CN115725761B - Core gene design-based Solanaceae Ralstonia detection primer and application thereof - Google Patents
Core gene design-based Solanaceae Ralstonia detection primer and application thereof Download PDFInfo
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Abstract
The invention discloses a core gene design-based detection primer for Ralstonia solanacearum and application thereof. The invention provides a primer pair for identifying or assisting in identifying Ralstonia solanaceae, which is designed according to CDC45_RS17435 genes in the Ralstonia solanaceae genome, and consists of two single-stranded DNA shown as SEQ ID No.1 and SEQ ID No. 2. The invention can be applied to a rapid quantitative detection method of the Solanaceae Ralstonia in actual production, and is helpful for solving the problem of bacterial wilt control in actual production in China.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer for detecting Ralstonia solanacearum based on core gene design and application thereof.
Background
Ralstonia solanaceae (Ralstonia solanacearum) is a gram-negative phytopathogenic bacterium, and bacterial wilt caused by it is a devastating soil-borne disease. The solanacearum has distribution in the global scope, and is mainly distributed in the south area in China. When no host plant exists, the Solanaceae Ralstonia can survive in soil or water for a plurality of years, when a proper host plant exists, the Solanaceae Ralstonia can infect the plant from the wound or natural opening of the root or stem base, and the Solanaceae Ralstonia is propagated in a large quantity on the xylem, so that the normal water transport path in the plant is blocked, and the plant wilts due to lack of water and even dies. The Solanaceae Ralstonia is a compound species and can be divided into 4 evolution types, wherein the distribution in China is mainly based on the evolution type I. The Ralstonia solanaceae can infect more than 200 plants in more than 50 families, and the disease speed is high, once the disease is generated, the crop yield is affected, and the agricultural product quality and the agricultural economy development in China are seriously affected. Therefore, the population density of the Solanaceae Ralstonia is detected before or at the early stage of onset, and the bacterial wilt can be effectively prevented from large-area outbreak by intervention of a biological control method or a chemical control method.
Along with the development of modern molecular biology, PCR technology has been widely used for monitoring, detecting and early warning the pathogenic bacteria population dynamics. Compared with the traditional plate coating counting method and plate scribing separation method, the PCR technology has the advantages of short detection time and high accuracy, and can greatly improve the detection efficiency. Currently, the main difficulty of applying this technology to practical production is to find target genes for specific primers.
Disclosure of Invention
The invention aims to design and verify a specific primer suitable for rapid quantitative detection of the solanacearum by using a conserved ortholog gene in the solanacearum as a target point, and solve the problem of bacterial wilt prevention in agricultural production.
orthoFinder is a commonly used tool in comparative genomics research at present, and has the advantage of fast, comprehensive and accurate operation. It can find orthologs and orthologs of orthogonal groups and infer root gene trees for all orthogonal groups, identifying gene duplication events present in these gene trees. Orthologous genes, also called orthologous genes, refer to genes that have evolved vertically from the same ancestor, and generally have the characteristics of coding sequences and function conservation. The invention utilizes the orthoFinder to analyze the ortholog gene existing in the Ralstonia solanacearum, searches a sequence-conserved gene target, designs a specific primer for rapid detection of the Ralstonia solanacearum according to the conserved nucleotide locus of the gene, and uses the NCBI database for performing specificity verification, thereby providing a rapid quantitative detection method of the Ralstonia solanacearum, which can be applied to actual production, and is beneficial to solving the bacterial wilt prevention and treatment problem in actual production in China.
In a first aspect, the invention claims a primer pair for use in or in assisting in the identification of Ralstonia solanacearum (Ralstonia solanacearum).
The primer pair claimed in the invention is designed according to the following (I) or (II):
(I) Cdc45_rs17435 gene in the genome of ralstonia solanacearum;
(II) amplification by using the genome of Ralstonia solanaceae as a template and adopting a primer pair consisting of two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 to obtain an amplification product.
Further, the primer pair consists of two single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No. 2.
In a second aspect, the invention claims a reagent or kit comprising a primer pair as described in the first aspect above.
