Gymnosporangium sabinae constant temperature amplification detection kit and application thereof

Abstract

The present application provides a kind of detection of pecan anthracnose fungus (Cryphonectria parasitica Murrill) Colletotrichum fructicola ) probe set, rapid detection kit and its method and application, belong to biological detection technical field.Aiming at the current pecan anthracnose fungus identification difficult, excessive dependence on expensive equipment and technical personnel and other limitations, carry out pecan fruit anthracnose fungus LFD-RPA detection technology research, the present application can simply, efficiently detect pecan fruit anthracnose fungus, and there is no cross response with 22 representative strains of anthracnose fungus genus, strong specificity, minimum detection limit is 10 pg genome DNA, high sensitivity.The present application is suitable for the on-site real-time rapid detection of the disease by port quarantine, scientific research units, agricultural and forestry management and planting households and other departments.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology, and particularly relates to a constant temperature amplification primer, probe, kit and method for detecting Colletotrichum fructicola, which is suitable for on-site real-time rapid detection of the disease by quarantine, scientific research units, agricultural and forestry management and planting households and other departments. BACKGROUND

[0002] Carya illinoinensis, also known as American pecan, is native to the United States and northern Mexico, and is a deciduous tall tree of the Juglandaceae family. It is a famous dried fruit in the world and is deeply loved by citizens as a kind of nutritional and healthy food. Due to its high economic value, it has been widely introduced to more than 20 countries around the world. China introduced Carya illinoinensis in the early 20th century, and the planting area is mainly distributed in Zhejiang, Jiangsu, Anhui and Yunnan, with a planting area of more than 1.2 million mu. However, with the continuous expansion of the planting area of Carya illinoinensis in China, Carya illinoinensis anthracnose has occurred in various planting areas, seriously affecting yield and quality, and is one of the main diseases of Carya illinoinensis. The disease has a long incubation period, and the infected plants do not show necrosis symptoms for a long time. When high temperature and high humidity conditions are encountered, large-scale necrosis and epidemic outbreaks will occur. Therefore, in the orchard, rapid detection at the initial stage of infection, mastering the infection situation of the disease and timely implementing prevention and control measures are the premise of controlling the outbreak of the disease. At present, there is a lack of on-site rapid detection method and kit for Carya illinoinensis anthracnose at home and abroad.

[0003] The dominant pathogen of Carya illinoinensis anthracnose is Colletotrichum fructicola, which can infect the leaves and fruits of Carya illinoinensis. The detection technology of Colletotrichum fructicola currently only stays on the laboratory detection technology platform, mainly relying on traditional pathogen detection technology and ITS sequence analysis. A large number of isolates of suspected pathogen-carrying materials or diseased plant tissues need to be isolated, cultured and purified in the laboratory, and the purified isolates need to be verified by "Koch's postulates". This detection process is tedious, time-consuming and labor-intensive, and the detection rate is not high. In addition, the detection or identification is also disturbed by other microorganisms such as saprophytic bacteria, parasitic bacteria and similar species, which also increases the difficulty of isolation, detection or identification. Therefore, the existing detection method cannot meet the detection needs of on-site scenes such as port quarantine, field quarantine and disease monitoring.

[0004] In order to overcome the detection problem of Carya illinoensis Karsten anthracnose fungus, the inventors designed and screened the Padlock probe and its detection method of Carya illinoensis Karsten anthracnose fungus according to the principle of Padlock probe (PLPs) detection technology and the sequence of pectin lyase (pl) of anthracnose fungus (patent number: ZL201910879229.X). The detection method can high-throughput and specifically detect Carya illinoensis Karsten anthracnose fungus (Colletotrichum gloeosporioides) in the strain to be detected, but it is not specific to other 16 species of anthracnose fungus genus. In addition, the detection technology depends on the laboratory environment, needs PCR instrument and molecular hybridization instrument, and cannot meet the demand of on-site rapid detection, so the existing rapid detection method of Carya illinoensis Karsten anthracnose fungus has certain defects in practicability.

