CN111100947A - LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof - Google Patents
LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof Download PDFInfo
- Publication number
- CN111100947A CN111100947A CN202010075036.1A CN202010075036A CN111100947A CN 111100947 A CN111100947 A CN 111100947A CN 202010075036 A CN202010075036 A CN 202010075036A CN 111100947 A CN111100947 A CN 111100947A
- Authority
- CN
- China
- Prior art keywords
- lamp
- primer
- detection
- seq
- solution
- 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.)
- Pending
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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an LAMP primer for rapidly detecting alternaria mali, a detection method and a kit thereof, wherein the LAMP primer consists of a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP. Wherein, the forward outer primer F3 is shown as SEQ ID No.1, the reverse outer primer B3 is shown as SEQ ID No.2, the forward inner primer FIP is shown as SEQ ID No.3, and the reverse inner primer BIP is shown as SEQ ID No. 4. The rapid detection of the apple brown spot pathogen can be realized by performing LAMP amplification on the sample to be detected by adopting the 2 pairs of specific LAMP primers, the technology has the advantages of simple operation, high sensitivity and specificity and rapid detection speed, does not need expensive instruments and equipment, and is suitable for the early diagnosis of the field apple brown spot and the detection and identification of the pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of apple brown spot germ detection methods, in particular to LAMP primers for rapidly detecting apple brown spot germ, and a detection method and a kit thereof.
Background
The doctor is far away from the apple one day in a colloquial way. The apple is sour, sweet, delicious and rich in nutrition, and is an essential nutritional fruit in daily life. At present, China has become the largest world producing country, and the planting area and the yield of the apples both account for more than 50 percent of the world. The cercospora brown spot of apple, also called as cercospora viridis, is caused by the fruit cone of apple Pantoea (Marssoninacoronaria), occurs in each apple planting area in China, and is a main disease causing early-stage leaf fall of fruit trees. In the years with serious apple brown spot disease, the disease can cause early leaf fall of fruit trees, weak tree vigor, abnormal fruit ripening and obviously reduced fruit yield and quality. In recent years, with the popularization of fruit bagging technology, the occurrence of brown spot is in an increasing trend. The brown spot is just like cancer, and once the brown spot is attacked, the brown spot indicates the middle and late stage, and the leaf pieces of the fruit tree can rapidly fall off. Therefore, brown spot must be prevented early in the next hand and early in the future, and fallen leaves are inevitable as long as germs are latent. However, the early stage of the cercospora leaf spot of the apple is difficult to be discovered by observing symptoms through naked eyes, so that the dynamic infection of pathogenic bacteria is rapidly and accurately monitored in the early stage or the initial stage of the disease, and effective prevention and control measures are timely taken, so that the method has important significance for controlling the occurrence of the disease.
Loop-mediated Isothermal Amplification (LAMP) technology is a constant temperature Amplification method developed by Notomi doctor of Japan Rongyan chemistry, and the method designs 4 primers aiming at 6 sites of a target gene, and utilizes a DNA Polymerase (Bst DNA Polymerase) with chain type displacement activity to complete Amplification reaction by heat preservation for 30-90 minutes under constant temperature (about 60 ℃), and the efficiency can reach 109～1010Several orders of magnitude. Because magnesium pyrophosphate precipitate is generated in the reaction process and generates a turbid phenomenon, the amplification result can be judged by naked eyes, and the dye can be added into the amplification product to judge through color change without professional instruments and equipment, and the method can be finished by using a common water bath kettle or other stable heat sources, and has the advantages of simplicity, rapidness, high efficiency and simple processAnd (4) the actions of tonifying qi. By virtue of the advantages of high speed and sensitivity, the LAMP technology is widely applied to the rapid detection of animal and plant diseases, the identification efficiency and the accuracy of pathogenic bacteria are greatly improved, and a basis is provided for further adopting scientific prevention and control means, but no relevant report exists on the application of the technology in the detection of the apple brown spot pathogen at present.