Further, the kit may contain other PCR conventional reagents in addition to the primer pair.
In a third aspect, the invention claims the use of a primer pair as described in the first aspect hereinbefore or a reagent or kit as described in the second aspect hereinbefore in any of the following:
(A1) Identifying or aiding in identifying Ralstonia solanacearum (Ralstonia solanacearum);
(A2) Identifying or assisting in identifying whether the strain to be tested is Ralstonia solanacearum (Ralstonia solanacearum);
(A3) Detecting or assisting in detecting whether the sample to be detected contains Solanaceae Ralstonia (Ralstonia solanacearum);
(A4) Preventing, monitoring and/or early warning the bacterial wilt;
(A5) And (3) quantitatively detecting the density of the Solanaceae Ralstonia strain group in the soil and/or plant samples.
In a fourth aspect, the invention claims a method for identifying or aiding in the identification of whether the strain to be tested is Ralstonia solanacearum (Ralstonia solanacearum).
The invention claims a method for identifying or assisting in identifying whether a strain to be tested is Ralstonia solanacearum (Ralstonia solanacearum), which can comprise the following steps:
PCR amplification is performed by using genomic DNA of a strain to be tested as a template and using the primer pair (SEQ ID No.1 and SEQ ID No. 2) described in the first aspect to obtain an amplification product, and then whether the strain to be tested is Solanaceae Ralstonia (Ralstonia solanacearum) is determined as follows: if the amplification product contains a 141bp target fragment, the strain to be detected is or is candidate to be solanaceae Ralstonia (Ralstonia solanacearum); otherwise, the strain to be tested is not or is not candidate for solanacearum (Ralstonia solanacearum).
In a fifth aspect, the invention claims a method for detecting or aiding in the detection of presence or absence of Ralstonia solanacearum (Ralstonia solanacearum) in a sample to be tested.
The method for detecting or assisting in detecting whether the sample to be detected contains the Ralstonia solanacearum (Ralstonia solanacearum) comprises the following steps:
extracting total DNA from a sample to be detected, carrying out PCR amplification by using the primer pair (SEQ ID No.1 and SEQ ID No. 2) according to the first aspect by taking the total DNA as a template to obtain an amplification product, and determining whether the sample to be detected contains Solanaceae Ralstonia (Ralstonia solanacearum) according to the following steps: if the amplification product contains a 141bp target fragment, the sample to be detected contains or is candidate to contain Ralstonia solanacearum (Ralstonia solanacearum); otherwise, the sample to be tested does not contain or is candidate to contain Ralstonia solanacearum (Ralstonia solanacearum).
In the methods of the fourth and fifth aspects, the annealing temperature at which the PCR amplification is performed may be 62 ℃.
Further, the reaction procedure for performing the PCR amplification is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 45s, annealing at 62℃for 45s, elongation at 72℃for 1min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
In the reaction systems in which the PCR amplification was carried out in (B1) and (C1), the concentrations of the forward primer (SEQ ID No. 1) and the reverse primer (SEQ ID No. 2) in the primer pair were 10. Mu.M.
Further, the reaction system for performing the PCR amplification was 20. Mu.L, which includes 10. Mu.L of 2X Taq PCR Master Mix (Kang Run organism), 1. Mu.L of forward primer, 1. Mu.L of reverse primer, 1. Mu.L of soil sample DNA, 7. Mu.L of ddH 2 O, wherein the concentration of the forward primer and the reverse primer is 10 mu M, and the total amount of DNA of the soil sample is 20ng-2 mu g.
In the present invention, the PCR may be either a general PCR (for qualitative detection) or a fluorescent quantitative PCR (for quantitative detection).
In the above-described related aspect, the strain to be tested may be selected from Ralstonia solanacearum (Ralstonia solanacearum), ralstonia other than Ralstonia solanacearum, or Ralstonia other than Ralstonia solanacearum.
