CN110607380B - Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma - Google Patents
Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma Download PDFInfo
- Publication number
- CN110607380B CN110607380B CN201910927209.5A CN201910927209A CN110607380B CN 110607380 B CN110607380 B CN 110607380B CN 201910927209 A CN201910927209 A CN 201910927209A CN 110607380 B CN110607380 B CN 110607380B
- Authority
- CN
- China
- Prior art keywords
- mulberry
- phytoplasma
- primer
- ltra
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma. The invention firstly provides an ltrA gene of mulberry phytoplasma, and the gene can be used for detecting the mulberry phytoplasma. And a group of specific detection primers of the mulberry phytoplasma are designed by taking the ltrA gene as a target gene, and a kit and a detection method for detecting the mulberry phytoplasma are further constructed‑ 6ng/mu L can monitor and control related diseases caused by the mulberry phytoplasma in the mulberry field in time before the diseases are obviously cured and outbreak, provides technical support for inspection and quarantine work, and has good popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of pathogen molecule detection. More particularly, relates to a mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma.
Background
Phytoplasma (Candidatus phytoplasma) classically belongs to the kingdom Bacteria (Bacteria), Thelenota (Firmicutes), the class of Flectomycetes (Mollicules), the order of Nostosphaeroides (Acholesalales), the family of Nostosphaeroidaceae (Acholesalaceae); sensitive to tetracycline antibiotics, cells are usually circular or oval, have no cell wall, have a single membrane outside the cytoplasm, and have granular ribosomes, soluble proteins, RNA, metabolites, and the like inside the cytoplasm. The phytoplasma cell has no nucleus, and the genome is double-stranded closed circular DNA, belonging to gram-positive bacteria. The phytoplasma is related to more than 1000 plant diseases, including some important horticultural crop diseases, such as grape yellowing disease, strawberry yellowing disease, onion yellowing disease and the like, and the main symptoms comprise arbuscular branch, yellowing, floral leaf change, floral organ fading and the like, and the early senescence of plants is caused in severe cases until the whole plant withers, so that a great deal of economic loss is caused. More than 100 plant diseases related to phytoplasma are also reported in China, and the more important plant diseases include jujube witches broom, paulownia witches broom, wheat blue dwarf and the like. Mulberry phytoplasma belongs to the group B of the Flaveria deltoides group (Aster yellows groups). Although the phytoplasma can be cultured in vitro in a micro-scale manner, the culture conditions are harsh, artificial culture is difficult, and a large amount of separation culture of the phytoplasma which has not been successfully carried out so far is caused, so that the development of rapid detection research of the phytoplasma is hindered.
At present, related diseases caused by mulberry phytoplasma are mostly judged by diseases after disease occurrence, and molecular detection means are less in application. With the development of molecular biology, molecular detection technology has become an important detection means due to its advantages of rapidness, sensitivity, etc. In the prior art, Liuqingshen and the like adopt common primers and nested primers designed by a phytoplasma 16S-23S rDNA sequence to establish a rapid and accurate nested PCR detection technology of mulberry phytoplasma; however, the nested PCR detection technology has the defects of complex detection process, low detection specificity and sensitivity and the like, and is not beneficial to popularization and use in basic detection units. Therefore, further research and exploration of a method for detecting the mulberry phytoplasma with wide application range, simplicity, rapidness and accuracy are needed.
Therefore, a method for detecting the mulberry phytoplasma, which has wide application range, is simple, rapid, accurate and specific, is established by selecting a proper gene segment as a target gene, and has important significance for monitoring and controlling related diseases caused by the mulberry phytoplasma.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing mulberry phytoplasma detection technology and provides a mulberry phytoplasma ltrA gene and application thereof in molecular detection of the mulberry phytoplasma.
The method can detect the existence of the mulberry phytoplasma before the related diseases caused by the mulberry phytoplasma appear obvious symptoms and the diseases are outbreak, and can accurately detect the existence of the mulberry phytoplasma under the condition of extremely low content of the mulberry phytoplasma, thereby timely monitoring and controlling the related diseases caused by the mulberry phytoplasma in the mulberry field. Specifically, the ltrA gene of the mulberry phytoplasma is used as a target gene, and a mulberry phytoplasma specificity detection primer is scientifically designed through a large amount of research and exploration, so that a method for rapidly detecting the mulberry phytoplasma is established; the method has the advantages of accurate and reliable detection result, simple and rapid operation, strong detection specificity and high sensitivity, and has wide application value and important significance in the actual monitoring and control of related diseases caused by the mulberry phytoplasma.
The first purpose of the invention is to provide an ltrA gene of mulberry phytoplasma.
The second purpose of the invention is to provide the application of the ltrA gene or the fragment thereof in the molecular detection of the mulberry phytoplasma or the preparation of a molecular detection product of the mulberry phytoplasma.
The third purpose of the invention is to provide a primer for detecting the specificity of the mulberry phytoplasma.