At present, the traditional classification and identification of the apple brown spot pathogen are mainly realized by separating and culturing pathogenic bacteria, and then performing morphological characteristic identification and pathogenicity determination results, the method has higher requirements on professional technologies and certain uncertainty, the identification method is time-consuming, tedious in procedure, low in sensitivity and strong in experience, the separation and identification of the pathogenic bacteria from diseased plants require several days, and the timely monitoring of the occurrence of diseases and the effective control of the spread and prevalence of the pathogenic bacteria are difficult to realize. The existing common PCR reaction is used for amplifying, recovering and sequencing the DNA of the pathogenic bacteria, has long detection time and high cost, and cannot meet the requirements of economic and efficient detection.
Therefore, the existing detection method for the apple brown spot germs greatly influences the timely discovery and real-time monitoring of the apple brown spot disease conditions in the fields, and further development is needed.
Disclosure of Invention
Aiming at the defects of long time consumption, complicated procedure, strong experience and low accuracy of the detection method for the apple brown spot pathogen in the prior art, the invention provides the LAMP primer for quickly detecting the apple brown spot pathogen, the detection method and the kit thereof.
The technical scheme of the invention is as follows:
the invention provides an LAMP primer for rapidly detecting apple brown spot pathogen, which consists of a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP.
Wherein, the forward outer primer F3 is shown as SEQ ID No.1, the reverse outer primer B3 is shown as SEQ ID No.2, the forward inner primer FIP is shown as SEQ ID No.3, and the reverse inner primer BIP is shown as SEQ ID No. 4.
Based on the technical principle of loop-mediated isothermal amplification (LAMP), the invention selects the ITS gene sequence of the apple brown spot pathogen as a detection target, designs the 4 specific primers F3, B3, FIP and BIP in 6 regions of the ITS gene sequence, and establishes the visual LAMP detection technology of the apple brown spot pathogen by taking HNB as a color development indicator through optimizing a reaction system and reaction conditions. The two pairs of primers have high specificity, nucleic acid amplification cannot be carried out as long as any one of the 6 regions is not matched with the corresponding primer, and the specificity is obviously improved compared with 2 independent regions of a PCR primer recognition target sequence. Moreover, the technology is simple to operate, high in sensitivity and specificity, does not need expensive instruments and equipment, is suitable for early diagnosis of field marssonina mali and detection and identification of pathogenic bacteria, has important significance for timely and effectively preventing and treating the marssonina mali, and provides a simple and easy detection primary screening means for quarantine departments such as port and the like.
In the present application, the use of the two pairs of specific primers (F3, B3, FIP, BIP) can achieve the desired amplification effect, and not only has high specificity, fast reaction speed, but also has low cost. And, experimentally, obtained: in the application, the reaction speed of LAMP reaction is not obviously improved under the condition of adding the loop primer, and the detection cost is also improved.
The invention also provides an LAMP detection method for rapidly detecting the apple brown spot pathogen, which adopts the LAMP primer to carry out LAMP amplification.
Preferably, the LAMP detection method for rapidly detecting Monilinia fructicola comprises the following steps:
(1) establishing an LAMP reaction system by using a DNA template of a sample to be detected and an LAMP primer, and carrying out LAMP amplification;
the 26 μ L LAMP reaction system comprises the following components:
10 μ M of F3: 0.5. mu.L
10 μ M of B3: 0.5. mu.L
10 μ M FIP: 2 μ L
10 μ M BIP: 2 μ L
10×ThermoPolReaction Buffer：2.5μL
100mM MgSO 4: 4 μ L
10mM dNTPs: 3.5 μ L
5M Betaine: 4 μ L
2.4mM HNB (hydroxynaphthol blue): 2 μ L
BstDNHA polymerase 8,000U/mL: 1 μ L
DNA template: 2 μ L.
