; C.orbiculare strain melon001; C.graminicola strain cp ; C.dematium strain xc01; C.capsici strain lj; C.musae strain xj003; C.coccodes strain 02; C.lini strain CB01; C.orchidearum strain 102; Glomerella acutata strain 002; G. lindemuthiana strain MK.

[0024] Figure 3 are the sensitivity test results, where 1 to 6 are template concentrations of 2 ng / μL, 200 pg / μL, 20 pg / μL, 2 pg / μL, 200 fg / μL, and a negative control (the template is water).

[0025] 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

[0026] The invention provides a primer and a probe for isothermal expansion of anthracnose fungus (Colletotrichum fructicola), 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.

[0027] In the present invention, the upstream primer Cf-RPA-F2 having a nucleotide sequence as shown in SEQ ID NO.1 is specifically: 5'-CGAATACTTGATTGGCCATGGAGCAGACGTC-3';

[0028] In the present invention, the nucleotide sequence of the downstream primer Cf-RPA-R1 shown in SEQ ID NO. 2 is specifically: 5'-CTCCGAGAGGTGTAGTTCCCACATTATCGCG-3';

[0029] In the present invention, the nucleotide sequence of the probe Cf-RPA-P1 shown in SEQ ID NO. 3 is specifically: 5'-GGCTTCTAATGAGGCAACGGTGCGATGCCTTCTCAAGCATGGAGC-3'.

[0030] 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.

[0031] In the present invention, the primers and probes are preferably isothermal amplification specific primers and probes designed and screened based on a specific anchoring protein (Ankyrin-1) gene (XM_032021514.2) sequence on the Colletotrichum fructicola genome (GenBank: GCA_000319635.2). 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 "SpacerC3" group, and the base at the 30th position from the 5' end is replaced with a "THF" group.

[0032] In the present invention, the primers and probes were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] The invention provides a constant temperature amplification detection kit for pecan anthracnose bacteria (Colletotrichum fructicola), comprising the primers and probes described in the above scheme, an RPA reaction reagent and a lateral flow chromatography test strip.

[0034] 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.

[0035] In the present invention, the lateral flow chromatography test strip is preferably purchased from Amp Future (Changzhou) Biotechnology Co., Ltd.

[0036] The present invention also provides the use of the primer probe or the kit described in the above scheme in detecting Colletotrichum fructicola.

[0037] In the present invention, the detection of thin-shell pecan anthracnose bacteria is preferably to detect Colletotrichum fructicola from the genus Anthracnose.

[0038] In the present invention, Colletotrichum fructicola is preferably detected from the genus Colletotrichum including Colletotrichum fructicola, other species of the genus Colletotrichum, preferably C.gloeosporioides, C.siamense, C.karsti, C.sojae, C.fioriniae, C.higginsianum, C.destructivum, C.linicola, C.caudatum, C.sublineolum, C.falcatum, C.orbiculare, C.graminicola, C.dematium, C.capsic, C.musae, C.coccodes, C.lini, C.orchidearum and preferred fungi of the genus Glomerella acutata and G.lindemuthiana.

[0039] The present invention also provides a method for detecting anthracnose bacteria of walnuts by combining RPA and lateral flow chromatography, comprising the following steps:

[0040] (1) extracting genomic DNA from the sample to be tested;

[0041] (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;

[0042] (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 thin-shelled pecan anthracnose bacteria; if the detection line does not have a detection band, the sample to be tested does not contain thin-shelled pecan anthracnose bacteria.

[0043] The present invention first extracts the genomic DNA of the sample to be detected.

[0044] 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.

[0045] 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.

[0046] After obtaining the genomic DNA of the sample to be tested, the present invention uses the genomic DNA as a template and adopts the primer probe in the above scheme to perform isothermal amplification on the template.

[0047] 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 detected contains thin-shell pecan anthracnose bacteria; if no positive detection band is observed, the sample to be detected does not contain thin-shell pecan anthracnose bacteria.

[0048] In the present invention, the isothermal amplification procedure is preferably: reacting at 32°C to 42°C for 10 to 40 minutes.

[0049] In the present invention, the isothermal amplification procedure is preferably: reacting at 37° C. for 25 to 40 minutes.

[0050] In the present invention, the reaction system of the isothermal amplification is 50 μL, and preferably includes the following components: 2 μL of upstream primer, 2 μL of downstream primer, 0.7 μL of probe, 29.4 μL of AD buffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 2.5 μL of Bbuffer (Anpu Future (Changzhou) Biotechnology Co., Ltd.), 5 μL of template, and 8.4 μL of dd H2O.

[0051] In the present invention, the final concentrations of the upstream primer and the downstream primer are preferably 0.4 to 0.6 μM, respectively; the final concentration of the probe is preferably 0.14 to 0.16 μM.