The fourth purpose of the invention is to provide a positive control plasmid for detecting mulberry phytoplasma.
The fifth object of the present invention is to provide a primer for constructing the positive control plasmid.
The sixth purpose of the invention is to provide the application of the specific detection primer, the positive control plasmid or the construction primer in the molecular detection of the mulberry phytoplasma or the preparation of a molecular detection product of the mulberry phytoplasma.
The seventh object of the present invention is to provide a kit for detecting mulberry phytoplasma.
The eighth purpose of the invention is to provide a mulberry phytoplasma detection method.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides an ltrA gene of mulberry phytoplasma, and the nucleotide sequence of the ltrA gene is shown as SEQ ID NO. 1.
The application of the ltrA gene or the fragment thereof in the molecular detection of the mulberry phytoplasma and the application in the preparation of the molecular detection product of the mulberry phytoplasma also belong to the protection scope of the invention.
Based on the research and the pertinence analysis summary of the invention on the ltrA gene of the mulberry phytoplasma, the ltrA gene is selected as a target gene, and specific detection primers of the mulberry phytoplasma are designed, wherein the specific detection primers are an upstream primer 927F and a downstream primer 1538R, and the nucleotide sequences of the specific detection primers are respectively shown as SEQ ID No.2 and SEQ ID No. 3.
Nucleotide sequence of upstream primer 927F (SEQ ID NO. 2): 5'-AAAGGTGGTGTCCTGGTTAG-3', respectively;
nucleotide sequence of downstream primer 1538R (SEQ ID NO. 3): 5'-GTGCGACCTAGGAAGGTATTT-3' are provided.
The invention also provides a positive control plasmid for detecting the mulberry phytoplasma, wherein the sequence of the positive control plasmid contains an ltrA gene sequence. The sequence may be a full-length sequence or a partial sequence. When the sequence is a partial sequence, the amplified region of the detection primer set is covered.
The vector used may be one commonly used in the art, such as pMDTM19。
Namely a positive control plasmid pMD for detecting mulberry phytoplasmaTM19-ltrA, the nucleotide sequence of which is shown in SEQ ID NO. 4.
The invention also provides primers for constructing the positive control plasmid, which are respectively an upstream primer ltrAF and a downstream primer ltrAR, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
Nucleotide sequence of the upstream primer ltrA (SEQ ID NO. 5): 5'-ATGCAAATGCAACCAAC-3', respectively;
nucleotide sequence of downstream primer ltrA R (SEQ ID NO. 6): 5'-TTATTCAGACGTATTTA TTTTTCTTT-3' are provided.
The specificity detection primer has strong specificity and high sensitivity, can simply, conveniently and quickly detect the mulberry phytoplasma based on the primer, and has important significance for monitoring and controlling related diseases caused by the mulberry phytoplasma.
Therefore, the application of the specific detection primer, the positive control plasmid or the construction primer in the molecular detection of the mulberry phytoplasma or the preparation of a molecular detection product of the mulberry phytoplasma is within the protection scope of the invention.
The invention also provides a kit for detecting the mulberry phytoplasma, which comprises the specific detection primer or the construction primer.
Preferably, the kit further comprises reagents required for DNA extraction or reagents required for PCR amplification reactions.
In addition, the invention also provides a mulberry phytoplasma detection method, which is used for detecting whether the ltrA gene exists in nucleic acid of a sample to be detected, and if so, the sample to be detected is positive for phytoplasma.
Preferably, a nucleic acid of a sample to be detected is used as a template, a primer which can be used for amplifying the ltrA gene is used for carrying out PCR amplification reaction, the positive control plasmid is used as a control, and if the amplification result is positive, the sample to be detected is positive of phytoplasma.
More preferably, the primer for amplifying the ltrA gene is a specific detection primer shown in SEQ ID NO.2 and SEQ ID NO.3 or a primer for construction shown in SEQ ID NO.5 and SEQ ID NO. 6.
Wherein, when the primers are specific detection primers shown in SEQ ID NO.2 and SEQ ID NO.3, the size of the amplified fragment is 611 bp; when the primers are the primers for construction shown in SEQ ID NO.5 and SEQ ID NO.6, the amplified fragment size is 1758 bp.
Wherein the sample to be detected is a mulberry leaf sample.
Preferably, the system of the PCR amplification reaction is as follows:
wherein, the 2 × reaction buffer comprises the following components: taq DNA polymerase, 160mM Tris-HCl, 40mM (NH)4)2SO4，3.0mM MgCl2，400μM dNTP。
Preferably, the procedure of the PCR amplification reaction is: 5min at 94 ℃; 30s at 94 ℃, 30s at 59 ℃, 45s at 72 ℃ and 32 cycles; 10min at 72 ℃.
