CN113528680A - Specific primer and probe for detecting corn bacterial leaf spot pathogen and real-time fluorescent quantitative PCR detection kit - Google Patents
Specific primer and probe for detecting corn bacterial leaf spot pathogen and real-time fluorescent quantitative PCR detection kit Download PDFInfo
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Abstract
The invention provides a specific primer and a probe for detecting corn bacterial leaf spot germs, wherein the specific primer is the following sequence or a complementary chain sequence of the following sequence: an upstream primer F: 5'-CTGCATGGCTGTCGTCA-3', respectively; a downstream primer R: 5'-ACCTTCCTCCGGTTTATCA-3', respectively; the probe sequence is as follows: 5'-ATCCTTTGTTGCCAGCGATTCGGT-3' are provided. The invention also provides a corresponding real-time fluorescent quantitative PCR kit. The specific primer, the probe and the corresponding kit can be used for real-time fluorescent quantitative PCR detection, and the established method has high sensitivity, high specificity and high stability for the corn bacterial leaf spot pathogen.
Description
Technical Field
The invention relates to a specific primer and a probe for detecting corn bacterial leaf spot bacteria and a real-time fluorescent quantitative PCR detection kit.
Background
Bacterial leaf spot (acidovaxaveasubsp. avenae) is caused by bacteria, and the disease mainly occurs on leaves and sometimes on petioles and stems. The disease symptoms are manifested as various spots, necrosis and even wilting of the whole plant. In recent years, the leaf spot of corn caused by some bacteria tends to be aggravated year by year, and susceptible varieties comprise common corn and sweet waxy corn. Bacterial leaf spot in corn has the characteristics of rapid spread, rapid onset of disease, great control difficulty and the like, and has already affected corn production in local areas. Due to the various symptoms of bacterial leaf spot, the difference of varieties and climatic conditions and the difference of the growth period of corn can influence the formation of symptom characteristics, thereby causing difficult field identification and increasing the identification difficulty of the diseases. Therefore, the research on the quick and efficient detection of the bacterial leaf spot pathogenic bacteria of corn in China is imperative.
At present, the detection method mainly comprises a separation culture method, phenotypic symptom identification, common PCR detection and other methods. The detection method of the separation culture method has complex operation, long time consumption and low detection efficiency, and is not suitable for rapid detection; the identification result of the phenotypic symptoms of field planting is unstable, the time consumption is long, and the like; the common PCR technology has low sensitivity and cannot identify the PCR process in real time. With the continuous development of molecular biology technology, real-time fluorescence quantitative PCR is widely applied to strain detection at present, and compared with the conventional isolation culture and the conventional PCR technology, the real-time fluorescence quantitative PCR has the advantages of short time consumption, simple and convenient operation, good specificity, direct observation of results, effective avoidance of cross contamination and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a specific primer and a probe for detecting corn bacterial leaf spot pathogen and a real-time fluorescent quantitative PCR detection kit. The invention aims to establish a QPCR (quantitative polymerase chain reaction) technology-based detection method for plant pathogenic bacteria, which comprises an upstream primer, a downstream primer and a fluorescent probe, and the method is based on the sequence with high conservation and high specificity and can selectively amplify the corn bacterial leaf spot pathogenic bacteria so as to separate the corn bacterial leaf spot pathogenic bacteria from other common corn disease strains. The real-time fluorescence quantitative PCR detection method based on the germs has the advantages of higher specificity, sensitivity, operation convenience and the like.
The invention provides a specific primer and a probe for detecting corn bacterial leaf spot germs, wherein the specific primer is the following sequence or a complementary chain sequence of the following sequence:
an upstream primer F: 5'-CTGCATGGCTGTCGTCA-3', respectively;
a downstream primer R: 5'-ACCTTCCTCCGGTTTATCA-3', respectively;
the probe sequence is as follows: 5'-ATCCTTTGTTGCCAGCGATTCGGT-3' are provided.
Preferably, the fluorescent reporter group marked at the 5' end in the probe is FAM; the fluorescence quenching group marked at the 3' end is TAMRA.
Preferably, the forward primer and the reverse primer are sequences obtained by extending one to several bases or deleting one to several bases in the directions of 5 'end and 3' end.
The invention provides a real-time fluorescent quantitative PCR kit for detecting corn bacterial leaf spot germs, which comprises the specific primer and the probe.