Further, the other Ralstonia bacteria other than Ralstonia solanacearum may be selected from the following: ralstonia eutropha, ralstonia respiraculi, ralstonia taiwanensis. The non-ralstonia may be selected from the following: psedomonas syringae, bacillus sp, pantoea dispersoa, xanthomonas sacchari, pseudomonas mediterranea, pseudomonas chlororapghis, pseudomonas fluorescens, ensifer sp, pseudomonas syringae, chryseobacter sp, acidovorax citrulli, pseudomonas fluorescens, pseudomonas fluorescens, bacillus tequilensis, pseudomonas mohnii, pseudomonas moraviensis, pseudomonas putida, streptomyces laurentii, bacillus subtilis.
In the above related aspect, the sample to be tested may be an environmental sample or a plant sample;
further, the environmental sample may be a soil sample.
In a sixth aspect, the invention claims the use of a method as described in the fourth or fifth aspect hereinbefore for any one of:
(B1) Preventing, monitoring and/or early warning the bacterial wilt;
(B2) And (3) quantitatively detecting the density of the Solanaceae Ralstonia strain group in the soil and/or plant samples.
The invention utilizes the orthoFinder to analyze the ortholog gene existing in the Ralstonia solanacearum, searches a sequence-conserved gene target, designs specific primers (SEQ ID No.1 and SEQ ID No. 2) for rapid detection of the Ralstonia solanacearum according to the conserved nucleotide locus of the gene, and uses an NCBI database for performing specificity verification, thereby providing a rapid quantitative detection method of the Ralstonia solanacearum, which can be applied to actual production, and is beneficial to solving the problem of bacterial wilt prevention in actual production in China.
Compared with the prior art, the invention has the following advantages:
(1) The method utilizes bioinformatics method to excavate core genes of the Solanaceae Ralstonia strain group, and finds out the highly conserved gene CDC45_RS17435 in the strain group, on the basis, the inventor designs a specific primer for PCR rapid detection, and the primer pair can specifically amplify a 141bp band in the Solanaceae Ralstonia strain group, and the band is clear and single, which indicates that the primer has good specificity.
(2) The specific primer designed by the invention has high sensitivity, and the DNA template is diluted in a gradient way and then is 59 multiplied by 10 -3 The specific primer can still stably detect the target band at the ng/. Mu.L level.
(3) The specific primer designed by the invention has good practicability, and can be used for rapidly identifying the Ralstonia solanacearum in actual field production. The traditional separation and coating plate identification method has long time consumption, high cost and complicated steps, and is not suitable for qualitative detection of soil samples with high flux. The primer designed by the invention can amplify an electrophoresis strip with expected size in a soil sample containing Solanaceae Ralstonia, which shows that the primer can be applied to prevention, monitoring and early warning of bacterial wilt in actual production, thereby providing scientific theoretical basis for prevention and control of bacterial wilt and protecting development of agricultural economy in China.
(4) The size of the primer amplification product designed by the invention is only 141bp, the extension time required in the PCR reaction program is short, most of Taq enzymes which can be obtained through commercial paths at present can be extended for only 10 seconds at most to complete the extension reaction for obtaining the specific band, the reaction time required by detection can be greatly shortened, and the detection efficiency is improved; the specific primer provided by the invention can be used for rapidly and simply completing qualitative detection of Solanaceae Ralstonia in a sample. The whole process from extraction of sample DNA to completion of agarose gel electrophoresis takes about 2.5 hours. Compared with the traditional plate separation method, the method can greatly shorten the time, reduce the required manpower and material resources and better meet the agricultural production requirements of China.
(5) The specific primer designed by the invention is also suitable for fluorescent quantitative PCR detection, can quantitatively detect the density of the Solanaceae Ralstonia bacterial flora in soil and plant samples, and can perform risk early warning on bacterial wilt.
Drawings
FIG. 1 shows the results of comparative analysis of orthologous genes specific to Ralstonia solanacearum in Ralstonia.
FIG. 2 shows the results of analysis of the comparison of ortholog genes specific to Ralstonia solanaceae with the ortholog genes of Pseudomonas.
