Test strip rpa primer and its detection kit for detecting potato brown stem nematode

Abstract

The application discloses a test strip RPA primer for detecting potato brown stem nematode, wherein the primer is composed of a nucleotide sequence shown in SEQ ID No:1 and SEQ ID No:2; a combination of the primer and a probe is also disclosed, wherein the probe is composed of a sequence shown in SEQ ID No:3. In addition, a detection kit comprising the primer and the probe combination is disclosed. The test strip RPA primer and the probe combination of the application have the advantages of high specificity and sensitivity for detecting potato brown stem nematode, low requirement for equipment and personnel quality, simple operation, fast detection speed and low cost, and are suitable for port quarantine and primary site detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nematode detection, and in particular relates to a test strip RPA primer for detecting Ditylenchus destructor; and also relates to a detection kit containing the primer, and application of the primer in detecting Ditylenchus destructor. Background Art

[0002] The potato stem nematode (Ditylenchus destructor; also known as the potato stem nematode, sweet potato stem nematode, or potato stem nematode) is a migratory plant endoparasitic nematode that primarily infests the underground parts of plants, particularly roots, tubers, and corms. This nematode is a serious pest not only to potatoes but also to sweet potatoes. In some areas of my country, sweet potato yield reductions due to this nematode typically range from 10% to 30%, but in severe cases, can reach 50% to 60%, or even total crop failure. Furthermore, this nematode has a wide range of infestations, affecting over 90 plant species, including carrots, ginseng, wheat, corn, and peanuts. This makes D. destructor a key quarantine nematode, and accurate and rapid identification of this nematode is essential for its effective prevention and control, as well as for port quarantine.

[0003] The traditional method for identifying potato stem nematodes is morphological. However, due to the limited number of effective distinguishing features and the instability of many phenotypic characteristics, this method is cumbersome, often subjective, and lacks reliability. With the advancement of molecular biology, molecular-level nematode identification has gained widespread application due to its high accuracy. PCR amplification technology, however, has gained widespread application due to its high sensitivity, specificity, accuracy, and efficiency. However, this technology has high requirements for instrumentation, experimental environment, and operator performance. Furthermore, it is costly, time-consuming, and difficult to implement on-site testing, significantly limiting its application.

[0004] The development and utilization of constant temperature amplification technology makes up for the shortcomings of PCR technology. This technology can complete amplification at a constant temperature. Compared with PCR technology, it has the advantages of low requirements for equipment and instruments, simple operation, and fast detection speed. Loop-mediated isothermal amplification (LAMP) is one of the constant temperature amplification technologies and has been applied to the detection of potato stem nematode (CN102260746A and CN107988383A and Ding Shanwen et al. (EurJ Plant Pathol, 2019, 153(4): 1165-1175), etc., and can complete the detection at a constant temperature of 65°C for 50 minutes. However, the reaction system of LAMP technology requires 6 primers, and has the disadvantages of complex primer design, cumbersome primer synthesis, easy false positives, and relatively high cost, which limits its application. Therefore, in order to meet the needs of port quarantine and on-site rapid detection, it is urgent to develop a method for detecting potato stem nematode that is accurate, fast, simple to operate, and has low requirements for instruments, equipment, and personnel.

[0005] Recombinase polymerase amplification (RPA) technology only requires a pair of 30bp to 35bp primers and a 5-20min amplification reaction at a constant temperature of 25-42°C to complete the detection. It has the advantages of strong specificity, high sensitivity, fast detection speed, low reaction temperature, and simple operation, and can meet the requirements of rapid detection such as port quarantine and on-site detection. At present, RPA technology has been widely used in the rapid detection of pathogens on humans, animals or plants, but its application in the field of plant parasitic nematode detection is relatively small. For example, there are only reports on the detection of plant root knot nematodes and pine wood nematodes (Deok Jea Cha et al. Forest Pathol, 2019, 49(3): e12503), but most of them are based on the detection of RPA gel electrophoresis system (CN109486960A; CN108559783A; Ju Yuliang et al. Eur J Plant Pathol, 2019, 155(4): 1155-1163). Real-time fluorescence RPA detection is one of the most widely used and mature RPA technologies, having already been applied to the detection of potato stem nematodes (CN110863058A). However, because real-time fluorescence RPA requires fluorescence detection equipment, it is costly and requires high-quality personnel, making it difficult to meet the on-site testing needs of grassroots farmers. Therefore, a detection system with lower equipment and personnel requirements is urgently needed to meet the testing needs of ordinary farmers. The test strip RPA detection system comes closest to meeting this need.