Preferably, in a 25ul PCR reaction system, the dosage of the upstream primer is 0.4ul, the dosage of the downstream primer is 0.4ul, and the dosage of the probe is 0.2 ul.
Preferably, the real-time fluorescent quantitative PCR kit further comprises a series of concentration standard products, and the nucleotide sequence of the standard products is SEQ ID NO.1 in the sequence table.
The invention provides the application of the specific primer, the probe and the real-time fluorescent quantitative PCR kit in any one of the following applications:
(1) qualitatively detecting or auxiliarily detecting bacterial leaf spot pathogen of corn;
(2) preparing a product for qualitatively detecting or assisting in detecting the corn bacterial leaf spot germs.
The invention provides a method for qualitatively detecting corn bacterial leaf spot pathogen, which respectively takes positive plasmid standard substance and sample DNA to be detected as templates and ddH2And O is blank control, the specific primers and the probes are utilized to carry out real-time fluorescence quantitative PCR, and whether the sample is positive or not is judged according to a fluorescence curve, wherein the method specifically comprises the following steps: if the Ct value is less than or equal to 35, the result is positive, otherwise, the result is negative.
Preferably, the reaction conditions of the real-time fluorescent quantitative PCR are as follows:
the invention has the beneficial effects that:
the invention provides a rake sequence of a highly conserved specific gene for detecting corn bacterial leaf spot pathogen, an upstream primer and a downstream primer are designed, the amplification length of the sequence is 133bp, the sequence has higher specificity to the corn bacterial leaf spot pathogen, and the sequence can be effectively distinguished from other disease and bacteria of corn; the probe corresponding to the upstream primer and the downstream primer is provided, and the probe is applied to a sample for qualitatively detecting the corn bacterial leaf spot bacteria by one-step fluorescence assay through the real-time fluorescence quantitative PCR reaction of the fluorescent probe, and has higher sensitivity and specificity.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph showing the results of NCBI Primer-Blast of the Primer of example 1.
FIG. 2 is an amplification curve of the system constructed according to the present invention.
FIG. 3 is a standard amplification curve for 4 dilution concentrations of the present invention.
FIG. 4 is a standard curve of the standard substance drawn by the software of the instrument.
FIG. 5 is a sensitivity test curve of the detection method of the present invention.
FIG. 6 is a test curve showing the specificity of the detection method of the present invention.
FIG. 7 is an amplification curve for stability evaluation of the detection method of the present invention.
FIG. 8 is an amplification curve of a sample detected by the detection method of the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The synthesis of the primer and the probe sequence is finished by Anhui general biotechnology limited; the rapid plasmid synthesis was performed by Kyama Kjeri.
Example 1 design and Synthesis of primers and probes
The present invention utilizes software Primer5.0 and Oligo 7 to design primers and probes based on the highly conserved and specific 16S rRNA gene sequence (Accession number: EU331415.1) of bacterial leaf spot pathogen of maize, disclosed in GeneBank, the nucleic acid sequence database of NCBI. Primer-specific alignments were performed at NCBI Primer-Blast and the results are shown in FIG. 1.2 pairs of primers with high specificity are selected and sent to a company for synthesis, and a dye method is utilized for primer screening. Synthesizing a specific probe according to the screened primer, designing a fluorescent reporter group of a probe sequence: the fluorescence reporter group marked at the 5 'end is FAM, and the fluorescence quencher group marked at the 3' end is TAMRA.
FIG. 1 is a graph showing the results of NCBI Primer-Blast of the Primer of example 1.
The fragments of interest are as follows:
the single underlined part indicates the position of the primer, and the dotted part indicates the position of the probe.
An upstream primer F: 5'-CTGCATGGCTGTCGTCA-3', respectively;
a downstream primer R: 5'-ACCTTCCTCCGGTTTATCA-3', respectively;
and (3) probe: FAM-ATCCTTTGTTGCCAGCGATTCGGT-TAMRA.
EXAMPLE 2 preparation of template DNA
1. The target fragment of example 1 was sent to Kyowa Kairy to rapidly synthesize a standard positive plasmid, thereby preparing a standard.