FIG. 3 is the result of the WP_011001652 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 4 is the result of the WP_011001652 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 5 is the result of the WP_011002720 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 6 is the result of the WP_011002720 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 7 is the result of the WP_011001985 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 8 is the result of the WP_011001985 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 9 is the result of the WP_011003273 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 10 is the result of the WP_011003273 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 11 is the result of the WP_011000296 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 12 is the result of the WP_011000296 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 13 is the result of the WP_011001553 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 14 is the result of the WP_011001553 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 15 is the result of the WP_011002155 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 16 is the result of the WP_011002155 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 17 is the result of the WP_011000032 alignment in Ralstonia solanacearum using NCBI Blastp tool.
FIG. 18 is the result of the WP_011000032 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 19 is the result of the WP_011000639 alignment in Ralstonia solanacearum using the NCBI Blastp tool.
FIG. 20 is the result of the WP_011000639 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 21 is the result of the WP_011002147 alignment in Ralstonia solanacearum using the NCBI Blastp tool.
FIG. 22 is the result of the WP_011002147 alignment in non-Solanaceae Ralstonia using NCBI Blastp tool.
FIG. 23 shows the result of a conservation analysis of CDC45_RS00335 gene encoding WP_011000032 using NCBI Blastn in non-Solanaceae Ralstonia.
FIG. 24 shows the result of a conservation analysis of CDC45_RS11410 gene encoding WP_011002155 using NCBI Blastn in non-Solanaceae Ralstonia.
FIG. 25 shows the result of a conservation analysis of CDC45_RS11370 gene encoding WP_011002147 using NCBI Blastn in non-Solanaceae Ralstonia.
FIG. 26 shows the result of a conservation analysis of CDC45_RS17435 gene encoding WP_011003273 using NCBI Blastn in non-Solanaceae Ralstonia.
FIG. 27 shows the specific primer design position of CDC45_RS17435 gene in Ralstonia solanacearum.
FIG. 28 shows the detection result of the optimal Tm value of the primers designed in the present invention.
FIG. 29 shows the results of specific detection of the primers designed in the present invention.
FIG. 30 shows the sensitivity of the primers designed in the present invention.
FIG. 31 shows the results of detection of the primers designed in accordance with the present invention in soil samples.
FIG. 32 is a qPCR standard curve constructed using the primers designed in accordance with the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 ortholog search for design of specific primers
This example provides a screening process for specific primer design targets. The genome of 106 strains of Ralstonia including 95 strains of Ralstonia solanacearum was downloaded via NCBI (https:// www.ncbi.nlm.nih.gov /) online website. The 106 strains of Ralstonia were subjected to ortholog gene comparison analysis by OrthoFinder (2.3.7) to obtain 1035 ortholog genes coexisting with 106 strains of Ralstonia, 1250 ortholog genes coexisting with 95 strains of Ralstonia solanacearum, and 259 ortholog genes present specifically with Ralstonia solanacearum were obtained by Venn analysis (FIG. 1). To further verify the specificity of the 259 orthologous genes in Ralstonia solanacearum, the inventors continued to compare and analyze the 88 strains of Pseudomonas consensus orthologous genes, which have been published by NCBI, that are closer to Ralstonia solanacearum, using OrthoFinder (2.3.7), and found 356 orthologous genes in total. The inventors have compared these 356 orthologous genes with 259 orthologous genes specific to Ralstonia solanaceae and found that there is no common gene between them (FIG. 2), further illustrating that the 259 orthologous genes are highly specific in Ralstonia solanaceae.
Example 2 conservation prediction of specific orthologous genes
In order to find a target gene more suitable for designing primers, the inventors deleted a gene having an excessively short sequence among the above 259 orthologous genes having specificity, and left 167 orthologous genes. Similarity was calculated for the single copy ortholog sequences (Single Copy Orthologue Sequences) corresponding to these 167 ortholog genes using dancan, the ortholog gene IDs obtained by software calculation were mapped to the actual protein IDs, and the top ten genes were taken for subsequent broad-spectrum, conservation verification (table 2).
Table 2, top ten similarity ranking protein IDs among 167 orthologous genes
The inventors predicted the conservation of the 10 proteins in Ralstonia and non-Ralstonia using the Blastp tool of NCBI website. The results are shown in FIGS. 3 to 22. The protein sequences of the 10 genes are highly conserved in Ralstonia solanaceae, while in Ralstonia solanaceae, the amino acid sequences of WP_01003273, WP_01102155, WP_01101032 and WP_01002147 are less conserved. The inventors then performed a conservative analysis of the nucleotide sequences of these four genes. The results are shown in FIGS. 23 to 26. Analysis revealed that the coding gene CDC45_RS17435 of WP_011003273 is least conserved in non-Ralstonia.
EXAMPLE 3 design of specific primers based on the guard site of CDC45_RS17435 in Ralstonia solanacearum
In view of the low conservation of the gene CDC45_RS1743 in non-Solanaceae Ralstonia, the inventor uses the gene as a target point to design a Solanaceae Ralstonia specific primer. The solanacearum can be divided into 4 evolution types, and the inventor selects representative strains from the 4 evolution types, so as to compare homologous genes of CDC45_RS17435 in the strains and analyze conserved nucleotide sites. The selected strains are shown in Table 3.
TABLE 3 homologous Gene alignment of CDC45_RS17435 in Ralstonia solanaceae selected strains
The result of comparison and analysis of the homologous genes of the CDC45_RS17435 of the Solanaceae Ralstonia in Table 3 shows that the genes have a plurality of nucleotide sites with higher conservation, and the result is shown in FIG. 27. The inventor designs a Solanaceae Ralstonia specificity detection primer based on CDC45_RS17435 according to a conserved nucleotide sequence, wherein the sequence is as follows:
an upstream primer: 5'-GCCAACATGGTCGAGCTGCAGT-3' (SEQ ID No. 1);
a downstream primer: 5'-CGAACGCCCGATCTTGAGCA-3' (SEQ ID No. 2).
The theoretical amplification product was 141bp.
Example 4 detection of optimal amplification conditions for primer specific for Ralstonia solanaceae
The specific detection primers (SEQ ID No.1 and SEQ ID No. 2) for Ralstonia solanaceae were synthesized by the biological company of the family Rhizoctonia in a manner of PAGE. The inventors detected the optimal Tm values of the pair of primers. The inventors have identified sterile ddH 2 O served as a control. PCR amplification system and detection of amplified products are described in the above summary, annealing temperature gradient in the reaction procedureSetting 60 ℃,62 ℃,64.3 ℃,65.5 ℃,67.8 ℃ and 70 ℃. The detection of the amplified products is shown in FIG. 28. At a Tm value of 60℃in sterile ddH 2 A specific band of 141bp in size can be amplified when O or R.solanacearum GMI1000 genomic DNA is used as a template. A clear 141bp size band was amplified using R.solace GMI1000 genomic DNA as a template at 62℃but with sterile ddH under the Tm 2 O is a template, and a band with the size of 141bp cannot be amplified. And the brightness of the amplified specific band gradually decreases when R.solaceae GMI1000 genomic DNA is used as a template at a Tm value of more than 64.3 ℃, and the amplified specific band is subjected to sterile ddH 2 The specific band cannot be amplified under the condition that O is taken as a template. Accordingly, the inventors considered that the optimum Tm value of the primer specific to Ralstonia solanacearum was 62 ℃. Thus, the optimal amplification conditions for this primer pair are: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 45s, annealing at 62℃for 45s, elongation at 72℃for 1min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
EXAMPLE 5 Solanaceae Ralstonia detection primer specificity verification designed based on CDC45_RS17435
The inventor has verified the specificity of the primer pair (SEQ ID No.1 and SEQ ID No. 2), selected 8 strains of Ralstonia solanacearum, including 12 strains of Ralstonia solanacearum, and 19 strains of Ralstonia non-Ralstonia, extracted the genome DNA of each strain, and then respectively subjected to PCR detection, sterile ddH 2 O is a negative control.
The 12 strains of Ralstonia are: ralstonia solanacearum GMI1000 (laboratory deposit), ralstonia solanacearum P380 (laboratory deposit), ralstonia solanacearum P381 (laboratory deposit), ralstonia solanacearum P382 (laboratory deposit), ralstonia solanacearum P383 (laboratory deposit), ralstonia solanacearum ACCC01472 (China agricultural microbiological culture Collection center), ralstonia solanacearum ACCC60146 (China agricultural microbiological culture collection center), ralstonia solanacearum ACCC60145 (China agricultural microbiological culture collection center), ralstonia eutropha ACCC01020 (China agricultural microbiological culture collection center), ralstonia respiraculi ACCC01045 (China agricultural microbiological culture collection center), ralstonia taiwanensis ACCC03117 (China agricultural microbiological culture collection center), ralstonia taiwanensis ACCC03254 (China agricultural microbiological culture collection center). Wherein Ralstonia solanacearum GMI and Ralstonia solanacearum P380 are described in "Shen Cong et al diversity interactions of five core type III effectors from Ralstonia solanacearum with plants.j Genet genomics.2022may 18; s1673-8527 (22) 00138-2.Doi:10.1016/j.jgg.2022.04.018, "A.C., available to the public from the applicant, is only useful in repeating the experiments of the present invention and is not useful.
The selected non-Solanaceae Ralstonia is: psedomonas syringae DC3000 s120 (laboratory deposit), pantoea dispersa PR834 (laboratory deposit), xanthomonas sacchari JT-9 (laboratory deposit), pseudomonas mediterranea S58 (laboratory deposit), pseudomonas chlororapghis S1130 (laboratory deposit), pseudomonas fluorescens P24 (laboratory deposit), ensifer sp.s1279 (laboratory deposit), pseudomonas syringae S1174 (laboratory deposit), chryseobacter sp.s1137 (laboratory deposit), acidovorax citrulli MH (laboratory deposit), pseudomonas fluorescens S823 (laboratory deposit), pseudomonas fluorescens S827 (laboratory deposit), bacillus tequilensis S878 (laboratory deposit), pseudomonas mohnii S891 (laboratory deposit), pseudomonas moraviensis S886 (laboratory deposit), pseudomonas putida S1250 (laboratory deposit), streptomyces laurentii S1238 (laboratory deposit), bacillus subtilis S1227 (laboratory deposit). Wherein Acidovorax citrulli MH is described in "Yi-Nan Ma et al identification of Benzyloxy Carbonimidoyl Dicyanide Derivatives as Novel Type III Secretion System Inhibitors via High-Throughput screening. Front Plant Sci.2019Sep 5; 1059.Doi:10.3389/fpls.2019.01059. ECallLectrolytion 2019, supra, pseudomonas fluorescens P24 is described in "Jing Wang et al, organization of the SPI-1Type III Secretion System in Pseudomonas fluorescens 2P24.Front Microbiol.2021Sep 21; "YIlin Gu et al Characterization of a Versatile Plant Growth-Promoting Rhizobacterium Pseudomonas mediterranea Strain S58.Microorganisms.2020Feb 27," U.S. Pat. No. 12,749037. Doi:10.3389/fmib.2021.749037. ECololation 2021; 8 (3): 334.doi:10.3390/microorganisms8030334, "one text, psedomonas syringae DC3000 is described in" Hai-Lei Wei et al Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv.tolato DC3000 Reveals a Matrix of Effector Interplay in Patogeneses.cell Rep.2018May 8;23 (6) 1630-1638.Doi:10.1016/j. Celep.2018.04.037, "A. Available to the public from applicant, can only be used for repeated experiments of the invention, and cannot be used for him.
PCR amplification System (20. Mu.L): 10. Mu.L 2X Taq PCR Master Mix (Kang Run organism), 1. Mu.L upstream primer (SEQ ID No. 1), 1. Mu.L downstream primer (SEQ ID No. 2), 1. Mu.L of genomic DNA of the strain to be tested, 7. Mu.L ddH 2 O, wherein the concentration of the upstream primer and the downstream primer is 10 mu M, and the total amount of genome DNA of the strain to be detected is 20ng-2 mu g.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 45s, annealing at 62℃for 45s, elongation at 72℃for 1min for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
Detecting by agarose gel electrophoresis after PCR amplification, separating 5 μL amplification product in 2% agarose gel, electrophoresis at 100V voltage for 30min, and observing the existence and the size of the band under ultraviolet lamp after GelRed staining.