(2) The detection result can be judged to be positive by directly observing the reaction product in the reaction tube through naked eyes to show sky blue, or by observing a trapezoidal strip through agarose electrophoresis to judge the detection result to be positive; the reaction product is purple, or the electrophoresis pattern has no strip, and the detection result is judged to be negative.
Optionally, the DNA of the sample to be detected as the template is the extracted genome of the alternaria mali, or the product of ITS gene amplification using the alternaria mali.
Preferably, the reaction conditions for LAMP amplification are: firstly, 60 ℃ for 70 min; then, 80 ℃ for 10 min.
The invention also provides an LAMP detection kit for the apple brown spot pathogen, which comprises the LAMP primer. When the kit is used, an operator can provide other reagents of the LAMP reaction system required by detection by himself.
Further, in order to improve the integrity of the LAMP detection kit and reduce the preparation work of operators, the kit further comprises: BstDNHA polymerase, HNB and Betaine. Further, the method can also comprise the following steps: ThermoPolReactionBuffer, MgSO4 and dNTPs.
The LAMP primer for rapidly detecting the alternaria mali, the detection method and the kit provided by the embodiment of the invention have the following beneficial effects:
1. the specificity is strong: the LAMP primer provided by the invention is 2 pairs of specific primers designed on the basis of 6 different regions in the ITS gene sequence of the Malus pumilus Valsa mali, any region in the 6 regions can not be matched with the primer for nucleic acid amplification, and the specificity is obviously improved compared with 2 independent regions of a PCR primer identification target sequence.
2. The sensitivity is high: the detection sensitivity of the invention to the apple brown spot pathogen can reach 100 fg/mu L on the DNA level, and the invention has very high sensitivity.
3. The practicability is good: compared with the defects of long period, high experience dependence, low accuracy, long detection time of common PCR reaction and high cost of the traditional pathogen detection, the LAMP reaction detection method provided by the invention only needs to carry out short-time reaction under the condition of constant temperature (60 ℃), and the result can be directly judged by naked eyes under normal light, so that the pathogen can be quickly detected, and the application value of the LAMP reaction detection method in agricultural production is increased.
4. The technology is simple to operate, high in sensitivity and specificity, does not need expensive instruments and equipment, is suitable for early diagnosis of field marssonina mali and detection and identification of pathogenic bacteria, has an important significance for timely and effectively preventing and treating the marssonina mali, and provides a simple and easy detection primary screening means for quarantine departments such as port and the like.
Drawings
FIG. 1 shows the result of comparison of LAMP primer specificity detection;
wherein A is an LAMP chromogenic change map; b is LAMP agarose gel electrophoresis picture;
m represents DL 2000DNA Marker, and the detection samples a1-a7 are apple brown spot pathogen (Marssoninacoronaria), apple rot pathogen (Valsa mali), apple ring rot pathogen (Botryosphaeridothidea), apple anthracnose pathogen (Colletotrichia malacosoides), apple Alternaria mali (Alternaria mali), apple southern blight (Sclerotiumrolfsii), apple scab (Venturian inaequalis) in sequence, and 8 is sterile double distilled water negative control; in Panel A, sample a1 is positive and samples a2-a7 are negative;
FIG. 2 is a sensitivity measurement of LAMP technique for detection of apple brown spot pathogen;
wherein A is an LAMP chromogenic change map; b is an electrophoresis picture of LAMP amplification products;
m represents DL 2000DNA Marker, the template DNA concentrations in the LAMP reaction system of b1-b7 are respectively 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL and 100 fg/muL, and 8 is a sterile double distilled water negative control; in Panel A, samples b1-b7 were positive and 8 was negative;
