CN116287354A - Method and kit for detecting bacterial fusarium wilt of corn - Google Patents
Method and kit for detecting bacterial fusarium wilt of corn Download PDFInfo
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- CN116287354A CN116287354A CN202310525899.8A CN202310525899A CN116287354A CN 116287354 A CN116287354 A CN 116287354A CN 202310525899 A CN202310525899 A CN 202310525899A CN 116287354 A CN116287354 A CN 116287354A
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Abstract
The invention relates to the technical field of molecular biology, in particular to a method and a kit for detecting corn bacterial wilt bacteria. The invention can be used for improving the rapid monitoring and quarantine of the maize bacterial wilt bacteria in customs and basic laboratories. The detection sensitivity of the CLL001F/R primer was determined to be 10 cfu/. Mu.L by a real-time fluorescent RPA reaction. The detection results of the simulation seed tape bacteria and the blade needling inoculation result are ideal. Compared with other detection methods, the RPA detection has high sensitivity, short detection time, easy operation and higher sensitivity and specificity, and is suitable for on-site quick quarantine of corn bacterial wilt bacteria.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method and a kit for detecting corn bacterial wilt bacteria.
Background
Bacterial fusarium wilt of cornPantoea stewartiisubsp.stewartiiPSS) is one of the infectious agents of the inbound plant, which is transmitted over a long distance mainly through seeds, and which causes bacterial wilt of corn, a disease on corn.
At present, PCR is widely applied to the rapid detection of PSS in imported corn, and a plurality of pairs of specific primers or probes have been reported. Due to PSS andP. stewartiisubsp. indologenesthe gene sequences of (PSI) are very similar, and a plurality of pairs of PSS specific primers can amplify PSI to cause false positive detection results, including primers adopted by PSS diagnosis standards of EPPO and primers adopted in national industry standard SN/T3756-2013 corn bacterial blight quarantine identification method PCR method.
Disclosure of Invention
According to the invention, based on a recombinase polymerase amplification technology (RPA), 12 pairs of PSS specific primers/probes are tested through 26 collected PSS and related strains thereof to evaluate the specificity of the primers/probes, so that a rapid detection method for corn bacterial wilt bacteria based on the RPA is finally established, and the requirements of rapid and accurate detection (on the port) are met.
One aspect of the invention relates to detection of bacterial blight germ of cornPantoea stewartiisubsp. stewartiiSubspecies based on recombinant polymerase amplification technology and used for exclusionP. stewartiisubsp.indologenesDetection of false yang caused by subspeciesThe method comprises the step of detecting the sample to be detected by adopting a primer pair shown in SEQ ID NO. 1 and SEQ ID NO. 2.
A further aspect of the invention relates to a method as described above for differentiating maize bacterial wilt bacteriaPantoea stewartiisubsp. stewartiiSubspecies andP. stewartiisubsp.indologenessubspecies, and the use thereof.
Yet another aspect of the invention relates to a method for detecting bacterial blight of cornPantoea stewartiisubsp. stewartiiA kit of subspecies comprising primers as shown in SEQ ID NO. 1 and SEQ ID NO. 2 and a probe.
The invention can be used for improving the rapid monitoring and quarantine of the maize bacterial wilt bacteria in customs and basic laboratories. The detection sensitivity of the CLL001F/R primer was determined to be 10 cfu/. Mu.L by a real-time fluorescent RPA reaction. The detection results of the simulation seed tape bacteria and the blade needling inoculation result are ideal. Compared with other detection methods, the RPA detection has high sensitivity, short detection time, easy operation and higher sensitivity and specificity, and is suitable for on-site quick quarantine of corn bacterial wilt bacteria.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a primer-specific RPA assay; 26 pathogenic bacteria on corn are taken for identification, the specificity of the primer and the probe is determined to be very good, and all five positive strains are detected.
FIG. 2 shows the primer sensitivity RPA detection results; wherein the bacterial liquid is diluted in a gradient way, and the minimum detection concentration is 10 cfu/. Mu.L.