[0006] The recombinase polymerase amplification assay combined with lateral flow dipstick (LFD-RPA) technology based on lateral flow chromatography test strips, also known as test strip RPA, can perform amplification reactions at a constant temperature of 37-42°C. It has low equipment requirements, such as a water bath, which is relatively inexpensive and can even be heated directly at human body temperature. The test results are read by lateral flow chromatography test strips, which is simple and reliable and can be used by ordinary personnel, requiring low personnel qualifications. Although the real-time fluorescence RPA and test strip RPA detection systems are the same in terms of primer design principles, the design principles of their probes are very different. Therefore, the primers for the two detection methods are not necessarily the same. The inventors have found that the primers for real-time fluorescence RPA (CN110863058A) cannot be used in test strip RPA detection. Therefore, it is necessary to redesign and develop primers for test strip RPA for detecting potato rot nematodes.

[0007] At present, there are no reports on the use of test strip RPA for the detection of potato destructor nematode. Summary of the Invention

[0008] To address the challenges of existing PCR, LAMP, and real-time fluorescence RPA technologies, which require high equipment and personnel qualifications and struggle to meet on-site real-time testing requirements, the present invention aims to utilize test strip RPA technology to detect Ditylenchus destructor. This technology requires simple equipment, and the test results are read via test strips, requiring minimal personnel qualifications. It can meet the real-time testing requirements of grassroots farmers, on-site testing, and quarantine inspections at ports of entry.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] The present invention provides a test strip RPA primer for detecting potato destructor stem nematode, comprising a pair of primers; the primers are composed of the nucleotide sequences shown in SEQ ID No: 1 and SEQ ID No: 2; the primers include an upstream primer Dt-LFITSF2 and a downstream primer Dt-LFITSR1:

[0011] Dt-LFITSF2: 5′-GCAAAAGTCGTAACAAGGTGGCTGTAGGTG-3′ (SEQ ID No: 1),

[0012] Dt-LFITSR1: 5′-Biotin-AACAAAGCCGTTTTTCGCCCACAAATTAGC-3′ (SEQ ID No: 2).

[0013] Among them, Biotin is to introduce a biotin group at the 5' end of the downstream primer.

[0014] The present invention also provides a test strip RPA primer and probe combination for detecting potato destructor, comprising the above pair of primers and a probe; wherein the primers are composed of the nucleotide sequences shown in SEQ ID No: 1 and SEQ ID No: 2; and the probe is composed of the sequence shown in SEQ ID No: 3; the sequence of the probe is as follows:

[0015] Dt-LFITSPs1: 5′-FAM-TAGTCCTCAAAGGTGGCATGCTTCTGCCATGC-THF-AGGCACAGGGTAGTTG-C3-Spacer-3′ (SEQ ID No: 3);

[0016] Among them, FAM is used to introduce a fluorescein group at the 5′ end; THF is tetrahydrofuran; and C3-Spacer is used to introduce a spacer arm at the 3′ end to prevent chain extension.

[0017] The present invention also provides the use of the test strip RPA primers in detecting Ditylenchus destructor.

[0018] The present invention also provides the use of the test strip RPA primer and probe combination in detecting potato destructor Ditylenchus destructor.