FIG. 3 shows that LAMP technology is used for detecting apple brown spot disease plants;
wherein A is an LAMP chromogenic change map; b is an electrophoresis picture of LAMP amplification products;
m represents DL 2000DNA Marker, c1-c7 are respectively different apple brown spot disease onset samples, and 8 is sterile double distilled water negative control; in Panel A, samples c1-c7 were positive and 8 was negative.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 primer design and Synthesis
According to the specificity of an ITS (internal TranscribedSpacer) sequence of the apple brown spot pathogen (Marssoninacoronaria) (the sequence is shown as SEQ ID No.5 in a sequence table), a set of LAMP primers specific to the apple brown spot pathogen is designed by using LAMP primer online design software PrimeExplorer (http:// Primexplor. jp/lampv5e/index. html), and the sequence synthesis is carried out, wherein the LAMP primers comprise outer primers F3 and B3, inner primers FIP and BIP, and the primer sequences are respectively:
F3：5’-GTGAATTTCTCCAAAAAAATGGT-3’；(SEQ ID No.1)
B3：5’-ATTGCGCGTGTTTGTTTG-3’；(SEQ ID No.2)
FIP：5’-AGGTGATACAAAGAGGCTATGTCAT-TGTATGAAAAAACCTTTTTTGCG-3’；(SEQ IDNo.3)
BIP：5’-AATGTATGAGTGGATGAGTGTCAG-CAGATGACAAAAGCACAAGA-3’；(SEQ IDNo.4)；
the synthesized primers are respectively diluted to 10 mu mol/L by using sterilized double distilled water, and the primers are placed in a refrigerator at the temperature of minus 20 ℃ and are kept away from light for later use.
Example 2 LAMP detection of Monilinia fructicola
1. Extraction of genome DNA of brown spot pathogen of apple to be detected
A small amount of sample to be tested is put into a centrifuge tube containing 400 mu of LDNA lysate (0.15M Tris-HCl, 0.04M EDTA-Na2, 0.2M NaCl, 3mM SDS, pH 8.0), a steel ball is added, shaking is carried out for 1min at 65Hz, centrifugation is carried out for 2min at 12000rpm, 200 mu L of supernatant is taken, 400 mu L of absolute ethyl alcohol is added, standing is carried out for 30min at the temperature of-20 ℃, centrifugation is carried out for 2min at 12000rpm, the supernatant is discarded, air drying is carried out, and 30 mu L of sterile double distilled water is added to dissolve DNA.
2. Establishment of LAMP reaction detection system:
taking the extracted genome DNA of the sample to be detected as a template, and performing LAMP amplification by using primers F3, B3, FIP and BIP, wherein the LAMP detection reaction system takes 26 mu L as an example, and comprises the following specific reaction systems:
10 μ M of F3: 0.5. mu.L
10 μ M of B3: 0.5. mu.L
10 μ M FIP: 2 μ L
10 μ M BIP: 2 μ L
10×ThermoPolReaction Buffer：2.5μL
100mM MgSO 4: 4 μ L
10mM dNTPs: 3.5 μ L
5M Betaine: 4 μ L
2.4mM HNB: 2 μ L
BstDNHA polymerase 8,000U/mL: 1 μ L
DNA template: 2 μ L.
The optimal reaction conditions of the above system are: firstly, reacting for 70min at 60 ℃; then, the mixture was kept at 80 ℃ for 10 min. The concentration of the DNA template is 100 fg/. mu.L to 100 ng/. mu.L.
3. Observation and analysis of detection results
The most intuitive judgment method comprises the following steps: and directly observing the reaction product in the reaction tube by naked eyes, if the reaction product is sky blue, judging that the detection result is positive, and if the reaction product is purple, judging that the detection result is negative. The method is the fastest, simple and the detection cost is the lowest.
Another method is to perform detection only by using the result of electrophoresis detection. The method comprises the following specific steps: carrying out agarose electrophoresis on the reaction product, and if a trapezoidal band appears in the electrophoresis result, judging that the detection result is positive; otherwise, the detection result is negative.
In order to improve the accuracy of the detection result, the best scheme is to combine the two results, namely: when the LAMP reaction product is displayed as sky blue, the electrophoresis pattern is a ladder-shaped strip, and the LAMP reaction product is judged to be positive; when the LAMP reaction product shows purple, and the electrophoresis pattern has no band, the result is judged to be negative.