[0005] In recent years, with the rapid development of molecular biology technology, scholars and enterprises in various countries focus on the research and development of molecular rapid detection technology and tools for pathogenic bacteria, and there are new molecular rapid detection technology research reports and applications. Isothermal amplification technology (isothermal amplification tachniques) is widely concerned and favored due to its high sensitivity, strong specificity, convenient operation, short time-consuming and other advantages. Recombinase Polymerase Amplification (RPA) is a new type of isothermal amplification technology developed by Piepenburg et al. in 2006, which is considered as the most potential isothermal amplification technology. Under isothermal conditions (37-42℃), specific primers combine with recombinase to form a complex Rec / ssDNA; with the help of auxiliary proteins and single-stranded DNA binding proteins (Singlestranded DNAbinding protein, SSB), it invades the double-stranded template DNA to form a D-Loop region, and the specific primers sequence recognition on the template DNA; the conformation of Rec / ssDNA complex changes on the target sequence, the recombinase dissociates, the 3' of the primer is exposed, and is recognized by DNA polymerase I (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 simultaneously unwinds the double-stranded 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, fluorescent-RPA and lateral flow dipstick RPA (LFD-RPA), among which LFD-RPA does not require complex and sophisticated instruments and equipment, is convenient and efficient, and is sensitive and efficient, which can meet the needs of on-site rapid detection technology. In the LFD-RPA system, endonuclease IV (nfo) and nfo probe are added, the 5' end of the nfo probe is labeled with a fluorescent group, the 3' end is labeled with a blocker, and a dealkylation site (THF) is labeled at a sequence position about 30 nt away from the 5' end. During the amplification process of RPA specific primers, nfo can specifically recognize the THF site on the probe and remove it, thereby generating a free hydroxyl end, and at the same time completing the extension of the target sequence 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 on the lateral flow chromatographic test strip, within 3-5 min, the biotin ligand is combined with the biotin at the 3' end to present a dark band on the detection line, and the untrapped immune complex continues to diffuse to the quality control line and is captured by the specific antibody to form a dark band, that is, a band appears on the test strip, and the detection result is positive, otherwise it is negative. The LFD-RPA detection has the advantages of high sensitivity, strong specificity, non-instrumentation, visualization and simple and fast, and has strong practicability and development potential in the fields of medicine, law and plant protection which need on-site rapid detection, but the LFD-RPA detection technology has not been researched and applied in the detection of Colletotrichum gloeosporioides of Carya illinoensis.

[0006] Based on the principle of LFD-RPA detection technology, on the basis of the previous invention of Padlock probe and detection method of Colletotrichum gloeosporioides of Carya illinoensis, the detection primer and probe of Colletotrichum gloeosporioides are designed based on pectin lyase gene sequence as a target, and sequence comparison analysis is carried out, it is found that pectin lyase gene is relatively conservative in Colletotrichum, which does not meet the design requirements of RPA detection primer and probe in theory. Although three groups of LFD-RPA detection primers and two probes of Colletotrichum gloeosporioides are designed by the inventors, but the specificity test shows that the candidate primers and probes do not have specificity to Colletotrichum gloeosporioides. Therefore, the LFD-RPA detection primer, probe and detection system for Colletotrichum gloeosporioides of Carya illinoensis still need to be researched and developed. SUMMARY

[0007] Therefore, the purpose of the present application is to solve the problems in the prior art that the detection method of Colletotrichum gloeosporioides of Carya illinoensis excessively depends on expensive instruments, laboratory environment and professional technical personnel, and to provide Colletotrichum gloeosporioides of Carya illinoensis constant temperature amplification detection primer probe group, kit and application method, which realizes simple, fast and accurate detection of Colletotrichum fructicola under the condition of no special instrument.

[0008] The present application provides a group of Colletotrichum fructicola constant temperature amplification detection 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 quenching gene; the 5' end of the downstream primer has a biotin label.

[0009] The present application also provides a Colletotrichum fructicola constant temperature amplification detection kit, which comprises the primer probe combination, constant temperature amplification detection reagent and lateral flow chromatographic test strip.

[0010] The application also provides application of the primer probe combination or the kit in preparation of a reagent for detecting Colletotrichum fructicola.

[0011] Preferably, the detection of the C. fructicola includes detection of Colletotrichum fructicola in the genus Colletotrichum.

[0012] The application also provides a method for detecting Colletotrichum fructicola by combining isothermal amplification and lateral flow chromatography, comprising the following steps:

[0013] (1) extracting genomic DNA of a sample to be detected;

[0014] (2) using the extracted DNA as a template, performing isothermal amplification on the template by using the primer probe combination, to obtain an isothermal amplification product;

[0015] (3) dropping the isothermal amplification product on a lateral flow chromatography test strip, if there is a band on the detection line and the quality control line, it indicates that the sample to be detected contains Colletotrichum fructicola, and if there is no band on the detection line, it indicates that the sample to be detected does not contain Colletotrichum fructicola.