FIG. 3 is a simulated seed and leaf carrier RPA test;
A. simulate seed tape experimentsAfter washing the overnight activated bacteria, the OD600 was adjusted to 0.6 (about 10 8 cfu/ml), then soaking seeds for 6 hours by using bacterial liquid after gradient dilution, drying, adding 1ml of water to soak the seeds for 4 hours, then kneading, and taking 2 mu L to carry out RPA experiment;
B. corn leaf bacteria detection, needle spot CT value 19, and CT value 29.9 at a slightly distant place.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.
Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.
In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Unless otherwise contradicted by purpose and/or technical solution of the present application, the cited documents related to the present invention are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are incorporated into the present application by reference, but are not limited to the embodiments that can be implemented. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.
The first aspect of the invention relates to the detection of bacterial blight germ of cornPantoea stewartiisubsp.stewartiiSubspecies based on recombinant polymerase amplification technology and used for exclusionP. stewartiisubsp.indologenesThe method comprises the step of detecting a sample to be detected by using primer pairs shown in SEQ ID NO. 1 and SEQ ID NO. 2.
In some embodiments, the method further employs a probe as shown below:
CATGGTGTCGTATTTTAGGTAAATAAGTTGTTTAG [ luminophore ] [ abasic nucleotide analog ] GT [ quencher ] TTTTTTCGCCATGCCG [ blocker ];
the blocking agent is used to block polymerase extension of the probe.
In some embodiments, the abasic nucleotide analog is tetrahydrofuran. It can act as a recognition site for exonucleases.
In some embodiments, the blocking agent is selected from a spacer, a phosphate group, biotin-TEG, or an amine (e.g., C6 amine).
In some embodiments, the arms are selected from any of ethylene glycol, C9 arms (Spacer 9), C18 arms (Spacer 18), dideoxy arms [1',2' -Dideoxyribose (dSpacer) ], C3 arms (C3 Spacer).
In some embodiments, the spacer is selected from a C3 spacer.
The Spacer (Spacer) can provide the necessary spacing for oligonucleotide labeling to reduce interactions between the labeling groups and the oligonucleotides, and is mainly used in DNA hairpin structure and double-stranded structure studies. C3 The spacer is used primarily to mimic the three carbon spacing between the 3 'and 5' hydroxyl groups of ribose, or "substitute" for an unknown base in a sequence. 3'-Spacer C3 is used to introduce a 3' Spacer to prevent the 3 'exonuclease and 3' polymerase from acting.
In some embodiments, the luminescent group is selected from any one of AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua phoor 593, texas Red, atto 590, cy5, quasar 670, cy5.5, and Cy 5.5.
In some embodiments, the quenching group is selected from any of BHQ1, BHQ2, BHQ3, dabcyl, eclipse, and MGB.
In some embodiments, the luminescent group is FAM and the quenching group is BHQ1.
In one aspect, useful primers and probes include nucleotide sequences that are greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the upstream primer, downstream primer or probe of the specific sequences provided above. Such primer and probe modifications are also contemplated and can be prepared according to standard techniques.
The term "% identity" in the context of two or more nucleotide sequences or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is the entire length of the coding region relative to the sequences to be compared.
For sequence comparison, typically one sequence is used as a reference sequence, and the test sequence is compared to that sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215:3, 403-410; altschul et al, 1997, nucleic Acids Res 25:17, 3389-402).
The primer and probe modification may be performed by a known method. Modified versions of these primer and/or probe sequences can include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, one or more nucleotides to the 3' end, one or more nucleotides to the 5 'and 3' ends, adding tails, shortening the sequence, extending the sequence, shifting the sequence several bases upstream and downstream, or any combination thereof. In addition to the modifications already mentioned above, other modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2, 6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) may be applied without significantly adversely affecting the function of the primer, and include at least one mismatched base at one of the bases, or at least one of the bases is replaced with an RNA base, to effect, for example, a nucleic acid interaction that increases the 3' end of the mutant specific primer to increase Tm. The addition of double-stranded stable base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. The modified probe should retain the ability to distinguish between the mutation site to be detected and the wild-type site.