Example 3 specificity test of LAMP primers
An LAMP reaction detection system is established according to the method of the embodiment 2, and LAMP detection is carried out on 7 samples to be detected, namely, apple brown spot pathogen (Marssoninacoronaria), apple rot pathogen (Valsa mali), apple ring rot pathogen (Botryosphaeriadonis), apple anthracnose pathogen (Colletotrichum gloeosporioides), apple Alternaria mali, apple southern blight (Sclerotiumrolfsii) and apple scab (Venturian inaaquaria) with the numbers of a1-a7 respectively, and synchronous experiments are carried out by taking sterile double distilled water as a negative control template.
And (3) testing results: as shown in FIG. 1, only the reaction product in the reaction tube of sample a1 (Malaria pumila) is sky blue, and the electrophoresis result is a trapezoidal strip, and the detection shows a positive result; the results of the other 6 samples and the negative control were negative. The results show that: the 3 pairs of primers provided by the invention have high specificity to the apple brown spot pathogen, can be well distinguished from other pathogens, and has accurate detection results.
EXAMPLE 4 sensitivity testing of the detection method
An LAMP reaction detection system is established according to the method of the embodiment 2, the genomic DNA of the Monilinia fructicola to be detected is diluted with sterilized double distilled water at different concentrations to obtain the samples b1-b7 to be detected with DNA concentrations of 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL and 100 fg/muL respectively, and the samples are respectively used as templates to carry out LAMP reaction, and meanwhile, the sterile double distilled water is used as a negative control template to carry out synchronous experiments.
And (3) testing results: as shown in FIG. 2, the samples b1-b7 all showed positive results, and only the negative control showed negative results. This indicates that the detection method of the present application can detect DNA concentrations as low as 100 fg/. mu.L with extremely high sensitivity.
Example 5 application of LAMP technology to detection of apple brown spot disease-causing plants
Respectively taking 7 apple brown spot disease-causing samples from different sources as samples to be detected, respectively numbering c1-c7, establishing an LAMP reaction detection system according to the method of the embodiment 2, carrying out LAMP reaction, and simultaneously carrying out synchronous experiments by taking sterile double-distilled water as a negative control template.
And (3) testing results: as shown in FIG. 3, the detection of 7 samples to be tested showed a positive result, the reaction product in the reaction tube was sky blue, and the electrophoresis result was a trapezoidal band. The experiment further shows that the method has high detection specificity on the apple brown spot germs, has good application prospect, and can be widely used for detecting the apple brown spot germs in different disease areas.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Sequence listing
<110> Qingdao agricultural university
<120> LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Synthetic sequence F3(Synthetic sequence F3)
<400>1
gtgaatttct ccaaaaaaat ggt 23
<210>2
<211>18
<212>DNA
<213> Synthetic sequence B3(Synthetic sequence B3)
<400>2
attgcgcgtg tttgtttg 18
<210>3
<211>48
<212>DNA
<213> synthetic sequence FIP
<400>3
aggtgataca aagaggctat gtcattgtat gaaaaaacct tttttgcg 48
<210>4
<211>44
<212>DNA
<213> synthetic sequence BIP
<400>4
aatgtatgag tggatgagtg tcagcagatg acaaaagcac aaga 44
<210>5
<211>276
<212>DNA
<213> ITS gene of Monascus aurantiaca (ITS of Marssoninacoronaria)
<400>5
ttgaacgcat cttgcgctcc ttggtattcc tcggagcatg cctgtttgag tgtcgtgaat 60
ttctccaaaa aaatggtttt ttttgtatga aaaaaccttt tttgcgggtc cttgggcttg 120
gtgatgacat agcctctttg tatcaccttg ccttaaatgt atgagtggat gagtgtcagc 180
catagatttt tgttaaggca aacccataaa aatcttgtgc ttttgtcatc tgcttccaaa 240
caaacacgcg caatatacac actttttgtg tgcata 276
Claims (6)
1. The LAMP primer for rapidly detecting the alternaria mali is characterized by consisting of a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP, wherein the forward outer primer F3 is shown as SEQ ID No.1, the reverse outer primer B3 is shown as SEQ ID No.2, the forward inner primer FIP is shown as SEQ ID No.3, and the reverse inner primer BIP is shown as SEQ ID No. 4.