The detection of the amplified products is shown in FIG. 29. The detection of the amplified products shows that the primer pair can amplify specific bands with the size of 141bp in the genome DNA of 8 strains of Ralstonia solanaceae. However, the specific band could not be amplified in the rest of non-Solanaceae Ralstonia, indicating that the primer pair specificity was good.
Example 6 specific primer pair sensitivity detection
Using sterile ddH 2 O gradient dilution of genomic DNA of Ralstonia solanacearum GMI1000 at an original concentration of 59 ng/. Mu.L 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -6 ,10 -7 ,10 -8 ,10 -9 Double, PCR amplification of diluted samples with specific primers (SEQ ID No.1 and SEQ ID No. 2) was performed as ddH 2 The amplification result with O as template served as negative control.The amplification system, reaction procedure and detection of amplification products were as described in example 5.
The detection results of the amplified products are shown in FIG. 30. It was found that the template concentration was 59X 10 -3 The primer pair was still able to amplify a 141bp specific band at ng/. Mu.L, whereas the template concentration was 59X 10 -4 At ng/. Mu.L or less, the primer pair cannot amplify a specific band of the desired size, so the detection threshold of the specific primer is 59X 10 -3 ng/. Mu.L and 59X 10 -4 Between ng/. Mu.L, this indicates that the specific primer pair has good sensitivity.
Example 7 specific detection of specific primers applied in samples of real Environment
In order to further verify the practicability of the primers (SEQ ID No.1 and SEQ ID No. 2) designed by the invention, the inventors performed PCR detection on the DNA gene of bacterial wilt soil. The initial concentration of bacterial wilt soil DNA is 62.7 ng/. Mu.L, and sterile ddH is utilized 2 Gradient dilution of soil DNA by O10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -6 The diluted sample was amplified by PCR using specific primers. The amplification system, reaction procedure and detection of amplification products were as described in example 5.
The detection results of the amplified products are shown in FIG. 31. It was found that the specific primer was unable to amplify a 141bp size band at either concentration in healthy plant soil DNA, and that the specific primer amplified a 141bp size band at concentrations of 62.7 ng/. Mu.L and 6.27 ng/. Mu.L in diseased plant soil DNA. This shows that the specific primer is suitable for qualitative detection of Solanaceae Ralstonia in soil sample, and the lowest detection threshold is between 0.627 ng/. Mu.L and 6.27 ng/. Mu.L.
Example 8 application of specific primers to quantitative detection of Ralstonia solanaceae in fluorescent quantitative PCR
In modern molecular biology, fluorescent quantitative PCR techniques have been widely used for quantitative detection of microorganisms in various samples. The specific primers (SEQ ID No.1 and SEQ ID No. 2) in the invention design target points as single copy genes, and are theoretically applicable to fluorescent quantitative PCR detection. To explore the inventionThe inventor utilizes the genome DNA of the Ralstonia solanaceae GMI1000 to construct a standard curve for fluorescent quantitative PCR detection. Genomic DNA of GMI1000 at a concentration of 7 ng/. Mu.L was diluted in a gradient of 10 -1 ,10 -2 ,10 -3 ,10 -4 Double, the diluted samples were subjected to fluorescent quantitative PCR detection using specific primers (SEQ ID No.1 and SEQ ID No. 2). The fluorescent quantitative PCR system was 20. Mu.L, including 10. Mu.L of 2 XSYBR Green (Ai Kerui Co.), 1. Mu.L of the upstream primer (SEQ ID No. 1), 1. Mu.L of the downstream primer (SEQ ID No. 2), 1. Mu.L of DNA (about 50 ng/. Mu.L), 7. Mu.L of ddH 2 O, wherein the concentration of the upstream and downstream primers is 10. Mu.M. The reaction conditions are as follows: pre-denaturation at 95℃for 10min; denaturation at 95℃for 10min, annealing at 62℃for 45s and extension at 72℃for 1min for 40 cycles. Construction of a feasible standard curve y=2.5427x+27.396, R, from the relation between Ct value (y) and sample concentration (x, unit: ng/. Mu.L) 2 =0.9999 (fig. 32). The correlation coefficient of the standard curve is larger than 0.98, which indicates that the specific primer can be applied to quantitative detection of Ralstonia solanacearum.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (11)
1. Use of a primer pair or kit in any of the following:
(A1) Identifying Ralstonia solanacearum;
(A2) Identifying whether the strain to be tested is Ralstonia solanaceae;
(A3) Detecting whether a sample to be detected contains Solanaceae Ralstonia or not;
(A4) Monitoring and/or early warning the bacterial wilt;
(A5) Quantitatively detecting the density of Solanaceae Ralstonia bacterial colony in soil and/or plant samples;
the kit contains the primer pair;
the primer pair consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2.