[0019] The present invention also provides a detection kit for potato destructor stem nematode, which comprises the above-mentioned test strip RPA primers and probes; wherein the primers are composed of nucleotide sequences shown in SEQ ID No: 1 and SEQ ID No: 1; and the probe is composed of the sequence shown in SEQ ID No: 3.

[0020] Furthermore, the above detection kit also includes rehydration buffer, 280mM magnesium acetate, RPA freeze-dried enzyme powder and ddH2O.

[0021] The RPA freeze-dried enzyme powder is a mixture of recombinase, single-strand binding protein and DNA polymerase required for the RPA amplification reaction, and exists in the RPA reaction tube in the form of RPA freeze-dried enzyme powder.

[0022] Furthermore, the detection kit includes: RPA freeze-dried enzyme powder (50 μL system dosage), rehydration buffer 29.5 μL, 10 μM upstream primer Dt-LFITSF2 2.1 μL, 10 μM downstream primer Dt-LFITSR1 2.1 μL, 10 μM probe Dt-LFITSPs1 0.6 μL, DNA template 2 μL, ddH2O 11.2 μL, and 280 mM magnesium acetate 2.5 μL.

[0023] Furthermore, the above detection kit also includes a lateral flow chromatography test strip and its matching test strip detection buffer PBST (1×PBS+0.1% Tween 20).

[0024] Except for the primers and probes in the above detection kit, which need to be synthesized separately, the other reagents and flow chromatographic test strips can be purchased on the market.

[0025] The present invention also provides the use of the detection kit in detecting Ditylenchus destructor.

[0026] The present invention also provides a method for detecting Ditylenchus destructor using the above-mentioned RPA primers or detection kit, comprising the following steps:

[0027] (1) extracting nematode DNA from the sample to be tested;

[0028] (2) Prepare the RPA reaction system: Add the following components in sequence to a reaction tube containing RPA freeze-dried enzyme powder: 29.5 μL of rehydration buffer, 2.1 μL of upstream primer Dt-LFITSF2 (10 μM), 2.1 μL of 10 μM downstream primer Dt-LFITSR1, 0.6 μL of 10 μM probe Dt-LFITSPs1, 2 μL of the test sample DNA, 11.2 μL of ddH2O, and 2.5 μL of 280 mM magnesium acetate; mix thoroughly.

[0029] (3) RPA amplification: Place the reaction tube of step (2) in a constant temperature device and react at 39°C for 12 minutes. After the reaction is completed, dilute the reaction product 50 times with PBST buffer, then take 10 μL of the diluted product and add it to the sample pad of the lateral flow chromatography test strip. Then insert one end of the sample pad of the test strip into the PBST buffer and react for 2-15 minutes. Observe the color reaction;

[0030] (4) Interpretation of results: If red strips appear on both the control line (C) and the test line (T) of the test strip, the sample is judged to be positive; if only the control line (C) appears red, the sample is judged to be negative; if neither the control line (C) nor the test line (T) shows red, it means that the test strip or the amplification reagent is damaged, invalid, or the operation is incorrect, and the result is invalid.