[0052] The technical solution of the present invention will be described in detail below in conjunction with the embodiments of the present invention.

[0053] Test materials, reagents and instruments

[0054] Test strains: Representative strains from the thin-shelled pecan host Colletotrichum fructicola strains B-5, W5-1, LHG-3901-9-3 and S3-5, C. gloeosporioides strain W5-2, C. siamense strain JS2, C. karsti strain HB-3726-15-8, C. sojae strain CZ-2916-7-2-3, C. fioriniae strain JR-3802-B5-1-2 were isolated and identified by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province; C. higginsianum strain cx101, C. destructivu Representative strains including C. m strain c-5, C. linicola strain mg02, C. caudatum strain bm, C. sublineolum strain gl, C. falcatum strain gz110, C. orbiculare strain melon001, C. graminicola strain cp, C. dematium strain xc01, C. capsici strain lj, C. musae strain xj003, C. coccodes strain 02, C. lini strain CB01, C. orchidearum strain 102, Glomerella acutata strain 002, and G. lindemuthiana strain MK were kindly donated by Professor Hu Baishi of Nanjing Agricultural University;

[0055] 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); DL2,000DNAMarker (Baori Medical Biotechnology 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.

[0056] Primer probe design and screening

[0057] The following genes were downloaded from the Genomic database of NCBI: Colletotrichum fructicola (GenBank: GCA_000319635.2), C. gloeosporioides (GenBank: GCA_029169055.1), C. siamense (GenBank: GCA_013390195.1), C. karsti (GenBank: GCA_011947395.2), C. sojae (GenBank: GCA_014235955.1), C. fioriniae (GenBank: GCA_026319145.1), C. higginsianum (GenBank: GCA_001672515.1), C. destructivum (GenBank: GCA_034447905.1), and C. caudatum (GenBank: GCA_013390195.1). The genomes of typical strains of the genus Colletotrichum, including C. sublineolum (GenBank: CA_020631755.1), C. falcatum (GenBank: GCA_019425465.1), C. orbiculare (GenBank: GCA_000350065.2), C. graminicola (GenBank: GCA_029226625.1), C. musae (GenBank: GCA_027406025.1), C. coccodes (GenBank: GCA_020466075.1), C. lini (GenBank: GCA_034638275.1) and C. truncatum (GenBank: GCA_014235925.2). Orthofinder software was used to perform comparative genomic analysis and screening of the coding genes in the genomes of these typical strains, and single-copy genes that only exist in the genome of Colletotrichum fructicola but not in the genomes of other strains were screened, and a total of 14 conserved genes unique to Colletotrichum fructicola were screened. The unique conserved gene (XM_032021514.2) was used as a candidate target. According to the RPA primer design principle, 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 about 31nt from the 5' end was replaced with dSpacer.Through orthogonal experimental design, a total of 27 combination patterns of upstream and downstream primers and probes were used. The optimal combination pattern of candidate target primers and probes was screened through specificity and sensitivity tests, and finally the constant temperature amplification primers and probes for anthracnose of thin-shelled drupe with strong specificity and high sensitivity were obtained.

[0058] Upstream primer Cf-RPA-F2: 5′-CGAATACTTGATTGGCCATGGAGCAGACGTC-3′;

[0059] Downstream primer Cf-RPA-R1: 5′-CTCCGAGAGGTGTAGTTCCCACATTATCGCG-3′;

[0060] Probe Cf-RPA-P1: 5′-GGCTTCTAATGAGGCAACGGTGCGATGCCTTCTCAAGCATGGAGC-3′.

[0061] Experimental Example 1 Primer and probe specificity determination

[0062] 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.

[0063] 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.

[0064] 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 probe at a concentration of 10μM, 5μL of DNA template at a concentration of 2ng / 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, C. fructicola strains B-5, W5-1, LHG-3901-9-3, and S3-5 showed clear detection bands, while other representative strains of the genus Colletotrichum, C. gloeosporioides strains W5-2, siamense strain JS2, C.karsti strain HB-3726-15-8, C.sojae strain CZ-2916-7-2-3, C.fioriniae strain JR-3802-B5-1-2, C.higginsianum strain cx101, C.destructivum strain c-5, C.linicola strain mg02, C.caudatum strain bm, C.sublineolum strain gl, C.falcatum strain gz110, C.orbiculare strain melon001, C.graminicola strain cp, C.dematium strain xc01, C.capsici strain lj, C.musae strain xj003, C.coccodes strain 02, C.lini strain CB01, C.orchidearum strain 102, Glomerella acutata strain 002 and G. lindemuthiana strain MK were not observed to detect bands, and the results showed that the primers and probes designed and screened by the present invention had strong specificity for C. fructicola.