The sample to be tested for use in the present invention may be any sample suspected of containing corn pathogen. In some embodiments, the sample to be tested comprises at least one of a corn sample, a soil sample, and a water sample (particularly a water source and soil near a corn planting environment).
As used herein, "corn sample" refers to a solid, viscous or liquid substance or formulation, typical examples of which may include cuttings, roots, leaves, stems, flowers, pollen, embryos, anthers, seeds, fruits or other parts of rice.
The corn sample may be subjected to conventional treatments such as grinding, compacting, degrading, drying, wetting, etc., in advance, provided that the treatment does not significantly interfere with the detection of the pathogenic bacteria.
In some embodiments, the method of detecting maize bacterial wilt further comprises isolating DNA (particularly genomic DNA) from a component suspected of containing the pathogenic bacteria. As used herein, the term "isolated" refers to: (1) substantially or essentially free of components that normally accompany or interact with the material in a naturally occurring environment, (2) substantially or essentially free of components of the material in a concomitantly or interacted processed form, such as in a beverage or food product or from substances used in the separation process, or (3) if the material is in its natural environment, the material has been altered and/or placed in a cell at a site different from the site inherent to the material by deliberate human intervention of the composition. When the term "substantially purified" is used, the designation will refer to a composition in which the protein or peptide forms the major component of the composition, e.g., about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more (i.e., e.g., weight/weight and/or weight/volume) of the composition. As used herein, the term "substantially purified" refers to nucleic acid molecules (particularly DNA) that are removed, isolated or separated from their natural environment or plant, and are at least 60% free, preferably 75% free, more preferably 90% free of other components with which they are naturally associated.
Isolation of the plant DNA fragments from the sample to be tested may involve the use of an isolation solvent such as methanol, ethanol, water, acetone or a combination thereof. In some embodiments, kits of DNA isolation kits may be used, including, for example, DNA isolation protocols using the Dneasy Mericon food kit (Qiagen, germanten, MD, USA) or cetyl trimethylammonium bromide (CTAB). Other separation techniques include lysis, heating, alcohol precipitation, salt precipitation, organic separation, solid phase separation, silica crude membrane separation, CSCL gradient purification, or any combination thereof. In some embodiments, the DNA in the sample to be detected is extracted and purified using the purification reagents described above.
A second aspect of the invention relates to a method as described above for differentiating maize bacterial wilt bacteriaPantoea stewartiisubsp. stewartiiSubspecies andP. stewartiisubsp.indologenessubspecies, and the use thereof.
A third aspect of the present invention relates to a method for detecting bacterial blight of cornPantoea stewartiisubsp. stewartiiA kit of subspecies comprising primers as defined in SEQ ID No. 1 and SEQ ID No. 2 and a probe as defined in the first aspect of the invention.
The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit further comprises one or more of a nucleic acid extraction reagent, a reagent for isothermal nucleic acid amplification, a positive control, and a negative control.
In some embodiments, the reagents used for isothermal nucleic acid amplification include one or more of a recombinase, a single-stranded DNA binding protein, a strand-displacement DNA polymerase, an accessory protein, an exonuclease III, a reverse transcriptase, ATP, reagents for ATP regeneration systems, pH adjusters, dntps, BSA and/or PEG of various molecular weight distributions, DTT, and water, which bind to single-stranded nucleic acids;
wherein the auxiliary protein is used for changing the reversible reaction process of dissociation and recombination of the recombinase-primer complex, so that the reaction is more favorable for isothermal nucleic acid amplification.
The pH adjuster may comprise acids and bases that do not significantly affect the progress of the reaction, as well as buffer components (e.g., tris and acetate, etc.). Further, the Tris buffer is Tris-tricine, which may be present at a working concentration of about 80mM to 120mM.