[0031] Compared with the prior art, the present application has the advantages that (1) the specificity is strong. The RPA primer of the present application can only obtain an amplification product when the DNA of potato stem rot nematode is as a template, and cannot amplify a product when other nematode DNA is as a template, which indicates that the RPA primer of the present application has strong specificity for potato stem rot nematode. In particular, the probe is used in the present application, and the specificity of detection is stronger; and the ordinary PCR and LAMP do not use the probe, and the specificity is relatively poor. Therefore, the result of identification of potato stem rot nematode by the RPA primer of the present application is accurate and reliable. (2) The sensitivity is high. The RPA primer of the present application has high sensitivity, and the DNA amount of 1 / 3125 nematodes is required to detect the potato stem rot nematode. (3) The reaction temperature is low, and the requirement for equipment is simple. The amplification reaction can be realized at 37-42 DEG C constant temperature in the present application, that is, the equipment capable of keeping constant temperature such as a water bath can be used to realize it, and professional instrument equipment is not required; and the existing PCR, LAMP, real-time fluorescent RPA detection and the like all require professional equipment, and may need to be constantly temperatureed to complete the detection; thus, the present application has simple requirement for equipment, and has low cost. (4) The detection speed is fast. The shortest time required for detection of potato stem rot nematode by the method of the present application is only about 15 min, while at least 50 min or even longer is required by the PCR and LAMP technology. (5) The operation is simple, and the on-site detection is convenient. The method of the present application only needs to mix several components, and place them in a temperature control equipment to react, and then the test paper strip is used to judge the reaction product to complete the detection. In addition, the test paper strip, RPA freeze-dried enzyme powder and other components can be stored at room temperature for a long time, and are convenient to carry to the site for detection. (6) The result judgment is simple, and the requirement for personnel quality is low. The result can be judged by visual inspection according to the color development of the quality control line and the detection line of the test paper strip, and the personnel quality requirement is not high; while the ordinary PCR, qPCR, LAMP and real-time fluorescent RPA all need to be read by special equipment before the result can be judged, and the general personnel need to be trained before the result can be correctly interpreted. (7) The detection cost is low. The instrument equipment used in the present application such as a water bath is the cheapest one with a price less than 1,000 yuan, while the equipment used for PCR or qPCR needs hundreds of thousands of yuan or even more expensive. In summary, compared with the PCR experiment, the RPA experiment can be carried out at constant temperature, has no complex operation, has low requirement for instruments and personnel, and is suitable for rapid diagnosis at the grassroots or on-site. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1. Electrophoresis pattern of RPA primer combination screening of potato destructor test strips; the primer combinations corresponding to each lane are: 1 is Dt-LFITSF1 / Dt-LFITSR1; 2 is Dt-LFITSF2 / Dt-LFITSR1, 3 is Dt-LFITSF3 / Dt-LFITSR1; 4 is Dt-LFITSF4 / Dt-LFITSR1; 5 is Dt-LFITSF5 / Dt-LFITSR1; 6 is Dt-LFITSF2 / Dt-LFITSR1; 7 is Dt-LFITSF2 / Dt-LFITSR2; 8 is Dt-LFITSF2 / Dt-LFITSR3; 9 is Dt-LFITSF2 / Dt-LFITSR4; 10 is Dt-LFITSF2 / Dt-LFITSR5; 11 is Dt-LFITSF2 / Dt-LFITSR6.

[0033] Figure 2 .Diagram of test strip results interpretation.

[0034] Figure 3 .Photographs showing the specific detection results of potato-destroying Ditylenchus destructor using real-time fluorescence RPA primers using the LFD-RPA method; 1 is a blank control; 2 is the southern root-knot nematode (Meloidogyne incognita); 3 is the elephant-ear bean root-knot nematode (Meloidogyne enterolobii); 4 is the bacteriotrophora (Heterorhabditis bacteriophora); 5 is the feltiae (Steinernema feltiae); and 6 is the potato-destructor Ditylenchus destructor.

[0035] Figure 4 Photographs showing the specific detection results of the RPA primers of the present invention for Ditylenchus destructor; 1 is a blank control; 2 is the southern root-knot nematode (M. incognita); 3 is the cereal cyst nematode (Heterodera avenae); 4 is the elephant-ear bean root-knot nematode (M. enterolobii); 5 is the northern root-knot nematode (M. hapla); 6 is the noctuid nematode (S. feltiae); 7 is the bacteriophora (H. bacteriophora); and 8 is the potato destructor (Ditylenchus destructor).

[0036] Figure 5Photographs showing the specific detection results of the RPA primers on the test strips of the present invention for Ditylenchus destructor. Figure 1 is a blank control; 2 is the southern root-knot nematode (M. incognita); 3 is the elephant-ear bean root-knot nematode (M. enterolobii); 4 is the Philippine cyst nematode (H. filipjevi); 5 is the corn nematode (Pratylenchus zeae); 6 is the dwarf nematode (Trichotylenchus changlingensis); 7 is the soil nematode (Ditylenchus spp.); and 8 is the potato nematode (D. destructor).