[0016] Preferably, the isothermal amplification program is 32-42°C for 10-40 min.

[0017] Preferably, the isothermal amplification program is 37°C for 25-40 min.

[0018] Preferably, the reaction system of the isothermal amplification is 50 μL, and includes the following components: 2 μL of an upstream primer, 2 μL of a downstream primer, 0.7 μL of a 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 a 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, and the concentration of the probe is 0.14-0.16 μM.

[0020] Advantages

[0021] The application has the advantages of high efficiency, sensitivity and strong specificity, and does not depend on expensive instruments and professional technicians, and can realize rapid on-site detection of C. gloeosporioides, has good application prospect in port quarantine, C. gloeosporioides monitoring and early warning and disease prevention measure guidance. It has been verified that the primer probe provided by the application can accurately detect Colletotrichum fructicola, has no cross reaction between different species of the same genus, has strong specificity, and the minimum detection limit is 10 pg of total DNA of the pathogenic bacteria, and has high detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a schematic diagram for target gene and primer probe design;

[0023] Figure 2 It is a specific detection result, wherein 1-4 are Colletotrichum fructicola strains B-5, W5-1, LHG-3901-9-3 and S3-5; 5-26 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. 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; G. lindemuthiana strain MK.

[0024] Figure 3 It is a sensitivity detection result, wherein 1-6 are respectively template concentrations of 2 ng / μL, 200 pg / μL, 20 pg / μL, 2 pg / μL, 200 fg / μL and negative control (template is water).

[0025] Figure 4 It is a field sample detection result, wherein 1-6 are C. gloeosporioides leaf samples; 7-12 are C. gloeosporioides fruit samples; and 13 is a negative control (template is water). DETAILED DESCRIPTION

[0026] The application provides a primer and a probe for constant temperature amplification of Colletotrichum fructicola, which 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 with a nucleotide sequence as shown in SEQ ID NO. 3; the 5' end of the probe is provided with a fluorescent reporter gene; and the 3' end of the probe contains a quenching gene.

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

[0028] In the application, the downstream primer Cf-RPA-R1 with a nucleotide sequence as shown in SEQ ID NO. 2 is specifically 5'-CTCCGAGAGGTGTAGTTCCCACATTATCGCG-3';

[0029] In the application, the probe Cf-RPA-P1 with a nucleotide sequence as shown in SEQ ID NO. 3 is specifically 5'-GGCTTCTAATGAGGCAACGGTGCGATGCCTTCTCAAGCATGGAGC-3'.

[0030] In the application, the fluorescent reporter gene preferably comprises a FAM fluorescent reporter gene; the quenching gene does not emit fluorescence, and the quenching gene preferably comprises a Spacer C3 quenching gene.

[0031] In the application, the primer and the probe are preferably a constant temperature amplification specific primer and a probe designed and screened according to a sequence of a specific Ankyrin-1 gene (XM_032021514.2) in a 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 sequence of the gene, and the 5' end of the downstream primer contains a 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 30th base from the 5' end is replaced with a "THF" group.

[0032] In the application, the primer and the probe are synthesized by GenScript Biotech (Shanghai) Co., Ltd.

[0033] The present application provides a constant temperature amplification detection kit for Colletotrichum fructicola, comprising the primers and probes described in the above scheme, RPA reaction reagents and a flow assay chromatographic test strip.

[0034] In the present application, the RPA reaction reagents are preferably purchased from Amp Future (Changzhou) Biotechnology Co., Ltd., and in addition, positive and negative controls are also included.

[0035] In the present application, the flow assay chromatographic test strip is preferably purchased from Amp Future (Changzhou) Biotechnology Co., Ltd.

[0036] The present application also provides the application of the primers and probes described in the above scheme or the kit in the detection of Colletotrichum fructicola.

[0037] In the present application, the detection of Colletotrichum fructicola is preferably the detection of Colletotrichum fructicola from the genus Colletotrichum.

[0038] In the present application, the detection of Colletotrichum fructicola from the genus Colletotrichum preferably includes Colletotrichum fructicola, other species of fungi in 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 fungi in the genus Glomerella, preferably Glomerella acutata and G.lindemuthiana.