(1) The synthesized plasmid DNA was measured by using an ultramicro ultraviolet-visible spectrophotometer (ND5000) of Beijing Baitach Biotechnology Ltd, and A was measured260、A280According to A260/A280Judging the purity of the plasmid;
(2) in real-time fluorescent quantitative PCR, the "copy number" is the unit required, and thus the unit is converted to copies/. mu.L. Plasmid concentration (copy number) calculation:
plasmid copies/. mu.L ═ avogalois number of moles of plasmid, where avogalois number is 6.02X 1023copies/mol; the molecular weight of the plasmid (target gene length + vector length) bp × 660 (average molecular weight per base pair); that is, the plasmid copies/ul ═ 6.02X 1023)×(？ng/ul×10-9)/(DNA length×660)。
(3) The plasmid concentration was found to be 3.0 ng/. mu.l, so that the plasmid (6.02X 10)23)×(2.5ng/ul×10-9)/(3090×660)＝0.89×109copies/ul。
(4) The concentration is 0.89X 109Plasmid of copies/ul 11.24ul, ddH added2O88.76 ul, diluted concentration of 1.0X 108copies/ul as standard. Then, 10-fold gradient dilution is carried out to obtain 1.0X 107～1.0×101copies/ul positive plasmid standards were pooled at-20 ℃ for future use.
2. Extracting corn bacterial leaf spot germ and corn bacterial wilt germ DNA, adopting bacterial genome extraction kit of Beijing Baitaike company, the specific extraction method is as follows:
(1) taking 1ml of culture solution, centrifuging at 10,000rpm for 30s, discarding supernatant, collecting thalli, and completely sucking the supernatant as far as possible;
(2) adding 200 mul of buffer RB for resuspending and washing the cells, centrifuging at 10,000rpm for 30 seconds, discarding the supernatant, and then shaking or blowing the cells to resuspend in 200 mul of buffer RB;
(3) adding 200 μ l binding solution CB, immediately turning upside down and shaking gently, mixing well, adding 20 μ l proteinase K (20mg/ml)) solution, mixing well, standing at 70 deg.C for 10 min;
(4) cooling, adding 100 μ l isopropanol, violently reversing, shaking gently, and mixing well, wherein flocculent precipitate may appear;
(5) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column AC, placing the adsorption column into a collecting pipe, centrifuging at 10,000rpm for 30s, and pouring off the waste liquid in the collecting pipe;
(6) adding 500ul inhibitor removing liquid IR, centrifuging at 10,000rpm for 30s, and discarding the waste liquid;
(7) adding 700ul of rinsing liquid WB, centrifuging at 10,000rpm for 30s, and discarding waste liquid;
(8) adding 500ul of rinsing liquid WB, centrifuging at 10,000rpm for 30s, and discarding the waste liquid;
(9) placing the adsorption column AC back into the empty collection tube, centrifuging at 10,000rpm for 2min, and removing rinsing liquid as much as possible;
(10) taking out the adsorption column AC, placing into a clean centrifuge tube, adding 100ul elution buffer EB (the elution buffer is preheated in water bath at 65-70 deg.C in advance) into the middle part of the adsorption membrane, standing at room temperature for 3min, and centrifuging at 12,000rpm for 1 min;
(11) the DNA may be stored at 2-8 ℃ and-20 ℃ if it is to be stored for a long period of time.
The DNA extraction of the maize chlorotic mottle virus adopts a fungus genome DNA kit of Beijing Baitaike company, and the specific extraction method is as follows:
(1) taking 1ml of bacterial suspension, adding the bacterial suspension into a 1.5ml centrifuge tube, centrifuging for 2 minutes at 8000r/min, and removing supernatant;
(2) adding 550ul of preheated Buffer FP1 at 65 ℃ and 4ul of RNaseA, violently whirling, shaking, mixing, uniformly mixing, putting into a water bath at 65 ℃ for 30min, and violently whirling and shaking for 3 times;
(3) adding 130ul Buffer P2, mixing, and centrifuging at 12000rmp for 3 min;
(4) carefully sucking the supernatant into a separation column A, taking care not to suck interfacial substances, centrifuging for 1min at 12000rmp, and collecting the supernatant;
(5) adding 1.5 times volume of Buffer P3, immediately and gently swirling, and fully mixing;
(6) adding the mixture obtained in the previous step into an adsorption column AC, centrifuging for 1min at 12000rmp, and pouring off waste liquid in a collecting pipe;
(7) adding 700 μ l of rinsing liquid WB (please check whether absolute ethanol is added or not), centrifuging at 12000rmp for 1min, and discarding the waste liquid;
(8) adding 500 μ l of rinsing liquid WB, centrifuging at 12000rmp for 1min, and discarding the waste liquid;
(9) placing the adsorption column AC back into an empty collection tube, and centrifuging at 12000rmp for 3-5 min;
(10) taking out the adsorption column AC, placing into a clean centrifuge tube, adding 50ul elution buffer EB (preheated in 65-70 deg.C water bath) in the middle part of the adsorption membrane, standing at room temperature for 3-5min, centrifuging at 12000rmp for 1min, and collecting DNA;
(11) the DNA is stored at 2-8 ℃ and, if it is to be stored for a long time, it can be stored at-20 ℃ for later use.
