RPA (recombinase polymerase amplification) primer pair, kit and method for detecting meloidogyne zeylanica

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to an RPA primer pair, a kit and a method for detecting elephant trunk bean root-knot nematode. According to the invention, rDNA-ITS of M. enterolobii is taken as a new target, a group of RPA primer pairs with strong specificity are obtained through optimization, when the RPA primer pairs are combined with an Exo probe and a real-time fluorescence detection technology is applied, strong fluorescence signals can be obtained within 20 minutes, the detection sensitivity can reach DNA of 10-4 female insects, and the RPA primer pairs have the characteristics of strong specificity, high sensitivity, simplicity and convenience in operation and the like, and can be applied to the field of fluorescence detection. The method is suitable for the detection of the elephant trunk bean root-knot nematode in a basic laboratory, and enriches the molecular detection means of the elephant trunk bean root-knot nematode.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid detection, and particularly relates to an RPA primer pair, a kit and a method for detecting root-knot nematodes of elephant ear beans. Background Art

[0002] Meloidogyne is one of the most serious plant parasitic nematode species that harms plants. 70% of the economic losses caused by plant parasitic nematode hosts to crops are caused by root-knot nematodes. There are many types of root-knot nematodes and they are widely distributed. At present, more than 105 effective species have been reported in this genus. The elephant ear bean root-knot nematode (Meloidogyne enterolobii) was first discovered and named on the elephant ear bean tree (Enterolobium sp.) in Danzhou City, Hainan Province, my country. After morphological characteristics and gene sequence comparison analysis, it is generally recognized that the two are synonyms of the Maya root-knot nematode (M.mayaguensis) discovered and named in Puerto Rico later. At present, this nematode is distributed in many countries in Asia, Africa, Europe, North and South America, and Oceania. The elephant ear bean root-knot nematode has a wide range of hosts. Because it can overcome the resistance of the host's Mi, N and other resistance genes, it has a high pathogenicity to a variety of crops and can cause large yield losses. Therefore, the European and Mediterranean Plant Protection Organization (EPPO) has regulated it as a Class A2 pest, and the United States, Japan, South Korea and other countries have listed it as a quarantine pest. The morphological identification of root-knot nematodes is complicated and requires the identification personnel to have a certain foundation in nematology. Therefore, it is necessary to develop a rapid and portable detection technology for elephant ear bean root-knot nematodes to achieve early monitoring of elephant ear bean root-knot nematodes and provide strong technical support for early warning and early prevention and control of diseases.

[0003] Compared with morphological, isoenzyme and other identification methods, molecular detection technology has gradually developed into the main means of identifying root-knot nematode species because it is not affected by external environmental conditions, nematode stage and gene expression. At present, based on the differences in IGS, mtDNA, ITS and other sequences of Meloidogyne enterolobii, PCR-RFLP (Xu JH, et al. Characterization of Meloidogyne species from China using isozyme phenotypes and amplified mitochondrial DNA restriction fragment length polymorphism. European Journal of Plant Pathology, 2004, 110 (3): 309-315.), PCR (Blok VC, et al. Comparison of sequences from the ribosomal DNA intergenic region of Meloidogyne mayaguensis and other major tropical root-knot nematodes. Journal of Nematology, 1997, 29: 16-22.; Tigano M, et al. Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for the guava-damaging species. Plant Pathology, 2010, 59(6): 1054-1061.), real-time fluorescence PCR (Kiewnick, S., et al. Development and validation of LNA-based quantitative realtime PCRassays for detection and identification of the root knot nematode Meloidogyneenterolobiiin complex DNA backgrounds. Phytopathology, 2015, 105: 1245-1249.), loop-mediated isothermal amplification (Niu JH, et al.Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots. Crop Protection, 2011, 30(8): 1063-1069.; He Xufeng, et al. Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots. Crop Protection, 2011, 30(8): 1063-1069.; He Xufeng, et al. Rapid detection of Meloidogyne spp. by LAMP in diseased plant tissues. Chinese Agricultural Science, 2013, 46(003): 534-544.) and other specific detection technologies are emerging in an endless stream.