In some embodiments, the recombinase is selected from uvsX and/or RecA;
in some embodiments, the single-stranded DNA binding protein is gp32;
in some embodiments, the strand displacement DNA polymerase is selected from BSu DNA polymerase and/or Sau DNA polymerase. The DNA polymerase used in the recombinase-mediated isothermal amplification of nucleic acids is bacillus subtilis DNA polymerase I (Bacillus subtilis Pol I, bsu) or staphylococcus aureus (Staphylococcus aureus Pol I, sau), both of which belong to the DNA polymerase I family. The DNA polymerase I family is a polymerase responsible for repair of lesions during DNA replication, and most of the DNA polymerases in this family have low continuous synthesis capacity, i.e., the polymerase in this family has a small number of polymerization reactions that can be catalyzed by a single binding of the polymerase to a template.
In some embodiments, the accessory protein is selected from uvsY;
in the case where a recombinase is used for the strand insertion step, the system may require an energy source. Most of these enzymes utilize ATP as an energy source, but because of the magnesium ion necessary for ATP-finishing (collate) enzyme activity, it is advantageous to provide an additional ATP regeneration system rather than to increase the concentration of ATP. In some embodiments, the reagent used in the ATP regeneration system is selected from one or more of magnesium ion, creatine phosphate and its counter ion, creatine kinase, pyrophosphatase, sucrose, and sucrose phosphorylase.
From the above components, the kits of the present invention may employ and preferably employ methods of Recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA), but may also employ improved methods over this technology, such as Recombinase-dependent amplification (RDA).
In some embodiments, the reagents used for isothermal nucleic acid amplification are lyophilized powder reagents or mixed liquid reagents.
The components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilization beads are generally understood to mean lyophilisates which are pressed into spheres after manufacture, after which the substance is usually present as a powder. Thus, the components required for the amplification reaction, in particular the various enzymes, nucleic acid components and reaction buffer components, may be provided in lyophilized form. In this way, the amplification process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is very advantageous for automated applications.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
Examples
1 materials and methods
1.1 materials
Test strain: the 26 strains of the strain are collected together, and the strain comprises 5 PSS, 2 PSI and 19 other strains. Wherein 5 PSS, 2 PSI and related species are confirmed by adopting a 16S sequencing method, and strain information is shown in Table 1.
TABLE 1 Strain information
Culture medium: NA medium (peptone 10 g, beef extract 3g, sodium chloride 5 g, agar powder 15 g, distilled water 1L).
Reagent: bacterial genomic DNA extraction kits were purchased from the root biochemistry technologies (beijing) limited company; DL2000 DNA marker, 2X Taq PCR Master Mix, premix Ex Taq (Probe qPCR) was purchased from TaKaRa, japan; the rest reagents are all made or imported analytically pure.
Instrument: t100 PCR instrument, bio-rad, USA; real-time fluorescence PCR instrument of Quan Studio 5, ABI company, USA; geldoc XR gel imaging System, bio-rad, inc., america, anpu future RPA rapid detector.
1.2 primer/Probe
The test primers/probes included 12 pairs of specific primers/probes for detecting PSS reported in the literature (table 2), which were synthesized by hua major genes limited company.
TABLE 2 primer/probe sequences
1.3DNA extraction
Purifying the strain to be tested on NA culture medium, taking single colony for propagation, eluting with sterile water, centrifuging the bacterial suspension, extracting bacterial genome DNA from the precipitate by using a bacterial genome DNA extraction kit, and preserving at-20 ℃ for later use.
1.4 primer test
The tested 26 strain DNA was tested with 12 pairs of primers, respectively, and the reaction procedure was referred to its original literature. The PCR amplified products were analyzed by agarose gel electrophoresis at 1.2%.
1.5 sensitivity test
2 pairs of specific primers CLL001F/R and cpsAB2313F/cpsR were selected by test, genomic DNA of strain 29277 was subjected to 10-fold serial dilutions at DNA concentrations of 50 ng/. Mu.L, 5 ng/. Mu.L, 0.5 ng/. Mu.L, 0.05 ng/. Mu.L, 0.005 ng/. Mu.L, 0.5 pg/. Mu.L, 0.05 pg/. Mu.L, 0.005 pg/. Mu.L and 0.0005 pg/. Mu.L, respectively, PCR amplification was performed with the above 2 pairs of primers, 2. Mu.L of DNA template was added, and the reaction procedure was as in the original document. The PCR amplified products were analyzed by 1% agarose gel electrophoresis.