The invention has the following beneficial effects:
the invention provides a mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma. The invention takes the ltrA gene of mulberry phytoplasma as the target baseTherefore, through a large amount of research and exploration, the specific detection primer of the mulberry phytoplasma is scientifically designed, the specificity of the primer is strong, the sensitivity is high, and the detection sensitivity is as high as 1.0 multiplied by 10-6ng/mu L, the primer can be used for simply, conveniently and quickly detecting the mulberry phytoplasma, and has important guiding significance for monitoring and controlling related diseases caused by the mulberry phytoplasma.
Based on the primers, the invention also provides a method for rapidly detecting the mulberry phytoplasma and a kit for detection, the method and the kit are simple and rapid to operate, the detection result is accurate and reliable, the existence of the mulberry phytoplasma can be detected before the related diseases caused by the mulberry phytoplasma appear obvious symptoms and the diseases are outbreak, the existence of the mulberry phytoplasma can also be accurately detected under the condition of extremely low content of the mulberry phytoplasma, the related diseases caused by the mulberry phytoplasma in a mulberry field can be timely monitored and controlled, technical support is provided for the detection of the mulberry phytoplasma, particularly the inspection and quarantine work, and the method and the kit have good popularization and application prospects in the actual monitoring and control of the related diseases caused by the mulberry phytoplasma.
Drawings
FIG. 1 is a result diagram of a phylogenetic tree constructed based on the amino acid sequence of the ltrA gene of Phytoplasma mori.
FIG. 2 is a graph showing the result of a phylogenetic tree constructed based on the nucleotide sequence of the 16s rRNA gene of Phytoplasma mori L.
FIG. 3 is a diagram showing the results of the specificity verification of the forward primer 927F and the backward primer 1538R for the detection of the mulberry phytoplasma; wherein, M represents TaKaRa DL1000 Marker; 1 represents the positive control plasmid pMDTM19-ltrA; 2 represents mulberry leaf type atrophy related virus; 3 represents mulberry vein associated virus; 4 represents pathogenic bacteria of mulberry bacterial wilt-Lauerella of Solanaceae; 5 represents pathogenic bacteria of mulberry wilt-enterobacter cloacae; 6 represents pathogenic bacteria of sclerotinia sclerotiorum-carumforbesii; 7 represents Mulberry bark disease pathogen-Mulberry ball needle shell; 8 represents DNA extracted from healthy mulberry leaves; 9 represents DNA extracted from mulberry leaves suffering from the phytoplasma of mulberry; 10 represents water.
FIG. 4 shows the sensitivity of forward primer 927F and reverse primer 1538R for detecting Mycoplasma moriA sexual verification result graph; wherein, M represents TaKaRa DL1000 Marker; 1 to 7 represent DNA concentrations of 1.0X 10, respectively-1ng/μL、1.0×10-2ng/μL、1.0×10-3ng/μL、1.0×10-4ng/μL、1.0×10-5ng/μL、1.0×10-6ng/μL、1.0×10- 7ng/μL、1.0×10-8ng/mu L; 8 represents ddH2O。
FIG. 5 is a graph showing the results of mulberry phytoplasma detection of morous mulberry leaves in different regions; wherein, M represents TaKaRa DL1000 Marker; 1 represents DNA extracted from healthy mulberry leaves; 2-10 respectively represent morbid leaves in a mulberry field of southern China agricultural university, morbid leaves in a silkworm research institute of Guangdong agricultural academy of sciences, morbid leaves in West county of Guangdong province, morbid leaves in Yinde City of Guangdong province, morbid leaves in Fengshan town demonstration area in Liucheng county of Guangxi province, morbid leaves in Bingyang county of Guangxi province, morbid leaves in Chongmai town of Liucheng city of Guangxi province, morbid leaves in Dianjing Zhenjin city of Qujing city of Yunnan province, and morbid leaves in Yunan Mongolian grass dam town of City; 11 represents the positive control plasmid pMDTM19-ltrA。
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 obtaining of the full-Length nucleotide sequence of the ltrA Gene of Morus alba Phytoplasma
1. Experimental methods
And obtaining the ltrA gene full-length nucleotide sequence of the mulberry phytoplasma by using a high-throughput sequencing method, annotating the ltrA gene, and performing sequence comparison analysis to construct a phylogenetic tree.
2. Results of the experiment
The full-length nucleotide sequence of the ltrA gene of the mulberry phytoplasma is shown as SEQ ID NO.1, the length of the full-length nucleotide sequence is 1758bp, and the results of the ltrA gene annotation are shown in Table 1.
TABLE 1 results of ltrA Gene annotation
Sequence comparison analysis is carried out on the full-length nucleotide sequence of the ltrA gene of the mulberry phytoplasma, and the consistency of the full-length nucleotide sequence of the ltrA gene of the mulberry phytoplasma and other nucleotide sequences in NCBI is lower than 75 percent, so that the ltrA gene has obvious difference; in addition, the amino acid sequence of the ltrA gene of the mulberry phytoplasma is subjected to sequence alignment analysis at the amino acid level, and the amino acid sequence of the ltrA gene of the mulberry phytoplasma is found to have homology with other amino acid sequences in NCBI, but the homology is less than 75 percent, and has remarkable difference, and the difference of the ltrA gene of the phytoplasma at the nucleic acid level and the amino acid level is presumed to have certain correlation with the selective infection of a host.
