CN114540468A - Method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR (polymerase chain reaction) - Google Patents
Method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention provides a method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR (polymerase chain reaction); a real-time fluorescent quantitative PCR detection method of colletotrichum gloeosporioides is established through a primer designed by an actin gene of colletotrichum gloeosporioides; the detection method has high specificity and sensitivity, can detect the colletotrichum gloeosporioides in the latent period, and has important significance for detecting potential colletotrichum gloeosporioides infection; the detection method of the invention also considers the normalization relation between the DNA of pathogenic bacteria and the plant DNA biomass, can accurately determine the growth conditions of the colletotrichum gloeosporioides in different host plants, can be used for screening and identifying the disease-resistant varieties of arabidopsis thaliana and stylosanthes guianensis, and provides a basis for early detection and timely prevention and control of the colletotrichum gloeosporioides.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for quantitatively detecting colletotrichum gloeosporioides by utilizing real-time fluorescent PCR.
Background
Traditionally, the diagnosis of pathogenic infection has relied on isolation, culture of the pathogenic bacteria, which is time consuming and labor intensive. The simplest method for detection of phytopathogens is assessment of visual disease symptoms, but visible lesions usually occur late in host-pathogen interactions, which makes early control of anthracnose difficult.
With the development of the genomics era, real-time fluorescent quantitative PCR has been developed as a method capable of rapidly identifying pathogens and diagnosing diseases. For example, the resistance of different wheat varieties to pathogenic bacteria can be identified by RT-PCR detection of the relative expression of actinin gene in wheat (Hongztao, Zhanyan, the relative biomass of Rhizoctonia cerealis (J) detected by fluorescent quantitative PCR (2015, 41(01): 116) and 121). However, compared with sclerotinia graminearum, hosts which can be infected by colletotrichum gloeosporioides are widely distributed, particularly in tropical and subtropical crop regions including strawberries, mangos, peppers, tomatoes, cassava, pomegranates, stylosana guianensis, hevea brasiliensis and the like, and the strain characteristics, the infection process and the like of the sclerotinia graminearum gloeosporioides and the colletotrichum gloeosporioides are obviously different. Colletotrichum gloeosporioides shows a semi-biological nutrition life style, the pathogenic bacteria invasion process comprises a biological nutrition stage and a destructive necrosis nutrition stage, the colletotrichum gloeosporioides can maintain a long-time latent state under the condition of not causing obvious symptoms in the biological nutrition stage, the conventional method can only identify the colletotrichum gloeosporioides after disease manifestation, and the detection sensitivity is low, for example, in patent CN111534621A primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides, the detection method has the sensitivity of 0.57 ng/mu L DNA, the sensitivity is not high enough, the best time for prevention and treatment is easily missed, and serious economic loss is caused. And the field hazard symptoms of the anthrax pathogenic bacteria of the colletotrichum such as colletotrichum gloeosporioides and colletotrichum oxysporum are basically similar, and the existing identification method is difficult to distinguish, so that the accurate detection of the potential colletotrichum gloeosporioides has important significance for reducing the spread of the colletotrichum gloeosporioides and preventing and controlling the diseases of the colletotrichum gloeosporioides.
Disclosure of Invention
In order to solve the problems, the invention provides a method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR.
The technical scheme of the invention is realized as follows:
a method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR comprises the following steps:
(1) two pairs of primers are designed according to the conserved region ACT gene sequence of colletotrichum gloeosporioides, namely ACT3F/ACT3R and ACT4F/ACT 4R;
(2) extracting gDNA of colletotrichum gloeosporioides to obtain colletotrichum gloeosporioides gDNA;
(3) diluting gDNA extracted in the step (2), taking the diluted gDNA as a template, performing real-time fluorescence PCR by using a primer, and establishing a linear regression curve between a threshold cycle (ct) and a template concentration logarithm;
(4) and (3) extracting DNA of a sample to be detected, detecting the sample to be detected by using a primer through real-time fluorescence quantitative PCR, and substituting the obtained CT value into the linear regression curve in the step (3) to obtain the content of colletotrichum gloeosporioides.
Further, the ACT3F is 5'-AGAGCTGTCTTCCGTAAGTCCC-3'.
