CN116515863B - Specific detection target Pve _contig_43 of Pythium clockii and application thereof - Google Patents

Specific detection target Pve _contig_43 of Pythium clockii and application thereof Download PDF

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CN116515863B
CN116515863B CN202310475296.1A CN202310475296A CN116515863B CN 116515863 B CN116515863 B CN 116515863B CN 202310475296 A CN202310475296 A CN 202310475296A CN 116515863 B CN116515863 B CN 116515863B
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戴婷婷
徐小娇
皋德馨
陆咸顺
还涛
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Nanjing Forestry University
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Abstract

The invention discloses a detection target Pve _contig_43 of Pythium clockii (phytopathosporum) and a detection primer thereof, wherein the DNA sequence of the detection target Pve _contig_43 is shown as SEQ ID NO:1, the protein sequence is shown as SEQ ID NO: 2. Meanwhile, a specific primer combination of a PCR detection technology of a specific detection target Pve _contig_43 is also disclosed, and a forward primer sequence of the specific primer combination is shown as SEQ ID NO:3, the reverse primer sequence is shown as SEQ ID NO: 4. Meanwhile, the detection target developed by the invention and the primer combination designed by the detection target are used for common PCR, and the detection target has strong specificity and high sensitivity, proves that Pve _contig_43 is suitable for the detection target of Pythium clockii, and provides a technical platform for the detection of Pp.Vexans.

Description

Specific detection target Pve _contig_43 of Pythium clockii and application thereof
Technical Field
The invention belongs to the technical field of Pp.vex ans detection and particularly relates to a specific detection target Pve _contig_43 of Pp.vex ans of Pp.p.Pp.Pp.Pp.Pp.Pp.Pp.m.Pp.m.m.m.
Background
Pythium clockii is genus Pythium, and has wide host plants, and can infect various vegetables, fruit tree, tea, sugarcane, potato, wheat and other plants. The colonies are radial on CMA, and the hyphae are branched and finer and have a thickness of 1.5-5.4 μm. Sporangia balls, pear shapes or oval shapes, acrogenesis or metaplasia, diameter 12-30 (average 16.7) μm, zoospore kidney shape, double flagella, resting ball shape, diameter about 12 μm. Egg collector sphere, smooth, top born, sometimes time born or cut born, diameter 14-27 (average 21.5) μm. The male is bell-shaped or irregular, top-born, co-filament-born, less heterogeneous filaments, 5.2-44.8 μm×5.1-8.6 μm, average 17.92 μm×6.99 μm. Each egg collector has 1-2 stamen. Oospore spheres, 12-27 (average 18.7) μm and wall thickness (1.5-1.7) - (2.2-3.0) μm.
More than 200 Pythium species are reported worldwide, wherein more than 80 species are important pathogenic bacteria of plants, harm various crops and cause symptoms such as damping-off, root rot and stem rot, and the like, for example, pythium aphanidermatum (Ph ytopythium aphanidermatum), pythium de-Li Fumei (Phytopythium debaryanum) and Pythium ultimum (Phytopythium ultimum) can infect tobacco to cause damping-off. Pythium clockii has been reported in 27 countries such as Malaysia, indonesia, india, japan, brazil, england, and the United states, in Yunnan, hainan, guangxi, ningxia, beijing, shaanxi, and the like, and has caused tobacco stem root rot in Henan province.
In order to prevent the spread of Pp. Vexans pathogenic bacteria, rapid and accurate detection is required, and the discovery of target genes with good specificity is the core of all detection technologies at present. The specificity and sensitivity of detection of different target sequences are different, so that the selected target sequences are different, the sizes of fragments are different, and the results are quite different. The target gene is selected to ensure that it is highly conserved among different strains within the species, while variability is high among species. Although there is also rapid molecular detection for pp.vexans in the prior art, its detection target sensitivity is not high.
In conclusion, the development of the high-reliability specific molecular detection target and the establishment of a sensitive and accurate detection technology system based on the new target play an important role in promoting early diagnosis of pathogenic injury of Pp.vexans rapid molecular detection research institute.
Disclosure of Invention
Aiming at the problems of long period, poor detection method specificity, low sensitivity and less specific detection targets of the Pp.vexans biological detection method in the prior art, the invention provides a novel Pp.vexans detection target Pve _contig_43 and a PCR detection primer composition based on the novel detection target.