In addition, the result of the phylogenetic tree constructed based on the amino acid sequence of the ltrA gene of the mulberry phytoplasma is shown in figure 1, and the result of the phylogenetic tree constructed based on the nucleotide sequence of the 16s rRNA gene of the mulberry phytoplasma is shown in figure 2, which confirms that the amino acid sequence of the ltrA gene of the phytoplasma has obvious branches and high specificity, and further explains that the amino acid sequence of the ltrA gene of the phytoplasma has obvious difference in selective infection of hosts.
Example 2 detection primer design and establishment of PCR amplification method
1. Primer design
On the basis of the ltrA gene full-length nucleotide sequence of the mulberry phytoplasma obtained in example 1, the ltrA gene is taken as a target gene, and a specific detection primer for detecting the mulberry phytoplasma is optimized and screened: the nucleotide sequences of the upstream primer 927F and the downstream primer 1538R are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3; and primers for constructing a positive control plasmid for detecting the mulberry phytoplasma are respectively an upstream primer ltrA F and a downstream primer ltrA R, and the nucleotide sequences of the primers are respectively shown as SEQ ID No.5 and SEQ ID No. 6.
Nucleotide sequence of upstream primer 927F (SEQ ID NO. 2): 5'-AAAGGTGGTGTC CTGGTTAG-3', respectively;
nucleotide sequence of downstream primer 1538R (SEQ ID NO. 3): 5'-GTGCGACCTAG GAAGGTATTT-3', respectively;
nucleotide sequence of the upstream primer ltrA (SEQ ID NO. 5): 5'-ATGCAAATGCAACCAAC-3', respectively;
nucleotide sequence of downstream primer ltrA R (SEQ ID NO. 6): 5'-TTATTCAGACGTATTTA TTTTTCTTT-3' are provided.
2. Establishment of PCR amplification method
Extracting total DNA of a mulberry leaf sample as a template, and respectively carrying out PCR amplification reaction by using the specific detection primers (an upstream primer 927F and a downstream primer 1538R, an upstream primer ltrA F and a downstream primer ltrA R).
(1) The PCR amplification reaction system and procedure of the upstream primer 927F and the downstream primer 1538R are as follows:
the PCR amplification reaction system is as follows:
wherein, the 2 × reaction buffer comprises the following components: taq DNA polymerase, 160mM Tris-HCl, 40mM (NH)4)2SO4，3.0mM MgCl2，400μM dNTP。
The procedure for the PCR amplification reaction was: 5min at 94 ℃; 30s at 94 ℃, 30s at 59 ℃, 45s at 72 ℃ and 32 cycles; 10min at 72 ℃.
(2) The PCR amplification reaction system and the procedure of the upstream primer ltrA F and the downstream primer ltrA R are as follows:
the PCR amplification reaction system is as follows:
wherein, the 2 × reaction buffer comprises the following components: taq DNA polymerase, 160mM Tris-HCl, 40mM (NH)4)2SO4，3.0mM MgCl2，400μM dNTP。
The procedure for the PCR amplification reaction was: 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃, 60s at 72 ℃ and 32 cycles; 10min at 72 ℃.
3. Result judgment
Detecting the PCR amplification reaction product by agarose gel electrophoresis, wherein if the fragment size of the PCR amplification reaction product of the upstream primer 927F and the downstream primer 1538R is 611bp, the sample to be detected contains the mulberry phytoplasma; otherwise, none; if the fragment size of the PCR amplification reaction product of the upstream primer ltrA F and the downstream primer ltrA R is 1758bp, the sample to be detected contains the mulberry phytoplasma; otherwise, it is not.
EXAMPLE 3 Positive control plasmid pMDTMConstruction of 19-ltrA
The research and the targeted analysis summary of the invention on the ltrA gene of the mulberry phytoplasma are designed and amplified by primers comprising the full-length nucleotide sequence of the ltrA gene of the mulberry phytoplasma, wherein the primers comprise an upstream primer 927F and a downstream primer 1538R, the nucleotide sequences of the primers are respectively shown as SEQ ID No.2 and SEQ ID No.3, a PCR amplification reaction system and a program are shown as example 2, and the pMD (polymerase chain reaction) of Takara biological company is adoptedTMConstruction of Positive control plasmid pMD with 19-T Vector Cloning KitTM19-ltrA, the nucleotide sequence of which is shown in SEQ ID NO. 4.