2. An LAMP detection method for rapidly detecting Monilinia fructicola, which is characterized in that LAMP amplification is carried out by adopting the LAMP primer as claimed in claim 1.
3. The LAMP detection method for rapidly detecting Monilinia fructicola according to claim 2, characterized by comprising the following steps:
(1) establishing an LAMP reaction system by using a DNA template of a sample to be detected and an LAMP primer, and carrying out LAMP amplification;
the 26 μ L LAMP reaction system comprises the following components:
10 μ M of F3: 0.5 mu L;
10 μ M of B3: 0.5 mu L;
10 μ M FIP: 2 mu L of the solution;
10 μ M BIP: 2 mu L of the solution;
10×Thermo Pol Reaction Buffer：2.5μL；
100mM MgSO 4: 4 mu L of the solution;
10mM dNTPs: 3.5 mu L;
5M Betaine: 4 mu L of the solution;
2.4mM HNB: 2 mu L of the solution;
bst DNHA polymerase 8,000U/mL: 1 mu L of the solution;
DNA template: 2 mu L of the solution;
adding sterilized double distilled water to 26 mu L;
(2) the detection result can be judged to be positive by directly observing the reaction product in the reaction tube through naked eyes to show sky blue, or by observing a trapezoidal strip through agarose electrophoresis to judge the detection result to be positive; the reaction product is purple, or the electrophoresis pattern has no strip, and the detection result is judged to be negative.
4. The LAMP detection method for rapidly detecting Monilinia fructicola according to claim 3, wherein the DNA of the sample to be detected as the template is the extracted genome of the Monilinia fructicola to be detected, or the product of ITS gene amplification of the Monilinia fructicola to be detected.
5. The LAMP detection method for detecting Monilinia fructicola according to claim 3, wherein the LAMP amplification reaction conditions are as follows: firstly, 60 ℃ for 70 min; then, 80 ℃ for 10 min.
6. An LAMP detection kit for brown spot pathogen apple, characterized by comprising the LAMP primer of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010075036.1A CN111100947A (en) | 2020-01-22 | 2020-01-22 | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010075036.1A CN111100947A (en) | 2020-01-22 | 2020-01-22 | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111100947A true CN111100947A (en) | 2020-05-05 |
Family
ID=70427886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010075036.1A Pending CN111100947A (en) | 2020-01-22 | 2020-01-22 | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111100947A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100948A (en) * | 2020-01-22 | 2020-05-05 | 青岛农业大学 | LAMP primer for rapidly detecting apple canker as well as detection method and kit thereof |
| CN113789406A (en) * | 2021-09-26 | 2021-12-14 | 海南大学 | Visualized mung bean leaf spot disease bacteria loop-mediated isothermal amplification detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892308A (en) * | 2010-05-13 | 2010-11-24 | 西北农林科技大学 | Specific amplification primer for detecting marssonina coronaria and detection method |
| KR20150099142A (en) * | 2014-02-21 | 2015-08-31 | 경북대학교 산학협력단 | Primer set for loop-mediated isothermal amplification reaction for detecting of Marssonina coronariae, and uses thereof |
| CN106434993A (en) * | 2016-11-30 | 2017-02-22 | 福建省农业科学院植物保护研究所 | LAMP primer composition for detecting fusarium wilt of cucumbers and application of LAMP primer composition |
-
2020
- 2020-01-22 CN CN202010075036.1A patent/CN111100947A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892308A (en) * | 2010-05-13 | 2010-11-24 | 西北农林科技大学 | Specific amplification primer for detecting marssonina coronaria and detection method |
| KR20150099142A (en) * | 2014-02-21 | 2015-08-31 | 경북대학교 산학협력단 | Primer set for loop-mediated isothermal amplification reaction for detecting of Marssonina coronariae, and uses thereof |
