Composition for detecting pear fire blight bacteria and application thereof

Abstract

The invention discloses a composition for detecting pear fire blight bacteria and application thereof, the composition comprises a primer pair for specifically amplifying a pear fire blight bacteria specific DNA fragment, the primer pair is composed of a primer Ea-F3 and a primer Ea-R1, the primer Ea-F3 is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID No.1, and the primer Ea-R1 is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID No.2. The primer Ea-R1 is a single-stranded DNA (deoxyribonucleic acid) molecule of which the nucleotide sequence is as shown in SEQ ID No. 2. The kit can be used for detecting pear fire blight bacteria, and has the characteristics of high sensitivity and strong specificity.

Description

Technical Field

[0001] The present application belongs to the technical field of microbial detection, and specifically relates to a composition for detecting Erwinia amylovora and its application. Background Art

[0002] Apricot fire blight is a devastating bacterial disease on Rosaceae plants such as pears, apples, and crabapples. It is caused by Erwinia amylovora and mainly harms flowers, young shoots, fruits, leaves, and branches. After being infected, the plants quickly wither and turn black, as if burned by fire, but they still hang on the trees without falling, hence the name. In 1780, the disease first broke out in the Hudson River Basin in New York, USA, and the early outbreak was limited to the northeastern United States. Due to the wide host range and multiple transmission routes of the disease, it has now spread to more than 60 countries and regions. Apricot fire blight is mainly transmitted over long distances or across borders through infected seedlings, scions, rootstocks, and fruits. In the disease-affected areas, it is mainly transmitted at close range by insects, wind and rain, pruning, and agricultural operations. Apricot fire blight is very destructive, not only greatly reducing fruit yields and affecting fruit quality, but also causing the death of fruit trees and even destroying orchards. Due to its extremely destructive nature and high risk of spreading and influx, the amylopectin pathogen has been listed as a quarantine pest by many countries and organizations, including China and the European Union.

[0003] Detection, interception and prevention with chemicals are the two main measures to control fire blight. Accurate, sensitive and simple pathogen detection technology is the main technical support for the effective implementation of the above measures. At present, a variety of detection technologies for fire blight pathogens have been established, mainly including traditional detection, immune detection and molecular detection. Traditional detection methods such as selective culture medium separation and pathogenicity determination have long detection cycles, low accuracy and low sensitivity. They can no longer meet current detection requirements and can only be used as auxiliary detection methods. The colloidal gold immunochromatographic test strip detection method only takes 5-10 minutes and has strong specificity, but the detection sensitivity is generally 1×10 6cfu / mL, and the detection results are easily affected by the sample material. In recent years, researchers have developed a variety of molecular biology methods, including species-specific PCR identification methods, Padlock probe combined with Macroarray detection methods, real-time fluorescence PCR identification methods, and isothermal amplification identification methods such as LAMP and RPA. Among the above methods, the species-specific PCR identification method requires PCR amplification and gel electrophoresis observation, which is a long process and cumbersome operation; Padlock probe combined with Macroarray technology can simultaneously detect Erwinia pyrifoliae and Erwinia pyrifoliae. This method includes three steps: linking, PCR amplification, and Macroarray multiple detection. It has high sensitivity and can be used for high-throughput screening of multiple targets. However, this method has many operating steps, is time-consuming, and requires equipment such as PCR amplifiers; real-time fluorescence PCR identification methods require expensive equipment and can generally only be completed in the laboratory. LAMP, RPA and other isothermal amplification methods currently have certain disadvantages, such as easy contamination, cumbersome operating steps, complex DNA extraction process, and some reagents required for detection need to be stored at -20℃.