Example 4 specificity verification of upstream primer 927F and downstream primer 1538R for detection of Phytoplasma sanguinea
1. Experimental methods
DNA extracted from mulberry leaves suffering from the mulberry phytoplasma disease, mulberry pathogenic DNA stored in the laboratory (pathogen available to those skilled in the art through other conventional sources): mulberry leaf atrophy-related Virus (Mulberry viral Associated Virus), Mulberry Vein zone-related Virus (Mulberry present binding Associated Virus), Mulberry bacterial wilt pathogen-Lauer type bacterium (Ralstonia solanacearum), Mulberry blight pathogen-Enterobacter cloacae (Enterobacter cloacae), Mulberry sclerotinia sclerotiorum pathogen-Carunchoides (Ciboria carunculoides), Mulberry powdery mildew pathogen-Mulberry needle shell (Phyllanthus mori), and DNA extracted from healthy Mulberry leaves were used as templates, and the positive pairs constructed in example 3 were used as templatesControl plasmid pMDTM19-ltrA as a positive control with ddH2O as a negative control; using the upstream primer 927F and the downstream primer 1538R, PCR amplification reaction was carried out according to the reaction system and procedure described in example 2, and the PCR amplification reaction product was detected by agarose gel electrophoresis.
2. Results of the experiment
The result of the specificity verification of the upstream primer 927F and the downstream primer 1538R for detecting the mulberry phytoplasma is shown in FIG. 3, and it can be seen that only the DNA extracted from the mulberry leaves with the mulberry phytoplasma disease can be amplified to obtain the plasmid pMD which is the positive control plasmidTM19-ltrA bands of the same length (611 bp). The results show that the forward primer 927F and the downstream primer 1538R can detect the mulberry phytoplasma and have strong detection specificity.
Example 5 sensitivity verification of upstream primer 927F and downstream primer 1538R for detection of Phytoplasma sanguinea
1. Experimental methods
Extracting DNA of mulberry leaves with phytoplasma disease of mulberry trees with original concentration of 1.0 × 10-1ng/. mu.L, the above DNA was diluted with 1 XTE, 10 and 10 respectively2、103、104、105、106、107Doubling to obtain DNA concentrations of 1.0 × 10-1ng/μL、1.0×10-2ng/μL、1.0×10-3ng/μL、1.0×10-4ng/μL、1.0×10-5ng/μL、1.0×10-6ng/μL、1.0×10-7ng/μL、1.0×10-8ng/mu L; DNA of the above-mentioned concentrations was used as a template, and ddH was used2O as a negative control, PCR amplification reaction was carried out using the reaction system and procedure described in example 2 using the forward primer 927F and the reverse primer 1538R, and the PCR amplification reaction product was detected by agarose gel electrophoresis.
2. Results of the experiment
The results of the sensitivity test of the forward primer 927F and the downstream primer 1538R for the detection of the mulberry phytoplasma are shown in FIG. 4, and it can be seen that when the DNA concentration is 1.0X 10-1～1.0×10-6ng/. mu.L, a 611bp band was amplified at a DNA concentration of 1.0X 10-7～1.0×10-8At ng/. mu.L, no band was amplified. The result shows that the upstream primer 927F and the downstream primer 1538R can detect the mulberry phytoplasma and the detection sensitivity is as high as 1.0 multiplied by 10-6ng/μL。
Example 6 Mulberry Phytoplasma detection of Mulberry diseased leaves in different regions
1. Selection of test materials
The method comprises the steps of randomly selecting mulberry field disease leaves of southern China agriculture university, Guangdong province agriculture academy of sciences silkworm research institute disease leaves, Guangdong province Wenyuan county mulberry disease leaves, Guangdong province Yingde City mulberry disease leaves, Guangxi province Liucheng county Fengshan town demonstration district mulberry disease leaves, Guangxi province Bingyang county mulberry disease leaves, Guangxi province Liucheng county Chongmai town mulberry disease leaves, Yunnan province Qujing City Pangjiang Zhen Mulberyun disease leaves, Yunnan Mongolian city grass dam Zhengyun mulberry disease leaves and healthy mulberry leaves in each district as experimental materials.
2. Extraction of Total DNA
Extraction of total DNA from the experimental material was performed using CTAB method, as follows:
fully grinding the experimental material to powder by using liquid nitrogen; adding 800 mu L of 2% CTAB solution into a 1.5mL centrifuge tube, and adding beta-mercaptoethanol to a final concentration of 0.1%; adding a powder sample ground by liquid nitrogen, and carrying out metal bath at a constant temperature of 65 ℃ for 4 hours or overnight; firstly, 500 mu L of phenol/chloroform is used for shaking and mixing uniformly for 5 minutes, then the mixture is centrifuged for 5 minutes at 12000r/min, and the supernatant is taken; adding 500 mu L chloroform/isoamyl alcohol, shaking and uniformly mixing for 5 minutes, centrifuging for 5 minutes at 12000r/min, and taking supernatant; sucking the supernatant into a new 1.5mL centrifuge tube, centrifuging for 5 minutes, and taking the supernatant; adding isopropanol with the same volume into the supernatant, placing the mixture at the temperature of minus 20 ℃ for 1 hour, taking out the mixture, centrifuging the mixture for 5 minutes at 12000r/min, pouring out the supernatant, adding 1mL of 70% ethanol solution, washing the mixture, uniformly mixing the mixture, and centrifuging the mixture for 5 minutes at 12000 r/min; removing supernatant, air drying, and removing isopropanol and ethanol. Then, 50. mu.L of TE Buffer was added thereto and the mixture was left at 4 ℃ for 1 hour to obtain total DNA.
