CN106978505B - Primer pair for detecting pedunculosis solani and application thereof - Google Patents
Primer pair for detecting pedunculosis solani and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair for detecting stemphylium solani and application thereof. The primer pair for detecting the stemphylium solani disclosed by the invention consists of single-stranded DNAs with the names of Stem-g7F and Stem-g7R respectively; stem-g7F is a single-stranded DNA with a nucleotide sequence of SEQ ID No.1 in a sequence table; stem-g7R is a single-stranded DNA with a nucleotide sequence of SEQ ID No.2 in the sequence list. The sensitivity of detecting the stemphylium solani by using the primer pair of the invention is improved by 1000 times, and reaches 4.285 multiplied by 102fg·μL‑1And accurate quantification can be realized. The primer pair can be used for quantitative detection at the early stage of disease occurrence and before disease occurrence, and can be applied to field soil bacterium-carrying detection and seed bacterium-carrying detection.
Description
Technical Field
The invention relates to a primer pair for detecting stemphylium solani and application thereof, belonging to the technical field of biology.
Background
Tomato (Lycopersicon esculentum Mill) is a perennial herbaceous plant of the genus Lycopersicon of the family Solanaceae, is native in south America, is a vegetable crop widely cultivated in the world, and is widely cultivated in south and north China. The tomato diseases are complex in types and mainly comprise various fungal diseases, bacterial diseases and viral diseases, wherein the types of the fungal diseases are more, early blight, late blight, gray mold, leaf mold and the like are all common tomato diseases in production, and reports on occurrence of tomato gray leaf spot disease are rare. Tomato gray leaf spot occurs worldwide and is severe in warm and humid areas. Tomato leaf spot disease caused by stemphylium species has been reported in the united states, israel, new zealand, etc., causing significant economic loss to local tomato production. In recent years, with the large-area popularization of imported tomato varieties in China, the tomato leaf spot disease is more and more serious. In 2002, the greenhouse disease rate of tomatoes planted on a Shandong fish table reaches 43%, and reaches more than 90% in the next year, the tomato yield reduction rate reaches about 20%, and the yield reduction rate of tomatoes in severely damaged greenhouses is more than 80%.
Tomato gray leaf spot is caused by the fungus Stemphylium (stemphyllium), which occurs on both young and old leaves. The leaves are full of round or irregular small spots at the early stage of disease attack, the center of the disease spot is light brown, the periphery is dark brown, and the later stage is easy to break and perforate. The edges of the leaves can also be attacked, irregular spots are formed along the edges of the leaves, the spots are connected with the leaves and are dark brown along with expansion, the spots gradually fade into grey white to grey brown along with the drying of the leaves, the symptoms change rapidly, and the yellow leaves of the whole plant are caused when the damage is serious.
The stoechium fungus (Stemphylium) is a common fungus of filamentous conidium and can cause diseases of various plants, so far, about 150 fungi have been reported in the world, and more than 40 fungi are recorded in China. Two species of botrytis cinerea in China, namely stolonium solani fungus and stolonium lycopersicum fungus, are mainly used for causing the botrytis cinerea in China, and recent research and research shows that the stolonium solani fungus becomes a main species harmful to tomatoes, hypha of the stolonium solani fungus is branched and separated, taupe is, conidiophores are not obviously differentiated, and the tomato is singly grown or 2-3 roots grow in a fascicular shape, cylindrical, brown and smooth. Conidiophores are terminal, multiple single, inverted stick-shaped, brown, smooth and brick-like, generally have 1-5 transverse septa and a plurality of longitudinal septa, and are contracted at the intermediate septa of the conidiophores.
The commonly used method for identifying and detecting the tomato gray leaf spot is mainly a morphological identification method, the morphology of conidia is observed in a microscopic examination mode, the type of disease damage is determined, the result is relatively accurate, but some problems exist, no spore is produced before disease manifestation occurs at the early stage of disease occurrence, the type of disease cannot be accurately determined through the traditional microscopic examination mode, and the disease is a key period of medicament prevention and treatment, and the medicament is taken at the time, so that the effect of achieving half the effort can be achieved. At present, a method for rapidly detecting the strain of the stemphylium solani, in particular the strain of the stemphylium solani at the initial stage of pathogenic bacteria infection, is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is how to detect the botrytis cinerea (stemphyllium solani).
In order to solve the technical problems, the invention firstly provides a primer pair which can be used for detecting or assisting in detecting the stemphylium solani or detecting or assisting in detecting the diseases caused by the stemphylium solani, wherein the primer pair consists of single-stranded DNAs with the names of Stem-g7F and Stem-g 7R;
the Stem-g7F is a single-stranded DNA with a nucleotide sequence of SEQ ID No.1 in a sequence table; the Stem-g7R is a single-stranded DNA with a nucleotide sequence of SEQ ID No.2 in a sequence table.
The molar ratio of the Stem-g7F to the Stem-g7R can be 1: 1. The Stem-g7F and the Stem-g7 can be packaged separately or together.
In order to solve the technical problems, the invention also provides a system for detecting or assisting in detecting the stolonifera mould or detecting or assisting in detecting the diseases caused by the stolonifera mould, wherein the system comprises the primer pair.
The system may also include reagents and/or instrumentation necessary to perform fluorescent quantitative PCR. The reagent required for performing the fluorescent quantitative PCR may be 2 XSSYBR Mix of Tiangen Biotechnology technology (Beijing) Ltd. The instrument required for performing fluorescent quantitative PCR may be a Real-Time PCR instrument such as an Applied Biosystems 7500Real Time PCRSystem (ABI, USA).
The system can be composed of the primer pair and the reagent and/or the instrument required for carrying out the fluorescence quantitative PCR.
The system may be a reagent or kit comprising the relevant reagents.
In order to solve the technical problem, the invention also provides any one of the following applications of the primer pair:
x1, in the preparation of products for detecting or assisting in detecting the botrytis cinerea;
x2, and application in preparation of products for detecting or assisting in detection of diseases caused by stemphylium solanacearum.
X3, in the detection or the auxiliary detection of the stolonifera abenanthus;
x4, and application in detection or auxiliary detection of diseases caused by stolonifera.
In order to solve the technical problem, the invention also provides any one of the following applications of the system:
x1, in the preparation of products for detecting or assisting in detecting the botrytis cinerea;
x2, and application in preparation of products for detecting or assisting in detection of diseases caused by stemphylium solanacearum.
X3, in the detection or the auxiliary detection of the stolonifera abenanthus;
x4, and application in detection or auxiliary detection of diseases caused by stolonifera.
The product may be a reagent or a kit.
In order to solve the technical problems, the invention also provides a preparation method of the primer pair, which comprises the following steps: and packaging the two single-stranded DNAs of the primer pair respectively.
In order to solve the technical problems, the invention also provides a method for detecting or assisting in detecting the stemphylium solani, which comprises the following steps: and (3) performing fluorescent quantitative PCR by using the genome DNA of the sample to be detected as a template and the primer pair, and determining whether the sample to be detected contains the botrytis cinerea or is the botrytis cinerea according to the change of substances in a fluorescent quantitative PCR reaction system.
The change of the substances in the fluorescent quantitative PCR reaction system can be determined by an amplification curve: the amplification curve of the fluorescent quantitative PCR reaction system is an S-shaped amplification curve, and the sample to be detected contains or is candidate to contain or is candidate to be the stolonifera mould; the amplification curve of the fluorescent quantitative PCR reaction system is a non-S-shaped amplification curve, and the sample to be detected does not contain or is candidate to contain the botrytis cinerea or is candidate to be non-botrytis cinerea.
The change of the substances in the fluorescent quantitative PCR reaction system can also be determined by detecting the size of the reaction product: the fluorescent quantitative PCR reaction product contains a DNA fragment of 150bp, and the sample to be detected contains or is candidate to contain or is candidate to be the stolonifera mould; the fluorescent quantitative PCR reaction product does not contain a DNA fragment of 150bp, and the sample to be detected does not contain or candidate does not contain the botrytis cinerea or non-candidate botrytis cinerea.
The size of the reaction product can be detected by electrophoresis. The 150bp DNA fragment can be embodied as a band between 100-200 bp.
In order to solve the technical problems, the invention also provides a method for detecting or assisting in detecting diseases caused by stemphylium solani, which comprises the following steps: and (3) performing fluorescent quantitative PCR by using the genome DNA of a sample to be detected as a template and the primer pair, and determining whether the disease of the sample to be detected is the disease caused by the stemphylium solanacearum or not according to the change of substances in a fluorescent quantitative PCR reaction system.
The change of the substances in the fluorescent quantitative PCR reaction system can be determined by an amplification curve: the amplification curve of the fluorescent quantitative PCR reaction system is an S-shaped amplification curve, and the disease of the sample to be detected is or is selected as a disease caused by the stolonifera abel; the amplification curve of the fluorescent quantitative PCR reaction system is not an S-shaped amplification curve, and the disease of the sample to be detected is not or is not selected from the disease caused by the stolonifera.
The change of the substances in the fluorescent quantitative PCR reaction system can also be determined by detecting the size of the reaction product: the fluorescent quantitative PCR reaction product contains a DNA fragment of 150bp, and the disease of the sample to be detected is or is selected as a disease caused by the stemphylium solani; the fluorescent quantitative PCR reaction product does not contain a DNA fragment of 150bp, and the disease of the sample to be detected is not or is not selected as a candidate to be the disease caused by the stolonifera.
The size of the reaction product can be detected by electrophoresis. The 150bp DNA fragment can be embodied as a band between 100-200 bp.
As described above, the concentrations of both strands of the primer pair in the fluorescent quantitative PCR reaction system were each 0.2. mu.M.
The annealing temperature of the fluorescent quantitative PCR reaction may be 60 ℃. The annealing condition may be annealing at 60 ℃ for 32 s. The reaction conditions of the fluorescent quantitative PCR reaction can be as follows: pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 32s, 40 cycles.
The primer pair for detecting the stemphylium solani has good specificity and rapid identification: in the environment, especially in a sunlight greenhouse, microorganisms are various, and the stemphylium solani is not sporulated in the early stage of tomato infection, so that the early warning and monitoring are difficult. And the botrytis cinerea is difficult to produce spores under the condition of artificial in-vitro culture, which also increases the difficulty of separation and identification. The primer pair of the invention overcomes the defects of microscopic observation and isolated cultureAnd the defects that the traditional plant pathology consumes long time and early warning monitoring cannot be realized are overcome. Compared with the traditional PCR detection technology, the sensitivity of the primer pair for carrying out fluorescence quantitative PCR detection on the ustilaginoidea solanacearum is improved by 1000 times, and reaches 4.285 multiplied by 102fg·μL-1And accurate quantification can be realized. The primer pair of the invention is used for carrying out fluorescence quantitative PCR, so that the quantitative detection can be carried out at the early stage of disease occurrence and before the disease is revealed, and meanwhile, the sensitivity is greatly improved compared with the traditional PCR method, so that the primer pair can be applied to the field soil bacterium-carrying detection and the seed bacterium-carrying detection.
Drawings
FIG. 1 shows the result of detecting the specificity of the primer pair. Wherein N is a negative control, 1-9 are respectively Aureobasidium solanacearum (YQZ11020901), Botrytis cinerea (HG09021201), Sclerotinia sclerotiorum (HG09021301), Alternaria alternata (FQ15051201), Alternaria solani (QZ08092301), Alternaria solani (JGFQ15080715), Alternaria cucurbitae (HG09042301), colletotrichum capsici (LJ10013001) and Psychotria polystachys (SD21), and M is a DNA molecular weight standard.
FIG. 2 is a standard curve for real-time fluorescent quantitative PCR constructed using recombinant plasmids.
FIG. 3 is a dissolution curve for the process of constructing a standard curve.
FIG. 4 is an amplification curve of a process of constructing a standard curve.
FIG. 5 shows the results of conventional PCR sensitivity detection.
FIG. 6 shows the real-time PCR sensitivity detection results.
FIG. 7 shows the phenotype of tomato leaves inoculated with A.solani at different inoculation times.
FIG. 8 shows the amplification curves of primer pair and primer pair Stem-g0 in example 1. Here, Stem-g7 represents the primer set of example 1.
FIG. 9 shows the melting curves of the primer set and the primer set Stem-g0 in example 1. Here, Stem-g7 represents the primer set of example 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
In the following examples, 2 XTAQQ PCR Master Mix was a product of Biotechnology Limited, Bomaide, Beijing, and 2 XSSYBR Mix was a product of Biotechnology, Ten-Gen Biotechnology (Beijing).
Example 1 preparation of primer set for detecting Phomopsis solani
The primer pair for detecting the stemphylium solani provided by the invention consists of single-stranded DNAs with the names of Stem-g7F and Stem-g7R respectively, wherein the Stem-g7F is the single-stranded DNA with the nucleotide sequence of SEQ ID No.1 in a sequence table; stem-g7R is a single-stranded DNA with a nucleotide sequence of SEQ ID No.2 in the sequence list.
The two single-stranded DNAs in the primer pair are independently packaged, and the molar ratio of Stem-g7F to Stem-g7R is 1: 1.
Example 2 detection of specificity of primer set for detection of Phomopsis solani
The test strains are: 1 strain of botrytis cinerea, 1 strain of phytophthora, 2 strains of alternaria alternata, 3 strains of alternaria solani and 1 strain of polyspora polystachya (table 1), wherein the test strains are stored in a vegetable disease comprehensive control task group of vegetable and flower institute of Chinese academy of agricultural sciences.
TABLE 1 test strains information
| Serial number | Numbering | Latin name | Name of Chinese | |
| 1 | YQZ11020901 | Stemphylium solani | Fungus of |
|
| 2 | HG09021201 | Botrytis cinerea | Botrytis cinerea (Fr.) |
|
| 3 | HG09021301 | Sclerotinia | Sclerotinia sclerotiorum | |
| 4 | FQ15051201 | Alternaria | Alternaria alternata | |
| 5 | QZ08092301 | Alternaria solani | Alternaria solani | |
| 6 | JGFQ15080715 | Alternaria solani | Alternaria solani | |
| 7 | HG09042301 | Alternaria | Alternaria cucumeriae | |
| 8 | LJ10013001 | Colletotrichum capsici | Pepper colletotrichum | |
| 9 | SD21 | Corynespora cassiicola | Exotidium torvum |
In Table 1, the strain A.solanacearum (Stemphylium solani) with the number YQZ11020901 is described in the literature (Xiiet al, First report of Stemphylium solani consuming leaf spot on gold egg plant China, Can.J. plant Pathol.,2016, Vol.38, No.4,517-one 521);
botrytis cinerea (Botrytis cinerea) with the number of HG09021201 is described in the literature (Tangming et al, comprehensive evaluation of Botrytis cinerea on control effect of cucumber gray mold, Chinese vegetables 2016 (2): 51-55);
sclerotinia sclerotiorum (Sclerotinia scleritum) with the number HG09021301, Alternaria solani (Alternaria solani) with the number QZ08092301, colletotrichum capsici (Colletotrichcapsicii) with the number LJ10013001, Alternaria cucumerina (Alternaria cuumera) with the number HG09042301 and Corynespora polyspora cassiicola (Corynespora cassiicola) with the number SD21 are all described in the literature (Gaoshi et al, Dot-ELISA detection technology study on cucumber clavospora leaf spot, plant protection, 2013,39 (6): 69-73).
2. Specificity detection
Extracting the genome DNA of each test strain in the step 1, respectively carrying out common PCR and real-time PCR amplification on the genome DNA of each strain by using the primer pair in the embodiment 1, detecting the specificity of the primers, and replacing the genome DNA with deionized water as a negative control.
1. General PCR reaction
General PCR reaction System:
general PCR reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was supplemented at 72 ℃ for 5 min. And (3) detecting the PCR product by 2% agarose gel electrophoresis, and analyzing the result by a gel imaging system.
2. Real-time PCR reaction
Real-time PCR reaction system:
real-time PCR reaction conditions: pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 32s, and automatically generating a dissolution curve by the system after 40 cycles. The Real-time PCR reaction was performed using the RealTime PCR System (ABI, USA) Applied Biosystems 7500. And (3) carrying out electrophoresis detection on the amplification product according to whether the peak value of the melting curve singly excludes the nonspecific amplification caused by the dimer, and judging whether the amplification product is the target.
The results of Real-time PCR reactions only showed that the strain A had a band of about 150bp amplified by the strain A, and no dimer, and the target product was obtained by the other strains without amplification with negative control (FIG. 1), and the same results were obtained by the ordinary PCR reactions. The Real-time PCR reaction result shows that an S-shaped amplification curve is generated when only the genomic DNA of the stemphylium solani is used as a template for carrying out Real-time PCR reaction, and other strains and negative control have no S-shaped amplification curve, which indicates that the primer can specifically detect the stemphylium solani.
When the primer pair in example 1 is used for Real-time PCR reaction to detect a sample to be detected, whether the sample to be detected contains or is not botrytis cinerea or not can be determined through the amplification curve, and whether the sample to be detected contains or is botrytis cinerea or not can be determined through detecting a Real-time PCR reaction product: if the curve obtained by the Real-time PCR reaction is an S-shaped amplification curve, or the Real-time PCR reaction product contains a 150bp target strip, the product to be detected contains the botrytis cinerea or the botrytis cinerea; if the curve obtained by the Real-time PCR reaction is a non-S-shaped amplification curve or the Real-time PCR reaction product does not contain a 150bp target strip, the product to be detected does not contain the botrytis cinerea or is not the botrytis cinerea.
Example 3 Standard Curve of primer set for detecting Phomopsis solani
Construction of recombinant plasmid: the genomic DNA of the strain A.solani of example 2 was used as a template, PCR amplification was carried out using the primer set of example 1, and the obtained PCR product having the correct sequence was ligated to pEASY-TI Cloning Vector (Cat. CT101-02, Okawa King Biotech Co., Ltd., Beijing) to obtain a recombinant plasmid, which was designated as pEASY-TI-Stem 7.
pEASY-TI-Stem7 was dissolved in deionized water to give a plasmid concentration of 1.57X 102ng/μL(3.7×1010Copies/. mu.L) of the pEASY-TI-Stem7 solution, and 10-fold gradient dilution of the pEASY-TI-Stem7 solution was performed to obtain a pEASY-TI-Stem7 concentration of 1.57X 10, respectively1ng/μL(3.7×109Copies/. mu.L), 1.57X 100ng/μL(3.7×108Copies/. mu.L), 1.57X 10-1ng/μL(3.7×107Copies/. mu.L), 1.57X 10-2ng/μL(3.7×106Copies/. mu.L), 1.57X 10-3ng/μL(3.7×105Copies/. mu.L), 1.57X 10-4ng/μL(3.7×104Copies/. mu.L), 1.57X 10-5ng/μL(3.7×103Copies/. mu.L) and 1.57X 10-6μg/μL(3.7×102Copies/. mu.L) of pEASY-TI-Stem 7.
The genomic DNA of the strain was replaced with the above-mentioned pEASY-TI-Stem7 solutions according to the reaction system of step 2 of example 2, and Real-time PCR was carried out under the reaction conditions of step 2 of example 2, in each of which three replicates were set.
Taking the logarithm value of the template concentration as an abscissa and the Ct value of Real-time PCR as an ordinate, constructing a Real-time PCR standard curve as follows: 30.789-3.2989x, coefficient of correlation (R)2) 0.9919, the automated amplification efficiency of the fluorescent quantitative PCR system was 100.974% (FIG. 2) and the dissolution curve was unimodal(FIG. 3), there was no nonspecific amplification of dimers or the like (FIG. 4).
Example 4 detection of sensitivity of primer set for detecting Phomopsis solani
Dissolving the genomic DNA of the strain No. YQZ11020901 of the stolonifera in deionized water to obtain the DNA concentration of 4.285 multiplied by 108fg·μL-1(1.12×1010The genomic DNA solution was diluted 10-fold in a gradient of copies/. mu.L to obtain a genomic DNA concentration of 4.285X 107fg·μL-1(1.12×109Genomic DNA copies/. mu.L), 4.285X 106fg·μL-1(1.12×108Genomic DNA copies/. mu.L), 4.285X 105fg·μL-1(1.12×107Genomic DNA copies/. mu.L), 4.285X 104fg·μL-1(1.12×106Genomic DNA copies/. mu.L), 4.285X 103fg·μL-1(1.12×105Genomic DNA copies/. mu.L), 4.285X 102fg·μL-1(1.12×104Genomic DNA copies/. mu.L), 4.285X 101fg·μL-1(1.12×103Genomic DNA copies/. mu.L), 4.285X 100fg·μL-1(1.12×102Genomic DNA copies/. mu.L) and 4.285X 10-1fg·μL-1(1.12×101Copies of genomic DNA/. mu.L).
The genomic DNA of the strain was replaced with the genomic DNA solution of each of the aforementioned stolonifera in accordance with the reaction system of step 2 of example 2, and the sensitivity of the primer set of example 1 was measured by Real-time PCR under the reaction conditions of step 2 of example 2, and the PCR product was detected by agarose gel electrophoresis, using deionized water as a negative control.
The sensitivity of the primer set of example 1 was measured by ordinary PCR using the reaction conditions of step 1 of example 2, using deionized water as a negative control, by replacing the genomic DNA of the strain with the genomic DNA solutions of the above-mentioned respective stolonifera, according to the reaction system of step 1 of example 2. Correlation coefficient R of standard curve of target gene and reference gene2More than 0.99, the PCR amplification efficiency E is between 0.9 and 1.05, and the amplification of the twoThe difference of the synergy rate is not more than 5%.
The results showed that the concentration of the genomic DNA solution of Tachybotrys solani was 4.285X 10 when it was subjected to ordinary PCR amplification5fg·μL-1The above all have specific bands, and the concentration of the genomic DNA of the Ractophyllum solani is less than 4.285X 105fg·μL-1No band of interest was observed in both the time and negative controls (FIG. 5), indicating that the sensitivity of the primer set of example 1 for ordinary PCR amplification was 4.285X 105fg·μL-1(ii) a The concentration of the genomic DNA solution of the A.solani was 4.285X 10 at the time of real-time PCR2fg·μL-1The above all have amplification curves, and the concentration of the genomic DNA solution of the Ractophora solanacearum is less than 4.285 × 105fg·μL-1No amplification curves were observed for both the time and negative controls (FIG. 6), indicating that the sensitivity of the primer set of example 1 of real-time PCR was 4.285X 102fg·μL-1。
In FIGS. 5 and 6, 1 to 10 represent the genomic DNA concentrations of 4.285X 10 for A.solani8fg·μL-1、4.285×107fg·μL-1、4.285×106fg·μL-1、4.285×105fg·μL-1、4.285×104fg·μL-1、4.285×103fg·μL-1、4.285×102fg·μL-1、4.285×101fg·μL-1、4.285×100fg·μL-1And 4.285 × 10- 1fg·μL-1The result of the genomic DNA solution of the stemphylium solani is that N is a negative control and M is a DNA molecular weight standard.
Example 5 Real-time PCR assay of tomato leaves inoculated with A.solani
Activating a strain of the botrytis cinerea with the strain number of YQZ11020901 stored at the low temperature of 4 ℃ by adopting a PDA (personal digital Assistant) plate, transferring the strain to a PD liquid culture medium, culturing for 7 days at the temperature of 28 ℃, breaking hypha by a soybean milk machine, inoculating tomato leaves by a fungus spraying suspension method to inoculate tomato plants in a 3-4 leaf stage, preserving heat and moisture, and photographing and sampling the tomato leaves in 6h, 12h, 24h, 48h, 72h, 96h and 108h periods respectively (figure 7). After extracting genomic DNA of each tomato leaf (concentration is 86.7 ng/. mu.l), the genomic DNA of the strain was replaced with the genomic DNA of each tomato leaf according to the reaction system of step 2 in example 2, and Real-timePCR was performed under the reaction conditions of step 2 in example 2, using deionized water as a negative control.
The results show that the stemphylium solani can be detected in the tomato leaves after the stemphylium solani is inoculated on the tomato leaves for 6 hours, and the bacterial load is continuously increased along with the increase of time; when the tomato leaves are inoculated with the stemphylium solanacearum for 108h, the whole leaves are infected with the stemphylium solanacearum, and the content of the genomic DNA of the stemphylium solanacearum in the genomic DNA obtained from the tomato leaves at the moment is calculated to be 1.16 multiplied by 10 according to the standard curve of the example 3-3ng/μl。
TABLE 2 dynamic variation of the content of stemphylium solani on living plants
Comparative example 1 comparison of detection of Phomopsis Solani with different primer pairs
The primer set and the primer set Stem-g0 in example 1 were used to perform Real-time PCR on genomic DNA of A.solani strain YQZ11020901, and the reaction system and reaction conditions were the same as those in example 2, step 2. Primer sequences for Stem-g0 are shown in Table 3, using deionized water as a negative control.
TABLE 3 primer sequences of Stem-g0
The results show that the primer pair of example 1 amplifies positively well, the negative control does not amplify, and the dissolution curve has a single peak without primer dimer; the primer pair Stem-g0 has good positive amplification, amplification is carried out in the later period of negative control, the dissolution curve is double peaks, and primer dimers are present (figure 8 and figure 9), which shows that the primer pair of the embodiment 1 has better effect on detecting the stemphylium solani than Stem-g 0.
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Claims (7)
1. Any one of the following uses of primer pairs:
x1, in the preparation of products for detecting or assisting in detecting the botrytis cinerea;
x2, in the preparation of products for detecting or assisting in detecting diseases caused by the stemphylium solani;
x3, in the detection or the auxiliary detection of the stolonifera abenanthus;
x4, in the detection or the auxiliary detection of diseases caused by the stemphylium solani;
the primer pair consists of single-stranded DNA with the names of Stem-g7F and Stem-g 7R;
the Stem-g7F is a single-stranded DNA with a nucleotide sequence of SEQ ID No.1 in a sequence table; the Stem-g7R is a single-stranded DNA with a nucleotide sequence of SEQ ID No.2 in a sequence table.
2. Use of any of the following systems for detecting or aiding in the detection of a strain of stemphylium solani or for detecting or aiding in the detection of a disease caused by stemphylium solani:
x1, in the preparation of products for detecting or assisting in detecting the botrytis cinerea;
x2, in the preparation of products for detecting or assisting in detecting diseases caused by the stemphylium solani;
x3, in the detection or the auxiliary detection of the stolonifera abenanthus;
x4, in the detection or the auxiliary detection of diseases caused by the stemphylium solani;
the system comprises a primer pair, wherein the primer pair consists of single-stranded DNA with the names of Stem-g7F and Stem-g 7R;
the Stem-g7F is a single-stranded DNA with a nucleotide sequence of SEQ ID No.1 in a sequence table; the Stem-g7R is a single-stranded DNA with a nucleotide sequence of SEQ ID No.2 in a sequence table.
3. Use according to claim 2, characterized in that: the system also includes reagents and/or instrumentation required to perform fluorescent quantitative PCR.
4. The method for preparing the primer set according to claim 1, comprising: and packaging the two single-stranded DNAs of the primer pair respectively.
5. A method of making the system of claim 2 or 3, comprising: and packaging the single-stranded DNA of the primer pair and/or reagents required for carrying out fluorescence quantitative PCR respectively and independently.
6. A method for detecting or aiding in the detection of stemphylium solani comprising: using the genome DNA of a sample to be detected as a template, carrying out fluorescent quantitative PCR by using the primer pair in claim 1, and determining whether the sample to be detected contains the stemphylium solanacearum or is the stemphylium solanacearum according to the change of substances in a fluorescent quantitative PCR reaction system.
7. A method for detecting or assisting in detecting diseases caused by stemphylium solani comprises the following steps: using the genome DNA of a sample to be detected as a template, carrying out fluorescence quantitative PCR by using the primer pair in claim 1, and determining whether the disease of the sample to be detected is the disease caused by the botrytis cinerea according to the change of substances in a fluorescence quantitative PCR reaction system.
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