Example 3 optimization of real-time fluorescent quantitative PCR reaction conditions and plotting of Standard Curve
(1) Optimization of real-time fluorescent quantitative PCR reaction conditions
The primers and probes screened in example 1 were diluted, deionized water was added to dilute the solution to 10uM, and real-time fluorescent quantitative PCR amplification was performed. And establishing a reaction system.
PCR reactionThe system is as follows: 2 × AceQU+master mix12.5uL, 10uM upstream primer 0.4uL, 10uM downstream primer 0.4uL, 10uM probe 0.2uL, template DNA2uL, ddH2O9.5 ul, total volume of reaction 25 ul. The positive plasmid standard of example 2 (concentration 1.0X 10)7copies/ul) as template, ddH2O is blank control.
The real-time fluorescent quantitative PCR reaction conditions are as follows:
FIG. 2 shows the amplification curve of the system constructed according to the present invention (3 parallel experiments were performed). Wherein, A: 1.0X 107copies/ul; b: blank control.
(2) Drawing of real-time fluorescence quantitative PCR standard curve
Positive plasmid standards were obtained in example 2 at the series of concentrations using the primers and probes of example 1 (concentrations: 1.0X 10, respectively)8、1.0×107、1.0×106、1.0×105copies/ul、1.0×104copies/ul) as template, ddH2And O is a blank control, and real-time fluorescent quantitative PCR amplification (3 parallel tests per concentration) is carried out by adopting the reaction system and the reaction conditions in the step (1) to obtain an amplification curve of the standard product, which is shown in figure 3. The logarithm of the concentration of the plasmid standard substance is taken as the abscissa, the Ct value is taken as the ordinate, and the analysis software carried by the real-time fluorescence quantitative PCR instrument is used for drawing a standard curve which is shown in figure 4. The standard curve equation of the invention is that Y is-3.38X + 39.94. The standard curve shows a good linear relationship, R2The correlation coefficient is high when the molecular weight is 0.9994, and the requirement of real-time fluorescent quantitative PCR detection is met.
FIG. 3 is a standard amplification curve for 5 concentration gradients of the invention. Wherein, A: 1.0X 108copies/ul；B：1.0×107copies/ul；C：1.0×106copies/ul；D：1.0×105copies/ul；E：1.0×104copies/ul; f: blank control.
FIG. 4 is a standard curve of the standard substance drawn by the software of the instrument.
Example 4 real-time fluorescent quantitative PCR kit for detecting bacterial leaf spot pathogen of corn
A kit for detecting corn bacterial leaf spot germs is prepared according to the following components: 2 × AceQU + master mix, 10uM upstream primer, 10uM downstream primer, 10uM probe, positive plasmid standard prepared in example 2 (concentration: 1.0 × 10)6copies/ul)，ddH2O。
The upstream primer, the downstream primer and the probe are all described in example 1.
The reaction system of the kit can be as follows: 2 × AceQU+master mix12.5ul, 10uM upstream primer 0.4ul, 10uM downstream primer 0.4ul, 10uM probe 0.2ul, template DNA2ul, ddH2O9.5 ul, total volume of reaction 25 ul.
The reaction conditions of the kit for carrying out real-time fluorescence quantitative PCR are as follows:
when the kit is used for detecting a sample, a fluorescence curve is obtained according to a fluorescence signal detected by an instrument, and whether the sample is positive or not is judged according to the fluorescence curve, which specifically comprises the following steps: if the Ct value is less than or equal to 35, the result is positive, otherwise, the result is negative.