[0004] Enzymes-mediated Duplex Exponential Amplification (EmDEA) nucleic acid rapid detection technology is a new detection technology that organically integrates nucleic acid exponential amplification and signal exponential amplification. It can complete the reaction in one tube and has great potential for on-site detection. Its reaction principle is mainly divided into three steps, namely DNA amplification, DNA transcription, and probe hydrolysis to generate signals. After the recombinase binds to the DNA primer, matches the homologous sequence, and forms a D-loop structure with the DNA double strand, it extends to produce a new strand under the action of the polymerase. This process is repeated to complete DNA replication; in DNA amplification, the forward primer carries a T7 promoter, which is transcribed to produce RNA single strands under the catalysis of T7 RNA polymerase; after the probe binds to the RNA single strand, it is specifically hydrolyzed by the signal amplifier enzyme, and the fluorescent groups at both ends of the probe are separated from the quenching groups to generate a fluorescent signal. Compared with various isothermal amplification methods such as LAMP and RPA, EmDEA nucleic acid rapid detection technology can not only achieve exponential amplification of target nucleic acids under constant temperature conditions of 42°C, but also use fluorescent probes for signal amplification, so it has extremely high detection accuracy and efficiency. At present, this technology has been applied to the detection of some pathogenic microorganisms, such as the published patents "Method for detecting tea cup fungus and its application, application number: 202410356936.1", "Rapid detection method of cocoa gall pathogen by enzyme-mediated dual exponential amplification nucleic acid detection technology, application number: 202410356937.6", "Composition for detecting azalea wilt pathogen and its application, application number: 202410356938.0", "Method for detecting grape stem blight pathogen and composition used, 202410356941.2". However, there is no report on the application of EmDEA nucleic acid rapid detection technology to amylovora. Summary of the invention

[0005] The technical problem to be solved by the present application is: how to quickly detect or identify the amylovora blight pathogen.

[0006] In order to solve the above technical problems, the present application provides a composition for specifically amplifying Erwinia amylovora (Latin name: Erwinia amylovora), the composition comprising a primer pair for specifically amplifying a specific DNA fragment of Erwinia amylovora, the primer pair consisting of primer Ea-F3 and primer Ea-R1, the primer Ea-F3 is a single-stranded DNA molecule whose nucleotide sequence is shown in SEQ ID No.1; the primer Ea-R1 is a single-stranded DNA molecule whose nucleotide sequence is shown in SEQ ID No.2.

[0007] Furthermore, the composition also includes RNA probe Ea-RNA3, and the nucleotide sequence of the RNA probe is SEQ ID No.3.

[0008] Furthermore, in the composition, the 5' end of the RNA probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

[0009] Furthermore, the fluorescent reporter group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas Red.

[0010] Furthermore, the quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB or Dabcy1.

[0011] Furthermore, the fluorescent reporter group can be labeled at the 5' end of the RNA probe.

[0012] Furthermore, the quencher group may be labeled at the 3' end of the RNA probe.

[0013] Furthermore, in the combination, the fluorescent reporter group of the RNA probe is FAM.

[0014] Furthermore, in the combination, the quenching group of the RNA probe is BHQ1.

[0015] The composition of the present invention can be used to detect samples of host plants susceptible to fire blight.

[0016] In some examples, the sample contains or may contain Erwinia amylovora.

[0017] In some examples, the sample may be various organs or tissues of a host plant susceptible to fire blight.

[0018] In some examples, the sample may also be a microbial culture.

[0019] The present application also provides an application, which is the application of the above composition in the following A1)-A6):

[0020] A1) Application in detecting or assisting in detecting Erwinia amylovora;

[0021] A2) Use in the preparation of a product for detecting or assisting in the detection of Erwinia amylovora;

[0022] A3) Application in identification or auxiliary identification of Erwinia amylovora;

[0023] A4) Use in the preparation of a product for identifying or assisting in the identification of Erwinia amylovora;

[0024] A5) Application in identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora;

[0025] A6) Use in the preparation of a product for identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora.

[0026] Furthermore, in the application, the product may be a reagent and / or a kit.

[0027] The present application also provides a kit for detecting Erwinia amylovora, wherein the kit comprises the above-mentioned composition.

[0028] Furthermore, the kit also includes detection reagents required for enzyme-mediated dual amplification nucleic acid detection technology (EmDEA).

[0029] Furthermore, the detection reagents required for the enzyme-mediated dual amplification nucleic acid detection technology include basic dry powder and activator. The basic dry powder includes the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplifier enzyme freeze-dried products and freeze-dried protective agents. The activator can be NTP (nucleoside triphosphate) and buffer.