In a first aspect, the invention provides a specific detection target Pve _contig_43 of pp.vexans, wherein the DNA sequence of the detection target is as shown in SEQ ID NO: 1. ATGCTGTATGGCTACGTGACCAACGACATCTTCCCGCTTCTCGTCACCTATGCCGTTGGCGATGTGCTCGGCGTGGTGTTCCTGATGGTCTACTTGCGGTGGACTACGCAGCGGAAAGCTGTGATCAAGACCATTGTCATCGCTCTCGCGTGTAATGCGGTCGCAGTCGTCTACACGGTGCTTGGCGCTCAAGGTGTGCTTCCTCAGTCCCACAGCAGCTTCAAACAGGTGATCGGGTATCTCTCCATCGCCTCGAGTCTGGTGCTCTACTCGTCCCCGCTCTCGACAATCAAGCAGGTGATCCAGACCAAGTCCTCAGTAACGATCCCAGCCACCATGGTCGTCGCTGGAGTGGTGAACAACGGTCTGTGGATTATTTACGGCTTTCTGCTGGAAGACATCATCCTGATCATCCCGACTTCCATCAACATCGTATTCGGGGTAGCCCAACTTGTTCTGTACATTGTCTACTCGCCCAAGAAGACCACGGCAGGACTCGCCGTCATTGAGCCACTTGAAATCGTTCCCTCGACACCAACGGGGGTCAAGACAGCTTTCGAGGCCATGTCGTCGCCAACATCAACCAACGGCGCGGTGGAACTGTACCGGATCACCATCGACCCGGTCTCGAAGCCGCCTCGTCCAGAGCTGGCACCGGTTCCAGCGCCAGTGCCATCCTCGTAG (SEQ ID NO: 1)
In a second aspect, the invention also provides a specific detection target Pve _contig_43 of Pp.vexans, and the coded protein sequence is shown as SEQ ID NO. 2.
MLYGYVTNDIFPLLVTYAVGDVLGVVFLMVYLRWTTQRKAVIKTIVIA
LACNAVAVVYTVLGAQGVLPQSHSSFKQVIGYLSIASSLVLYSSPLSTIK
QVIQTKSSVTIPATMVVAGVVNNGLWIIYGFLLEDIILIIPTSINIVFGVAQ
LVLYIVYSPKKTTAGLAVIEPLEIVPSTPTGVKTAFEAMSSPTSTNGAVELYRITIDPVSKPPRPELAPVPAPVPSS*(SEQ ID NO.2)
In a third aspect, the invention also provides a primer combination for detecting Pp.vexans, wherein the forward primer Pve _contig_43-F1 has a sequence as shown in SEQ ID NO:3, the sequence of the reverse primer Pve _contig_43-R1 is shown in SEQ ID NO: 4.
Pve_contig_43-F1:CGACATCTTCCCGCTTCT(SEQ ID NO：3)
Pve_contig_43-R1:GACCGTTGTTCACCACTCC(SEQ ID NO：4)
In a fourth aspect, the invention also provides a kit for detecting Pythium clockii, said kit comprising a detection solution comprising 20. Mu.M of the primer combination of claim 3.
Further, the detection solution further comprises: 4 dNTPs 2000. Mu.M each, 100. Mu.L of 10 XPCR reaction buffer, 80mM Mg 2+ 100. Mu.L of 1% BSA,50 units Taq enzyme (TaKaRa).
In a fifth aspect, the invention also provides the use of the primer combination or the kit in the detection of Pythium clockii.
In a sixth aspect, the present invention also provides a method for detecting Pythium clockii, comprising the steps of: taking 1 mu L of DNA solution of a detection object, adding 23 mu L of the detection solution and 1 mu L of sterilized deionized water, wherein the total volume is 25 mu L; the PCR amplification procedure was denaturation at 94℃for 3 min, denaturation at 94℃for 30 sec; annealing at 58 ℃ for 30 seconds; extending at 72 ℃ for 45 seconds; 33 cycles, last 72℃extension for 10 minutes.
Compared with the prior art, the invention has the advantages that:
1) The invention discloses a high-reliability specificity molecular detection target Pve _contig_43 for the first time, and establishes a sensitive and accurate PCR detection technology system based on the new target, thereby having important effect on promoting rapid molecular detection research of Pp.vexans and early diagnosis of pathogenic injury during detection.
2) The detection primer combination provided by the invention can amplify specific bands aiming at Pp.vexans, and the sizes of the bands are 344bp; meanwhile, the specificity experiment proves that the sensitivity of the PCR method for detecting the Pp.vexans genome DNA is 100 pg.mu.L -1 。
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 will be briefly described below.
FIG. 1 is a schematic diagram of a PCR specificity verification electrophoresis pattern of specific primers Pve _confg_43-F1/Pve _confg_43-R1 designed based on Pp.vexans new detection target Pve _confg_43 between Pythium species; specific primers the upstream primer Pve _contig_43-F1 and the downstream primer Pve _contig_43-R1 can only specifically amplify a 344bp band from the tested Pp.vexan strain, while the remaining Phytophthora species do not produce the band of interest; wherein, from left to right, it is in proper order: 1, marker;2 Pythium clockii (Phytopythium vexans); 3, pythium gracile (Phytopythium helicoides) and 4,Phytopythium plurisporium; 5, pythium aphanidermatum (Phytopythium aphanidermatum); 6, pythium spinosum (Phytopythium spinosum); pythium lakeside (Phytopythium littorals); pythium gracile (Phytopythium dissotocum); pythium isox (Phytopythium diclinum); pythium ultimum (Phytopythium ultimum); 11, n negative control.
FIG. 2 is a diagram of a general PCR-specific verification electrophoresis of specific primers designed for Pp.vexans new detection target Pve _confg_43 in other fungi and oomycetes; specific primers the upstream primer Pve _contig_43-F1 and the downstream primer Pve _contig_43-R1 can only specifically amplify one 344bp band from the tested Pp.vexans strain, while the remaining fungi or oomycetes do not produce the band of interest;
FIG. 3 is a sensitivity verification electrophoresis chart of a detection primer combination designed based on Pp.vexans new detection target Pve _contig_43, and the result shows that the detection sensitivity of the primer can reach 100pg. Mu.L -1 。
FIG. 4 shows a pattern of specific verification electrophoresis of detection primer combinations designed based on Pp. Vexans new detection target Pve _contig_768. The results showed that one 301bp band was amplified specifically from the tested pp.vexans strain, while the remaining fungi or oomycetes did not produce the desired band.
FIG. 5 is a sensitivity verification electrophoresis chart of a detection primer combination designed based on Pp. Vexans new detection target Pve _contig_768, and the result shows that the detection sensitivity of the primer can reach 1ng. Mu.L -1 。
Fig. 6 is a view of the onset of the Pp.vexans-based new detection target Pve _contig_43 in artificially inoculated plants and the detection result. Fig. 6A is a view of rhododendron leaves for artificially inoculating Pp.vexans, CK is a view of rhododendron leaves for artificially inoculating blank agar, 1 is a view of rhododendron leaves for artificially inoculating the first day of the occurrence of Pp.vexans, 2 is a view of rhododendron leaves for artificially inoculating the second day of the occurrence of Pp.vexans, 3 is a view of rhododendron leaves for artificially inoculating the third day of the occurrence of Pp.vexans, and fig. 6B is a result of PCR detection of rhododendron leaves for artificially inoculating the pathogenic bacteria Pp.vexans. In the PCR detection result graph, marker (500 bp), pp. Vexans pathogen, NC (healthy plant as negative control), first day of disease, second day of disease and third day of disease are arranged from left to right. The results show that the Pp. Vexans strain and the DNA extracted from the rhododendron by artificially inoculating Pp. Vexans can specifically amplify a 344bp band, while the DNA extracted from the rhododendron by artificially inoculating agar fast and the negative control have no amplified band.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, which should not be construed as limiting the scope of the present invention. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
Example 1
Through whole genome comparison, more than 1000 specific detection targets of Pp.vexans are obtained in total; a part of genes were randomly selected from 1000 specific genes of Pp.vexans as candidate genes, specific primers were designed and screened, and 4 target genes are listed in Table 1. And verifying the designed specific primer by adopting a PCR technology. Specific evaluation Pythium species different from Pythium bellum (Phytopythium vexans), pythium gracile (Phytopythium helicoides); phytopythium plurisporium Pythium ultimum; pythium aphanidermatum (Phytopythium aphanidermatum); pythium spinosum (Phytopythium spinosum); pythium lakeside (Phytopythium littorals); pythium gracile (Phytopythium dissotocum); pythium isox (Phytopythium diclinum); pythium ultimum (Phytopythium ultimum) and bacteria of different genus (pine wood nematodes (Bursaphelenchus xylophillus), fusarium graminearum (Fusarium graminearum), populus canker (Botryospaeria dothidea), apple anthracnose (Colletotrichum aenigma), phytophthora winter (Phytophthora hibernalis), fusarium putrescentiae (Fusarium solani), alternaria (Alternaria alternata), botrytis cinerea) and the like are used as templates for PCR specificity and sensitivity verification, and finally 1 new target gene for detecting Pp.vexans is obtained.
Table 14 Gene sequence listing of Pp. Vexans
The DNA sequence of the novel target gene Pve _contig_43 is shown as SEQ ID NO:1, the CDS sequence is shown as SEQ ID NO:1, the protein sequence of which is shown in SEQ ID NO:2, and based on the new target, a sensitive and accurate PCR detection technology system is established.
PCR detection primer composition used by the detection technology system: the sequence of each primer is specifically as follows from an upstream primer Pve _contig_43-F1 and a downstream primer Pve _contg_43-R1:
Pve_contig_43-F1：CGACATCTTCCCGCTTCT(SEQ ID NO：3)
Pve_contig_43-R1：GACCGTTGTTCACCACTCC(SEQ ID NO：4)
extracting DNA of a microorganism to be detected, taking 1 mu L of DNA solution, adding 23 mu L of detection solution in a kit and 1 mu L of sterilized deionized water, wherein the total volume is 25 mu L; the PCR amplification procedure was denaturation at 94℃for 3 min, denaturation at 94℃for 30 sec; annealing at 58 ℃ for 30 seconds; extending at 72 ℃ for 45 seconds; 33 cycles, last 72℃extension for 10 minutes. Wherein 1mL of the detection solution comprises: 4 dNTPs 2000. Mu.M each, 100. Mu.L of 10 XPCR reaction buffer, 80mM Mg 2+ mu.L of 1% BSA,50 units of Taq enzyme (TaKaRa), specific primers Pve _contig_43-F1 and Pve _contig_43-R120. Mu.M were added to prepare 1mL of a detection solution.
After the reaction, 10. Mu.L of the amplified product was electrophoresed in 1.0% agarose gel for 30min (150V), and detected and photographed on a gel imaging system. Each experiment was repeated at least 3 times.
Example 2
To verify the specific primer sequences of Pp. Vexans, 3 Pp. Vexans strains of Pp. Vexans, pathogenic fungi and other oomycetes were used as test materials in this example, and DNA of Pp. Vexans in the diseased tissue was extracted by CTAB method. The specific method comprises the following steps: a small amount of mycelium powder was taken, 900. Mu.L of 2% CTAB extract and 90. Mu.L of 10% SDS were added, mixed with vortexing, and put in a water bath at 60℃for 1 hour with the middle turned upside down several times every 10 min. Centrifuging at 12000rpm for 10min, adding equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) into the supernatant, mixing, and centrifuging at 12000rpm for 10min; the supernatant was transferred to a fresh tube, an equal volume of chloroform was added, mixed gently upside down, and centrifuged at 12000rpm for 5min. The supernatant was transferred to a new tube, 2 volumes of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2) were added, -precipitated at 20 ℃ (> 1 h). Centrifuging at 12000rpm for 10min, decanting the supernatant, washing the precipitate twice with 70% ethanol, and air drying at room temperature. Adding a proper amount of sterilized ultrapure water or TE (pH 8.0) to dissolve precipitate (containing 20 mug/mL RNase), treating at 37 ℃ for 1h, and preserving at-20 ℃ for later use.
TABLE 2 fungi and oomycetes for Pp. Vexans PCR detection
As shown in FIG. 1 and FIG. 2, the Pp. Vexans strain can specifically amplify a 344bp band, and the other pathogenic fungi and oomycete agarose gel electrophoresis do not generate amplified bands. Selecting Pythium gracile (Phytopythium helicoides) of a different species than Pythium clockii (Phytopythium vexans); phytopythium plurisporium Pythium ultimum; pythium aphanidermatum (Phytopythium aphanidermatum); pythium spinosum (Phytopythium spinosum); pythium lakeside (Phytopythium littorals); pythium gracile (Phytopythium dissotocum); pythium isox (Phytopythium diclinum); pythium ultimum (Phytopythium ultimum) and bacteria of different genus (pine wood nematodes (Bursaphelenchus xylophillus), fusarium graminearum (Fusarium graminearum), populus canker (Botryospaeria dothidea), apple anthrax (Colletotri chum aenigma), phytophthora winter (Phytophthora hibernalis), fusarium putrescentiae (Fusarium solani), alternaria (Alternaria alternata), botrytis cinerea (Botrytis ci nerea) and the like are taken as templates, 1 μl of DNA solution is taken, 23 μl of kit detection solution and 1 μl of sterilized deionized water are added, the total volume is 25 μl, the PCR amplification procedure is 94 ℃ denaturation for 3 minutes, 94 ℃ denaturation for 30 seconds, 58 ℃ annealing for 30 seconds, 72 ℃ extension for 45 seconds, 33 cycles, and finally 72 ℃ extension for 10 minutes.
The designed specific primer upstream primer and the designed specific primer downstream primer PCR have species specificity, and Pve _contig_43 is a new detection target with stronger specificity.
Example 3
PCR amplification was performed using genomic DNA of Pp. Vexans strains at various concentrations as amplification templates, and the concentration of the extracted DNA of example 1 was measured to be 100ng. Mu.L using a Nanodro 2000 micro-spectrophotometer -1 . Sequentially diluting it to 10ng. Mu.L -1 、1ng.μL -1 、100pg.μL -1 、10pg.μL -1 、1pg.μL -1 、100fg.μL -1 PCR was performed on DNA of different concentrations according to the primers, reaction system and reaction conditions used in examples 1-2. As a result, as shown in FIG. 3, 100ng. Mu.L of each of the 25. Mu.L reaction systems was contained -1 、10ng.μL -1 、1ng.μL -1 、100pg.μL -1 Pp.vexans DNA showed 344bp specific positive bands, which were positive, and the 25. Mu.L reaction system contained 10pg. Mu.L -1 、1pg.μL -1 、100fg.μL -1 Negative reaction occurs when specific bands of Pp.ve xans DNA do not appear; the result shows that the sensitivity of the PCR detection reaches 100pg. Mu.L -1 (FIG. 3).
Comparative example
For the selection of specific detection targets of Pp.vexans and the design of PCR primer sets, 4 targets (Pve _confg_43, pve _confg_1116pve_confg_1509 and pve _confg_768) are initially selected and primers meeting the conditions are designed based on the 4 targets, and finally 1 specific detection new target Pve _confg_43 is selected based on the primersThe targets were designed with 1 set of most specific and extremely sensitive primers, namely the primer composition used in example 1 (upstream primer Pve _contig_43-F1 and downstream primer Pve _contig_43-R1). The specificity of the primers designed by adopting the other 3 detection targets is not high as shown by the PCR detection result, and the actual primer sequences of the primers are as follows: pve-contig 768-F1 (SEQ ID NO. 12) GTCGGTGACATGCACAACA; pve-contig 768-R1 (SEQ ID NO. 13) TGATTGAGTGCTCCGAGG; the strains used in example 2 were Pp. Vexans and other Pp. Vexans as the test materials and various pathogenic fungi, and the PCR detection showed high specificity of the remaining selected primers, as shown in FIG. 4, but with poor sensitivity, only 1ng. Mu.L -1 The results are shown in FIG. 5.
The target Pve _contig_43 for high-reliability specific molecular detection and the specific primers (Pve _contig_43-F1, pve _contig_43-R1) designed based on the target are finally screened out, and the sensitivity detection shows that the concentration of the target is different from that of the target in a 25 mu L reaction system: 100ng. Mu.L -1 、10ng.μL -1 、1ng.μL -1 、100pg.μL -1 、10pg.μL -1 、1pg.μL -1 、100fg.μL -1 Pp.vexans) DNA; PCR amplification was performed using genomic DNA of these standard strains of Pp. Vexans at various concentrations as the amplification template, and the reaction was repeated 3 times. The results of 3 replicates were consistent and the amplification results are shown in FIG. 3. At a genomic DNA concentration of 10pg. Mu.L -1 When the PCR detection is carried out, the specificity band can be detected, the specificity amplification is proved to occur, and the detection result is judged to be positive. And the concentration of DNA in the genome was 10pg. Mu.L -1 When the PCR product is detected, no specific band is found, and the detection result is negative, namely the sensitivity of the PCR method for detecting Pp. Vexans genome DNA is 100pg. Mu.L -1 。
Example 4
When Pp.vexans exists in the pathogenic tissues, the DNA of Pythium belleville is extracted by adopting a NaOH rapid lysis method, and the specific process is as follows: a section of diseased plant tissue is taken, 10 mu L of 0.5M NaOH is added to each milligram of tissue, the mixture is fully ground in a mortar and transferred into an EP tube with 1.5mL, the mixture is centrifuged at 12000rpm for 5min, 495 mu L of 0.1mM Tris (pH 8.0) is added to 5 mu L of supernatant, and 1 mu L of supernatant is directly used for PCR reaction after uniform mixing. Each reaction was repeated at least three times, with no PCR inhibitor present in the plants to be determined.
NaOH alkaline lysis is used for extracting DNA of the pathogenic rhododendron tissue inoculated with Pp. Vexans, and the DNA is used as a template for PCR amplification. A1 uL DNA solution was taken and subjected to PCR as in example 2. FIG. 6A is a diagram of rhododendron leaf artificially inoculated with Pp. Vexans, wherein CK is a blank agar fast rhododendron leaf artificially inoculated with Pp. Vexans, 6A-1 is a rhododendron leaf artificially inoculated with Pp. Vexans for the first day, 6A-2 is a rhododendron leaf artificially inoculated with Pp. Vexans for the second day, and 3 is a rhododendron leaf artificially inoculated with Pp. Vexans for the third day. FIG. 6B shows the results of PCR detection of the pathogenic azalea of Pp.vexans inoculated artificially. In the PCR detection result diagram, from left to right, marker (500 bp), pp. Vexans, NC (negative control), rhododendron leaf of artificial inoculation Pp. Vex ans, the first day of disease, the second day of artificial inoculation of rhododendron leaf of Pp. Vexans, the third day of artificial inoculation of rhododendron leaf, the result shows that DNA extracted from Pp. Vexans strain and Pp. Vexans can specifically amplify a 344bp band, while DNA extracted from rhododendron leaf of artificial inoculation agar block and DNA extracted from negative control do not have amplification band.
The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.

Claims (6)

1. Pythium clockworkPhytopythium vexans) The specific molecular marker Pve _contig_43 of (2) is characterized in that the DNA sequence of the molecular marker Pve _contig_43 is shown in SEQ ID NO: 1.
2. Detecting Pythium species in clockPhytopythium vexans) The primer combination is characterized by comprising a forward primer and a reverse primer, wherein the forward primer Pve _contig_43-F1 has a sequence shown in SEQ ID NO:3, the sequence of the reverse primer Pve _contig_43-R1 is shown in SEQ ID NO: 4.
3. Detecting Pythium species in clockPhytopythium vexans) Is characterized in that the kit comprises a detection solution comprising 20. Mu.M of the primer combination according to claim 2.
4. The kit of claim 3, wherein the detection solution further comprises: 4 dNTPs 2000. Mu.M each, 100. Mu.L of 10 XPCR reaction buffer, 80mM Mg 2+ 100. Mu.L of 1% BSA,50 units Taq enzyme.
5. The primer combination of claim 2 or the kit of any one of claims 3 to 4 in the detection of Pythium bellumPhytopythium vexans) Is used in the field of applications.
6. Detecting Pythium species in clockPhytopythium vexans) Is characterized by comprising the following steps: taking 1 μl of the DNA solution of the test subject, adding 23 μl of the test solution described in claim 4 and 1 μl of sterilized deionized water, and making the total volume 25 μl; the PCR amplification procedure was denaturation at 94℃for 3 min, denaturation at 94℃for 30 sec; annealing at 58 ℃ for 30 seconds; extending at 72 ℃ for 45 seconds; 33 cycles, last 72℃extension for 10 minutes.
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