3. PCR detection
The positive control plasmid pMD constructed in example 3 was used as a template for the total DNA of the experimental materials obtained aboveTM19-ltrA as a positive control, using forward 927F and reverse 1538R primers, as described in example 2And carrying out PCR amplification reaction by the system and the program, and detecting the PCR amplification reaction product by agarose gel electrophoresis.
4. Results of the experiment
The result of detecting the mulberry phytoplasma on the mulberry diseased leaves in different areas is shown in FIG. 5, and it can be seen that the DNAs extracted from the mulberry diseased leaves in different areas can be amplified to form the positive control plasmid pMDTM19-ltrA band with the same length (611bp), while DNA extracted from healthy mulberry leaves has no band amplified. The result shows that the upstream primer 927F and the downstream primer 1538R have specific detection effect on the mulberry phytoplasma of the mulberry diseased leaves in different regions.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1758
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcaaatgc aaccaactga agcagataaa gcttctaagc tttcaaactt actgaatagg 60
attcagacaa aatcattaaa caagataacc ttgaaaaagg aactgcaaca aggaatgaac 120
tccctggata acatcacatt cgcctttaac aaggttgcag ggaatgacgg tgcaggaaca 180
catgggacgg acggtcaaac tattgacgga aaagattgga actacattat caagttaaac 240
gataaatata gaaacaatca ataccgaccc aagcccctca ggagagttta tattcccaaa 300
cctaacggag acaaaagacc cttaggtatc cccaccattg atgacagaat aatccaacag 360
gcaatgtacc aactactcaa cccgttttat gaagtaaaat tctcaaaatg gagctatgga 420
tttagacctg acaaatcacc acatgatgca atcaaacgca ttaaggatag attcaaagga 480
ataaaatggt taataaaaat cgatatcaaa ggattctttg acacaattaa tcatgacata 540
ttgttaaaaa ttttcaacga agacatccgt aaagccaaaa ccctcaaaac cataaaacaa 600
tggctggagg caggcataat ggataactgg gtttttcaga agaccttttc gggtactccc 660
caaggaggta ttatatctcc tttattagct aacgtttatc ttcatgaagt tgacgagaaa 720
atggaaaaac tagtagtaaa aggaacacca aaaaggaagt ctaatcctaa atataaaaaa 780
atacaacgat taggattttt aagaaaagcc aaagtagcaa gcgatattaa cataaatcca 840
aacattaggg ttgagtacat acgctatgcg gatgacttca tcatcggatg tacgggaaca 900
atagaacagg ccaatgaatt gaaacaaaag gtggtgtcct ggttagtacg ggacttgggg 960
ttgtcaatat cttatgacaa atccaaaatc gtaccagccc ataaaggaac aaaatttctt 1020
tcatacctca taagagtcaa tccaacggca tcgtctaaaa ataaatctgc aaggaaatct 1080
cttaacggaa aaaccgatat cagaatcccg aaggaggaaa tagacaatag atgccaaaga 1140
tgggtgaggg tctctaagaa gaagacaaag attatacata acagtcaact ccattatagg 1200
gacgaattag aaataatcca gtcatataaa agaatagttg aaggtataat tagatacttc 1260
gcatatggaa gaaacctaag tcagttaata aaattggcct attttgcaga atactcatgt 1320
ttaatgacga tagcggggaa atacaaaacc tccatcgcta gagtccgtaa gaagctgaac 1380
cgaggccagg ggacttgggg tgtcaagtac acgaagaaaa caaaaccaag atatgaaacc 1440
tggccgaagt acacctggga taggattaaa aggatgagaa gatataagga tattaaccct 1500
gatatcatac caaatccaaa taccttccta ggtcgcacac gtctcacaga ccgcctcaaa 1560
gcggaaaaat gtgaagggtg ccaaaggatt gacaataagt tggaaataca ccacgcgaag 1620
acagtgcgca atcgcaactg gcagagtgtg atgaataaat tgacaatggt attatgtaaa 1680
aaatgccata gaagaaaaac caacgaacaa atcaaaagct ttaagaatat aaaaagaaaa 1740
ataaatacgt ctgaataa 1758
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaaggtggtg tcctggtta 19
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtgcgaccta ggaaggtatt t 21
<210> 4
<211> 4450
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420
cttccagaga tatgcaaatg caaccaactg aagcagataa agcttctaag ctttcaaact 480
tactgaatag gattcagaca aaatcattaa acaagataac cttgaaaaag gaactgcaac 540
aaggaatgaa ctccctggat aacatcacat tcgcctttaa caaggttgca gggaatgacg 600
gtgcaggaac acatgggacg gacggtcaaa ctattgacgg aaaagattgg aactacatta 660
tcaagttaaa cgataaatat agaaacaatc aataccgacc caagcccctc aggagagttt 720
atattcccaa acctaacgga gacaaaagac ccttaggtat ccccaccatt gatgacagaa 780
taatccaaca ggcaatgtac caactactca acccgtttta tgaagtaaaa ttctcaaaat 840
ggagctatgg atttagacct gacaaatcac cacatgatgc aatcaaacgc attaaggata 900
gattcaaagg aataaaatgg ttaataaaaa tcgatatcaa aggattcttt gacacaatta 960
atcatgacat attgttaaaa attttcaacg aagacatccg taaagccaaa accctcaaaa 1020
ccataaaaca atggctggag gcaggcataa tggataactg ggtttttcag aagacctttt 1080
cgggtactcc ccaaggaggt attatatctc ctttattagc taacgtttat cttcatgaag 1140
ttgacgagaa aatggaaaaa ctagtagtaa aaggaacacc aaaaaggaag tctaatccta 1200
aatataaaaa aatacaacga ttaggatttt taagaaaagc caaagtagca agcgatatta 1260
acataaatcc aaacattagg gttgagtaca tacgctatgc ggatgacttc atcatcggat 1320
gtacgggaac aatagaacag gccaatgaat tgaaacaaaa ggtggtgtcc tggttagtac 1380