[0039] The present application also provides a method for detecting Colletotrichum fructicola by combining RPA and flow assay chromatography, comprising the following steps:

[0040] (1) extracting the genomic DNA of the sample to be detected;

[0041] (2) using the genomic DNA of the sample to be detected as a template, performing isothermal amplification on the template using the primer probe combination described in the above scheme to obtain an isothermal amplification product;

[0042] (3) dropping the isothermal amplification product on a flow-through chromatographic test strip, if a detection line and a quality control line have a band, then the sample to be detected contains C. glaucum; if no detection band is observed on the detection line, then the sample to be detected does not contain C. glaucum.

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

[0044] The present application extracts the genomic DNA of the sample to be detected using a fungal DNA extraction kit or a rapid nucleic acid extraction method.

[0045] In the present application, the fungal DNA extraction kit is preferably purchased from Omega Company; the rapid extraction method uses an ultrafast nucleic acid release agent, which is preferably purchased from Ampure Future (Changzhou) Biotechnology Co., Ltd.

[0046] After obtaining the genomic DNA of the sample to be detected, the present application uses the genomic DNA as a template and performs isothermal amplification on the template using the primers and probes in the above scheme.

[0047] After obtaining the isothermal amplification product, the present application drops the amplification product on a flow-through chromatographic test strip, if a positive detection band is observed, then the sample to be detected contains C. glaucum; if no positive detection band is observed, then the sample to be detected does not contain C. glaucum.

[0048] In the present application, the isothermal amplification program is preferably 32-42°C for 10-40 min.

[0049] In the present application, the isothermal amplification program is preferably 37°C for 25-40 min.

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

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

[0052] The technical solutions of the present application will be described in detail below with reference to the embodiments in the present application.

[0053] Test materials, reagents and instruments

[0054] Test strains: Representative strains of Colletotrichum fructicola strains B-5, W5-1, LHG-3901-9-3 and S3-5, C. gloeosporioides strain W5-2, C. siamense strain JS2, C. karstii strain HB-3726-15-8, C. sojae strain CZ-2916-7-2-3, C. fioriniae strain JR-3802-B5-1-2, etc. were isolated and identified by Jiangsu Institute of Botany, Chinese Academy of Sciences; Representative strains of 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, G. lindemuthiana strain MK, etc. were presented by Professor Hu Baishi of Nanjing Agricultural University;

[0055] Test reagents and instruments: Fungal DNA Kit (50) fungal DNA extraction kit (Omega Company, item number: D3390-01), ultra-fast nucleic acid release agent (Anfu Future (Changzhou) Biotechnology Co., Ltd., DNA type); RPA reaction reagent (Anfu Future (Changzhou) Biotechnology Co., Ltd., item number: WLN8203KIT); Flow-through chromatography test strip (Anfu Future (Changzhou) Biotechnology Co., Ltd., item number: WLFS8204); DL2,000 DNA Marker (Baori Biotechnology Co., Ltd., item number: 3427Q); 2x Taq Master Mix (Novozyme Biotechnology Co., Ltd., item number: P111-01); Primers and probes were synthesized by Shengong Biotechnology (Shanghai) Co., Ltd.

[0056] Design and screening of primers and probes

[0057] The genomes of typical strains of Colletotrichum fructicola (GenBank: GCA_000319635.2), C. gloeosporioides (GenBank: GCA_029169055.1), C. siamense (GenBank: GCA_013390195.1), C. karstii (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), C. caudatum (GenBank: GCA_030867225.1), 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) were downloaded from the Genomic database in NCBI. Comparative genomics analysis and screening of the coding genes of the genomes of these typical strains were performed using Orthofinder software, and single-copy genes that only exist in the genome of Colletotrichum fructicola and do not exist in the genomes of other strains were screened, and a total of 14 conserved genes specific to Colletotrichum fructicola were screened. Taking the unique conserved gene (XM_032021514.2) as a candidate target, three candidate upstream primers, three candidate downstream primers, and three candidate probes were designed on the candidate target gene according to the RPA primer design principle, the 5' end of the downstream primer contains a biotin label, the 5' end of the probe contains a "FAM" group, the 3' end contains a "Spacer C3" group, and any base at a position of about 31 nt from the 5' end is replaced with dSpacer.Through orthogonal test design, 27 combinations of upstream and downstream primers and probes were screened for the best combination of candidate target primers and probes through specificity and sensitivity tests. Finally, the specific and sensitive constant temperature amplification primers and probes for the detection of Prunus humilis Canker 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] Specificity determination of primers and probes in test example 1

[0062] The genomic DNA of the test strains was extracted by a fungal genomic DNA small amount preparation kit (OMEGA company) according to the kit instructions. The extracted genomic DNA was stored in a -20℃ refrigerator for later use.