[0030] Furthermore, the kit may also include a nucleic acid extraction reagent, which is used to extract nucleic acid from a biological sample (test sample) from a subject.

[0031] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0032] Furthermore, the kit may also include a readable carrier recording the method for detecting Erwinia amylovora described herein. The readable carrier may be a kit instruction sheet (e.g., a printed instruction sheet) for practicing the method of the present invention or a computer-readable medium (e.g., a floppy disk, a CD, etc.) on which information has been recorded.

[0033] The kit may be a detection kit based on enzyme-mediated dual amplification nucleic acid detection technology (EmDEA).

[0034] The present application also provides the use of the above-mentioned kit in any of the following items:

[0035] A1) Application in detecting or assisting in detecting Erwinia amylovora;

[0036] A2) Application in identification or auxiliary identification of Erwinia amylovora;

[0037] A3) Application in identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora.

[0038] The present application also provides a method for detecting Erwinia amylovora, the method comprising the following steps:

[0039] B1) extracting DNA from the sample to be tested;

[0040] B2) using the DNA as a template, performing enzyme-mediated dual amplification nucleic acid detection using the composition of the present invention or the kit of the present invention to form an amplification curve;

[0041] B3) Determine the test result based on the Tt value.

[0042] Furthermore, the detection method described in B2) comprises: adding the composition (primer Ea-F3, primer Ea-R1 and RNA probe Ea-RNA3), template DNA, and activation solution (a solution composed of NTP (nucleoside triphosphate) and buffer) to the basic dry powder (a lyophilized product containing DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplifier enzyme, and a lyophilized protective agent) to carry out an enzyme-mediated dual amplification nucleic acid detection reaction.

[0043] Furthermore, the reaction conditions for enzyme-mediated dual amplification nucleic acid detection in B2) may be: the temperature of the handheld fluorescence detector is set to 42° C., one cycle is performed for 30 seconds, a total of 40 cycles (20 minutes), and a signal is collected once in each cycle.

[0044] Further, B3) is: within 40 cycles (20 minutes), the positive control should have a typical amplification curve and a Tt value ≤ 10, the blank control should have no fluorescence and Tt value, and on the premise that the positive control and the blank control are correct: if the machine detects no Tt value within 20 minutes, the test result is judged to be negative, indicating that the amylovora is not present; if the Tt value appears, the test result is judged to be positive, indicating that the amylovora is present.

[0045] Furthermore, the smaller the Tt value and the larger the peak value, the better the detection effect or the higher the DNA concentration.

[0046] The sample may be various organs or tissues from a host plant susceptible to fire blight.

[0047] The sample may also be a culture of microorganisms.

[0048] Compared with the prior art, the present invention has the following beneficial technical effects:

[0049] 1. High specificity: Through specific detection, it can be found that the primer-probe combination of the present invention has no cross-reaction with common plant pathogens and hosts of other species except the amylovora species, and has high specificity.

[0050] 2. High sensitivity: The primer and probe combination of the present invention has higher sensitivity than other primers and probes, and can detect 1.5 copies / μL of plasmid template or 100 fg / μL of DNA template.

[0051] 3. Quick detection: Rapid nucleic acid preparation can be completed within 10 minutes, nucleic acid detection can be completed within 20 minutes, and detection can be completed within half an hour.

[0052] 4. Easy to operate and suitable for on-site detection: The present invention does not require precise temperature-changing equipment, and the target gene can be effectively amplified under constant temperature conditions; the interpretation of the amplification results is performed by a handheld fluorescence detector (which can be powered by a lithium battery), which not only realizes the visualization of the results, but also avoids false positive results caused by aerosols generated by opening the cover; the entire amplification and detection process can be completed within 20 minutes, greatly shortening the detection time of conventional PCR, Realtime PCR, and conventional RPA. At the same time, the kit of the present invention is combined with freeze-drying technology and can be stored at 4°C. The storage conditions are relatively simple, and the handheld fluorescence detector can be powered by a lithium battery, so it is very suitable for on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shown are the results of the RNA probe for Erwinia amylovora compared with EmDEA.

[0054] Figure 2 Shown is the comparison of the EmDEA detection results of the upstream / downstream primer combinations of the amylovora DNA.