[0065] Experimental Example 2 Optimization of Constant Temperature Amplification Reaction System

[0066] 1) Primer concentration optimization

[0067] In the reaction system, the template DNA (2ng / 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. The test results were observed after 15 minutes, 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 0.4μM, 0.5μM and 0.6μM, there was no obvious difference in the clarity of the band. Therefore, the optimal reaction concentration of primers in the reaction system is 0.4μM, that is, the amount of upstream and downstream primers (10μM) added to the reaction system is 2μL.

[0068] 2) Optimization of probe concentration

[0069] In the reaction system, the template DNA (2ng / μL) was added in an amount of 5μL, the upstream and downstream primers (10μM) were added in an amount of 2μL, 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℃ for 30min, and the LFD-RPA was used for detection. 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 the probe concentration, the clarity of the positive detection band was significantly improved. When the concentration reached 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 was 0.14μM, that is, the amount of the probe (10μM) added in the reaction system was 0.7μL.

[0070] 3) Reaction temperature optimization

[0071] Based on the optimization of primer and probe concentrations, the reaction system was set to react for 30 min at 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 32°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 37°C, the clarity of the detection bands was most obvious, so the optimal reaction temperature was 37°C.

[0072] 4) Optimize response time

[0073] 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 37 °C. The reaction results were detected by LFD-RPA. The results showed that as the reaction time increased, the clarity of the detection band gradually increased. After 25 min, there was no obvious difference in the detection band, so the optimal reaction time was 25 min.

[0074] Through the LFD-RPA detection system optimization experiment, the LFD-RPA detection system for thin-shelled pecan anthracnose (C. fructicola) was finally determined as: 2 μL of upstream and downstream primers (10 μM) each, 0.7 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, 8.4 μL of dd H2O, and a total reaction system of 50 μL; the total reaction system was reacted at a constant temperature of 37°C for 25 minutes.

[0075] Experimental Example 3 Primer and probe sensitivity determination

[0076] The strain C. fructicola DNA was diluted in series to make its concentration 2ng / μL, 200pg / μL, 20pg / μL, 2pg / μL and 200fg / μL respectively, and the sensitivity test 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 2pg / μL, that is, the content of template DNA is 10pg, there is a detection band, but the clarity of the detection band is not obvious; but when the concentration of template DNA is 200fg / μL, that is, the content of template DNA is 1pg, there is no detection band. The sensitivity test results show that the minimum detection limit of the detection system of the present invention is 10pg, which is about 6 copies of the genome of the strain Venturia effusa.

[0077] Test Example 4: Actual Sample Testing

[0078] This experiment selected 12 samples from different thin-shelled pecan planting areas in China for actual sample testing. An appropriate amount of tissue (1-2 g) was selected to 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 100 μL 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 5 minutes, take 5 μL of supernatant and add 495 μL 0.1M Tris (pH 8.0), mix well and take 5 μL as the detection template.

[0079] The results are as follows Figure 4As shown, the detection system of the present invention can detect 4 positive samples, namely samples 2, 3, 6 and 12. 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 analyzed by ITS sequence sequencing. The results showed that the results of the constant temperature rapid detection system established by the present invention were all correct.

[0080] In summary, the present invention takes 9 pathogenic species of Anthracnose on thin-shelled pecans and 16 pathogenic species of Anthracnose on other hosts as research objects, and conducts research on the LFD-RPA detection technology system for anthracnose in thin-shelled pecans. 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 anthracnose in thin-shelled pecans, and has good application prospects in port quarantine, monitoring and early warning of anthracnose in thin-shelled pecans, and guidance of disease prevention measures.

[0081] 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 anthracnose bacteria in thin-shelled pecans ( Colletotrichum fructicola ) 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 anthracnose pathogens of pecan nuts, 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 anthracnose bacteria of walnuts or preparing a reagent for diagnosing anthracnose disease of walnuts.

4. The use according to claim 3, characterized in that: The detection of thin-shell pecan anthracnose bacteria includes detecting fruit anthracnose bacteria in the genus Anthracnose ( Colletotrichum fructicola ).

5. A method for detecting anthracnose bacteria of walnuts 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 thin-shelled pecan anthracnose bacteria; if no detection band appears on the detection line, the sample to be tested does not contain thin-shelled pecan anthracnose bacteria.

6. The method according to claim 5, characterized in that The procedure of the isothermal amplification is: reacting at 32°C to 42°C for 10 to 40 min.

7. The method according to claim 6, characterized in that The isothermal amplification procedure is: react at 37°C for 25-40 min.

8. The method according to claim 5, characterized in that The isothermal amplification reaction system is 50 μL, including the following components: 2 μL of upstream primer, 2 μL of downstream primer, 0.7 μL of probe, 29.4 μL of AD buffer, 2.5 μL of B buffer, 5 μL of template, and 8.4 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 0.4-0.6 μM; the concentration of the probe is both 0.14-0.16 μM.

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