Further, the ACT3R is 5'-AGCACACGCCGGTTGATGACC-3'.
Further, the ACT4F is 5'-GCCCAGAGCTGTCTTCCGT-3'.
Further, the ACT4R is 5'-GCGAGAAGCAGACATACCCAT-3'.
Further, in the step (2), the extraction is to extract the DNA by a CTAB method (cetyl trimethyl ammonium bromide method), specifically, a fungus culture or a plant material of colletotrichum gloeosporioides is ground by using liquid nitrogen, and then the genomic DNA is extracted by the CTAB method.
Further, the total volume of the reaction system of the real-time fluorescent PCR is 20 μ l, and the method specifically comprises the following steps: 2 × ChamQ Universal SYBR Qpcr Master Mix 10 μ L; sterile deinized water 7.2. mu.L; primer mixture, molarity 10 μ M each, final molarity 0.4 μ M each, 4 μ L; the template DNA was 2. mu.L.
Further, the reaction procedure of the real-time fluorescent PCR is as follows: preheating at 50 deg.C for 2min, pre-denaturing at 95 deg.C for 2min, and circularly amplifying at 95 deg.C for 15s and 60s for 40 times.
Further, the primers are ACT3F/ACT3R or ACT4F/ACT 4R.
Further, in step (3), when the primers are ACT3F/ACT3R, the linear regression curve is that y is-3.3027 x +44.292, and R is20.9982, where y is the CT value of the real-time fluorescent quantitative PCR reaction and x is the log of the colletotrichum gloeosporioides DNA concentration.
Further, in step (3), when the primers are ACT4F/ACT4R, the linear regression curve is that y is-3.1951 x +44.058, and R is20.9947, where y is the CT value of the real-time fluorescent quantitative PCR reaction and x is the logarithm of the DNA concentration of colletotrichum gloeosporioides.
Furthermore, the invention also provides a method for accurately measuring the growth condition of colletotrichum gloeosporioides in a host plant, which specifically comprises the following steps:
s1, extracting gDNA of a plant material sample to obtain gDNA of the plant material sample;
s2, diluting the gDNA in the step S1, taking the diluted gDNA of the plant material sample as a template, performing real-time fluorescence PCR by using an internal reference gene primer, and establishing a linear regression curve between threshold cycle and template concentration logarithm;
s3, extracting DNA of the plant material sample to be detected, detecting the plant material sample to be detected through real-time fluorescence quantitative PCR by using an internal reference gene primer, and substituting the obtained CT value into the linear regression curve of the step S2 to obtain the DNA biomass of the plant material sample;
s4, carrying out normalization treatment on the DNA biomass of the plant material sample obtained in the step S3 and the content of colletotrichum gloeosporioides obtained by the method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR to obtain the relative content of the colletotrichum gloeosporioides content in the plant material sample, and thus obtaining the growth condition of the colletotrichum gloeosporioides in the host plant.
Further, the dilution is a ten-fold gradient dilution method.
Further, the plant material is one of arabidopsis thaliana and stylosanthes guianensis.
Further, when the plant material is arabidopsis thaliana, the reference gene primer is QACTF/QACTR.
Further, when the plant material is stylosanthes guianensis, the reference gene primer is UBCE1F/UBCE 1R.
Compared with the prior art, the invention has the beneficial effects that:
the method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR provided by the invention is characterized in that primers with colletotrichum gloeosporioides specificity are designed according to actin gene sequences in conserved regions of colletotrichum gloeosporioides, and then a linear regression curve between threshold cycle and template concentration logarithm is established by using the real-time fluorescent quantitative PCR method, so that the DNA content of colletotrichum gloeosporioides is quantitatively detected; the method can detect the content of colletotrichum gloeosporioides in different periods in plants, can effectively eliminate the interference of similar pathogenic bacteria such as colletotrichum gloeosporioides and the like, has the detection limit of gDNA of 100fg, pDNA of 100 copy number and DNA of 20 conidia, and has high specificity and sensitivity.
The invention also establishes a rapid and accurate real-time fluorescence quantitative detection method, which can measure the growth conditions of colletotrichum gloeosporioides in different host plants by utilizing reference genes of different plants to quantify the DNA content of the plants and then carrying out normalization treatment on the DNA biomass of the plants and the DNA content of colletotrichum gloeosporioides; the method considers the normalized relation between the DNA of pathogenic bacteria and the plant DNA biomass, can accurately determine the growth condition of the colletotrichum gloeosporioides in different host plants, can be used for researching the dynamic change of the colletotrichum gloeosporioides, can be used for comparing the reaction of plant germplasm resources such as arabidopsis thaliana, stylosana guianensis and the like to the colletotrichum gloeosporioides, detecting disease-resistant or susceptible arabidopsis thaliana and stylosana guianensis varieties, and provides a basis for the early detection and disease control of the colletotrichum gloeosporioides and the screening of the resistant varieties.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an agarose gel electrophoresis chart showing PCR amplification products using ACT3 as a primer and DNA of colletotrichum gloeosporioides, colletotrichum oxysporum, karst colletotrichum and Botrytis cinerea as a template.
FIG. 2 is an agarose gel electrophoresis image of PCR amplification products using ACT4 as a primer and DNA of colletotrichum gloeosporioides, colletotrichum oxysporum, karst colletotrichum and Botrytis cinerea as a template.
FIG. 3 is a linear regression curve established with ACT3 as primer and colletotrichum gloeosporioides gDNA as template, where the ordinate is CT value and the abscissa is logarithm of colletotrichum gloeosporioides gDNA template concentration.
FIG. 4 is a linear regression curve constructed using ACT4 as a primer and colletotrichum gloeosporioides gDNA as a template, wherein the ordinate is the CT value and the abscissa is the logarithm of colletotrichum gloeosporioides gDNA template concentration.
FIG. 5 shows a linear regression curve constructed using ACT3 as a primer and plasmid DNA (pDNA) containing the ACT region of Colletotrichum gloeosporioides as a template, in which the ordinate represents the CT value and the abscissa represents the logarithm of the copy number of the target gene.
FIG. 6 shows a linear regression curve constructed using ACT4 as a primer and plasmid DNA (pDNA) containing the ACT region of Colletotrichum gloeosporioides as a template, in which the ordinate represents the CT value and the abscissa represents the logarithm of the copy number of the target gene.
FIG. 7 is a linear regression curve constructed using ACT3 as a primer and DNA of conidia of colletotrichum gloeosporioides as a template, in which the ordinate is CT value and the abscissa is the logarithm of the number of conidia of colletotrichum gloeosporioides.
FIG. 8 is a linear regression curve constructed using ACT3 as a primer and DNA of conidia of colletotrichum gloeosporioides as a template, in which the ordinate is CT value and the abscissa is the logarithm of the number of conidia of colletotrichum gloeosporioides.
FIG. 9 is a linear regression curve established with the internal reference gene UBCE1 of Stylosanthes guianensis RY2 as primer and the gDNA of Stylosanthes guianensis RY2 as template, wherein the ordinate is CT value and the abscissa is the logarithm of concentration of the gDNA template of Stylosanthes guianensis RY 2.
FIG. 10 is a linear regression curve established with the internal reference gene UBCE1 of Stylosanthes guianensis RY5 as primer and the gDNA of Stylosanthes guianensis RY5 as template, wherein the ordinate is CT value and the abscissa is the logarithm of concentration of the gDNA template of Stylosanthes guianensis RY 5.
FIG. 11 is a linear regression curve established with Arabidopsis thaliana internal reference gene QACT as primer and Arabidopsis thaliana gDNA as template, where the ordinate is CT value and the abscissa is the logarithm of concentration of Arabidopsis thaliana gDNA template.
FIG. 12 is a photograph showing the onset phenotype of anthrax in different time periods after inoculating wild type Arabidopsis thaliana and mutant Arabidopsis thaliana with anthrax.
FIG. 13 is a graph of the relative content of colletotrichum gloeosporioides in two Arabidopsis varieties measured by real-time fluorescent PCR with ACT3 as primer to detect the Arabidopsis material infected with colletotrichum gloeosporioides; wherein Col-0 is wild type, cpk5,6,11 are mutant Arabidopsis; in the figure, the ordinate represents the relative content of colletotrichum gloeosporioides in 100ng leaf DNA, and the abscissa represents different time points after inoculation.
FIG. 14 is a graph of the relative content of colletotrichum gloeosporioides in two Arabidopsis varieties measured by real-time fluorescent PCR with ACT4 as a primer to detect the Arabidopsis material infected with colletotrichum gloeosporioides; wherein Col-0 is wild type, cpk5,6,11 are mutant Arabidopsis; in the figure, the ordinate represents the content of colletotrichum gloeosporioides in 100ng leaf DNA, and the abscissa represents different time points after inoculation.
FIG. 15 is the picture of the onset phenotype of the Stylosanthes guianensis RY5 and Stylosanthes guianensis RY2 inoculated with anthrax at different times.
FIG. 16 is a graph of the relative content of Colletotrichum gloeosporioides in two species of Stylocentrotus obliquus measured by real-time fluorescence PCR using ACT3 as a primer to detect Colletotrichum gloeosporioides infected Stylocentrotus; wherein, the ordinate of the picture is the relative content of colletotrichum gloeosporioides in 100ng leaf DNA, and the abscissa is different time points after inoculation.
FIG. 17 is a graph of the relative content of Colletotrichum gloeosporioides in two species of Stylocentrotus obliquus measured by real-time fluorescence PCR using ACT4 as a primer to detect Colletotrichum gloeosporioides infected Stylocentrotus; wherein, the ordinate of the picture is the relative content of colletotrichum gloeosporioides in 100ng leaf DNA, and the abscissa is different time points after inoculation.
Detailed Description
In order to clearly and completely describe the technical solutions of the present invention, it is obvious that the inventors describe the embodiments in combination, but the following embodiments describe only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The plant materials used in the test of the invention are arabidopsis thaliana, stylosanthes guianensis No. RY2 and stylosanthes guianensis No. RY 5;
example 1 method for quantitative determination of colletotrichum gloeosporioides by real-time fluorescent PCR
1) Design of specific primers
The method comprises the steps of carrying out multi-sequence alignment on conserved region ACT gene sequences of colletotrichum gloeosporioides by using DNAMAN software, selecting conserved regions in colletotrichum gloeosporioides, and designing specific primers ACT3F/ACT3R and ACT4F/ACT4R by using OLIGO7 software.
2) Grinding a biological sample using liquid nitrogen, and then extracting genomic DNA using a CTAB method [ Yan, l.; zhang, c.; ding, l.; ma, Z.development of a real-time PCR assay for the detection of Cladosporum fulvum in tomato leaves J Appl Microbiol 2008,104, 1417-.
3) Real-time fluorescence PCR establishment of a Linear regression Curve
Carrying out ten-fold gradient dilution on the obtained gDNA, taking the diluted gDNA with different concentrations as a template, and respectively taking colletotrichum gloeosporioides specific primers ACT3F/ACT3R and ACT4F/ACT4R as primers to carry out real-time fluorescence PCR to obtain a CT value, and establishing a linear regression curve between the CT value and the template concentration (see fig. 3-4);
ACT3F is 5'-AGAGCTGTCTTCCGTAAGTCCC-3'.
ACT3R is 5'-AGCACACGCCGGTTGATGACC-3'.
ACT4F is 5'-GCCCAGAGCTGTCTTCCGT-3'.
ACT4R No. 5'-GCGAGAAGCAGACATACCCAT-3'
4) Extracting DNA of a sample to be detected, respectively using ACT3F/ACT3R and ACT4F/ACT4R as primers, detecting the plant material sample through real-time fluorescent quantitative PCR, and substituting the obtained CT value into the linear regression curve in the step 3) to obtain the content of colletotrichum gloeosporioides in the plant material sample;
the reaction system of the real-time fluorescent PCR is as follows:
the reaction procedure is as follows:
example 2 primer specificity test
(1) Extracting DNA of colletotrichum gloeosporioides, karst gloeosporioides and botrytis cinerea and DNA of plant materials infected by colletotrichum gloeosporioides by using a CTAB method;
(2) PCR amplification was carried out using 1% agarose gel electrophoresis using DNA from colletotrichum gloeosporioides, colletotrichum oxysporum, karst gloeosporioides, and Botrytis cinerea as templates, and ACT3F/ACT3R and ACT4F/ACT4R as primers, respectively, and the amplification products were subjected to 1% agarose gel electrophoresis.