[0055] Figure 3 Shown are the results of specific detection of EmDEA of Erwinia amylovora.

[0056] Figure 4 The results are shown for the sensitivity test of EmDEA for Erwinia amylovora. Figure A shows the sensitivity test of EmDEA using a 10-fold gradient dilution of a plasmid as a DNA template, and Figure B shows the sensitivity test of EmDEA using a 10-fold gradient dilution of genomic DNA as a template.

[0057] Figure 5 Shown are the results of EmDEA testing of diseased samples from the field. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with specific embodiments.

[0059] Those skilled in the art will appreciate that these embodiments are only used to illustrate the present invention and do not constitute any limitation to the scope of the present invention. Unless otherwise specified, all scientific and technological terms in this application have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0060] Test culture medium: NB medium: peptone 10g / L, beef extract 3g / L, NaCl 5g / L, pH 7.2.

[0061] Test drugs: Bacterial genomic DNA extraction kit (TIANamp Bacteria DNA Kit), pMD TM 19T, TIANgel Midi Purification Kit, and TIANprep Mini Plasmid Kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. Basic fluorescence isothermal amplification detection kit (Cat. No. RR032) and DNA lysis buffer were purchased from Suzhou Jingrui Biotechnology Co., Ltd.

[0062] Instruments and equipment: Palm fluorescence detector (model uReader 1600, Suzhou Jingrui Biotechnology Co., Ltd.), nucleic acid / protein detector (NanoDrop TM One, Thermo).

[0063] All reagents and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.

[0064] Example 1 Design of test strains and primers and probes

[0065] 1.1 Test strains

[0066] There are 20 test strains (Table 1) involved in the present invention, including 16 bacterial strains and 4 fungal strains, all of which are stored in the Agricultural Microbiology and Biotechnology Laboratory of Xinjiang Agricultural University for a long time. All test strains can be obtained from the applicant to repeat the experiments of the present invention.

[0067] Table 1 Test strains

[0068] Strain number Species name Latin name E.a001 Erwinia amylovora Erwinia amylovora E.a015 Erwinia amylovora Erwinia amylovora E.a017 Erwinia amylovora Erwinia amylovora E.a018 Erwinia amylovora Erwinia amylovora E.a020 Erwinia amylovora Erwinia amylovora Pcc Chinese cabbage soft rot pathogen Pectobacterium carotovorum subsp.carotovorum Pss Bacterial leaf spot of pepper Pseudomonas syringae pv.syringae Df Pear rust pathogen Dickeya Fangzhongda FC440 Bacterial fruit spot of watermelon Acidovorax citrulli CL112 Erwinia amylovora Erwinia pyrifoliae PSL Cucumis sativa L. Pseudomonas amygdali pv.lachrymans ECZ Bacterial rot of rice Erwinia chrysanthemi pv.zeae Pst Pseudomonas syringae Pseudomonas syringan pv.tabaci Psg Soybean bacterial spot Pseudomonas syringae pv.glycinea DH5α Escherichia coli Escherichia coli FX1 Bacillus Velez Bacillus velezensis NC40 Colletotrichum sphaeroides Colletrichum gloeosporioides VpK Pear rot Valsa pyri HB55 Alternaria obesa Alternaria alternata JR19 Pear ring spot Botryosphaeria dothidea

[0069] 1.2 Synthesis of different primers and probes

[0070] The present invention selects and refers to the hypothetical protein gene sequence of Erwinia amylovora published in GenBank (Accession No.: NC_013961), and designs DNA primers and RNA probes, wherein the DNA primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the RNA probes are provided by Suzhou Jingrui Biotechnology Co., Ltd. The specific sequences of the primers and probes are shown in Table 2.

[0071] Table 2 Primer and probe sequences

[0072]

[0073]

[0074] Note: The 5' end of the RNA probe is labeled with the reporter group FAM, and the 3' end is labeled with the quencher group BHQ1. The underlined sequence in Table 1 is the RNA polymerase promoter sequence. The U in the RNA probe sequence is represented by T in the sequence table.

