Primers and probes for rapid detection of tobacco target spot pathogen and application, kit and tobacco target spot pathogen rapid detection method

Abstract

The application provides primers and probes for rapid detection of tobacco target spot pathogen, application, kit and a rapid detection method of tobacco target spot pathogen, and relates to the technical field of tobacco prevention and treatment. The primers and probes for rapid detection of tobacco target spot pathogen are screened out through test optimization, have high specificity for the tobacco target spot pathogen, the amplification product is a single band, and there is no non-specific amplification and obvious primer dimer. The primers and probes for rapid detection of tobacco target spot pathogen are used for RPA amplification of the target gene of the tobacco target spot pathogen, and the lateral flow chromatographic test strip is used for visual detection of the target gene, and has the advantages of high sensitivity, good specificity and short operation time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco prevention and treatment, and particularly relates to primers and probes for rapidly detecting tobacco target spot pathogen, application, kit and a rapid detection method of tobacco target spot pathogen. BACKGROUND

[0002] Tobacco target spot is a kind of fungal disease caused by Rhizoctonia solani, and the anamorphic state of Guanyu Myceliophthora is Rhizoctonia solani. Tobacco target spot can occur from the seedling stage to the maturity of tobacco leaves, and it not only infects leaf blades, but also damages stems. The initial infection of leaf blades is round water spot-like spots, and if the temperature is higher, the humidity is larger, and the leaf wetting time is longer, the disease spot will rapidly expand to form irregular spots with a diameter of 2-20 centimeters, with concentric ring patterns, forming a faded green halo spot. The necrotic part of the disease spot is easy to break and form perforations, which looks like the holes left on the target after being shot by a gun, so it is called target spot.

[0003] Tobacco target spot is not easy to be found, and once it spreads rapidly, it is difficult to prevent and control, which causes great losses to farmers and hinders the development of the tobacco planting industry. At present, the detection methods of the disease mainly include pathogen isolation and identification, as well as ordinary PCR and fluorescent quantitative PCR identification. These methods have relatively high professional requirements, and the process is complicated and time-consuming, which cannot meet the requirements of efficient and economical detection. SUMMARY

[0004] The present application aims to provide primers and probes for rapidly detecting tobacco target spot pathogen, application, kit and a rapid detection method of tobacco target spot pathogen, and solve the problems of complicated process and long time consumption in the existing detection of tobacco target spot pathogen by using ordinary PCR.

[0005] To achieve the above purpose, the present application provides primers and probes for rapidly detecting tobacco target spot pathogen, wherein the primers comprise an upstream primer and a downstream primer.

[0006] The nucleotide sequence of the upstream primer is shown in SEQ ID NO. 1.

[0007] The nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2.

[0008] The nucleotide sequence of the probe is shown in SEQ ID NO. 3.

[0009] The 5' end of the downstream primer and the 5' end of the probe have antigen labels, respectively.

[0010] Preferably, the 5' end of the downstream primer has a biotin label, and the 5' end of the probe has a fluorescent label.

[0011] The fluorescent label comprises any one of AMC, FAM and FITC.

[0012] Preferably, the probe comprises at least one of the following features:

[0013] a. the middle sequence of the probe contains nucleotide analogues;

[0014] b. the nucleotide analogues comprise tetrahydrofuran residues;

[0015] c. the 3' end of the probe has a polymerase extension blocking group;

[0016] d. the polymerase extension blocking group comprises any one of C3-spacer, phosphate group, dideoxynucleotides.

[0017] The application also provides the use of the above-mentioned primer and probe for rapid detection of P. nicotiana in the detection of P. nicotiana.

[0018] The application also provides a kit for rapid detection of P. nicotiana, comprising the above-mentioned primer and probe for rapid detection of P. nicotiana.

[0019] Preferably, the kit further comprises a recombinase, a single-stranded DNA binding protein, and a strand displacement DNA polymerase.

[0020] Preferably, the kit further comprises a lateral flow chromatographic test strip, which comprises a detection band and a quality control band, the detection band is fixed with a biotin antibody, and the quality control band is fixed with a fluorescently labeled antibody.

[0021] The application also provides a rapid detection method of P. nicotiana, comprising:

[0022] using the above-mentioned primer and probe for rapid detection of P. nicotiana to perform RPA amplification on the DNA of P. nicotiana to obtain an amplification product;

[0023] using the lateral flow chromatographic test strip to visually detect the amplification product.