[0037] Figure 6 Photo of the sensitivity test results of the test strip RPA primers of the present invention to potato destructor Ditylenchus destructor; from right to left, the concentration of the extracted single nematode gDNA was diluted to 1 / 5 of the original concentration, 1 / 5 2 , 1 / 5 3 , 1 / 5 4 , 1 / 5 5 , 1 / 5 6 , 1 / 5 7 , CK is the blank control.

[0038] Figure 7 Photo of the strip RPA test results for Ditylenchus destructor in diseased soil; 1 is a blank control, 2 to 7 are six replicates of soil samples collected from the diseased field, and 8 is a positive control.

[0039] Figure 8 Photo of the strip RPA test results for Ditylenchus destructor in soil from a non-diseased area; 1 is a blank control, 2 to 7 are six replicates of soil samples collected from a non-diseased area, and 8 is a positive control.

[0040] Figure 9 Photo of the strip RPA test results for Ditylenchus destructor in sweet potato stem tissue; 1 is a blank control; 2 is a healthy stem tissue control; 3 and 4 are sweet potato stem tissues with one Ditylenchus infested; 5 and 6 are sweet potato stem tissues with three Ditylenchus infested; 7 and 8 are sweet potato stem tissues with five Ditylenchus infested. DETAILED DESCRIPTION

[0041] Example 1 Design and screening of RPA primers and probes for Ditylenchus destructor

[0042] (1) According to the design principles of RPA primers and probes for test strips, the following five upstream primers, six downstream primers, and one probe (all synthesized by Shanghai Bioengineering) were designed based on the conserved region of the rDNA-ITS sequence of Ditylenchus destructor (see Table 1):

[0043] Table 1. LFD-RPA primer and probe sequence design list

[0044]

[0045] Among them, Biotin is used to introduce a biotin group at the 5' end of the downstream primer; FAM is used to introduce a fluorescein group at the 5' end of the probe; THF is tetrahydrofuran; C3-Spacer is used to introduce a spacer arm at the 3' end to prevent chain extension.

[0046] (2) Extraction of Ditylenchus destructor DNA:

[0047] (a) Extraction of single nematode DNA: Extraction was performed according to the method of invention patent CN109750034A.

[0048] (b) Extraction of large amounts of nematode DNA: TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 was used for extraction and the operation was performed according to the instruction manual.

[0049] (3) RPA reaction system and conditions: according to The following components were added to the reaction tube containing the RPA freeze-dried powder according to the instructions of the basic kit: 29.5 μL of rehydration buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, 2 μL of Ditylenchus destructor DNA, 11.8 μL of ddH2O, and finally 2.5 μL of 280 mM magnesium acetate. After thorough mixing, the RPA reaction tube with various components was placed in a water bath and reacted at 39°C for 20 min.

[0050] (4) Purify the RPA amplification product obtained in step (3) using a PCR product purification kit (commercially available kits can be used). 5 μl of the product is then electrophoresed on a 1% agarose gel. After staining with nucleic acid dye (Gelred), the gel is imaged and photographed. If a band is present, the target fragment has been amplified.

[0051] Results (see Figure 1): Based on the cross-matching method, Dt-LFITSR1 was used as the downstream primer to screen the best upstream primer. The result showed that the band was brightest when Dt-LFITSF2 was used as the upstream primer, that is, this primer was the best upstream primer; then Dt-LFITSF2 was used as the upstream primer to screen the best downstream primer. The result showed that the band was brightest when Dt-LFITSR1 was used as the downstream primer, that is, the best downstream primer was Dt-LFITSR1; thus, the best primer pair Dt-LFITSF2 / Dt-LFITSR1 was obtained through preliminary screening, and the probe Dt-LFITSPs1 was designed within the amplified fragment of the primers and downstream testing was performed to screen out the test strip RPA primers and probe for potato rot nematode of the present invention.