[0075] Example 2 Comparison of probes and primers

[0076] 2.1 DNA extraction

[0077] The amylovora strain E.a001 was inoculated into 100 mL NB medium, cultured overnight at 30°C and 160 rpm, and 1 mL of bacterial solution was centrifuged at 12000 rpm for 1 min to collect the bacteria, and genomic DNA was extracted using a bacterial genomic DNA extraction kit. After the DNA concentration was determined using a nucleic acid / protein detector, the extracted genomic DNA was adjusted to a concentration of 100 pg / μL using sterile deionized water and stored at -20°C for later use.

[0078] 2.2 RNA probe comparison

[0079] The ultraRADAR fluorescence isothermal amplification detection blank kit (Cat. No. RR032) of Suzhou Jingrui Biotechnology Co., Ltd. was used for the experiment. The reaction system was 20 μL: 1.0 μL of upstream primer and downstream primer at a concentration of 10 μmol / L, 1.0 μL of RNA probe at 1.0 μmol / L, and 7.0 μL of DNA template were added to the eight-tube containing basic dry powder (including the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplifier enzyme freeze-dried products and freeze-dried protective agents, components in the basic fluorescence isothermal amplification detection kit), and the mixture was allowed to stand at room temperature for 2 minutes; then 10 μL of activation solution (a solution composed of NTP (nucleoside triphosphate) and buffer, a component in the basic fluorescence isothermal amplification detection kit) was added, the lid was covered, the mixture was shaken and mixed for 10 seconds, and the mixture was placed in a handheld fluorescence detector for detection after instant centrifugation. The temperature of the handheld fluorescence detector was set to 42°C, and one cycle was performed for 30 seconds, and a signal was collected once in each cycle.

[0080] Specifically, the DNA of E.a001 of E. amylovora (concentration of 100 pg / μL) was used as the amplification template of the test group. Three upstream primers Ea-F1, Ea-F2, Ea-F3 and three downstream primers Ea-R1, Ea-R2, Ea-R3 were taken for cross-combination test, and 9 groups of DNA primer combinations Ea-F1 / Ea-R1, Ea-F1 / Ea-R2, Ea-F1 / Ea-R3, Ea-F2 / Ea-R1, Ea-F2 / Ea-R2, Ea-F2 / Ea-R3, Ea-F3 / Ea-R1, Ea-F3 / Ea-R2, Ea-F3 / Ea-R3 were obtained, and three RNA probes Ea-RNA1, Ea-RNA2, Ea-RNA3 were added to each primer combination, for a total of 27 test combinations (Table 3). The test was performed according to the above reaction system and reaction procedure, and 3 replicates were set for each combination in the test group. The test results of 27 combinations were compared.

[0081] The comparison results of RNA probes are shown in Table 3 and Figure 1 As shown, it can be seen that the Tt value of the RNA3F3R1 combination of the present invention is relatively low, which is 6.83, and the end point fluorescence value is the highest, indicating that the RNA probe Ea-RNA3 of the present invention has the best detection effect.

[0082] Table 3 RNA probe comparison results

[0083]

[0084]

[0085] 2.3 Comparison of primer combinations

[0086] Using the DNA of E. amylovora amylovora E.a001 as the amplification template (DNA concentration of 100 pg / μL), and Ea-RNA3 as the probe, the six upstream primers Ea-F1 to Ea-F6 and the six downstream primers Ea-R1 to Ea-R6 were further cross-combined, with a total of 36 combinations (see Table 4). The detection was performed according to the reaction system and reaction procedure in 2.2, and 3 replicates were set for each combination. The detection results of the 36 combinations were compared.

[0087] The comparison results of primer combinations are shown in Table 4 and Figure 2 As shown, among the 36 combinations, the RNA3F3R1 combination of the present invention has a lower Tt value of 6.83 and the highest endpoint fluorescence value, indicating that the primer-probe combination of the RNA probe Ea-RNA3, the upstream primer Ea-F3 and the downstream primer Ea-R1 of the present invention has the best effect.