[0024] Preferably, the reaction conditions of the RPA amplification comprise a reaction temperature of 37-39℃ and constant temperature incubation for 10-30min.

[0025] Preferably, using the above-mentioned primer and probe for rapid detection of P. nicotiana to perform RPA amplification on the DNA of P. nicotiana to obtain an amplification product comprises:

[0026] mixing the primer and probe for rapid detection of P. nicotiana with Rehydration Buffer and the DNA of P. nicotiana to obtain a mixture;

[0027] MgAc was added to the mixture, mixed thoroughly, and then RPA amplification was performed.

[0028] Compared with the prior art, the beneficial effects of the present application include:

[0029] The primers and probes for rapidly detecting Phytophthora nicotianae provided by the present application are screened through test optimization, have high specificity for Phytophthora nicotianae, the amplification product is a single band, there is no non-specific amplification and obvious primer dimer. The primers and probes for rapidly detecting Phytophthora nicotianae are used to amplify the target gene of Phytophthora nicotianae through RPA, and the target gene is visually detected through the lateral flow chromatographic test strip, which has the advantages of high sensitivity, good specificity, and short operation time. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope of the present application.

[0031] Figure 1 The RPA detection result of Phytophthora nicotianae is shown in the figure, wherein 1 is the DNA of Phytophthora nicotianae as a template, and 2 is the negative control of ultrapure sterile water as a template;

[0032] Figure 2a The specificity result of RPA detection of Phytophthora nicotianae is shown in the figure;

[0033] Figure 2b The specificity result of ordinary PCR detection of Phytophthora nicotianae is shown in the figure;

[0034] Figure 3a The sensitivity result of RPA detection of Phytophthora nicotianae is shown in the figure;

[0035] Figure 3b The sensitivity result of ordinary PCR detection of Phytophthora nicotianae is shown in the figure;

[0036] Figure 4a The detection result of the RPA detection method of Phytophthora nicotianae on the sample of Phytophthora nicotianae is shown in the figure;

[0037] Figure 4b The detection result of the ordinary PCR detection method of Phytophthora nicotianae on the sample of Phytophthora nicotianae is shown in the figure. DETAILED DESCRIPTION

[0038] As used herein:

[0039] "comprising," "having," "including," "containing," "characterized by" or any other variation thereof is intended to cover non-exclusive inclusion, such that a composition, step, method, article, or apparatus that comprises, has, includes, contains, characterizes, or is characterized by a list of elements can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus. "Consisting essentially of, when

[0040] The conjunctive term "consisting of' excludes any element, step, or ingredient not specified. If used in the claims, this phrase shall

[0041] When numerical ranges are disclosed, such ranges are inclusive of the numbers defining the range and include each integer within the defined range. Unless otherwise specified, the ranges are inclusive of the recited endpoints. When a range is disclosed, the disclosure is intended to include the range and any and all individual values within the range. In addition, whole numbers within a range are intended to be recited even if the whole number is not explicitly listed. For example, a range of "1 to 5" is intended to be a disclosure of the whole numbers 1, 2, 3, 4, and 5, even if these are not explicitly listed. When a range is disclosed, the disclosure is intended to include the range and any and all individual values within the range. In addition, whole numbers within a range are intended to be recited even if the whole number is not explicitly listed. For example, a range of "1 to 5" is intended to be a disclosure of the whole numbers 1, 2, 3, 4, and 5, even if these are not explicitly listed.

[0042] In these embodiments, the parts and percentages are by mass unless otherwise indicated.

[0043] "Parts by mass" refers to a basic unit of measurement that represents the proportional relationship of the mass of multiple components, 1 part can represent any unit mass, such as 1 g, 2.689 g, etc. If we say that the mass of component A is a parts and the mass of component B is b parts, it means that the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number representing a multiple factor). It must not be misunderstood that, unlike parts by mass, the sum of the parts by mass of all components is not limited to 100 parts.

[0044] "and / or" is used to indicate one or both of the stated circumstances can occur, for example, A and / or B includes (A and B) and (A or B).

[0045] The application provides primers and probes for quickly detecting Peronospora tabacina, which are used for recombinase polymerase amplification (RPA). The RPA technology mainly relies on three enzymes: a recombinase capable of combining single-stranded nucleic acids (oligonucleotide primers), a single-stranded DNA binding protein (SSB), and a strand displacement DNA polymerase. The mixture of the three enzymes is also active at room temperature, preferably at a reaction temperature of about 37°C. At room temperature, the recombinase can be tightly combined with the primer DNA to form an enzyme and primer polymer, when the primer searches for a completely matched complementary sequence on the template DNA, the double-stranded structure of the template DNA is opened with the help of the single-stranded DNA binding protein, and a new DNA complementary strand is formed under the action of the DNA polymerase, and the amplification product grows exponentially. The use of fluorescent probe labeling, biotin-labeled primers and endonuclease digestion can realize the timed result analysis.

[0046] Among them, the RPA nucleic acid amplification technology has certain differences from the conventional PCR primer design. A pair of primers is composed of two oligonucleotides, which specifically recognize the upstream and downstream nucleotide sequences of a nucleic acid target, respectively; the length is between 30-35 nucleotides (nt), and there is no palindromic sequence, continuous single-base repeat sequence and internal secondary structure region in the sequence; the primer Tm value is not considered as a main factor in design;

[0047] The nucleotide sequence of the upstream primer obtained through screening is Rs-RPAF: 5'-GGACTGGTCGGGACTCAAGGTTACGGGTGGCA-3'(SEQ ID NO. 1); and the nucleotide sequence of the downstream primer is Rs-RPAR: 5'-TGACAGTGACAGTAACAAACTAAGCAACGAATAT-3'(SEQ ID NO. 2). The amplification product is a single band, without non-specific amplification and obvious primer dimer. Meanwhile, the 5' end of the downstream primer for RPA is usually labeled with an antigenic marker, which is usually biotin.

[0048] Among them, the probe is composed of an oligonucleotide backbone with a 5'-antigenic label, which is usually a fluorescent label, and contains base nucleotide analog replacement nucleotides and 3' terminal polymerase extension blocking groups in the interior. The nucleotide sequence of the probe is Rs-LF-Probe: 5'-GTTCTTGTGATAGTATTC GTTATATGTGTTNAGCTGTTGGACGTAGACCG-3'(SEQ ID NO. 3), wherein N is a nucleotide analog, which can be a tetrahydrofuran residue (THF).

[0049] The fluorescent label comprises any one of AMC, FAM, and FITC. AMC is 7-amino-4-methylcoumarin, FAM is carboxyfluorescein, and FITC is fluorescein isothiocyanate.

[0050] The nucleotide analogue comprises any one of a tetrahydrofuran residue (THF) and a C3-spacer, a phosphate group, and a dideoxynucleotide.

[0051] The application also provides an application of the primer and the probe for rapidly detecting the tobacco target spot pathogen in detection of the tobacco target spot pathogen.

[0052] The application also provides a kit for rapidly detecting the tobacco target spot pathogen, comprising the primer and the probe for rapidly detecting the tobacco target spot pathogen.

[0053] Preferably, the kit further comprises a recombinase, a single-stranded DNA binding protein, and a strand displacement DNA polymerase.

[0054] Preferably, the kit further comprises a lateral flow chromatographic test strip, wherein the lateral flow chromatographic test strip comprises a detection zone and a quality control zone, the detection zone is fixed with a biotin antibody, and the quality control zone is fixed with a fluorescently labeled antibody.

[0055] The application also provides a rapid detection method of the tobacco target spot pathogen, comprising:

[0056] The DNA of the tobacco target spot pathogen is subjected to RPA amplification using the primer and the probe for rapidly detecting the tobacco target spot pathogen to obtain an amplification product.

[0057] The amplification product is subjected to visual detection using a lateral flow chromatographic test strip.

[0058] Preferably, the reaction condition of the RPA amplification comprises a reaction temperature of 37-39℃ and constant temperature incubation for 10-30 min.

[0059] Preferably, the RPA amplification of the DNA of the tobacco target spot pathogen using the primer and the probe for rapidly detecting the tobacco target spot pathogen comprises:

[0060] The primer and the probe for rapidly detecting the tobacco target spot pathogen are mixed with Rehydration Buffer and the DNA of the tobacco target spot pathogen to obtain a mixture.

[0061] MgAc is added to the mixture and mixed thoroughly, and then RPA amplification is performed.

[0062] The primers and probes for rapidly detecting target spot of tobacco provided by the application are screened through test optimization, and have high specificity for target spot of tobacco, and the amplification product is a single band, without non-specific amplification and obvious primer dimer. The primers and probes for rapidly detecting target spot of tobacco are used for RPA amplification of the target gene of target spot of tobacco, and the target gene is visually detected by using a lateral flow chromatographic test strip, and has the advantages of high sensitivity, good specificity, and short operation time.

[0063] The embodiments of the application will be described in detail below with specific examples, but those skilled in the art will understand that the following examples are only used to illustrate the application, and should not be regarded as limiting the scope of the application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0064] Example 1: Establishment of RPA detection system for target spot of tobacco

[0065] The DNA of target spot of tobacco was used as a template, and the reaction solution was configured according to the reaction system shown in Table 1 below. 40.9 μL of Rehydration Buffer, 4 μL of primers, and 0.6 μL of probes were added to a sterile detection unit tube, 2.0 μL of sample DNA to be detected was added to the detection unit tube, 2.5 μL of MgAc was added to the detection unit tube, the tube cap was covered, and the mixture was mixed for 5-6 times by inverting up and down, and centrifuged at low speed for 10 seconds. The detection unit tube was reacted at 38°C for 20 min.

[0066] Table 1: RPA detection reaction system for target spot of tobacco

[0067] Rehydration Buffer 40.9 μL Primer F+R 4 μL Probe 0.6 μL Template DNA 2.0 μL MgAc 2.5 μL Total volume 50.0 μL

[0068] After the reaction was completed, the RPA amplification product was visually detected using a lateral flow chromatographic test strip (purchased from Milenia-Biotec GmbH), and two bands were presented on the lateral flow chromatographic test strip, i.e. a detection band and a quality control band; the negative control with ultrapure sterile water as a template only presented a quality control band, indicating that the RPA technology can be used for rapid detection of target spot of tobacco, and the detection results are shown in Figure 1 , wherein 1 is the detection result of the DNA of target spot of tobacco as a template, and 2 is the negative control with sterile water as a template.

[0069] Example 2: Specificity of RPA detection of target spot of tobacco

[0070] The genomic DNA of tobacco target spot pathogen, tobacco black shank pathogen, tobacco anthracnose pathogen, and rice sheath blight pathogen occurring on tobacco was used as a template to perform RPA amplification reaction in the manner of Example 1. After 20 min of constant temperature reaction at 38℃, lateral flow chromatographic test strip detection found that only the RPA amplification product of tobacco target spot pathogen genomic DNA as a template appeared detection band and quality control band on the lateral flow chromatographic test strip, and the detection results of other strains and ultrapure water as a template only had quality control band, and the detection results are shown in Figure 2a , wherein 1 is tobacco target spot pathogen, 2 is tobacco black shank pathogen, 3 is tobacco anthracnose pathogen, 4 is rice sheath blight pathogen, and 5 is ultrapure water.

[0071] In addition, ordinary PCR was performed using primer pair Rs-RPAF / Rs-RPAR, and the results showed that, as shown in Figure 2b , wherein 1 is tobacco target spot pathogen, 2 is tobacco black shank pathogen, 3 is tobacco anthracnose pathogen, 4 is rice sheath blight pathogen, and 5 is ultrapure water, only the tobacco target spot pathogen template can amplify the 208 bp target band, and the templates of other strains have no amplification band.

[0072] It is illustrated that the primer and probe designed for detection of tobacco target spot pathogen have the same specificity as ordinary PCR in RPA detection of tobacco target spot pathogen, but the operation time is greatly shortened.

[0073] Example 3 Sensitivity of RPA detection of tobacco target spot pathogen

[0074] In order to verify the sensitivity of RPA detection of tobacco target spot pathogen, tobacco target spot pathogen genomic DNA was diluted by 10 times to obtain genomic DNA with concentrations of 1000 ng / μL, 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL, and RPA and PCR amplification reactions were performed using the templates of each gradient dilution.

[0075] As shown in Figure 3a , the RPA detection results of tobacco target spot pathogen are shown, wherein No. 1 to No. 8 test strips are the detection results of genomic DNA with concentrations of 1000 ng / μL, 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL, respectively. The amplification results show that No. 1 to No. 6 test strips are obviously positive, No. 7 test strip has weak positive detection results, and No. 8 lateral flow chromatographic test strip has negative detection results, which indicates that the limit value of the RPA detection amplification method for tobacco target spot pathogen genomic DNA detection is 1 pg / μL.

[0076] At the same time, the common PCR amplification product was detected by agarose gel electrophoresis, and the results are shown in Figure 3b Lane 1 to Lane 8 are detection results of 1000 ng / μL, 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 100 fg / μL of genomic DNA, respectively, and the results show that Lane 1 to Lane 6 all present clear bands of 208 bp, and the amplification product of Lane 7 is lighter than that of Lane 1 to Lane 6, which indicates that the detection limit of common PCR for tobacco target spot pathogen genomic DNA is also 1 pg / μL. It is shown that the sensitivity of the tobacco target spot pathogen RPA detection method of the present application is comparable to that of common PCR technology, but the operation time is greatly shortened.

[0077] Example 4: Detection of tobacco target spot pathogen RPA detection method on tobacco target spot pathogen samples

[0078] The DNA of the tobacco target spot pathogen of the tobacco field in Chenzhou was extracted, and RPA amplification was performed, and the test strip detection results are shown in Figure 4a Lane 1 is a positive control tobacco target spot pathogen strain; Lanes 2 to 9 are field plant samples; and Lane 10 is a negative control. The results show that RPA can successfully detect tobacco target spot pathogen from 6 plants, and no tobacco target spot pathogen is detected from 2 healthy plants and the negative control.

[0079] In order to verify the RPA detection results, the field plant samples were detected by common PCR, and the gel electrophoresis results are shown in Figure 4b Lane 1 is a positive control tobacco target spot pathogen strain; Lanes 2 to 9 are field plant samples; and Lane 10 is a negative control. It is found that the PCR detection results are consistent with the RPA detection results, indicating that the RPA detection technology can quickly and accurately detect tobacco target spot pathogen from tobacco plants.

[0080] Finally, it should be pointed out that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

[0081] Furthermore, to the extent that the terms "comprises", "comprising", "includes", "including" and "has" or any variation thereof are used in the following description and / or claims, such terms are intended to include a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Also, where appropriate to context, the above description and / or claims can refer to actions to be taken by a person or apparatus. Such actions are sometimes referred to as being taken by an "operator" or "user" where it is contextualually appropriate. However, such actions, or steps, are taken autonomously and automatically by an apparatus, without intervention of a person.

Claims

1. Primers and probes for rapid detection of Pseudomonas syringae pv. tabaci, characterized in that, The primers comprise an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO. 1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2; The nucleotide sequence of the probe is shown in SEQ ID NO. 3; The 5' end of the downstream primer is labeled with biotin, and the 5' end of the probe is labeled with a fluorescent label; the fluorescent label comprises any one of AMC, FAM, and FITC; N in the probe is a nucleotide analogue; the nucleotide analogue is a tetrahydrofuran residue; the 3' end of the probe has a polymerase extension blocking group; the polymerase extension blocking group comprises any one of a C3 spacer, a phosphate group, and a dideoxynucleotide.

2. The primers and probes for rapidly detecting Pseudomonas syringae pv. tabaci according to claim 1 in the detection of Pseudomonas syringae pv. tabaci.

3. A kit for rapid detection of Pseudomonas syringae pv. tabaci, characterized by, The primers and probes for rapidly detecting Pseudomonas syringae pv. tabaci according to claim 1.

4. The kit for rapid detection of target spot of tobacco according to claim 3, characterized in that, The kit further comprises a recombinase, a single-stranded DNA binding protein, and a strand displacement DNA polymerase.

5. The kit for rapid detection of target spot of tobacco according to claim 4, characterized in that, The kit further comprises a lateral flow chromatographic test strip, which comprises a detection band and a quality control band, the detection band being fixed with a biotin antibody, and the quality control band being fixed with a fluorescently labeled antibody.

6. A rapid detection method of Pseudomonas syringae pv. tabaci, characterized by, Comprise: RPA amplification of the DNA of the Pseudomonas syringae pv. tabaci using the primers and probes for rapidly detecting Pseudomonas syringae pv. tabaci according to claim 1 to obtain an amplification product; Visual detection of the amplification product using a lateral flow chromatographic test strip.

7. The rapid detection method of the tobacco target spot pathogen according to claim 6, characterized in that, The reaction conditions of the RPA amplification comprise a reaction temperature of 37-39℃ and constant temperature incubation for 10-30 min.

8. The rapid detection method of the tobacco target spot pathogen according to claim 6, characterized in that, RPA amplification of the DNA of the Pseudomonas syringae pv. tabaci using the primers and probes for rapidly detecting Pseudomonas syringae pv. tabaci according to claim 1 to obtain an amplification product, comprising: Mixing the primers and probes for rapidly detecting Pseudomonas syringae pv. tabaci with Rehydration Buffer and the DNA of the Pseudomonas syringae pv. tabaci to obtain a mixture; Adding MgAc to the mixture and mixing thoroughly, followed by RPA amplification.

Images