gggacttggg gttgtcaata tcttatgaca aatccaaaat cgtaccagcc cataaaggaa 1440
caaaatttct ttcatacctc ataagagtca atccaacggc atcgtctaaa aataaatctg 1500
caaggaaatc tcttaacgga aaaaccgata tcagaatccc gaaggaggaa atagacaata 1560
gatgccaaag atgggtgagg gtctctaaga agaagacaaa gattatacat aacagtcaac 1620
tccattatag ggacgaatta gaaataatcc agtcatataa aagaatagtt gaaggtataa 1680
ttagatactt cgcatatgga agaaacctaa gtcagttaat aaaattggcc tattttgcag 1740
aatactcatg tttaatgacg atagcgggga aatacaaaac ctccatcgct agagtccgta 1800
agaagctgaa ccgaggccag gggacttggg gtgtcaagta cacgaagaaa acaaaaccaa 1860
gatatgaaac ctggccgaag tacacctggg ataggattaa aaggatgaga agatataagg 1920
atattaaccc tgatatcata ccaaatccaa ataccttcct aggtcgcaca cgtctcacag 1980
accgcctcaa agcggaaaaa tgtgaagggt gccaaaggat tgacaataag ttggaaatac 2040
accacgcgaa gacagtgcgc aatcgcaact ggcagagtgt gatgaataaa ttgacaatgg 2100
tattatgtaa aaaatgccat agaagaaaaa ccaacgaaca aatcaaaagc tttaagaata 2160
taaaaagaaa aataaatacg tctgaataaa tcgtcgaacg gcaggcgtgc aaacttggcg 2220
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 2280
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 2340
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 2400
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 2460
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2520
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 2580
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 2640
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 2700
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 2760
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 2820
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 2880
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 2940
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3000
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3060
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3120
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3180
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 3240
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 3300
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 3360
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 3420
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 3480
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 3540
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 3600
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 3660
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 3720
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 3780
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 3840
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 3900
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 3960
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 4020
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 4080
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 4140
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 4200
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 4260
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 4320
tgtatttaga aaaataaaca aatgggggtt ccgcgcacat ttccccgaaa agtgccacct 4380
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 4440
ccctttcgtc 4450
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgcaaatgc aaccaac 17
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttattcagac gtatttattt ttcttt 26
Claims (8)
1. An ltrA gene of mulberry phytoplasma, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The specific detection primer for the mulberry phytoplasma is characterized by consisting of an upstream primer 927F and a downstream primer 1538R, wherein the nucleotide sequences of the specific detection primer are respectively shown as SEQ ID No.2 and SEQ ID No. 3.
3. A positive control plasmid for detecting mulberry phytoplasma is characterized in that the positive control plasmid is a plasmid pMD (plasmid-based plasmid-.
4. A primer for construction of the positive control plasmid according to claim 3, wherein the primer for construction consists of an upstream primer ltrA F and a downstream primer ltrA R, and the nucleotide sequences thereof are respectively shown as SEQ ID No.5 and SEQ ID No. 6.
5. The use of the combination of the specific detection primer of claim 2 and the positive control plasmid of claim 3 in molecular detection of mulberry phytoplasma or preparation of a molecular detection product of mulberry phytoplasma.
6. A kit for detecting mulberry phytoplasma, comprising the specific detection primer according to claim 2 and the positive control plasmid according to claim 3.