[0088] Table 4 DNA primer combination comparison results

[0089]

[0090]

[0091] Example 3 Specificity and sensitivity verification

[0092] 3.1 Specificity Verification

[0093] Using the test strain DNA in Table 1 as a template, the total DNA of healthy leaves of Pyrus communis, Malus, and Pyrus betulaeformis as a negative control, and sterile deionized water as an amplification template (NTC) for the blank control group, the primer-probe combination RNA3F3R1 of Example 2 (i.e., the primer-probe combination of RNA probe Ea-RNA3, upstream primer Ea-F3, and downstream primer Ea-R1) was detected using a handheld fluorescence detector with reference to the reaction system and reaction procedure described in Example 2 to test its specificity.

[0094] The specificity verification results are as follows Figure 3 As shown, all the tested strains of Erwinia amylovora can be detected by RNA3F3R1 (i.e., the primer-probe combination of RNA probe Ea-RNA2, upstream primer Ea-F3, and downstream primer Ea-R1) (the amplified fragment is shown in SEQ ID NO.4), while other tested strains and total DNA of healthy plants cannot be detected, indicating that the primer-probe combination RNA3F3R1 can better detect Erwinia amylovora in actual detection, and does not cross-react with common plant pathogens and hosts of other species except Erwinia amylovora species, and has high specificity.

[0095] 3.2 Sensitivity Verification

[0096] 3.2.1 Preparation of standard plasmid

[0097] The PCR primers were designed based on the hypothetical protein gene sequence of Erwinia amylovora. The primer sequences were hp-F1: 5'-TATTCTCAGTGGTCAAAGCC-3'; hp-R1: 5'-TATTCTCAGTGGTCAAAGCC-3'. The DNA of strain E.a001 was used as a template, and the primers hp-F1 / hp-R1 were used for PCR amplification to obtain an amplified fragment size of 855 bp. The specific bands in the PCR amplification product were cut from the gel, recovered using an agarose gel recovery kit, and purified. The recovered PCR product was mixed with the vector pMD TM-19T was connected at 16°C for 2h. The ligation product was transformed into DH5α competent cells and cultured at 37°C for 14-16h on LB agar plate medium containing X-Gal (20g / L), IPTG (50g / L), and Amp (100mg / L) to form single colonies. The selected positive single colonies were inoculated into LB liquid culture medium containing Amp (100mg / L), cultured overnight at 37°C, and the plasmid was extracted. The sequencing identification was performed by the sequencing department of Sangon Biotech (Shanghai) Co., Ltd. to confirm that the target gene had been correctly cloned into the vector. The plasmid concentration was determined using a micro-UV-visible spectrophotometer, and the plasmid concentration was calculated according to the formula to be 1.5×10 11 copies / μL, calculation formula: 6.02×10 23 ×Plasmid DNA mass concentration (ng / μL)×10 -9 / (plasmid DNA length × 660). Store at -20℃ for future use.

[0098] 3.2.2 Sensitivity verification method

[0099] The standard plasmid (1.5×10 11 The DNA of strain E.a001 (concentration of 10 ng / μL) was diluted 10-fold with sterile deionized water. 6 copies / μL, 1.5×10 5 copies / μL, 1.5×10 4 copies / μL, 1.5×10 3 copies / μL, 1.5×10 2 The plasmids with 7 concentration gradients of 100 copies / μL, 1.5×10 copies / μL, and 1.5 copies / μL and the DNA with 7 concentration gradients of 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL were used as templates, and sterile deionized water was used as the amplification template (NTC) of the blank control, wherein 3 replicates were set for each test group and 1 replicate was set for the blank control group. The primer-probe combination RNA3F3R1 of Example 2 (i.e., the primer-probe combination of RNA probe Ea-RNA3, upstream primer Ea-F3, and downstream primer Ea-R1) was used with reference to the reaction system and reaction procedure described in Example 2, and the sensitivity was determined by using a handheld fluorescence detector for detection.

[0100] 3.2.2 Sensitivity Verification Results

[0101] The sensitivity verification results are as follows Figure 4As shown, the primer-probe combination RNA3F3R1 can detect 1.5 copies / μL of plasmid template and 100 fg / μL of DNA template.