2. The use according to claim 1, characterized in that: the strain to be tested is selected from Ralstonia solanaceae, other Ralstonia except Ralstonia solanaceae or non-Ralstonia.
3. The use according to claim 1, characterized in that: the sample to be measured is an environmental sample or a plant sample.
4. A use according to claim 3, characterized in that: the environmental sample is a soil sample.
5. A method for identifying whether a strain to be tested is solanacearum or not, comprising the steps of: taking genome DNA of a strain to be detected as a template, carrying out PCR amplification by adopting a primer pair to obtain an amplification product, and then determining whether the strain to be detected is Solanaceae Ralstonia according to the following steps: if the amplification product contains a 141bp target fragment, the strain to be detected is or is candidate to be Solanaceae Ralstonia; otherwise, the strain to be detected is not or is not candidate of Solanaceae Ralstonia;
the primer pair consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2.
6. A method for detecting whether a sample to be detected contains Solanaceae Ralstonia or not comprises the following steps: extracting total DNA from a sample to be detected, carrying out PCR (polymerase chain reaction) amplification by using the total DNA as a template and adopting a primer pair to obtain an amplification product, and determining whether the sample to be detected contains Solanaceae Ralstonia according to the following steps: if the amplification product contains a 141bp target fragment, the sample to be detected contains or is candidate to contain Solanaceae Ralstonia; otherwise, the sample to be detected does not contain or is candidate to contain Ralstonia solanacearum;
the primer pair consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2.
7. The method according to claim 5 or 6, characterized in that: the annealing temperature at the time of performing the PCR amplification was 62 ℃.
8. The method according to claim 5, wherein: the strain to be tested is selected from Ralstonia solanaceae, other Ralstonia except Ralstonia solanaceae or non-Ralstonia.
9. The method according to claim 6, wherein: the sample to be measured is an environmental sample or a plant sample.
10. The method according to claim 9, wherein: the environmental sample is a soil sample.
11. Use of the method of any one of claims 5-10 in any one of the following:
(B1) Monitoring and/or early warning the bacterial wilt;
(B2) And (3) quantitatively detecting the density of the Solanaceae Ralstonia strain group in the soil and/or plant samples.
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| KR20100041256A (en) * | 2008-10-13 | 2010-04-22 | 대한민국 농촌진흥청 | Race specific primers for detecting bacterial wilt of ralstonia solanacearum |
| CN113151523A (en) * | 2021-05-13 | 2021-07-23 | 长江大学 | PCR detection method for ralstonia solanacearum |
| CN114686609A (en) * | 2022-03-30 | 2022-07-01 | 中国农业科学院农业资源与农业区划研究所 | Specific primer for identifying ralstonia solanacearum and application thereof |
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| KR20100041256A (en) * | 2008-10-13 | 2010-04-22 | 대한민국 농촌진흥청 | Race specific primers for detecting bacterial wilt of ralstonia solanacearum |
| CN113151523A (en) * | 2021-05-13 | 2021-07-23 | 长江大学 | PCR detection method for ralstonia solanacearum |
| CN114686609A (en) * | 2022-03-30 | 2022-07-01 | 中国农业科学院农业资源与农业区划研究所 | Specific primer for identifying ralstonia solanacearum and application thereof |
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