1.6 simulation seed and leaf area detection
Taking PSS strain29277 preparation 10 8 And (3) carrying out gradient dilution on CFU/mL bacterial suspension, taking 1mL bacterial suspension into 6 corn seeds, uniformly mixing, naturally airing to serve as an artificial bacteria sample, and then extracting DNA after soaking treatment for RPA experiments. Meanwhile, 10≡8 CFU/mL bacterial suspension of the strain 29277 is inoculated with tender leaves by a needle punching method, and after two weeks of water injection as negative control, the disease condition is observed, and RPA experiments are carried out. The primers cpsAB2313F/cpsR and cpsF/cpsR were used to detect the above sample DNA, respectively, and the primer CLL001F/R and probe were also used for RPA detection.
2 results and analysis
2.1 primer specificity test
The genomic DNA of 26 strains was tested by PCR or qPCR using 12 pairs of specific primer/probe pairs, and the test results showed that the primers CLL001F/R and cpsAB2313F/cpsR were both positive for the tested PSS and negative for the other test strains, and the specificity of 2 pairs of primers was superior to that of the other test primers/probes (Table 3). The primer cpsAB2313F/cpsR has weak nonspecific amplification when part of strains are amplified, but the size of a nonspecific band is far different from that of a target band, and the judgment of the result is not influenced.
TABLE 3 primer/probe detection results
Note that: positive for "+"; "-" is negative;
"-" indicates that the result is negative, but that there is non-specific amplification, which does not affect the judgment of the result.
Experimental results show that 10 pairs of test primers have different degrees of false positive or weak false positive amplification, and the strain which is easy to cause the false positive or weak false positive amplification mainly comprises PSI,P. stewartii、P. alliiEtc. (Table 3). Primer cpsF/cpsR can amplify 4 strains of PSI and 3 strainsP. stewartiiAn unexpected band of 193 bp was obtained, whereas the band size upon expansion of PSS was 375 bp. Sequencing results of amplified products are shown (data not shown) due to PSI andP. stewartiithe amplified region lacks 182 bases, resulting in yieldThe sizes of the substances differ by 182bp. In addition, the primer can be used for nonspecific amplificationP. alliiThe amplified band is very close to the target band (only a fraction slightly larger than the target band), which easily affects the outcome determination.
2.2 sample-specific and sensitive RPA detection
The screened primer CLL001F/R and the probe are used for carrying out RPA specificity detection on 26 strains, and experimental results show that the primer and the probe have strong specificity, the corresponding data of A, B, C are shown in the table 4, the table 5 and the table 6 in the figure 1, and PSS and other strains can be successfully distinguished. Sensitivity tests of the primers CLL001F/R and probes were performed using genomic DNA series gradient concentrations of strain 29277 at 50 ng/. Mu.L, 5 ng/. Mu.L, 0.5 ng/. Mu.L, 0.05 ng/. Mu.L, 0.005 ng/. Mu.L, 0.5 pg/. Mu.L, 0.05 pg/. Mu.L, 0.005 pg/. Mu.L, 0.0005 pg/. Mu.L. The results of the RPA experiments showed that the lowest concentration of DNA detected by the RPA method was 0.0005 pg/. Mu.L (FIG. 2, see Table 7 for corresponding data).
TABLE 4 Table 4
TABLE 5
TABLE 6
TABLE 7
2.3 analog seed sample detection with bacteria
The invention prepares 6 parts of corn seeds with different bacterial loads (inoculum size is 1 multiplied by 10) 8 ~1×10 2 CFU/ml), RPA experiments using CLL001F/R and probes showed a detection limit of 1X 10 for seed samples 2 CFU/ml, i.e. in a single reaction system2 cells (see table 8 for corresponding data in fig. 3 a) can be detected. After the young corn leaves are subjected to needling inoculation, sampling is carried out after the occurrence of the disease is confirmed, DNA is extracted, RPA detection is carried out, the CT value of a sample at an inoculation position is found to be 19.3, the same piece is about 27 a little away, the sample is positive, the result of the control sample is negative (B in fig. 3, corresponding data are shown in table 9).