[0102] Example 4 Establishment and application of a method for detecting Erwinia amylovora based on enzyme-mediated dual amplification nucleic acid detection technology

[0103] 4.1 DNA extraction

[0104] About 100 mg of the sample to be tested was placed in a 2 mL centrifuge tube. 500 μL of lysis solution (the lysis solution was produced by Suzhou Jingrui Biotechnology Co., Ltd.) and 2 stainless steel grinding beads (5 mm in diameter) were added to the centrifuge tube. The plant tissue was ground by hand-shaking the centrifuge tube for 10 seconds and heated at 95°C for 10 minutes to obtain a DNA template.

[0105] 4.2 Detection steps based on enzyme-mediated dual amplification nucleic acid detection technology

[0106] 1.0 μL of upstream primer (Ea-F3) and downstream primer (Ea-R1) at a concentration of 10 μmol / L, 1.0 μL of 1.0 μmol / L RNA probe Ea-RNA3, a total of 3 μL, were added to an eight-tube containing basic dry powder (including enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, freeze-dried products of reverse transcriptase and signal amplifier enzyme and freeze-dried protective agent, and components of the basic fluorescent constant temperature amplification detection kit (Suzhou Jingrui Biotechnology Co., Ltd., catalog number RR032)), and centrifuged instantly; after centrifugation, 7 μL of DNA, centrifuge instantaneously, and stand at room temperature for 2 minutes; add 10 μL of activation solution (a solution composed of NTP (nucleoside triphosphate) and buffer, a component of the basic fluorescence constant temperature amplification detection kit (Suzhou Jingrui Biotechnology Co., Ltd., Cat. No. RR032)) along the middle and lower part of the wall of the eight-tube, cover the lid, shake and mix for 10 seconds, centrifuge instantaneously, and put it into a handheld fluorescence detector (Suzhou Jingrui Biotechnology Co., Ltd., model uReader 1600) for detection. The temperature of the handheld fluorescence detector was set to 42°C, 30 seconds for one cycle, and a signal was collected once in each cycle. At the same time, a positive control was set (using the standard plasmid of amylovora amylovora as a template, with a concentration of 1.5×10 5 copies / μL) and blank control (sterile deionized water was used as the amplification template of the blank control)

[0107] 4.3 Result determination

[0108] The results of EmDEA detection are determined by the fluorescence curve and Tt value (the time when the fluorescence value exceeds the internal threshold). The positive control should have a typical amplification curve and a Tt value ≤ 10, and the blank control should have no fluorescence and Tt value. Under the premise that the positive control and blank control are correct: if the machine detects no Tt value within 20 minutes, the test result is determined to be negative, and if a Tt value appears, the test result is determined to be positive, and the smaller the Tt value and the larger the peak value, the higher the DNA concentration in the sample or the higher the pathogen content in the sample.

[0109] 4.4 Testing of actual samples

[0110] A total of 18 samples of pear, hawthorn and crabapple infected by E. amylovora were collected in Korla, Urumqi, Xinjiang, and Zhangye, Gansu. The sample DNA was extracted as a template according to the method described in 4.1, Ea-RNA3 was used as the RNA probe, Ea-F3 and Ea-R1 were used as the upstream and downstream DNA primer combinations, and EmDEA detection was performed according to the steps in 4.2. The standard plasmid of E. amylovora was used as the positive control, and sterile deionized water was used as the blank control (NTC) to determine the effect of this method in actual sample detection.

[0111] From Table 5 and Figure 5 It can be seen that in the actual sample testing, the DNA and positive control tests of crabapple, hawthorn and pear samples infected with fire blight were all positive, while the blank control test results were negative, indicating that this method can be used to detect whether plants are infected with fire blight.

[0112] Table 5 Test results of actual field samples

[0113]

Claims

1. A composition for specifically amplifying Erwinia amylovora, characterized in that: The composition includes a primer pair for specifically amplifying a specific DNA fragment of the amylovora blight pathogen, the primer pair consists of primer Ea-F3 and primer Ea-R1, the primer Ea-F3 is a single-stranded DNA molecule whose nucleotide sequence is shown in SEQ ID No.1; the primer Ea-R1 is a single-stranded DNA molecule whose nucleotide sequence is shown in SEQ ID No.2; preferably, the composition also includes an RNA probe Ea-RNA3, and the nucleotide sequence of the RNA probe is SEQ ID No.

3.