[0063] The dry powder of the primers and probes synthesized by the entrusted company was dissolved in sterile deionized water to 10 μM as working solution according to the specific parameters on the synthesis report for standby.

[0064] According to the reaction system (50 μL reaction system) of the RPA kit (AmplifX (Changzhou) Biotechnology Co., Ltd.), 29.4 μL of AD buffer, 2 μL of 10 μM upstream primer, 0.6 μL of 10 μM probe, 5 μL of 2 ng / μL DNA template, and 8.5 μL of ddH2O were added into the dry powder tube of the kit, and finally 2.5 μL of B buffer was added into the reaction tube. After shaking and mixing thoroughly, the reaction tube was placed in a 39℃ metal bath for 30 min. After the reaction, the amplification product was diluted 10 times with sterile deionized water, mixed thoroughly, and 80 μL of the reaction solution was dropped into the sample well of the test chromatography test strip. After 15 min, the detection results were observed. The results are shown in Table 1. Figure 2As shown, clear detection bands were observed for C. fructicola strains B-5, W5-1, LHG-3901-9-3 and S3-5, while no detection bands were observed for other representative strains of Colletotrichum genus, C. gloeosporioides strain W5-2, Colletotrichum 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, indicating that the primers and probes designed and screened in the application have strong specificity for C. fructicola.

[0065] Test Example 2 Optimization of the constant temperature amplification reaction system

[0066] 1) Optimization of the primer concentration

[0067] In the reaction system, the addition amount of template DNA (2 ng / μL) was 5 μL, the addition amount of probe (10 μM) was 0.6 μL, and the addition amount of upstream and downstream primers (10 μM) was set to be 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL and 3.5 μL, respectively, i.e. the final concentration of primers was 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM and 0.7 μM, respectively. The reaction system was placed in a constant temperature of 39°C for 30 min; the reaction solution was diluted by 10 times, 80 μL of the diluted solution was dropped into the sample well of the test flow chromatography test strip, and the detection result was observed after 15 min; according to the clarity of the detection band, the optimal reaction concentration of primers in the reaction system was screened. The results of the primer concentration optimization test showed that with the increase of the primer concentration, the clarity of the positive detection band gradually increased, and when the final concentration reached 0.4 μM, 0.5 μM and 0.6 μM, there was no obvious difference in the clarity of the band, so the optimal reaction concentration of primers in the reaction system was 0.4 μM, i.e. the addition amount of upstream and downstream primers (10 μM) in the reaction system was 2 μL.

[0068] 2) Probe concentration optimization

[0069] The amount of template DNA (2 ng / μL) added in the reaction system was 5 μL, the amount of upstream and downstream primers (10 μM) added was 2 μL, and the amount of probe added was set at 0.2 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, and 0.8 μL, i.e. the final concentration of the probe was 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°C for 30 min, and LFD-RPA detection was performed. According to the clarity and appearance time of the detection band, the optimal reaction concentration of the probe in the reaction system was screened. The results showed that the clarity of the positive detection band was significantly improved with the increase of the probe concentration. When the concentration reached 0.14 μM and 0.16 μM, 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 0.14 μM, i.e. the amount of probe (10 μM) added in the reaction system was 0.7 μL.

[0070] 3) Reaction temperature optimization

[0071] On the basis of the optimization of the concentrations of primers and probes, the reaction system was set at 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C for 30 min. The reaction results were detected by LFD-RPA. The results showed that the detection band appeared under the conditions of 32°C to 42°C, but the clarity of the detection band first increased and then decreased with the increase of the temperature. The clarity of the detection band was most obvious at 37°C, and therefore the optimal reaction temperature was 37°C.

[0072] 4) Reaction time optimization

[0073] On the basis of the optimization of 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 the clarity of the detection band gradually increased with the increase of the reaction time, and there was no significant difference in the detection band after 25 min. Therefore, the optimal reaction time was 25 min.

[0074] Through the LFD-RPA detection system optimization test, finally determine the LFD-RPA detection system of C. fructicola as follows: 2 μL of upstream primer (10 μM) and 2 μL of downstream primer (10 μM), 0.7 μL of probe (10 μM), 29.4 μL of AD buffer (Anfu Future (Changzhou) Biotechnology Co., Ltd.), 2.5 μL of B buffer (Anfu Future (Changzhou) Biotechnology Co., Ltd.), 5 μL of DNA template, 8.4 μL of dd H2O, and the total reaction system is 50 μL; the total reaction system is reacted at 37℃ constant temperature for 25 min.

[0075] Test Example 3: Sensitivity determination of primer and probe

[0076] The DNA of the strain C. fructicola is diluted by times in turn, so that the concentration is 2 ng / μL, 200 pg / μL, 20 pg / μL, 2 pg / μL and 200 fg / μL, and the sensitivity is detected by the LFD-RPA detection system optimized by the application. The results are shown in Figure 3 As shown in the table, when the concentration of the template DNA is 2 pg / μL, that is, the content of the template DNA is 10 pg, there is a detection band, but the detection band is not clear; but when the concentration of the template DNA is 200 fg / μL, that is, the content of the template DNA is 1 pg, there is no detection band. The sensitivity test results show that the minimum detection limit of the detection system of the application is 10 pg, which is about 6 copies of the genome of the strain Venturia effusa.

[0077] Test Example 4: Actual sample detection

[0078] In this experiment, 12 samples from different domestic P. monticola planting areas were selected for actual sample detection. A part of the tissue (1-2 g) was selected, and the genomic DNA was extracted according to the instruction of the ultra-fast nucleic acid release agent (DNA type) (Anfu Future (Changzhou) Biotechnology Co., Ltd.), or the genomic DNA was quickly extracted according to the method of Wang et al. (1993), and the specific method is as follows: 100 μL of 0.5M NaOH was added to each gram of tissue, and then it was ground in a mortar and transferred to a 1.5 ml EP tube, and centrifuged at 12000 rpm for 5 min, 5 μL of supernatant was taken and added to 495 μL of 0.1M Tris (pH 8.0), and then 5 μL was taken as a detection template.

[0079] The results are shown in Figure 4As shown, 4 positive samples, samples 2, 3, 6 and 12, can be detected by the detection system of the application. In order to verify the accuracy of the detection system of the application in detecting actual samples, the traditional pathogenic bacteria of the 12 collected samples are separated and purified, and the ITS sequence is sequenced and analyzed, and the results show that the results of the constant temperature rapid detection system established by the application are correct.

[0080] In summary, the application takes 9 pathogenic species of Guignardia on Carya illinoinensis and 16 pathogenic species of Guignardia on other hosts as research objects, and studies the LFD-RPA detection technology system of Guignardia on Carya illinoinensis fruit. The application has the advantages of high efficiency, sensitivity and strong specificity, and does not depend on expensive instruments and professional technical personnel, and can quickly realize the on-site detection of Carya illinoinensis anthracnose, has good application prospect in port quarantine, Carya illinoinensis anthracnose monitoring and early warning and disease prevention measure guidance.

[0081] Although the above embodiments make a detailed description of the application, they are only part of the embodiments of the application but not all the embodiments, researchers can also obtain other embodiments under the premise of no creativity according to the embodiments, and these embodiments all belong to the protection scope of the application.

Claims

1. The use of a primer probe combination in the preparation of a reagent for detecting the pathogen of Carya illinoinensis anthracnose, characterized in that, The primer probe combination is 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; the 5' end of the downstream primer has a biotin label; and the C. fructicola is Colletotrichum fructicola.

2. A method for detecting C. gloeoporium by RPA and flow-through chromatography, characterized in that, The C. fructicola is Colletotrichum fructicola; The method comprises the following steps: (1) extracting genomic DNA of a sample to be detected; (2) using the extracted DNA as a template, performing isothermal amplification on the template by using the primer probe combination according to claim 1 to obtain an isothermal amplification product; (3) dropping the isothermal amplification product on a flow chromatography test strip, if there is a band on the detection line and the quality control line, then the sample to be detected contains the C. fructicola, and if there is no band on the detection line, then the sample to be detected does not contain the C. fructicola.

3. The method of claim 2, wherein, The isothermal amplification program is 32-42℃ for 10-40 min.

4. The method of claim 2, wherein, The isothermal amplification program is 37℃ for 25-40 min.

5. The method of claim 2, wherein, In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.4-0.6 μM; and the concentration of the probe is 0.14-0.16 μM. In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.4-0.6 μM; and the concentration of the probe is 0.14-0.16 μM.

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