[0052] The specific sequence is as follows:

[0053] Dt-LFITSF2: 5′-GCAAAAGTCGTAACAAGGTGGCTGTAGGTG-3′ (SEQ ID No: 1),

[0054] Dt-LFITSR1: 5′-Biotin-AACAAAGCCGTTTTTCGCCCACAAATTAGC-3′ (SEQ ID No: 2),

[0055] Dt-LFITSPs1: 5′-FAM-TAGTCCTCAAAGGTGGCATGCTTCTGCCATGC-THF-AGGCACAGGGTAGTTG-C3-Spacer-3′ (SEQ ID No: 3).

[0056] Example 2 Specificity test of test strip RPA candidate primer combinations for potato destructor Ditylenchus destructor

[0057] (1) The present inventors previously disclosed a set of primers and probes for real-time fluorescence RPA detection of Ditylenchus destructor in patent CN110863058A. To verify the suitability of this primer-probe combination for strip RPA, the present inventors used the sequence of this primer-probe combination as a basis and modified the bases according to the requirements of strip RPA (see Table 2).

[0058] Table 2 Primer and probe combinations after base modification

[0059]

[0060] (2) Test methods:

[0061] (1) Nematodes to be tested: Ditylenchus destructor, Meloidogyne enterolobii, Meloidogyne hapla, Meloidogyne incognita, Heterodera filipjevi, Heterodera avenae, Pratylenchus zeae, Steinernemafeltiae, Heterorhabditis bacteriophora, Trichotylenchus changlingensis, and Ditylenchus spp. in soil. All of the above nematodes are stored at the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences.

[0062] (2) DNA preparation:

[0063] (a) Extraction of single nematode DNA: Extraction was performed according to the method described in invention patent CN109750034A.

[0064] (b) Extraction of large amounts of nematode DNA: TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 was used for extraction and the operation was performed according to the instruction manual.

[0065] (3) RPA reaction system: The primer probe combination Dt-LFITSF2 / Dt-LFITSR1, Dt-LFITSPs1 obtained by screening in Example 1 and the primer probe combination modified in (1): DtLF-F4 / DtLF-R1, DtITS-LFPs2 were tested according to nfo kit were used. The following components were added sequentially to the reaction tube containing RPA lyophilized powder: 29.5 μL of rehydration buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, 0.6 μL of 10 μM probe, 2 μL of DNA template, 11.2 μL of ddH2O, and finally 2.5 μL of 280 mM magnesium acetate; the mixture was thoroughly mixed.

[0066] Note: The addition of magnesium acetate will immediately initiate the entire RPA reaction. Therefore, if multiple samples are tested simultaneously, 2.5 μL of magnesium acetate can be added to the cap of each reaction tube in sequence. Finally, the magnesium acetate can be added to each reaction tube simultaneously by centrifugation to start the RPA reaction in multiple tubes at the same time.

[0067] (4) RPA reaction conditions: Place the RPA reaction tube to which various components have been added in step (3) in a water bath and react at 39°C for 12 min. After the reaction is completed, dilute the reaction product 50 times with PBST, and then take 10 μL of the diluted product and add it to the sample pad of the lateral flow chromatography test strip. Then, insert one end of the test strip sample pad into the PBST buffer for 2-15 min, and observe the color reaction of the test strip.

[0068] (5) Interpretation of results: See the schematic diagram of result interpretation. Figure 2 If red stripes appear on both the control line (C) and the test line (T) of the test strip, the sample is judged to be positive, that is, the nematode to be tested is Ditylenchus destructor; if only the control line (C) of the test strip shows a red strip, the sample is judged to be negative, that is, the nematode to be tested is not Ditylenchus destructor; if both the C and T lines do not show color, it indicates that the test strip and amplification reagent may be damaged, ineffective, or operated incorrectly.