TABLE 8
TABLE 9
Discussion of the invention
At presentP. stewartiiThe subspecies have 2 subspecies, namely PSI and PSS, and the genetic relationship among subspecies is similar, but PSI is not pathogenic to corn, and can cause millet to be treatedSetaria italica) Andpennisetum amer- icanumleaf spot and pineapple of (a)Ananas comosus) Is decomposed. Based on the belief of the learnerP. stewartiiCan be separated into 2 different species. Because of the high sequence similarity of these 2 subspecies, many PSS specific primers can amplify PSI, thereby resulting in false positive amplification, affecting the accuracy of the detection results. Wang, etc ［1］ 5 pairs of reported PSS specific primers are verified by 39 strains, and 3 pairs of primers can be found to amplify PSI; block et al ［2］ 9 pairs of reported primers are verified, and the false positive amplification phenomena with different degrees are found; pal et al ［3］ 8 pairs of reported primers were verified and each of these 8 pairs of primers was found to amplify PSI. Based on previous researches, 26 strains including similar strains separated from corn seed samples are collected, the specificity of 18 pairs of primers reported at present is evaluated, the test results are consistent with the previous researches, most of the tested primers have false positive amplification, and only the DC283galE/DC283galEc and cpsAB2313F/cpsR primers have good specificity.
[1] Wang Ying, zhou Guoliang, yin Liping, et al, maize bacterial wilt bacteria PCR detection, plant pathology journal, 2009, 39 (4): 368-376.
［2］ Block C C，Shepherd L M，Munkvold G. Comparison of nine PCR primer sets designed to detect Pantoea stewartii subsp. stewartii in maize［EB/OL］.（2021-06-06）［2021-06-07］.https://www.ars.usda.gov/research/publications/publication/?seqNo115=266266.
［3］ Pal N，Block C C，Gardner C A G. A real-time PCR differentiation Pantoea stewartii subsp. stewartii from P.stewartii subsp. indologenes in corn seed. Plant Disease，2019，103（7）：1474-1486.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (10)
1. Detection of bacterial fusarium wilt of cornPantoea stewartii subsp. stewartiiSubspecies based on recombinant polymerase amplification technology and used for exclusionP. stewartii subsp. indologenesThe method comprises the step of detecting a sample to be detected by using primer pairs shown in SEQ ID NO. 1 and SEQ ID NO. 2.
2. The method of claim 1, further employing a probe as follows:
CATGGTGTCGTATTTTAGGTAAATAAGTTGTTTAG [ luminophore ] [ abasic nucleotide analog ] GT [ quencher ] TTTTTTCGCCATGCCG [ blocker ];
the blocking agent is used to block polymerase extension of the probe.
3. The method of claim 2, wherein the abasic nucleotide analog is tetrahydrofuran.
4. The method of claim 2, wherein the blocking agent is a spacer.
5. The method of claim 4, wherein the arm is a C3 arm.
6. The method of claim 2, wherein the luminescent group is FAM and the quenching group is BHQ1.
7. The method of any one of claims 1-6, wherein the sample to be tested comprises at least one of a corn sample, a soil sample, and a water sample.
8. The method of any one of claims 1 to 7 for differentiating maize bacterial wilt bacteriaPantoea stewartii subsp. stewartiiSubspecies andP. stewartii subsp. indologenessubspecies, and the use thereof.
9. Is used for detecting bacterial fusarium wilt of cornPantoea stewartii subsp. stewartiiA kit of subspecies comprising primers as defined in any one of claims 2 to 6 as set forth in SEQ ID No. 1 and SEQ ID No. 2.
10. The kit of claim 9, further comprising one or more of a nucleic acid extraction reagent, a reagent for isothermal nucleic acid amplification, a positive control, and a negative control.
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