| CN106434993A (en) * | 2016-11-30 | 2017-02-22 | 福建省农业科学院植物保护研究所 | LAMP primer composition for detecting fusarium wilt of cucumbers and application of LAMP primer composition |
Non-Patent Citations (1)
| Title |
|---|
| T. OBERHÄNSLI等: "Development of a quantitative PCR for improved detection of Marssonina coronaria in field samples", 《ECOFRUIT. 16TH INTERNATIONAL CONFERENCE ON ORGANIC-FRUIT GROWING: PROCEEDINGS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100948A (en) * | 2020-01-22 | 2020-05-05 | 青岛农业大学 | LAMP primer for rapidly detecting apple canker as well as detection method and kit thereof |
| CN113789406A (en) * | 2021-09-26 | 2021-12-14 | 海南大学 | Visualized mung bean leaf spot disease bacteria loop-mediated isothermal amplification detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106434884A (en) | Method, primer and kit for fast detecting Listeria monocytogenes at constant temperature | |
| Williams et al. | The application of species-specific assays based on the polymerase chain reaction to analyse Fusarium crown rot of durum wheat | |
| CN106434993B (en) | For detecting LAMP primer composition object and its application of cucumber fusarium axysporum | |
| CN111100947A (en) | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof | |
| CN108220474A (en) | A kind of LAMP detection primer of Fusarium graminearum and its application | |
| CN103276057B (en) | LAMP technology based rapid Botrytis cinerea detection method | |
| CN112126702A (en) | White beech mushroom GJ7 strain and SSR marker primer and application thereof | |
| CN110982922B (en) | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP | |
| CN114196766B (en) | Molecular marker, primer pair, kit and method for specifically identifying rice ralstonia solanacearum Xoo | |
| CN108796118B (en) | Potato early blight bacterium loop-mediated amplification detection primer and detection method thereof | |
| CN107447036B (en) | Method for identifying fusarium cucurbitacearum | |
| CN107828906B (en) | Method for detecting maize smut bacteria and special kit | |
| Zou et al. | Rapid detection of Colletotrichum siamense from infected tea plants using filter-disc DNA extraction and loop-mediated isothermal amplification | |
| Ren et al. | Development and application of a LAMP method for rapid detection of apple blotch caused by Marssonina coronaria | |
| CN111850155A (en) | Application of specific target primer in simultaneous and rapid identification of two pathogenic bacteria of strawberry infection | |
| CN111893208A (en) | PCR primer for rapidly detecting strawberry colletotrichum gloeosporioides and quantitative detection method | |
| CN111100948A (en) | LAMP primer for rapidly detecting apple canker as well as detection method and kit thereof | |
| CN108624655A (en) | Real-time fluorescence L AMP detection method and kit for potato late blight bacteria | |
| CN116516036A (en) | Primer and probe combination for detecting Pantoea ananatis by LAMP method and application | |
| CN103131780B (en) | Molecular detection method used for rapidly identifying sclerotinia sclerotiorum | |
| CN114015799B (en) | LAMP (loop-mediated isothermal amplification) detection primer group, kit and LAMP detection method for Helminthosporium putrescens | |
| CN111334601B (en) | Early diagnosis method for northern millet leaf blight | |
| CN104059909A (en) | Globodera rostochiensis SCAR marker as well as LAMP fast detection method and application of method | |
| CN111500765B (en) | LAMP primer, kit and detection method for rapidly detecting apple scab | |
| CN117511935B (en) | LAMP (loop-mediated isothermal amplification) detection primer and rapid detection method for pepper anthracnose based on TUB2 |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200505 |
|
| WD01 | Invention patent application deemed withdrawn after publication |