2. The composition according to claim 1, characterized in that The 5' end of the RNA probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. Preferably, the fluorescent reporter group is labeled at the 5' end of the RNA probe, and the quencher group is labeled at the 3' end of the RNA probe; preferably, the fluorescent reporter group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas Red, and more preferably, the fluorescent reporter group of the RNA probe is FAM; preferably, the quencher group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB or Dabcy1; more preferably, the quencher group of the RNA probe is BHQ1.

3. A kit comprising the composition according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit is a detection kit based on enzyme-mediated dual amplification nucleic acid detection technology.

5. The kit according to claim 3, characterized in that The kit also includes detection reagents required for enzyme-mediated dual amplification nucleic acid detection technology; preferably, the detection reagents required for enzyme-mediated dual amplification nucleic acid detection technology include basic dry powder and activator; preferably, the basic dry powder includes enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplifier enzyme lyophilized product and lyophilization protectant; preferably, the activator is NTP (nucleoside triphosphate) and buffer.

6. The kit according to claim 3, characterized in that The kit may also include a nucleic acid extraction reagent, which is used to extract nucleic acid from a biological sample from a subject; preferably, the various reagent components of the kit are present in separate containers, or are pre-combined in whole or in part into a reagent mixture; preferably, the kit may also include a readable carrier recording a method for detecting fire blight; preferably, the readable carrier is a kit instruction manual or a computer-readable medium on which information has been recorded.

7. Use of the composition according to claim 1 or 2, characterized in that: The application is any of the following applications A1)-A6): A1) Application in detecting or assisting in detecting Erwinia amylovora; A2) Use in the preparation of a product for detecting or assisting in the detection of Erwinia amylovora; A3) Application in identification or auxiliary identification of Erwinia amylovora; A4) Use in the preparation of a product for identifying or assisting in the identification of Erwinia amylovora; A5) Application in identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora; A6) Application in the preparation of a product for identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora.

8. Use of the kit according to any one of claims 3 to 6, characterized in that: The application is any one of the following A1), A3) or A5): A1) for detecting or assisting in detecting Erwinia amylovora; A3) Application in identification or auxiliary identification of Erwinia amylovora; A5) Application in identifying or assisting in identifying whether a sample to be tested is or contains Erwinia amylovora.

9. A method for detecting Erwinia amylovora, characterized in that: The method comprises the following steps: B1) extracting DNA from the sample to be tested; B2) using the DNA as a template, performing enzyme-mediated dual amplification nucleic acid detection using the composition of claim 3 or 4 or the kit of any one of claims 5 to 7 to form an amplification curve; B3) Determine the test result based on the Tt value, provided that the positive control and blank control are correct: if the machine detects no Tt value within 20 minutes, the test result is considered negative, indicating that the organism does not contain the amylovora pathogen; if the Tt value appears, the test result is considered positive, indicating that the organism contains the amylovora pathogen.

10. The method according to claim 9, characterized in that B2) The detection method described in the above-mentioned step comprises: in a solution containing DNA recombinase, shrimp alkaline phosphatase, T7 An activation solution consisting of the composition according to claim 3 or 4, template DNA, NTP (nucleoside triphosphate) and buffer is added to the freeze-dried products of RNA polymerase, reverse transcriptase and signal amplifier and the basic dry powder of freeze-dried protective agent to carry out enzyme-mediated dual amplification nucleic acid detection reaction; preferably, the reaction conditions for enzyme-mediated dual amplification nucleic acid detection in B2) can be: the temperature of the handheld fluorescence detector is set to 42°C, one cycle is carried out for 30 seconds, a total of 40 cycles, and a signal is collected once in each cycle; preferably, B3) is: within 40 cycles, the positive control should have a typical amplification curve and a Tt value ≤ 10, the blank control should have no fluorescence and Tt value, and on the premise that the positive control and the blank control are correct: if the machine detects no Tt value within 20 minutes, the test result is judged to be negative, indicating that the amylovora pathogen is not present; if the Tt value appears, the test result is judged to be positive, indicating that the amylovora pathogen is present; preferably, the sample to be tested is various organs or tissues or microbial cultures of a host plant susceptible to amylovora pathogen.

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