Example 5 real-time fluorescent quantitative PCR sensitivity assay for detecting bacterial leaf spot pathogen in maize
Using the kit, reaction System and reaction conditions described in example 4, the positive plasmid standard 1.0X 10 prepared in example 2 was used4～1.0×101copies/ul as template (3 replicates per concentration) and qPCR amplification assay was performed to determine the lowest detection limit for the detection method of the invention. The fluorescence curve was obtained from the fluorescence signal detected by the instrument and the results are shown in FIG. 5. As is clear from FIG. 5, the lower limit of detection of real-time fluorescent quantitative PCR was 1.0X 102copies/ul。
FIG. 5 is a sensitivity test curve of the detection method of the present invention. Wherein, A: 1.0X 104copies/ul；B：1.0×103copies/ul；C：1.0×102copies/ul；D：1.0×101copies/ul; e: blank control.
Example 6 specificity assay for real-time fluorescent quantitative PCR for detection of bacterial leaf spot pathogen in maize
The DNA of the bacterial leaf spot bacteria, bacterial blight bacteria and chlorotic mottle virus of maize described in step 2 of example 2 was used as a template, ddH2O as blank, using the kit described in example 4, at a concentration of 1.0X 10 as described in step 1 of example 27The positive plasmid standard of copies/ul is used as a positive control, and 3 parallel tests qPCR amplifications are carried out by adopting the method of example 4 to carry out specific test detection. The fluorescence curve is obtained from the fluorescence signal detected by the instrument, and the detection result is shown in FIG. 6. As can be seen from FIG. 6, the DNA detection of the bacterial leaf spot of maize is positive, and the DNA detection of the bacterial blight of maize and the chlorotic mottle virus of maize are negative, which indicates that the invention has good specificity.
FIG. 6 is a test curve showing the specificity of the detection method of the present invention. Wherein, A: a positive control; b: bacterial leaf spot pathogen of corn; c: bacterial wilt of maize, chlorotic mottle virus of maize, ddH2O。
Example 7 stability assay for real-time fluorescent quantitative PCR for detection of bacterial leaf spot pathogen in maize
The real-time fluorescent quantitative PCR detection method established in example 4 was used to perform 3-lot repeat assay detection, each of which was repeated 3 times, and the stability of the detection method was evaluated using diluted standards, and the results are shown in FIG. 7. The errors of Ct values calculated between 3 parallel and 3 repeated detection batches in the same batch are both less than 0.5, and the variation coefficients are both less than 5% (Table 1 and Table 2), which indicates that the method of the invention has higher stability and repeatability.
FIG. 7 is an amplification curve for stability evaluation of the detection method of the present invention. Wherein a, b and c are 3-time stability repeated amplification curves; in the figure, A to E are respectively: a: 1.0X 108copies/ul；B：1.0×107copies/ul；C：1.0×106copies/ul；D：1.0×105copies/ul; e: blank control.
TABLE 1 stability test results of the method of the invention (same batch)
TABLE 2 stability test results of the method of the invention (different batches)
Example 8 sample identification test for bacterial leaf spot pathogen in maize
1. Preparation of corn bacterial leaf spot pathogen DNA: DNA was extracted using the bacterial genome extraction kit of Baitach, Beijing as described in step 2 of example 2.
2. The sample was detected using the real-time fluorescent quantitative PCR detection kit for bacterial leaf spot pathogen of maize as described in example 4. The method specifically comprises the following steps:
(1) the DNA of the sample to be tested and the plasmid (1.0X 10) as the standard prepared in example 2 were used7copies/ul) as template, ddH2O is a blank control and 3 replicates were run.
(2) The real-time fluorescent quantitative PCR reaction system described in example 4 was adopted: 2 × AceQU+master mix12.5ul, 10uM upstream primer 0.4ul, 10uM downstream primer 0.4ul, 10uM probe 0.2ul, template DNA2ul, ddH2O9.5 ul, total volume of reaction 25 ul.
Wherein, the upstream primer F: 5'-CTGCATGGCTGTCGTCA-3', respectively;
a downstream primer R: 5'-ACCTTCCTCCGGTTTATCA-3', respectively;
and (3) probe: FAM-ATCCTTTGTTGCCAGCGATTCGGT-TAMRA.
The real-time fluorescent quantitative PCR reaction conditions are as follows:
the fluorescence curve was obtained from the fluorescence signal detected by the instrument, as shown in FIG. 8. The results are shown in Table 3 below.