The reaction system of PCR is:
the reaction procedure is as follows:
as a result, it was found that: ACT3 and ACT4 only amplified in colletotrichum gloeosporioides, and none of the other strains.
Example 3 sensitivity test
The gDNA, pDNA and conidium DNA of colletotrichum gloeosporioides are respectively diluted ten times, the diluted gDNA, pDNA and conidium DNA are respectively used as templates, real-time fluorescence PCR is carried out by using primers, the amplification conditions of the real-time fluorescence quantitative PCR are the same as those of the embodiment 1, and a linear regression curve between the threshold cycle (ct) and the logarithm of the template concentration is established (see the chart of 3-8).
As a result, it was found that: ACT3 and ACT4 were able to detect 100fg of colletotrichum gloeosporioides gDNA, 100 copies of pDNA, and 20 conidia of DNA.
Example 4 real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides content in Arabidopsis thaliana
Will be 1 × 106The colletotrichum gloeosporioides DZ-19 with conidia/mL is inoculated into wild type arabidopsis (col-0) and mutant arabidopsis (cpk5,6,11) which are not infected with colletotrichum gloeosporioides by spraying, and the colletotrichum gloeosporioides content in arabidopsis material is determined respectively in 0h, 36h and 60h of inoculation, and the detection method is as follows:
(1) grinding a wild type arabidopsis material by using liquid nitrogen, and extracting genome DNA by using a CTAB method;
(2) diluting the gDNA obtained in the step (1) by adopting a ten-fold gradient dilution method, carrying out real-time fluorescence quantitative PCR by using the diluted gDNA as a template and QACTF/QACTR as a primer, establishing a linear regression curve between the CT value and the concentration of the Arabidopsis material template in the same way as in example 1 under the amplification condition of the real-time fluorescence quantitative PCR, and obtaining a graph shown in FIG. 11;
QACTF of 5'-GGTAACATTGTGCTCAGTGGTGG-3';
QACTR is 5'-GGTGCAACGACCTTAATCTTCAT-3';
(3) extracting DNA of a to-be-detected sample of arabidopsis thaliana by adopting a CTAB method, detecting the arabidopsis thaliana material infected by colletotrichum gloeosporioides by using ACT3F/ACT3R, ACT4F/ACT4R and QACTF/QACTR as primers through real-time fluorescent quantitative PCR, wherein the amplification conditions of the real-time fluorescent quantitative PCR are the same as those of example 1 to obtain a CT value;
(4) and (3) respectively substituting CT values obtained by the real-time fluorescent quantitative PCR in the step (3) into corresponding linear regression curves, calculating the relative content of colletotrichum gloeosporioides in arabidopsis thaliana by normalizing the obtained values, and observing the growth condition of colletotrichum gloeosporioides in arabidopsis thaliana, wherein the calculation formula of the relative content is shown as follows.
Relative content of fungal DNA in 100ng of plant DNA (fungal DNA content (ng)/plant DNA content (ng)) x 100
The results are shown in fig. 13-14, the detection method can detect the colletotrichum gloeosporioides when the colletotrichum gloeosporioides is infected with arabidopsis thaliana, and plants do not show obvious infection symptoms in the first 3 days of colletotrichum gloeosporioides infection, but the detection result of the method shows that arabidopsis thaliana materials are obviously increased after being infected for 36 hours, and the content of colletotrichum gloeosporioides in plant materials is correspondingly increased along with the increase of infection time, so that the detection method can detect the colletotrichum gloeosporioides in the latent period in arabidopsis thaliana; the mutant arabidopsis is more susceptible than wild arabidopsis, and the detection method disclosed by the invention can be used for accurately detecting the content of colletotrichum gloeosporioides in arabidopsis material, accurately reflecting the infection condition of arabidopsis, researching the dynamic change of colletotrichum gloeosporioides and screening disease-resistant or disease-susceptible varieties, and providing a basis for early detection and disease control of colletotrichum gloeosporioides in arabidopsis and screening of resistant varieties.
Example 5 real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides content in Styrax japonicus
Will be 1 × 107The colletotrichum gloeosporioides DZ-19 with conidia/mL is inoculated into No. RY2 and No. RY5 of stylosanthes guianensis which are not infected with colletotrichum gloeosporioides by spraying, and the colletotrichum gloeosporioides content in the stylosanthes guianensis material is respectively measured after inoculation of 0h, 36h and 72h, and the detection method is as follows:
(1) grinding the No. RY2 and No. RY5 of the stylosanthes guianensis respectively by using liquid nitrogen, and then extracting genome DNA by using a CTAB method;
(2) diluting the gDNA obtained in the step (1) by adopting a ten-fold gradient dilution method, respectively taking the diluted gDNA as a template and UBCE1F/UBCE1R as primers, performing real-time fluorescence quantitative PCR, and respectively establishing linear regression curves between the CT value and the concentrations of the templates of No. RY2 and No. RY5 of the stylosanthes guianensis in the same way as the example 1 under the amplification conditions of the real-time fluorescence quantitative PCR, wherein the linear regression curves are shown in the graph 9-10;
UBCE1F is: 5'-GGTAACATTGTGCTCAGTGGTGG-3', respectively;
UBCE1R is: 5'-GGTGCAACGACCTTAATCTTCAT-3', respectively;
(3) extracting DNA of a sample to be detected by adopting a CTAB method, respectively taking ACT3F/ACT3R, ACT4F/ACT4R and UBCE1F/UBCE1R as primers, detecting an arabidopsis thaliana material infected by colletotrichum gloeosporioides by using real-time fluorescence quantitative PCR, wherein the amplification condition of the real-time fluorescence quantitative PCR is the same as that of example 1, and obtaining a CT value;
(4) and (3) respectively substituting the CT values obtained by the real-time fluorescent quantitative PCR in the step (3) into corresponding linear regression curves, calculating the relative content of the colletotrichum gloeosporioides in the stylosanthes guianensis by normalizing the obtained values, and observing the growth condition of the colletotrichum gloeosporioides in the stylosanthes guianensis.
The results are shown in fig. 16-17, the detection method can detect the colletotrichum gloeosporioides when the colletotrichum gloeosporioides is infected with the stylosanthes guianensis, and plants do not show obvious infection symptoms in the first 3 days of colletotrichum gloeosporioides infection, but the detection result of the detection method shows that the colletotrichum gloeosporioides is increased after 36 hours of infection of stylosanthes guianensis materials, and is obviously increased after 72 hours, which indicates that the detection method can detect the colletotrichum gloeosporioides in the latency stage in the stylosanthes guianensis; the RY2 of stylosanthes guianensis is easier to feel than the RY5 of stylosanthes guianensis, which shows that the detection method of the invention can also accurately detect the content of colletotrichum gloeosporioides in stylosanthes guianensis materials, accurately reflect the infection condition of the stylosanthes guianensis, can be used for researching the dynamic change of the colletotrichum gloeosporioides and screening disease-resistant or disease-sensitive varieties, and provides a basis for the early detection and disease control of the colletotrichum gloeosporioides in the stylosanthes guianensis and the screening of resistant varieties
In conclusion, the method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR can detect the content of colletotrichum gloeosporioides in different periods in plants, effectively eliminates the interference of similar pathogenic bacteria such as colletotrichum gloeosporioides and has high specificity and sensitivity, and the method can also detect the colletotrichum gloeosporioides in a latent period, accurately determine the growth conditions of the colletotrichum gloeosporioides in different host plants, can be used for researching the dynamic change of the colletotrichum gloeosporioides, can be used for comparing the reaction of plant germplasm resources such as arabidopsis thaliana, stylosana guianensis and the like to the colletotrichum gloeosporioides, and can be used for detecting disease-resistant or disease-sensitive varieties, thereby providing a basis for the early detection and control of the colletotrichum gloeosporioides and the screening of resistant varieties.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. A method for quantitatively detecting colletotrichum gloeosporioides by using real-time fluorescent PCR is characterized by comprising the following steps:
(1) two pairs of primers are designed according to the conserved region ACT gene sequence of colletotrichum gloeosporioides, namely ACT3F/ACT3R and ACT4F/ACT 4R;
(2) extracting gDNA of colletotrichum gloeosporioides to obtain colletotrichum gloeosporioides gDNA;
(3) diluting gDNA extracted in the step (2), taking the diluted gDNA as a template, performing real-time fluorescence PCR by using a primer, and establishing a linear regression curve between threshold cycle and template concentration logarithm;
(4) and (3) extracting DNA of a sample to be detected, detecting the sample to be detected by using a primer through real-time fluorescence quantitative PCR, and substituting the obtained CT value into the linear regression curve in the step (3) to obtain the content of colletotrichum gloeosporioides.
2. The method for quantitative detection of colletotrichum gloeosporioides using real-time fluorescent PCR as claimed in claim 1, wherein ACT3F is 5'-AGAGCTGTCTTCCGTAAGTCCC-3'; the ACT3R is 5'-AGCACACGCCGGTTGATGACC-3'.
3. The method for quantitative detection of colletotrichum gloeosporioides using real-time fluorescent PCR as claimed in claim 1, wherein ACT4F is 5'-GCCCAGAGCTGTCTTCCGT-3'; the ACT4R is 5'-GCGAGAAGCAGACATACCCAT-3'.
4. The method for quantitative detection of colletotrichum gloeosporioides by using real-time fluorescent PCR according to claim 1, wherein the total volume of the reaction system of the real-time fluorescent PCR is 20 μ l, and specifically comprises: 2 × ChamQ Universal SYBR Qpcr Master Mix 10 μ L; sterile deinized water 7.2. mu.L; primer mixture, molarity 10 μ M each, final molarity 0.4 μ M each, 4 μ L; the template DNA was 2. mu.L.
5. The method for quantitative detection of colletotrichum gloeosporioides by real-time fluorescent PCR according to claim 1, wherein the reaction procedure of real-time fluorescent PCR is as follows: preheating at 50 deg.C for 2min, pre-denaturing at 95 deg.C for 2min, and circularly amplifying at 95 deg.C for 15s and 60s for 40 times.
6. The method for quantitative detection of colletotrichum gloeosporioides by real-time fluorescent PCR as claimed in claim 4, wherein the primer is ACT3F/ACT3R or ACT4F/ACT 4R.
7. The method for quantitative detection of colletotrichum gloeosporioides by real-time fluorescent PCR as claimed in claim 6, wherein in step (3), when the primers are ACT3F/ACT3R, the linear regression curve is-3.3027 x +44.292, R20.9982, wherein y is the CT value of the real-time fluorescent quantitative PCR reaction and x is the logarithm of the DNA concentration of colletotrichum gloeosporioidesThe value is obtained.
8. The method for quantitative detection of colletotrichum gloeosporioides by real-time fluorescent PCR as claimed in claim 6, wherein in step (3), when the primers are ACT4F/ACT4R, the linear regression curve is-3.1951 x +44.058, R20.9947, where y is the CT value of the real-time fluorescent quantitative PCR reaction and x is the logarithm of the DNA concentration of colletotrichum gloeosporioides.
9. The method for quantitative determination of colletotrichum gloeosporioides by real-time fluorescent PCR according to any one of claims 1 to 8, wherein the method for precisely measuring the growth of colletotrichum gloeosporioides in a host plant comprises the following steps:
s1, extracting gDNA of a plant material sample to obtain gDNA of the plant material sample;
s2, diluting the gDNA in the step S1, taking the diluted gDNA of the plant material sample as a template, performing real-time fluorescence PCR by using an internal reference gene primer, and establishing a linear regression curve between threshold cycle and template concentration logarithm;
s3, extracting DNA of the plant material sample to be detected, detecting the plant material sample to be detected through real-time fluorescence quantitative PCR by using an internal reference gene primer, and substituting the obtained CT value into the linear regression curve of the step S2 to obtain the DNA biomass of the plant material sample;
s4, carrying out normalization treatment on the DNA biomass of the plant material sample obtained in the step S3 and the content of colletotrichum gloeosporioides obtained in the claim 1 to obtain the relative content of the colletotrichum gloeosporioides content in the plant material sample, and thus obtaining the growth condition of the colletotrichum gloeosporioides in the host plant.
10. The method according to claim 9, wherein the plant material is selected from one of arabidopsis thaliana and stylosanthes guianensis.
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