[0004] In recent years, nucleic acid isothermal amplification detection technology has attracted much attention due to its short reaction time, simple operation, and low requirements for instruments and equipment. Among them, recombinase polymerase (RPA) nucleic acid isothermal amplification technology has been widely used in the rapid detection of plant nematodes due to its advantages such as low reaction temperature (37-42°C) and fast reaction time (20min). Subbotin established an RPA rapid detection system based on the IGS sequence differences of the root-knot nematode Meloidogyne enterolobii (Subbotin S A. Recombinase polymerase amplification assay for rapid detection of the root-knot nematode Meloidogyne enterolobii. Nematology, 2019, 21: 243-251.). Chi et al., Subbotin et al. established the RPA-Exo detection system for Javan root-knot nematode (M. javanica) and northern root-knot nematode (M. hapla) (Chi Y, et al. Evaluation of recomb inase polymerase amplification assay for detecting Meloidogyne javanica. Plant disease, 2020, 104(3): 801-807.; Subbotin SA, et al. Sensitive, accurate and rapid detection of the northern root-knot nematode, Meloidogyne hapla, using recombinase polymerase amplification assays. Plants, 2021, 10: 336.). Lin Yu et al., Ma Jukui et al. established the RPA electrophoresis detection system for Aphelenchoides ritzem abosi and Ditylenchus destructor (Lin Yu, et al. Research on RPA detection method of Aphelenchoides ritzem abosi. Hunan Agricultural Sciences, 2021(1): 1-3, 10.; Ma Jukui, et al. Establishment and application of recombinase polymerase amplification method for sweet potato Ditylenchus destructor. Journal of Southwest Agriculture, 2021, 34(02): 293-298.).Yao et al. established the RPA-CRISPR / CAS12a detection system for beet cyst nematode (Heterodera schachtii) (Yao, K. et al. Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology. Int. J. Mol. Sci. 2021, 22, 12577.). Gao Bo et al., Wang et al. established the RPA-LFD detection system for potato destructor stem nematode (D. destructor) and potato golden nematode (G. rostochiensis) (Gao Bo, et al. Establishment of RPA-LFD rapid visualization detection method for potato destructor stem nematode. Acta Phytopathologica Sinica, 2022, 52(06): 984-992.; Wang, X. et al. Rapid Diagnosis and Visual Detection of Potato Cyst Nematode (Globodera rostochiensis) Using Recombinase Polymerase Amplification Combination with Lateral Flow Assay Method (RPA-LFA). Agronomy 2022, 12, 2580.). Summary of the invention

[0005] The present invention first provides an RPA primer pair for detecting Meloidogyne enterolobii. The RPA primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is as follows: 5'-GTTTTGCGAACTGCAGCAGCCTTGAGCATAA-3'; the nucleotide sequence of the downstream primer is as follows: 5'-CCACAAAAGCAATTATACAATAAAGC-3'.

[0006] The present invention also provides an RPA primer-probe combination, which comprises: the primer pair and the probe; the nucleotide sequence of the probe is as follows: 5'-AACCCTTTGCTATGTCTGGTTCAGGGTCAT / fluorescent group-dT / / basic spacer / / fluorescent quencher group-dT / CTTTCAAAGCGA / alkyl carbon spacer / -3'.

[0007] The present invention also provides a reagent, which contains the RPA primer pair or the RPA primer-probe combination.

[0008] The present invention also provides a kit, which contains the RPA primer pair, the RPA primer-probe combination or the reagent.

[0009] The present invention also provides the use of the RPA primer pair, the RPA primer probe combination, the reagent or the kit in detecting root-knot nematodes of elephant ear beans.

[0010] The present invention also provides a method for detecting root-knot nematodes of elephant ear beans, which comprises: using the RPA primer pair, the RPA primer-probe combination, the reagent or the kit to perform RPA amplification on the nucleic acid of the sample to be tested, and analyzing the reaction result.

[0011] The present invention uses rDNA-ITS of the root-knot nematode M. enterolobii as a new target and optimizes a set of RPA primer pairs with strong specificity. When the primers are combined with Exo probes and real-time fluorescence detection technology is applied, a strong fluorescence signal can be obtained in as low as 20 minutes, and the detection sensitivity can reach 10 -4 The DNA of the female worm has the characteristics of strong specificity, high sensitivity and simple operation. It is suitable for grassroots laboratories to carry out the detection of elephant ear bean root-knot nematodes, enriching the molecular detection methods of elephant ear bean root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 This is an agarose gel electrophoresis diagram for screening RPA primers for root-knot nematode of elephant ear bean in an example of the present invention; in the diagram, M: DL2000 Marker; 1: ME1F / ME1R; 2: ME1F / ME2R; 3: ME1F / ME3R; 4: ME2F / ME2R; 5: ME2F / ME3R; 6: ME3F / ME3R; 7: blank control.

[0014] Figure 2The sites for designing the RPA-specific primers and fluorescent probes for the elephant ear bean root-knot nematode in the embodiments of the present invention are shown in the figure. In the figure, MH756128.1: elephant ear bean root-knot nematode M. enterolobii; MT951634.1: northern root-knot nematode M. hapla; KT258981.1: southern root-knot nematode M. incognita; KJ572384.1: peanut root-knot nematode M. arenaria; KX646187.1: Java root-knot nematode M. javanica; MK212184.1: short root-knot nematode M. exigua.

[0015] Figure 3 It is the reaction time optimization result of the RPA detection system of the root-knot nematode of the elephant ear bean in the embodiment of the present invention; In this figure, M: DL2000 Marker; 1-5 correspond to the detection results of reaction times of 20min, 25min, 30min, 35min, and 40min, respectively; NC: negative control.

[0016] Figure 4 The results of reaction temperature optimization of the RPA detection system for root-knot nematodes of the bean ear in the embodiment of the present invention are shown in the figure. In the figure, M: DL2000 Marker; 1: 41°C; 2: 40°C; 3: 39°C; 4: 38°C; 5: 37°C; NC: negative control.

[0017] Figure 5 : The specificity verification result of the RPA detection system of the elephant ear bean root-knot nematode in the embodiment of the present invention; in the figure, M: DL2000 Marker; 1-3 correspond to sample numbers MeHN1, MeHN2, and MeGX, respectively, which are all elephant ear bean root-knot nematodes; 4-5 correspond to sample numbers MiLN and MiLY, respectively, which are all southern root-knot nematodes; 6 corresponds to sample number MhLN, which is northern root-knot nematodes; 7-8 correspond to sample numbers MjBE1 and MjHN, respectively, which are all Javan root-knot nematodes; 9 corresponds to sample number MaNT, which is peanut root-knot nematodes; 10 corresponds to sample number MgJX, which is a root-knot nematode of the Poaceae family; 11 corresponds to sample number HgHLJ, which is a soybean cyst nematode; 12: blank control.

[0018] Figure 6 : This is the RPA-Exo detection result of the elephant ear bean root knot nematode in the example of the present invention; in the figure, A to C correspond to sample numbers MeHN1, MeHN2, and MeGX, respectively, all of which are elephant ear bean root knot nematodes; NC is a blank control.

[0019] Figure 7: The sensitivity results of RPA-Exo detection of the root-knot nematode of the elephant ear bean in the embodiment of the present invention; in the figure, A is the RPA detection result, wherein M: DL2000 Marker; 1 to 6 correspond to 1, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ; NC: blank control; B is the RPA-Exo detection result. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0021] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0023] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0024] The present invention relates to concentration values, which include fluctuations within a certain range. For example, it can fluctuate within a corresponding precision range (such as ±0.1%). For values ​​that are large or do not require too fine control, it is also allowed to include greater fluctuations, such as fluctuations within the range of ±1%, ±2%, ±5%, etc.

[0025] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0026] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0028] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0029] The present invention first provides an RPA primer pair for detecting Meloidogyne enterolobii, wherein the RPA primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is as follows: 5'-GTTTTGCGAACTGCAGCAGCCTTGAGCATAA-3' (SEQ ID No. 2); the nucleotide sequence of the downstream primer is as follows: 5'-CCACAAAAGCAATTATACAATAAAGC-3' (SEQ ID No. 5). The primer pair of the present invention can detect the elephant ear bean root knot nematode with high specificity and high sensitivity, and when applied to RPA detection, efficient and convenient detection can be achieved.

[0030] The present invention also provides an RPA primer-probe combination, which contains: the primer pair and the probe; the nucleotide sequence of the probe is as follows: 5'-AACCCTTTGCTATGTCTGGT TCAGGGTCAT / fluorescent group-dT / / basic spacer / / fluorescent quencher group-dT / CTTTCAA AGCGA / alkyl carbon spacer / -3'. The RPA primer-probe combination of the present invention has high specificity and high sensitivity in fluorescence detection, and can quickly detect the root-knot nematode of the elephant ear bean.

[0031] In some embodiments, the fluorescent group is selected from one of FAM (Fluorescein amidite), HEX (Hexachloro-fluorescein), TET (Tetrachlorofluorescein), Cyanine dye, ROX (Carboxy-X-rhodamine), and Texas Red.

[0032] In some embodiments, the abasic spacer is selected from one of THF (tetrahydrofuran) and dSp (dSpacer).

[0033] In some embodiments, the fluorescence quenching group is selected from one of BHQ (Black Hole Quencher), IowaBlack, and BBQ (Blackberry Quencher). In some specific embodiments, the fluorescence quenching group is selected from one of BHQ1, BHQ2, and BHQ3.

[0034] In some embodiments, the alkyl carbon spacer is selected from one of C2 Spacer, C3 Spacer, and C4 Spacer.

[0035] In some specific embodiments, the nucleotide sequence of the probe is as follows: 5'-AACCCTTTGCTATGTCTGGTTCAGGGTCAT / iHEXdT / / idSp / / iBHQ1dT / CTTTCAAAGCGA / C3 spacer / -3' (SEQ ID No. 7).

[0036] The present invention also provides a reagent, which contains the RPA primer pair or the RPA primer-probe combination.

[0037] In some specific embodiments, the reagent further contains a solvent for dissolving the primer pair or the primer-probe combination as described above, such as TE buffer (Tris-EDTA buffer) or other solvents suitable for DNA stabilization.

[0038] The present invention also provides a kit, which contains the RPA primer pair, the RPA primer-probe combination or the reagent.

[0039] In some embodiments, the kit further comprises: a recombinase, a single-stranded DNA binding protein, a strand displacement DNA polymerase, dNTPs, Mg 2+ At least one of a source, a buffer, and a positive standard.

[0040] In some specific embodiments, Mg is provided in the form of magnesium acetate. 2+ source.

[0041] In some specific embodiments, the kit further contains: a stabilizer and a preservative to ensure the stability of the kit during storage and use.

[0042] In some specific embodiments, the kit further comprises: a detergent to help reduce inhibitors in the sample and improve amplification efficiency.

[0043] In some embodiments, the kit further comprises: extraction and dilution buffers. If the kit is designed to detect directly from complex samples, it may contain buffers for sample lysis and DNA release.

[0044] In some specific embodiments, the kit further comprises: instructions for providing detailed operating steps, reaction conditions, usage guidelines and precautions.

[0045] The other components in the kit mentioned in the present invention can be obtained through commercial channels, and can be provided in multiple or one reagent alone or in combination. For example, in some embodiments, the recombinase, single-stranded DNA binding protein, and strand displacement DNA polymerase can be provided separately. In some embodiments, the kit also contains: an RPA enzyme mixture; the RPA enzyme mixture comprises a recombinase, a single-stranded binding protein (SSB) and a DNA polymerase.

[0046] The present invention also provides the use of the RPA primer pair, the RPA primer probe combination, the reagent or the kit in detecting root-knot nematodes of elephant ear beans.

[0047] The present invention also provides a method for detecting root-knot nematodes of elephant ear beans, which comprises: using the RPA primer pair, the RPA primer-probe combination, the reagent or the kit to perform RPA amplification on the nucleic acid of the sample to be tested, and analyzing the reaction result.

[0048] In specific implementation, the above-mentioned RPA amplification reaction can be combined with real-time fluorescence detection technology, agarose gel electrophoresis, lateral flow chromatography test strips, CRISPR / Cas system and other methods to achieve rapid detection of samples to be tested.

[0049] During specific implementation, those skilled in the art can confirm the reaction system and reaction conditions of RPA detection based on common sense.

[0050] In some embodiments, each 50 μL RPA reaction system contains 21±3 pmoL (such as 18 pmoL, 19 pmoL, 20 pmoL, 21 pmoL, 22 pmoL or 23 pmoL, etc.) of upstream primers, 21±3 pmoL (such as 18 pmoL, 19 pmoL, 20 pmoL, 21 pmoL, 22 pmoL or 23 pmoL, etc.) of downstream primers, and optionally 6±1 pmoL (such as 5 pmoL, 5.5 pmoL, 6 pmoL, 6.5 pmoL or 7 pmoL, etc.) of probes. Those skilled in the art can set the concentrations of upstream primers, downstream primers and probes in the RPA reaction system within the above range (or slightly deviate from the above range, especially greater than or equal to the upper limit) in combination with common sense, which also belongs to the protection scope of the present invention.

[0051] In some embodiments, the reaction temperature of the RPA amplification is 39±2° C. For example, the reaction temperature of the RPA amplification can be 37° C., 38° C., 39° C., 40° C., 41° C., etc.

[0052] In some embodiments, when using fluorescence detection technology, the reaction time of the RPA amplification is more than 5 minutes. As an example, the reaction time of the RPA reaction can be 5 minutes, 7 minutes, 9 minutes, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, or any value greater than or equal to 5 minutes.

[0053] In some embodiments, when the electrophoresis detection method is used, the reaction time of the RPA amplification is more than 20 minutes. As an example, the reaction time of the RPA reaction can be 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or any value greater than or equal to 20 minutes.

[0054] Based on comprehensive consideration of detection efficiency and detection effect, it is more preferred that the reaction time of the RPA amplification is 30±10 min.

[0055] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0056] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0057] 1 Materials and methods

[0058] 1 Materials

[0059] 1.1.1 Test materials

[0060] Nematode species tested: Bean root-knot nematode (M. enterolobii) and other plant nematode populations were collected or collected from all over the country (Table 1) and propagated or frozen in the greenhouse of the China Institute of Inspection and Quarantine.

[0061] Table 1 Nematode sample codes and population sources

[0062]

[0063] 1.1.2 Main reagents and instruments

[0064] Nematode DNA was extracted using a nematode nucleic acid extraction kit (Baichuan Biological, Zhenhai, Ningbo); RPA amplification kit TwistAmp TM Basic Kit and RPA Amplification Fluorescence Detection Kit TwistAmp TM Exo Kit (TwistDx, UK); DL2000 DNA marker (TaKaRa). Centrifuge (Sigma1-14); Tanon DYY-11 electrophoresis instrument (Beijing Liuyi Instrument); LightCycler480 real-time fluorescence PCR instrument (Roche, Switzerland).

[0065] 1.2 Primer design

[0066] The rDNA-ITS sequence of R. elegans (No. MH756128.1) and ITS sequences of five other common root-knot nematodes, such as R. incognita, were downloaded from NCBI GenBank. The sequence analysis software DNAMAN was used to compare the differential sites between R. elegans and other similar species, and the software Primer6.0 was used to assist in the design of primers. A total of 6 primers were obtained through optimization and screening (Table 2). They were synthesized by Shanghai Bioengineering, and the upstream and downstream primers were combined into 6 pairs: ME1F / ME1R, ME1F / ME2R, ME1F / ME3R, ME2F / ME2R, ME2F / ME3R, and ME3F / ME3R.

[0067] 1.3 Probe design

[0068] Based on the results of RPA electrophoresis, one primer specific for R. sphaeroides was selected from the six primer pairs. According to the requirements of RPA-Exo fluorescent probe, a specific Exo fluorescent probe for R. sphaeroides was designed within the RPA amplification fragment (Table 2) and synthesized by Shanghai Bioengineering.

[0069] Table 2 Specific primers and probe sequences for R. elegans

[0070]

[0071] 1.4 Extraction of nematode DNA

[0072] DNA was extracted from the root-knot nematode of the elephant ear bean and other plant nematodes using a nematode nucleic acid extraction kit (Baichuan Biological, Zhenhai, Ningbo). 10 μL of lysis solution A was aspirated onto a glass slide, and the nematodes were picked into lysis solution A. After the nematode body was cut or pierced with a pick, it was aspirated into a 200 μL EP tube, and 2 μL of lysis solution B was added to mix; the EP tube was heated at 95°C for 15 min, and 20 μL of sterilized water was added after instant centrifugation. The supernatant was used as a DNA template for direct detection or frozen at -20°C for later use.

[0073] 1.5 Establishment of RPA reaction system for root-knot nematodes of Elephant ear bean

[0074] The DNA of the root-knot nematode in Table 1 was used as a template and the RPA amplification kit TwistAmp TM According to the operating instructions of the Basic Kit, prepare the RPA reaction system. The 50μL reaction system includes 29.5μL of Rehydration Buffer, 2.4μL of upstream and downstream primers (10μmol / L), added to the enzyme powder, 2μL of DNA template, 11.2μL of DEPC water, and mixed thoroughly with a pipette; add 2.5μL of magnesium acetate (280nmol / L). Place in a 37-41℃ constant temperature metal bath for 20-40 minutes. After the RPA product is purified by DNA extraction solution, it is detected by 3.5% agarose gel electrophoresis to screen the best primer pair, and the RPA reaction conditions are optimized for reaction temperature and reaction time, with DEPC water as a blank control.

[0075] 1.5.1 Optimization of RPA reaction conditions

[0076] Reaction time optimization: Set the reaction time to 20min, 25min, 30min, 35min, and 40min, and perform RPA reaction at 37°C to optimize the reaction time. Reaction temperature optimization: Set the reaction temperature to 37°C, 38°C, 39°C, 40°C, and 41°C. Select the best reaction time and screen the five reaction temperatures. Determine the best reaction conditions for RPA and conduct subsequent specificity and sensitivity tests.

[0077] 1.5.2 Evaluation of the specificity of the RPA reaction system

[0078] Using the nematode DNA in Table 1 as a template, referring to the reaction system in 1.5, the optimal reaction conditions in 1.5.1 were used for RPA reaction and electrophoresis detection, and DEPC water was used as a blank control.

[0079] 1.5.3 Sensitivity test of RPA reaction system

[0080] DNA from a single female of the root-knot nematode was extracted and diluted 10-fold to 5 concentrations (10-1 ~10 -5 ), using the above 6 concentrations as templates, referring to the reaction system in 1.5, adopting the optimal reaction conditions in 1.5.1 for RPA reaction and electrophoresis detection, and using DEPC water as blank control.

[0081] 1.6 Establishment of RPA-Exo fluorescence reaction system

[0082] Using the root-knot nematode DNA in Table 1 as a template, the RPA amplification fluorescence detection kit TwistAmp TM According to the operating instructions of ExoKit (TwistDx, UK), prepare the RPA fluorescence reaction system. The 50μL reaction system includes 29.5μL RehydrationBuffer, 2.1μL upstream and downstream primers (10μmol / L), 0.6μL probe (10μmol / L), added to the enzyme powder, 2μL DNA template, 11.2μL DEPC water, and mixed thoroughly with a pipette tip; add 2.5μL magnesium acetate (280nmol / L). Place the above reaction tube in a constant temperature real-time fluorescence PCR instrument and detect the HEX fluorescence value every 30s. Use DEPC water as a blank control, and refer to 1.5.1 for reaction conditions.

[0083] 1.6.1 Fluorescence sensitivity test of RPA-Exo reaction system

[0084] Prepare the template according to 1.5.3, perform the sensitivity test of RPA-Exo detection system, use DEPC water as blank control, and compare the sensitivity with RPA reaction system.

[0085] 2 Results and analysis

[0086] 2.1 Screening of RPA primers for R.

[0087] The RPA primer screening test was performed using the RPA basic kit. The electrophoresis test results are shown in the figure ( Figure 1 ): The amplified bands of the three primer pairs ME1F / ME2R, ME2F / ME2R, and ME3F / ME3R were all obvious, with wider and brighter bands. However, ME1F / ME2R (whose design site is as follows Figure 2 This pair of primers has non-specific amplification, and the band amplified by ME2F / ME2R primers has the best effect, with a fragment size of 142bp.

[0088] 2.2 Optimization of the reaction time of the RPA detection system for root-knot nematodes of elephant ear beans

[0089] Using the DNA of the root-knot nematode Elephant bean as a template and ME2F / ME2R as primers, the RPA reaction time was optimized at 37°C. The results showed that ( Figure 3 ), when the reaction time is 20 minutes, a small amount of RPA amplification products can be detected. As time goes on, the amplification products increase significantly. The brightness and width of a specific band detected at 30 minutes are optimal, and there is no obvious difference in the amplification products after 30 minutes. The present invention finally selects a reaction time of 30 minutes.

[0090] 2.3 Optimization of reaction temperature of RPA detection system for root-knot nematodes of elephant ear bean

[0091] Using the DNA of the root-knot nematode Elephant bean as a template and ME2F / ME2R as primers, the RPA reaction temperature was optimized under a reaction time of 30 min. Figure 4 ), the amplified products of the DNA of the root-knot nematode of Elephant bean could be detected at the five reaction temperatures. The brightness of the specific bands at 37℃ and 38℃ was weak, while the bands at 39℃-41℃ were clear, with little difference in brightness and width. Therefore, the reaction temperature of 39℃ was selected for subsequent experiments.

[0092] 2.4 Results of RPA specificity evaluation of Elephant ear bean root knot nematode detection

[0093] Based on the previously established RPA detection system and optimized reaction conditions, the specificity of the RPA detection primers ME2F / ME2R for the root-knot nematode of Elephant bean was evaluated using the nematode DNA in Table 1 as a template. The electrophoresis results showed that ( Figure 5 ), only the DNA templates of three groups of R. elegans were amplified and electrophoresed, and clear specific bands could be observed, while other samples and negative controls had no bands. This indicates that the designed and screened RPA detection primers for R. elegans have good specificity.

[0094] 2.4 Establishment of RPA-Exo detection system for root-knot nematodes of Elephant ear bean

[0095] Using the DNA of the root-knot nematode Elephant bean as a template, ME2F / ME2R as primers, and MePe as a probe, RPA fluorescence detection was performed using the RPA-Exo detection kit at 39°C. The results showed that ( Figure 6 ): The amplification curves of the three elephant ear bean DNA began to peak at around 5 minutes, and the amplification rate slowed down slightly after 20 minutes. No amplification curve was observed in the blank control.

[0096] 2.5 Sensitivity results of RPA and RPA-Exo detection of root-knot nematodes of Elephant ear bean

[0097] Using ME2F / ME2R as primers, at 39°C, and 6 different concentrations of R. philadelphica DNA as templates, the sensitivity test of RPA and RPA-Exo for R. philadelphica was carried out. The RPA test results showed that ( Figure 7 A): DNA template diluted 10-3 When the band is blurred, dilute 10 -4 There is no amplification band after the RPA-Exo fluorescence amplification curve shows ( Figure 7 B): dilution 10 -4 The amplification curve was significant, and the results showed that the sensitivity of RPA-Exo fluorescence detection was 10 times higher than that of RPA electrophoresis detection.

[0098] 3 Conclusion and discussion

[0099] Root-knot nematodes are the most common and most harmful plant parasitic nematode group. Compared with the widely distributed southern root-knot nematodes, the root-knot nematodes of elephant ear beans have stronger pathogenicity and fecundity. The development and establishment of rapid detection technology for root-knot nematodes of elephant ear beans play a vital role in the early warning and comprehensive prevention and control of nematode diseases. The present invention uses RPA-Exo fluorescence rapid detection technology to develop a rapid detection system for root-knot nematodes of elephant ear beans, which can obtain test results within 20 minutes. This method has the advantages of strong specificity, high sensitivity, and simple operation, and is suitable for use in grassroots laboratories.