7. A method for detecting mulberry phytoplasma, which is characterized in that the method is used for detecting whether the ltrA gene of claim 1 exists in nucleic acid of a sample to be detected, and if the ltrA gene exists, the sample to be detected is positive for the mulberry phytoplasma.
8. The method according to claim 7, wherein the nucleic acid of the sample is used as a template, the specific detection primer according to claim 2 is used for PCR amplification reaction, the positive control plasmid according to claim 3 is used as a control, and if the amplification result is positive, the sample is positive for the mulberry phytoplasma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910927209.5A CN110607380B (en) | 2019-09-27 | 2019-09-27 | Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910927209.5A CN110607380B (en) | 2019-09-27 | 2019-09-27 | Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110607380A CN110607380A (en) | 2019-12-24 |
| CN110607380B true CN110607380B (en) | 2021-07-09 |
Family
ID=68893836
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910927209.5A Active CN110607380B (en) | 2019-09-27 | 2019-09-27 | Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110607380B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100064970A (en) * | 2008-12-05 | 2010-06-15 | 경북대학교 산학협력단 | Primer for detecting phytoplasma |
| CN104017890A (en) * | 2014-06-20 | 2014-09-03 | 华南农业大学 | Application of EB1 gene to detection of nosema bombycis |
| CN109457046A (en) * | 2018-12-28 | 2019-03-12 | 华南农业大学 | Application of the ITS2 gene in detection powdery mildew cause of disease Erysiphe alphitoides |
-
2019
- 2019-09-27 CN CN201910927209.5A patent/CN110607380B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100064970A (en) * | 2008-12-05 | 2010-06-15 | 경북대학교 산학협력단 | Primer for detecting phytoplasma |
| CN104017890A (en) * | 2014-06-20 | 2014-09-03 | 华南农业大学 | Application of EB1 gene to detection of nosema bombycis |
| CN109457046A (en) * | 2018-12-28 | 2019-03-12 | 华南农业大学 | Application of the ITS2 gene in detection powdery mildew cause of disease Erysiphe alphitoides |
Non-Patent Citations (2)
| Title |
|---|
| Comparison of the complete genome sequence of two closely related isolates of "Candidatus Phytoplasma australiense" reveals genome plasticity;Mark T Andersen等;《BMC Genomics》;20130802;第14卷;第1-15页 * |
| 模拟感染家蚕微粒子病的蚕卵、蚕蛾PCR检测的初步研究;刘吉平等;《蚕业科学》;20041231;第30卷(第4期);第367-370页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110607380A (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113549618B (en) | SARS-CoV-2 nucleic acid detection method based on RAA amplification and CRISPR-Cas13a system | |
| CN113481327B (en) | Novel coronavirus ORF1ab gene detection method based on RAA amplification and CRISPR-Cas12a | |
| US20020025561A1 (en) | Vectors for gene-self-assembly | |
| CN108285886A (en) | The method that recombined bacillus subtilis resting cell produces N-acetyl-neuraminate | |
| CN108395996B (en) | Classical swine fever virus subunit vaccine and preparation method and application thereof | |
| CN109609579B (en) | Genetically engineered bacterium for producing beta-carotene and construction method thereof | |
| CN108531471A (en) | A kind of long method for synthesizing gene | |
| CN113604505A (en) | pSFV-p32 virus-like particle and preparation method and application thereof | |
| CN109652352B (en) | Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method | |
| CN114933970B (en) | Toxoplasma gene knock-out strain lacking 6-phosphogluconate dehydrogenase 1 gene | |
| CN113584223B (en) | Identification method of D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a | |
| CN111321163B (en) | Construction and application of bacillus subtilis linear plasmid system | |
| CN114292864B (en) | Bacillus bailii mutant strain with high surfactant yield, construction method and application thereof | |
| CN110607380B (en) | Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma | |
| CN111979134B (en) | Construction and application of recombinant saccharomyces cerevisiae for synthesizing carminic acid | |
| CN112322706A (en) | Specific human gene fragment, primer probe and application thereof | |
| CN107661496A (en) | A kind of pig parvoviral immune composition and preparation method and application | |
| CN111378718A (en) | Construction method of gene sequencing library | |
| CN114540345B (en) | Label fluorescent probe with hairpin structure and fluorescent detection method | |
| CN113073097B (en) | CHO cell endogenous temperature-sensitive promoter and application thereof | |
| CN112626116B (en) | Method for site-specific integration of large-fragment exogenous DNA | |
| CN113718047B (en) | Kit for detecting 10 bacteria in human breast milk by fluorescence quantitative method and application thereof | |
| CN108699596A (en) | The method of detection, positioning and monitoring hydraulic structure leakage and leakage | |
| CN114214346B (en) | Plasmid system for targeting liver precursor cells and application | |
| CN114214459A (en) | African swine fever virus and porcine circovirus type 2 dual digital PCR detection primer composition and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |