CN114015804B - Specific detection target Psyrin _ s00018g00015.1 of phytophthora syringae and application of specific detection target Psyrin _ s00018g00015.1 - Google Patents
Specific detection target Psyrin _ s00018g00015.1 of phytophthora syringae and application of specific detection target Psyrin _ s00018g00015.1 Download PDFInfo
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Abstract
The invention discloses a new detection target Psyrin _ s00018g00015.1 of Phytophthora syringae (Phytophthora syringa syinga and a detection primer, a detection kit and a detection method, wherein a protein sequence of the detection target Psyrin _ s00018g00015.1 is shown as SEQ ID NO: 1, and the DNA sequence for coding the protein is shown as SEQ ID NO: 2, respectively. Meanwhile, the invention also discloses a specific primer and probe combination of the RPA detection technology for specifically detecting the target Psyri _ s00018g00015.1, wherein the forward primer sequence is shown as SEQ ID NO: 3, the reverse primer sequence is shown as SEQ ID NO.4, and the probe sequence is shown as SEQ ID NO: 5, respectively. The invention discovers a new detection target of the phytophthora syringae, provides a new detection way for the detection of the phytophthora syringae, and simultaneously can realize the specific detection of the phytophthora syringae and has high sensitivity based on the RPA-LFD detection primer and probe developed by the target.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a specific detection target of phytophthora syringaePsyri_ s00018g00015.1And applications thereof.
Background
Phytophthora (A), (B) and (C)Phytophthora) More than 100 species are formally reported, and many phytophthora have records seriously harming agricultural production safety, so that each species is a crop destructor and causes defectsThe seedlings are broken and heavy, so that serious yield loss is caused, and even the seedlings are out of production. Therefore, the method has important significance for preventing the introduction of the foreign harmful quarantine phytophthora into China. Phytophthora syringae (A), (B), (C)P. syringae) The phytophthora pathogenic bacteria can cause root rot, branch gummosis and other symptoms to host plants to cause citrus fruit brown rot, are widely distributed in various citrus-rich areas in the world at present, are not found in China, and are important entry quarantine objects in China. A great amount of citrus fruits including grapefruit, navel orange and the like are imported from foreign countries in China every year, and pathogenic bacteria have risks accompanying the import of products into China. A2011 year Tianjin port captures phytophthora syringae from American navel orange (P. syringae) The quarantine authorities in various regions found the phytophthora from imported fruits many times, and 6 and 14 batches were respectively intercepted from the imported fruits only in the united states in 2013 and 2015.
The specificity and sensitivity of different target sequence detection have certain difference, and the result has great difference due to different selected target sequences and different fragment sizes. The target gene with good specificity is discovered and is the current phytophthora syringaeP. syringae) The core of the detection technology. The target gene is selected to ensure that it is highly conserved across different strains within a species, while being highly variable across species. Discovering high-reliability specific molecular detection target and establishing sensitive and accurate phytophthora syringae based on new target: (P. syringae) The detection technology system plays an important role in promoting the rapid molecular detection research on the phytophthora syringae and the early diagnosis of diseases caused by the detection.
Disclosure of Invention
Aiming at the problems of long required period, poor specificity and low sensitivity of the detection method of the phytophthora syringae in the prior art, the invention aims to provide a novel target for detecting the phytophthora syringaePsyri_s00018g00015.1And an RPA-LFD detection primer composition established by the target. The invention also aims to provide the RPA-LFD detection method of the phytophthora syringae. The method for rapidly detecting the phytophthora syringae, which is established by the invention, can be used for detecting actual samples through specificity and sensitivity evaluation, and provides a sensitive and sensitive method for the field detection of the phytophthora syringaeThe new method is provided.
In order to achieve the purpose, the invention adopts the following technical scheme:
the study was based on published Phytophthora genomic sequences (Phytophthora cinnamomi)P. cinnamomiPhytophthora sojaeP. sojaePhytophthora oakP. ramorumPhytophthora infestansP. infestansPhytophthora capsiciP. capsici) And a phytophthora genome sequence obtained from CGRB sequencing center, including phytophthora syringae: (P. syringae) Phytophthora hibernalis: (A), (B), (C)P. hibernalis) Phytophthora nicotianae (A) and (B)P. nicotianae) Phytophthora cowpea (A) and (B)P. vignae) Phytophthora nigra (A) and (B) in chestnutP. cambivora) Phytophthora camphora (A), (B) and (C)P. cinnamomi) And the phytophthora cinnamomum variants and the like, and the whole genome sequence of 15 kinds of phytophthora is analyzed, and a large-scale genome database is excavated through Blast sequence search, sequence extraction, comparison and analysis, so that the detection target of the phytophthora cinnamomum is excavated. 1131 specific detection targets of the phytophthora syringae are obtained in total through whole genome comparison; randomly selecting partial genes from 1131 specific genes of phytophthora syringae as candidate genes, and designing and screening specific primers. And verifying the designed specific primer by adopting an RPA-LFD technology. The specificity evaluation selects DNA of different species (phytophthora hibernalis; phytophthora black-water, phytophthora parasitica; phytophthora infestans; phytophthora banksiae; phytophthora strawberry; phytophthora ramie, etc.) and bacteria (colletotrichum truncatum; fusarium solani; rice blast, rhizoctonia solani; verticillium dahliae; pythium ultimum) of different genera as templates, and the DNA is verified to finally obtain 1 new target gene for detecting phytophthora lilacinumPsyri_s00018g00015.1。
In a first aspect, the invention provides a specific detection target of phytophthora syringaePsyri_ s00018g00015.1The protein sequence of the detection target is shown as SEQ ID NO: 1, and the following components:
MTSTREASDQSSPTTHTPERLRSQQQEEEEDIAPPLEYVHLKDTILPPSILALSHITKELNLLLMTPDEAAVEAAATGDTEWMIYLLARFKYFDNEETVAETAAVHGHLQMVKMVAFHFYDYGSFIKI（SEQ ID NO.1）。
in another aspect, the invention provides a specific detection target of phytophthora syringaePsyri_ s00018g00015.1The nucleotide sequence of the detection target is shown as SEQ ID NO: 2, as shown in the figure:
ATGACGTCCACGCGTGAGGCGTCTGACCAGAGCAGCCCCACCACCCACACCCCAGAGCGTCTACGCTCACAGCAGCAAGAAGAAGAAGAAGATATCGCCCCTCCCCTCGAATATGTCCACCTCAAAGACACCATTTTGCCCCCCTCCATTCTGGCTCTGTCACATATCACAAAAGAGCTCAATCTTTTGCTCATGACCCCAGACGAAGCGGCTGTTGAAGCCGCAGCAACGGGCGACACTGAGTGGATGATTTACCTATTGGCCAGATTTAAGTACTTTGACAACGAAGAGACGGTTGCCGAGACGGCGGCTGTCCATGGGCACCTCCAGATGGTGAAAATGGTGGCATTCCACTTTTACGACTACGGAAGTTTCATCAAGATATGA（SEQ ID NO.2）。
on the other hand, the invention also provides a primer and probe combination for detecting the phytophthora syringae, wherein the primer comprises a forward primer and a reverse primer, and the sequence of the forward primer is shown as SEQ ID NO: 3, and the reverse primer sequence is shown as SEQ ID NO:4, and the probe sequence is shown as SEQ ID NO: 5, respectively.
Psyri015.1F:CCAGAGCGTCTACGCTCACAGCAGCAAGAAGAAGA（SEQ ID NO：3）；
Psyri015.1R:TGATGAAACTTCCGTAGTCGTAAAAGTGGAATGC（SEQ ID NO：4）；
Psyri 015.1P: 5'-CGAAGCGGCTGTTGAAGCCGCAGCAACGGGGACACTGAGTGGATG-3', respectively; (SEQ ID NO: 5) which is labeled with FAM at the 5' end, modified with C3Spacer at the 3' end, and THF-modified at 30bp apart from the 5' end in the sequence of the probe.
On the other hand, the invention also provides a kit for detecting the phytophthora syringae, which at least comprises 1 dosage of detection solution containing the combination of the primer and the probe.
Further, the kit further comprises: a Twist Amp reaction unit tube filled with freeze-dried enzyme powder, a Buffer solution Buffer, MgAc, deionized water, a HybriDetect assay Buffer and a lateral flow chromatography test strip.
In another aspect, the invention also provides the specific detection targetPsyri_s00018g00015.1The primer and probe combination and the application of the kit in detecting phytophthora syringae.
On the other hand, the invention also provides a method for detecting the phytophthora syringae, which is characterized in that,
1) extracting DNA of a sample to be detected;
2) using DNA as a template, and carrying out RPA amplification by using the combination of the primers (SEQ ID NO.3 and SEQ ID NO. 4) and the probe (SEQ ID NO. 5) or the kit;
wherein the RPA amplification: to a 0.2 mL Twist Amp reaction unit tube (Twist Amp nfo kits, Twist) containing lyophilized enzyme powder, 29.5. mu.L of Buffer, 2.1. mu.L of 10. mu.M upstream primer (SEQ ID NO. 3), 2.1. mu.L of 10. mu.M downstream primer (SEQ ID NO. 4), 0.6. mu.L of probe (SEQ ID NO. 5), 2.0. mu.L of DNA, and 2.5. mu.L of MgAc were added inside the PCR tube cap, and deionized water was made up to 50. mu.L; fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5,000 Xg, placing on a metal bath at 39 ℃ for reaction for 30min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5 s, and placing in a water bath kettle at 39 ℃ for further reaction for 30 min.
3) Detecting the RPA amplification product by using a lateral flow chromatography test strip;
mu.L of the RPA reaction product was diluted by adding 190. mu.L of HybriDetect assay buffer (Milenia Biotec, Giessen, Germany), and 10. mu.L of the diluted product was dropped onto a HybriDetect 1 strip of a test strip. The other strip was inserted vertically into 100. mu.L of HybriDetect assay buffer and left at room temperature for 5 min.
When two brown strips appear on the test strip, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the sample contains the phytophthora syringae; when only the quality control area of the test strip has a brown strip and the detection area has no strip, the result is negative, which indicates that the sample does not contain the phytophthora syringae.
Unlike conventional PCR reactions, the length of the primers required for the RPA reaction is usually 30-35bp, the length of the probe sequence is 46-52bp, and the increase of the length of the primers increases the difficulty of designing and selecting the primers in order to avoid the formation of secondary structures inside and between the primers during the design of the primers, so the design and selection of the primers are very important for the result of the RPA. The RPA technology is in the initial research stage, no special primer and probe design software exists, and no large amount of data provides basis for the primer design principle. Therefore, the primer and probe combination of the invention can be obtained only by designing a plurality of pairs of primers from both ends of a target sequence and optimizing and screening the primers.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention provides a novel and high-reliability specific phytophthora syringae molecular detection targetPsyri_ s00018g00015.1Provides a new detection way for the detection of the phytophthora syringae.
2) A sensitive, accurate and high-flux RPA-LFD detection technology system is established based on the new target, and the problems that the biological detection method of the phytophthora syringae needs a long period, wastes time and labor, is complicated and has poor specificity in the prior art and a thermal cycler is needed in a PCR detection technology, so that the phytophthora syringae cannot be detected quickly are solved. Compared with the conventional PCR, the detection method has the advantages of high detection speed, no need of three steps of denaturation, annealing and extension, the optimum temperature of the RPA reaction is between 25 and 40 ℃, no need of denaturation, and the reaction can be completed at normal temperature for about 20 min. Does not need complex instruments, can better meet the field detection of the phytophthora syringae, and has important effects on promoting the rapid molecular detection research of the phytophthora syringae and the early diagnosis of diseases caused by the detection of the phytophthora syringae.
3) The detection method provided by the invention has high accuracy: the invention detects a new target according to the phytophthora syringaePsyri_ s00018g00015.1The sequence of (1), the specific detection primer and probe combination is designed, the target band of the LFD lateral flow chromatography test strip detection result is cleared, and the RPA detection concentration is 10 pg. mu.L-1The sensitivity of the RPA lateral flow chromatography test strip detection method is 100 times higher than that of the PCR method.
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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 shows a target for detection based on new excavation of Phytophthora syringaePsyri_s00001g00003. 1The primer RPA-LFD lateral flow chromatography test strip sensitivity detection result chart;
FIG. 2 shows the new growth of phytophthora syringaeDigging detection targetPsyri_s00018g00015.1The detection result graph of the interspecies specificity of the remaining primer RPA-LFD lateral flow chromatography test strip is shown;
FIG. 3 shows a new detection target based on phytophthora syringaePsyri_s00018g00015.1The result of the specific detection of the RPA-LFD lateral flow chromatography test strip among species is shown;
FIG. 4 shows a detection target based on the new discovery of Phytophthora syringaePsyri_s00018g00015.1The test result chart of the specificity test result chart of the lateral flow chromatography test strip of the RPA-LFD among the categories;
FIG. 5 shows novel detection targets based on Phytophthora syringaePsyri_s00018g00015.1The designed specific primer is used for verifying an electrophoretogram by using the common PCR sensitivity;
FIG. 6 shows novel detection targets based on Phytophthora syringaePsyri_s00018g00015.1The lateral flow chromatography test strip sensitivity of the RPA-LFD verifies the electrophoretogram;
FIG. 7 is a graph showing the result of detection by an RPA-LFD lateral flow test strip of Phytophthora syringae in a living tissue.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the present invention belongs.
Example 1
1100 specific genes of phytophthora syringae are obtained by whole genome sequence alignment, and part of genes are randomly selected from 1000 specific genes of phytophthora syringae (a) to serve as candidate genes: (Psyri_s00018g00015.1、Psyri_ s00001g00003.1、Psyri_s00001g00023.1Psyri_s00016g00009.1、Psyri_ s00024g00013.1) Specific primers were designed and screened, and table 1 lists 5 target genes and upstream and downstream detection primers (table 1).
TABLE 1 Phytophthora syringae 5 specific gene sequences and primer tables
Different species (phytophthora cedrata, phytophthora hibernalis, phytophthora blackcurrants, phytophthora melonis, phytophthora infestans and the like) with phytophthora syringae are selected to carry out RPA-LFD detection by using each designed target primer.
Sample detection: adding Buffer 29.5. mu.L, 10. mu.M upstream primer 2.1. mu.L, 10. mu.M downstream primer 2.1. mu.L, probe 0.6. mu.L, DNA 2.0. mu.L, MgAc 2.5. mu.L into a 0.2 mL Twist reaction unit tube (Twist Basic kits, Twist) filled with freeze-dried enzyme powder, and adding deionized water to 50. mu.L; fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5,000 Xg, placing on a metal bath at 39 ℃ for reaction for 30min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5 s, and placing in a water bath kettle at 39 ℃ for further reaction for 30 min.
Negative control: the operation steps are the same as those of sample detection, 2.0 mu L of template DNA is changed into 2.0 mu L of sterilized ddH2And O. And after the RPA reaction is finished, detecting the amplification product by using a lateral flow chromatography test strip. When two brown strips appear on the Test strip, one is located in a quality Control area (Control line) and the other is located in a Test area (Test line), the result is positive, and the sample contains phytophthora syringae; when only the quality control area of the Test strip has a brown strip and the detection area (Test line) has no strip, the result is negative, which indicates that the sample does not contain the phytophthora syringae.
With a targetPsyri_s00001g00003. 1For example, as，Detection of targets using moleculesPsyri_s00001g00003. 1The designed primers have the following sequences: the upstream primer Psyri003.1F and the downstream primer Psyri003.1R have the following actual primer sequences: psyri 003.1F: ATGCCCCGGCTCACAGTCAGACATAATGTTTTGG (SEQ ID NO: 8); Psyri 003.1R: TTATTGGCAAACGTGTGGTGTGGTGGTCAGATGA (SEQ ID NO: 9); the result of selecting a species different from that of the phytophthora syringae (phytophthora cedira; phytophthora hibernalis; phytophthora cassolensis; phytophthora melonis; phytophthora infestans, etc.) shows that the specificity of the primer is high, but the sensitivity is poor, and is only 10 ng/uL, and the result is shown in figure 1.
With a targetPsyri_s00018g00015.1For example, the actual primer sequences are: psyrin 015.1F: CCAGAGCGTCTACGCTCACAGCAGCAAGAAGAAGA (SEQ ID NO: 3); psyrin 015.1R: TGATGAAACTTCCGTAGTCGTAAAAGTGGAATGC (SEQ ID NO: 4); different species (phytophthora cedar, phytophthora hibernalis, phytophthora black currant, phytophthora melonis, phytophthora infestans and the like) from phytophthora syringae are selected, and the results of the RPA-LFD detection show that the specificity of the rest of the selected primers is poor, and the results are shown in figure 2.
The molecular detection target is obtained by screening by integrating the consideration of the detection specificity and the sensitivity of the molecular targetPsyri_ s00018g00015.1The forward primer sequence of the primer is shown as SEQ ID NO: 3, the reverse primer sequence is shown as SEQ ID NO.4, and the probe sequence is shown as SEQ ID NO: 5, respectively.
Example 2
The specificity of the RPA lateral flow chromatography Test strip detection method is further verified, an epidemic lilyturf disease strain, other phytophthora and pathogenic bacteria are used as Test materials (table 2), the result of the RPA lateral flow chromatography Test strip detection method shows that the Test strip of the epidemic lilyturf disease has two brown strips, one is located in a quality Control area (Control Line), the other is located in a detection area (Test Line), the result is positive, only one brown strip is located in the quality Control area of the Test strip of the other phytophthora and pathogenic bacteria, and the result is negative, so that the sample does not contain the epidemic lilyturf disease.
The DNA of a strain(s) different from that of Phytophthora syringae (Phytophthora cedrata; Phytophthora hibernalis; Phytophthora blackcurrants; Phytophthora melonis; Phytophthora infestans; Phytophthora equi; Fusarium equiseti; Anthragmarius, Verticillium dahliae; Rhizoctonia solani; Magnaporthe oryzae; etc.) and a strain(s) different from that of Phytophthora syringae are selected as templates and RPA-LFD (lateral flow chromatography test strip detection) is carried out.
TABLE 2 fungus and oomycete strains for PCR detection of Phytophthora syringae and detection results
FIG. 3 shows the new target for detecting phytophthora syringae based on new discoveryPsyri_s00018g00015.1The specific detection result diagram of the RPA-LFD lateral flow chromatography test strip is shown; in the figure 1: phytophthora syringae (A), (B), (C)P. syringae) (ii) a 2: phytophthora cedar: (A. cedar)P. lateralis) (ii) a 3: phytophthora hibernalis: (A. hibernalis)P. hibernalis) (ii) a 4: phytophthora melonis (A), (B), (C)P. melonis) (ii) a 5: phytophthora infestans (A), (B), (C) P. infestans) (ii) a 6: phytophthora infestans (A), (B) and (C)P. cactorum) (ii) a 7: phytophthora sojae (A.sojae) ((B.))P. sojae) (ii) a 8: and (5) negative control. FIG. 3 shows No. 1 with 2 bands, wherein one band is a Control Line (Control Line), one Line is a detection Line (Test Line), and thus positive, and the other bands with only one Control Line (Control Line) are negative, indicating that the sample contains Phytophthora syringae; explaining the newly discovered detection targetPsyri_s00018g00015.1Has specificity among species.
FIG. 4 is a diagram of the detection of novel targets based on high confidence specific moleculesPsyri_s00018g00015.1The specific detection result graph of the RPA-LFD lateral flow chromatography test strip among the categories is a detection result graph; 1: phytophthora syringae (A), (B), (C)P. syringae) (ii) a 2: pythium ultimum: (A. ultimum)Pythium ultimum) (ii) a 3: fusarium equiseti (F.), (Fusarium equiseti) (ii) a 4: anthrax bacteria (B) of FlankiaColletotrichum truncatum) (ii) a 5: verticillium dahliae (C.), (Verticilium dahliae) (ii) a 6: rhizoctonia solani (A), (B), (C)Rhizoctonia solani) (ii) a 7: pyricularia oryzae (A. oryzae) on riceMagnaporthe grisea) (ii) a 8: negative control; FIG. 4 shows 2 bands in lane 1, wherein one band is a Control line (Control line) and one line is a detection line, and thus the positive result indicates that the sample contains Phytophthora syringae; the remaining bands with only one Control line (Control line) were negative. Explaining the newly discovered detection targetPsyri_s00018g00015.1Has intergeneric specificity.
Example 3
The genome DNA of the standard strain of the phytophthora syringae with the same concentration is used as an amplification template, an upstream primer Psyri015.1F/Psyri015.1R and a downstream primer Psyri015.1R are adopted to carry out PCR amplification reaction, and the sensitivity of the two methods is compared; the experiment was repeated 3 times to confirm the PCR assayDetecting the sensitivity of phytophthora syringae genome DNA; the results of 3 replicates were consistent. The concentration of genomic DNA was 1 ng. mu.L-1In this case, the PCR detection enables detection of a specific band, and the result is shown in FIG. 5, which proves that specific amplification occurs and the detection result is positive. After the RPA amplification reaction of the present invention was performed, the detection result of the LFD lateral flow chromatography test strip was analyzed as a target band shown in fig. 6. It can be seen that the same new excavation target is based onPsyri_s00018g00015.1The sensitivity of the RPA lateral flow chromatography test strip detection method is 100 times higher than that of the PCR method. The result shows that the detection concentration of RPA is 10 pg. mu.L-1And the PCR detection concentration is 1 ng. mu.L-1The target band can be seen, which indicates that the sensitivity of RPA detection is higher than that of PCR. But the PCR detection process needs 2.5h, the RPA detection time only needs 30min, expensive instruments such as a PCR instrument and the like are not needed, the operation procedure is simple and convenient, and the method is more favorable for popularization and application in production.
Example 4
The DNA of the pathogenic citrus inoculated with phytophthora syringae is extracted by adopting an NaOH alkaline cracking method and is used as a template for PCR amplification. Using 1uL of the DNA solution, the RPA-LFD assay was performed in the same manner as in example 3. The results are shown in FIG. 7, where the LFD lateral flow test strip test results after the RPA amplification reaction. The result shows that the diseased citrus (2,3 and 4) inoculated with the phytophthora syringae can display 2 brown bands, the target bands are analyzed, and the phytophthora syringae can be effectively detected. While other fungi and oomycetes only have a brown band in the quality control area, and the negative control only has a brown band in the quality control area.
Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.
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<110> Nanjing university of forestry
<120> specific detection target Psyrin _ s00018g00015.1 of phytophthora syringae and application thereof
<130> 2021
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<211> 128
<212> PRT
<213> Phytophthora syringae (Phytophthora syringae)
<400> 1
Met Thr Ser Thr Arg Glu Ala Ser Asp Gln Ser Ser Pro Thr Thr His
1 5 10 15
Thr Pro Glu Arg Leu Arg Ser Gln Gln Gln Glu Glu Glu Glu Asp Ile
20 25 30
Ala Pro Pro Leu Glu Tyr Val His Leu Lys Asp Thr Ile Leu Pro Pro
35 40 45
Ser Ile Leu Ala Leu Ser His Ile Thr Lys Glu Leu Asn Leu Leu Leu
50 55 60
Met Thr Pro Asp Glu Ala Ala Val Glu Ala Ala Ala Thr Gly Asp Thr
65 70 75 80
Glu Trp Met Ile Tyr Leu Leu Ala Arg Phe Lys Tyr Phe Asp Asn Glu
85 90 95
Glu Thr Val Ala Glu Thr Ala Ala Val His Gly His Leu Gln Met Val
100 105 110
Lys Met Val Ala Phe His Phe Tyr Asp Tyr Gly Ser Phe Ile Lys Ile
115 120 125
<210> 2
<211> 387
<212> DNA
<213> Phytophthora syringae (Phytophthora syringae)
<400> 2
atgacgtcca cgcgtgaggc gtctgaccag agcagcccca ccacccacac cccagagcgt 60
ctacgctcac agcagcaaga agaagaagaa gatatcgccc ctcccctcga atatgtccac 120
ctcaaagaca ccattttgcc cccctccatt ctggctctgt cacatatcac aaaagagctc 180
aatcttttgc tcatgacccc agacgaagcg gctgttgaag ccgcagcaac gggcgacact 240
gagtggatga tttacctatt ggccagattt aagtactttg acaacgaaga gacggttgcc 300
gagacggcgg ctgtccatgg gcacctccag atggtgaaaa tggtggcatt ccacttttac 360
gactacggaa gtttcatcaa gatatga 387
<210> 3
<211> 35
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
ccagagcgtc tacgctcaca gcagcaagaa gaaga 35
<210> 4
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
tgatgaaact tccgtagtcg taaaagtgga atgc 34
<210> 5
<211> 45
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
cgaagcggct gttgaagccg cagcaacggg gacactgagt ggatg 45
<210> 6
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
aagaagatat cgcccctccc ctcgaatat 29
<210> 7
<211> 192
<212> DNA
<213> Phytophthora syringae (Phytophthora syringae)
<400> 7
atgccccggc tcacagtcag acataatgtt ttggggcgtg agcaggaaga cctggtgctt 60
ctccagctga cacctcattc tccaatgtgc aacgcaattg aaggtgagtt agacgaattt 120
attgctattg catgcgtcct caactcaacc atcaagcgtc atctgaccac cacaccacac 180
gtttgccaat aa 192
<210> 8
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
atgccccggc tcacagtcag acataatgtt ttgg 34
<210> 9
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
ttattggcaa acgtgtggtg tggtggtcag atga 34
<210> 10
<211> 204
<212> DNA
<213> Phytophthora syringae (Phytophthora syringae)
<400> 10
atggtcaagt ccgagcgacg ctacatctcc atggaaaggc tctatttttg cgcggcgtac 60
atttgtgctc agcagagttt gacctttcgt gtgacggtgt ggggtgctac gttgagcgac 120
cccaagtacg tcgtcagctc taatctccgt ctgcgcaata tccgcgacat cactgtctgg 180
aatgacattt tggtcgacaa ttga 204
<210> 11
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
atggtcaagt ccgagcgacg ctacatctcc atgg 34
<210> 12
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
tcaattgtcg accaaaatgt cattccagac agtg 34
<210> 13
<211> 516
<212> DNA
<213> Phytophthora syringae (Phytophthora syringae)
<400> 13
atgacgcaca tcagctggaa ttgtatccga gctcagcatt tttggagaag atacttggag 60
cactggctgg ggtatgaagt ctccaatcta cgactcgagc agtacaagca ctacattgcg 120
gcacgacggg cgccaccaat cgggactcga ctgaagcgca aaataacgga acgatacggg 180
acatggagac aagagaatgc agacgcacta ccatcggtgg cgcactattg tgactcgtac 240
gcaaccaagt ggtccatgaa gacgtggcga gctgctccgc aactggaata tatgtggaca 300
acctgtctac ggcagctgca cgggatagcg cggcgagaac gaaccaggtc cgcgacaaag 360
cttgaaggag ttcagttcca gctcagcctc gactgctacg cggatatagg aacggaaatg 420
gacccgcagg atcctcctcc agctccagcc aagtggctcc gaacagagtc gcagttgggc 480
aaacgactta cacaatacca ggaagctatt aactaa 516
<210> 14
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
gcactggctg gggtatgaag tctccaatct acga 34
<210> 15
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 15
cagttgcgga gcagctcgcc acgtcttcat ggac 34
<210> 16
<211> 423
<212> DNA
<213> Phytophthora syringae (Phytophthora syringae)
<400> 16
atgcttggtg gcgccccacc gccgacatac aaagtggctt gtcgaaagcc acaattgatg 60
gatcaactgg ctaagtattt gctgagtaca ttgataccgt gggagttgga gggtaagcaa 120
cttcccttcg actttagcgg tgatggttta cttgaattgt gtagactgtg ggaccgttcg 180
gatgatgttc tcattaatcg acagcgttat cgtttaatag ccaatattcg tcgacgaggt 240
aaccaaaaga atgcagctgg tgacacgttt atgcattggc gtgctcgcaa tgttgactgg 300
tggaccgatg cttctcgacg tactgtcaac gaggacgaca ataatgttgt ttccaatgat 360
ggtctggaat ttgataccga gttccaggga gctaacacgt tgctgaagct ttgtttcaat 420
tag 423
<210> 17
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
acaattgatg gatcaactgg ctaagtattt gctg 34
<210> 18
<211> 34
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 18
attgtcgtcc tcgttgacag tacgtcgaga agca 34
Claims (6)
1. A specific detection target Psyrin _ s00018g00015.1 of phytophthora syringae is characterized in that the nucleotide sequence of the detection target is as shown in SEQ ID NO: 2, respectively.
2. The primer and probe combination for detecting the phytophthora syringae is characterized in that the primer comprises a forward primer and a reverse primer, and the sequence of the forward primer is shown as SEQ ID NO: 3, and the reverse primer sequence is shown as SEQ ID NO:4, and the probe sequence is shown as SEQ ID NO: 5, respectively.
3. A kit for detecting Phytophthora syringae, comprising at least 1 use of a detection solution comprising the primer and probe combination of claim 2.
4. The kit of claim 3, further comprising: a Twist Amp reaction unit tube filled with freeze-dried enzyme powder, a Buffer solution Buffer, MgAc, deionized water, a HybriDetect assay Buffer and a lateral flow chromatography test strip.
5. The use of the specific detection target Psyrin _ s00018g00015.1 of claim 1, the primer and probe combination of claim 2, and the kit of claim 3 or 4 for detecting Phytophthora syringae.
6. A method for detecting phytophthora syringae is characterized in that,
1) extracting DNA of a sample to be detected;
2) performing RPA amplification using the primer and probe combination of claim 2 or the kit of claim 4 using DNA as a template;
3) detecting the RPA amplification product by using a lateral flow chromatography test strip; when two brown strips appear on the test strip, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the sample contains the phytophthora syringae; when only the quality control area of the test strip has a brown strip and the detection area has no strip, the result is negative, which indicates that the sample does not contain the phytophthora syringae.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130100090A (en) * | 2013-08-30 | 2013-09-09 | 충남대학교산학협력단 | Primers of polymerase chain reactions for the detection of phytophthora species broken out on kind of fruit tree or seedling, and detection kits and methods thereof |
| CN104099404A (en) * | 2013-04-08 | 2014-10-15 | 中国检验检疫科学研究院 | Primer pair and probe for detecting Phytophthora syringae and detection method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099404A (en) * | 2013-04-08 | 2014-10-15 | 中国检验检疫科学研究院 | Primer pair and probe for detecting Phytophthora syringae and detection method thereof |
| KR20130100090A (en) * | 2013-08-30 | 2013-09-09 | 충남대학교산학협력단 | Primers of polymerase chain reactions for the detection of phytophthora species broken out on kind of fruit tree or seedling, and detection kits and methods thereof |
Non-Patent Citations (2)
| Title |
|---|
| Tingting Dai等.A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis.《PEERJ》.2019,(第7期),第1-14页. * |
| 朱林慧等.检疫性柑橘冬生疫霉和丁香疫霉的三重PCR同步检测.《西南大学学报(自然科学版)》.2015,第37卷(第05期),第1-8页. * |
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