TABLE 3 sample identification results
FIG. 8 is an amplification curve of a sample detected by the detection method of the present invention. Wherein, A: 1.0X 107copies/ul; b: a sample; c: blank control.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Baiyuan Gene technology of Lanzhou Ltd
<120> specific primer and probe for detecting corn bacterial leaf spot pathogen and real-time fluorescent quantitative PCR detection kit
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 380
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggaggttgt tcccttgagg agtggcttcc ggagctaacg cgttaagtcg accgcctggg 60
gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag 120
catgtggttt aattcgatgc aacgcgaaga accttaccta ctcttgacat ccagagaact 180
tagcagagat gctttggtgc cttcgggaac tctgagacag gtgctgcatg gctgtcgtca 240
gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg 300
ccagcgattc ggtcgggaac tcaaaggaga ctgccggtga taaaccggag gaaggtgggg 360
atgacgtcaa gtcatcatgg 380
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atcctttgtt gccagcgatt cggt 24
Claims (9)
1. The specific primer and probe for detecting the corn bacterial leaf spot pathogen are characterized in that: the specific primer is the following sequence or a complementary strand sequence of the following sequence:
an upstream primer F: 5'-CTGCATGGCTGTCGTCA-3', respectively;
a downstream primer R: 5'-ACCTTCCTCCGGTTTATCA-3', respectively;
the probe sequence is as follows: 5'-ATCCTTTGTTGCCAGCGATTCGGT-3' are provided.
2. Specific primers and probes according to claim 1, characterized in that: the fluorescence reporter group marked at the 5' end in the probe is FAM; the fluorescence quenching group marked at the 3' end is TAMRA.
3. Specific primers and probes according to claim 1, characterized in that: the upstream primer and the downstream primer are sequences obtained by extending one to several bases or deleting one to several bases in the directions of 5 'end and 3' end.
4. A real-time fluorescent quantitative PCR kit for detecting corn bacterial leaf spot bacteria is characterized in that: the real-time fluorescent quantitative PCR kit comprises the specific primer and the probe of any one of claims 1 to 3.
5. The real-time fluorescent quantitative PCR kit according to claim 4, wherein: in a 25ul PCR reaction system, the dosage of the upstream primer is 0.4ul, the dosage of the downstream primer is 0.4ul, and the dosage of the probe is 0.2 ul.
6. The real-time fluorescent quantitative PCR kit according to claim 4 or 5, characterized in that: the real-time fluorescent quantitative PCR kit also comprises a series of concentration standard products, and the nucleotide sequence of the standard products is SEQ ID NO.1 in the sequence table.
7. The use of the specific primers and probes as claimed in any one of claims 1 to 3 and the real-time fluorescent quantitative PCR kit as claimed in any one of claims 4 to 6 in any one of the following applications:
(1) qualitatively detecting or auxiliarily detecting bacterial leaf spot pathogen of corn;
(2) preparing a product for qualitatively detecting or assisting in detecting the corn bacterial leaf spot germs.
8. A method for qualitatively detecting corn bacterial leaf spot bacteria is characterized in that: respectively taking positive plasmid standard substance and DNA of sample to be detected as templates, and ddH2And O is blank control, the specific primer and the probe in the claim 1 are utilized to carry out real-time fluorescence quantitative PCR, and whether the sample is positive or not is judged according to a fluorescence curve, and the method specifically comprises the following steps: if the Ct value is less than or equal to 35, the result is positive, otherwise, the result is negative.
9. The method of claim 8, wherein: the reaction conditions of the real-time fluorescent quantitative PCR are as follows:
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|---|---|---|---|---|
| US6146834A (en) * | 1999-09-10 | 2000-11-14 | The United States Of America As Represented By The Secretary Of Agriculture | PCR primers for detection of plant pathogenic species and subspecies of acidovorax |
| CN108342498A (en) * | 2018-03-13 | 2018-07-31 | 中国农业科学院饲料研究所 | A kind of PCR detection method of the general bacterium of pineapple |
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2021
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6146834A (en) * | 1999-09-10 | 2000-11-14 | The United States Of America As Represented By The Secretary Of Agriculture | PCR primers for detection of plant pathogenic species and subspecies of acidovorax |
| CN108342498A (en) * | 2018-03-13 | 2018-07-31 | 中国农业科学院饲料研究所 | A kind of PCR detection method of the general bacterium of pineapple |
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