[0100] The RPA-Exo fluorescence detection system for root-knot nematodes of the elephant ear bean established in the present invention has high sensitivity, and the detection limit can be as low as 10 -4 The sensitivity of the present invention is at least 10 times higher than that of the RPA electrophoresis detection method in the present invention, and nearly 10,000 times higher than that of the specific PCR detection sensitivity (single female insect) using SCAR markers by Tigano et al. It is 5 times higher than the sensitivity of the LAMP method with IGS as the target reported by Niu et al. (1 / 2000 female insect). The detection sensitivity of this method is lower than that of the LAMP detection sensitivity with ITS as the target reported by He Xufeng et al. (1 / 200 000 female insect), but the method using PCR buffer and proteinase K to extract DNA requires 2 hours to complete, while the nucleic acid extraction kit used in the present invention can start amplification detection after heating at 95°C for 15 minutes. Compared with the elephant ear bean RPA fluorescence detection method designed with IGS as the target gene reported by Subbotin (amplified fragment size 240bp), the present invention has a faster reaction and the fluorescence signal can be detected after 5 minutes, which may be related to the shorter target fragment amplified in the present invention (142bp). At the same time, compared with PCR technology, RPA-Exo detection technology requires simpler instruments and equipment, and can be completed by a portable fluorescent constant temperature detector. The reaction process does not require repeated temperature rise and fall, and the amplification speed is faster. Compared with other nucleic acid isothermal amplification technologies such as LAMP, RPA detection technology has the advantages of lower reaction temperature, short running time, and primers not being easily cross-contaminated. Based on this, the present invention develops the RPA-Exo detection system for elephant ear bean root knot nematodes, which can be completed within 1 hour from nucleic acid extraction to obtaining test results, and the detection process does not require large and complex instruments and equipment, and has a wide range of application prospects in grassroots laboratories.

[0101] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the protection scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An RPA primer pair for detecting Meloidogyne enterolobii, the RPA primer pair comprising an upstream primer and a downstream primer, in, The nucleotide sequence of the upstream primer is as follows: 5'-GTTTTGCGAACTGCAGCAGCCTTGAGCATAA-3'; The nucleotide sequence of the downstream primer is as follows: 5'-CCACAAAAGCAATTATACAATAAAGC-3'.

2. An RPA primer-probe combination, comprising: The primer pair according to claim 1, and Probe; The nucleotide sequence of the probe is as follows: 5'-AACCCTTTGCTATGTCTGGTTCAGGGTCAT / fluorescent group-dT / / abasic spacer / / fluorescent quencher-dT / CTTTCAAAGCGA / alkyl carbon spacer / -3'; in, The fluorescent group is selected from one of FAM, HEX, TET, Cyanine dye, ROX, and Te xas Red; The abasic spacer is selected from one of THF and dSp; The fluorescence quenching group is selected from one of BHQ, Iowa Black and BBQ; The alkyl carbon spacer is selected from one of C2 Spacer, C3 Spacer and C4 Spacer.

3. The RPA primer-probe combination according to claim 2, wherein: The nucleotide sequence of the probe is as follows: 5'-AACCCTTTGCTATGTCTGGTTCAGGGTCAT / iHEXdT / / idSp / / iBHQ 1dT / CTTTCAAAGCGA / C3spacer / -3'.

4. A reagent comprising the RPA primer pair according to claim 1 or the RPA primer-probe combination according to claim 2 or 3.

5. A kit comprising the RPA primer pair according to claim 1, the RPA primer-probe combination according to claim 2 or 3, or the reagent according to claim 4.

6. The kit according to claim 5, further comprising: a recombinase, a single-stranded DNA binding protein, a strand displacement DNA polymerase, dNTPs, Mg2 + At least one of a source, a buffer, and a positive standard.

7. Use of the RPA primer pair according to claim 1, the RPA primer-probe combination according to claim 2 or 3, the reagent according to claim 4 or the kit according to claim 5 or 6 in detecting root-knot nematodes of Elephant ear bean.

8. A method for detecting root-knot nematodes of Elephant's ear bean, comprising: Using the RPA primer pair described in claim 1, the RPA primer-probe combination described in claim 2 or 3, the reagent described in claim 4 or the kit described in claim 5 or 6, RPA amplification is performed on the nucleic acid of the sample to be tested, and the reaction results are analyzed.

9. The method for detecting root-knot nematodes of Elephant's ear bean according to claim 8, wherein: Each 50 μL RPA reaction system contains 21±3 pmoL of upstream primer, 21±3 pmoL of downstream primer, and optionally 6±1 pmoL of probe.

10. The method for detecting root-knot nematodes of Elephant's ear bean according to claim 8 or 9, wherein: The reaction temperature of the RPA amplification is 39±2°C; the reaction time of the RPA amplification is more than 5 min, preferably more than 20 min, and more preferably 30±10 min.

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