[0069] Results: The specificity test results of the modified test strip RPA primer probe combination DtLF-F4 / DtLF-R1 and probe DtITS-LFPs2 based on the primer sequence in patent application CN110863058A showed (see Figure 3 ) The T lines of the root-knot nematode, Steinernema carinii, and the target potato rot nematode all showed color bands, that is, the primer probe set is not specific to potato rot nematode, that is, the primer probe set cannot be directly used in the detection of test strip RPA. The specificity test results of the primer probe combination Dt-LFITSF2 / Dt-LFITSR1 and probe Dt-LFITSPs1 obtained by screening in Example 1 of the present invention (see Figure 4 and Figure 5 ) showed that the T lines of ten nematode species, including the root-knot nematode (Elephant bean root-knot nematode), the northern root-knot nematode, the southern root-knot nematode, and the cereal cyst nematode, as well as the blank control (CK (water), showed no color bands, indicating a negative test result. However, the T lines of the potato destructor Ditylenchus destructor all showed color bands, indicating a positive test result. This indicates that the primer set Dt-LFITSF2 / Dt-LFITSR1 and the probe Dt-LFITSPs1 of the present invention has excellent specificity for Ditylenchus destructor and can be used for strip RPA detection of Ditylenchus destructor.

[0070] Example 3 Sensitivity test of the test strip RPA primer set of the present invention

[0071] Proceed as follows:

[0072] (1) DNA preparation: The genomic DNA of a single nematode of Ditylenchus destructor was extracted using the method described in step (2) of Example 2. The total volume was 10 μL, i.e., the initial concentration was 0.1 head / μL. The DNA was diluted in a 5-fold gradient dilution method to 1 / 5, 1 / 5 of the original concentration, respectively. 2 , 1 / 5 3 , 1 / 5 4 , 1 / 5 5 , 1 / 5 6 , 1 / 5 7 .

[0073] (2) RPA reaction system and reaction conditions: refer to steps (3) and (4) of Example 2, respectively. 2 μL of genomic DNA of each gradient concentration was added to the template.

[0074] Results (see Figure 6 ) at 1 / 5, 1 / 5 2 , 1 / 5 3 , 1 / 5 4 The T line showed obvious red stripes at the dilution multiples, indicating that the detection limit of the detection method of the present invention is 0.1 head / μL×1 / 5 4 ×2μL=1 / 5 5 The above results show that the test strip RPA primers and detection method of the present invention have high detection sensitivity and can meet the requirements of port quarantine and field real-time detection.

[0075] Example 4 Identification test of Ditylenchus destructor in field soil using the test strip RPA primer-probe combination of the present invention

[0076] Proceed as follows:

[0077] (1) Collection of test soil: Five-point sampling method was used to collect 5 soil samples from the diseased land (the plot where sweet potato stem nematode disease has occurred for many years), all of which were from the farm of the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. 100 g of soil was taken from each of the 5 soil samples and mixed well before use. Five soil samples were collected from the non-disease land (the plot where sweet potato had never been planted), which was from a plot in Funing, Qinhuangdao where ginger was planted all year round. Similarly, 100 g of soil was taken from each of the 5 soil samples and mixed well before use.

[0078] (2) Investigation of Ditylenchus destructans in soil: Soil nematodes were isolated from the soil to be tested using the shallow dish method, and the presence of Ditylenchus destructans in the soil was investigated under a stereo microscope.

[0079] (3) Preparation of total soil DNA: Extraction was performed according to the instructions of the Spin Kit for Soil, with 6 replicates for each sample.

[0080] (4) RPA reaction system and reaction conditions: The reaction system and conditions were respectively referred to steps (3) and (4) of Example 2. The total soil DNA extracted in step (3) was added as the template, the potato destructor DNA was used as the positive control, and water was used as the blank control.

[0081] The test results showed that 5 out of 6 replicates of samples collected from the diseased area were positive (see Figure 7 ), while all 6 replicates of samples collected from non-disease areas were negative (see Figure 8 ). Investigation of nematodes isolated from soil using the shallow dish method revealed that all five samples collected from diseased sites contained Ditylenchus destructor, while none of the five samples from non-disease sites were found. This finding is consistent with the results of the RPA test strips presented herein, demonstrating that the RPA primers and detection method presented herein are accurate and reliable for detecting Ditylenchus destructor. However, one replicate, NC6, from the diseased site did not show a positive result. This may be related to the sampling used. Since the kit extracts only 0.5 g of soil per sample, and nematodes are unevenly distributed in soil, it is possible that target nematodes may not be captured in a single soil sample used for DNA extraction. This suggests that multiple replicates should be used in practical applications to ensure capture of the target nematode.

[0082] Example 5 Identification test of Ditylenchus destructor in sweet potato stem tissue using the method of the present invention

[0083] Proceed as follows:

[0084] (1) Preparation of sweet potato stem tissue: 7 portions of healthy sweet potato stem tissue were cut at 0.1 g / portion, one portion was taken out as a negative control, and the other 6 portions were divided into three groups of two, each group was added with 1, 3 and 5 potato rot nematodes.

[0085] (2) Extraction of sweet potato stem tissue DNA: Genomic DNA was extracted using a universal genomic DNA extraction kit (TaKaRa MiniBESTUniversal Genomic DNA Extraction Kit Ver.5.0, purchased from Dalian Takara Biotechnology) and the operation was performed according to the instructions.

[0086] (3) Test strip RPA reaction system and reaction conditions: refer to steps (3) and (4) of Example 2, respectively, and add the stem tissue DNA extracted in step (2) as the template, use healthy seedling tissue DNA as the negative control, and water as the blank control.

[0087] Results (see Figure 9All six sweet potato stem tissues inoculated with Ditylenchus destructor tested positive, indicating that amplification products were obtained. In contrast, no color bands appeared on the T lines of healthy sweet potato stem tissue and the water control, indicating that no amplification products were produced. This demonstrates that the RPA primer and probe combination of the test strips of the present invention can sensitively detect Ditylenchus destructor in sweet potato stem tissue.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the same. Those skilled in the art may modify or make equivalent substitutions to the technical solutions of the present invention based on the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are intended to be protected by the claims of the present invention. Sequence Listing <110> Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences <120> Test strip RPA primers and detection kit for detecting potato destructor nematode <130> 2020S1765IHCY <141> 2020-06-17 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <400> 1 gcaaaagtcg taacaaggtg gctgtaggtg 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(1) <223> n is Biotin <400> 2 naacaaagcc gtttttcgcc cacaaattag c 31 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(51) <223> n is FAM;THF;C3-Spacer <400> 3 ntagtcctca aaggtggcat gcttctgcca tgcnaggcac agggtagttg n 51

Claims

1. A combination of RPA primers and probes for detecting potato destructor nematodes, characterized in that The combination consists of a pair of primers and a probe; wherein the primers consist of the nucleotide sequences shown in SEQ ID No: 1 and SEQ ID No: 2; and the probe consists of the nucleotide sequence shown in SEQ ID No:

3.

2. Use of the paper strip RPA primer and probe combination according to claim 1 in detecting Ditylenchus destructor.

3. A detection kit for potato destructor nematode, characterized in that The kit comprises the test strip RPA primers and probes according to claim 1.

4. The detection kit according to claim 3, characterized in that The method further comprises rehydration buffer, magnesium acetate, RPA freeze-dried enzyme powder and ddH2O; wherein the RPA freeze-dried enzyme powder is a mixture of recombinase, single-strand binding protein and DNA polymerase.

5. The detection kit according to claim 4, characterized in that The detection kit includes: RPA freeze-dried enzyme powder, 29.5 μL of rehydration buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, 0.6 μL of 10 μM probe, 2 μL of DNA template, 11.2 μL of ddH2O, and 2.5 μL of 280 mM magnesium acetate; it also includes lateral flow chromatography test strips and detection buffer PBST.

6. Use of the detection kit according to any one of claims 3 to 5 for detecting Ditylenchus destructor.

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