CN113373258A - LAMP detection primer for alternaria solanacearum on thin shell, and establishment method and application of detection system - Google Patents
LAMP detection primer for alternaria solanacearum on thin shell, and establishment method and application of detection system Download PDFInfo
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Abstract
The invention discloses a LAMP detection primer of alternaria solani on thin shells, a method for establishing a detection system and application of the LAMP detection primer, and belongs to the technical field of crop disease detection, identification and prevention. The LAMP detection primer for the pecan alternaria alternata comprises 2 pairs of outer primers F3 and B3, 2 pairs of inner primers FIP and BIP, and 2 pairs of loop primers F2 and B2. The method is suitable for rapid and reliable monitoring and identification of pecan alternaria alternata in diseased tissues, the designed LAMP primer is used for rapid detection of pecan alternata, LAMP nucleic acid amplification is carried out under an isothermal condition, only a water bath kettle is needed, complex instruments and expensive molecular reagents are not needed, the method has the advantages of strong practicability and simplicity and rapidness in operation, and has very important significance for early monitoring and determining the optimal period of disease control before disease manifestation caused by pecan alternata.
Description
Technical Field
The invention belongs to the technical field of crop disease detection, identification and prevention and control, and particularly relates to LAMP detection primers of pecan alternaria solani and an establishment method and application of a detection system.
Background
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique reported by Notomi et al in 2000. The kit can identify 4 forward and reverse outward primers and forward and reverse inner primers of 6 specific regions on a target sequence, and specifically, efficiently and quickly amplify a target gene by using high-activity strand displacement DNA polymerase (Bst DNA polymerase) at the constant temperature of 59-67 ℃. The sensitivity of the detection system is 100 times that of the common PCR. And researches show that the LAMP reaction speed can be increased by adding 2 loop primers, so that the whole reaction time is shortened from 1/3 to 1/2.
Carya illinoinensis (also called as american hickory), is a deciduous tree of hickory of juglandaceae, has a scientific name of hickory, is pleased with light and warm humid climate, has strong adaptability, can grow in hilly mountain areas, coastal mudflats and low-lying wetlands, has dual purposes of fruits and materials, and is a preferable tree species for plain greening (penfang ben, etc., 2012; juyun, etc., 2014; yanjianhua, etc., 2007; tianaimei, etc., 2002). The carya illinoensis is one of famous dry fruits in the world and is increasingly popular as a nutritional health food. The apocarya is introduced from the beginning of the 20 th century in China, and is mainly distributed in Zhejiang, Jiangsu, Yunnan and the like at present.
The black spot of the apocarya is mainly harmful to fruits, leaves, twigs, petioles and inflorescences, once the diseases occur, the fruit yield can reach 10-40 percent, and the yield and the quality of the apocarya are seriously influenced. At present, the disease has already occurred in Zhejiang and Jiangsu provinces (chiffon, Qian, etc., 2016) in China. The pecan black spot disease pathogenic bacteria are Pestalotiopsis microspora (Speg.) and Neoestolopsis calcispora, the identification method of Pestalotiopsis microspora and New Pestalotiopsis is mainly judged by the culture and morphological characteristics of pathogenic bacteria and ITS sequence sequencing, the traditional pathogenic bacteria quarantine detection technology is the traditional detection method mainly based on the morphological characteristics of pathogenic organisms, the method is long in time consumption and low in efficiency, materials suspected to carry pathogenic bacteria or pathogenic plant tissues are usually required to be separated, cultured and purified, then the purely cultured pathogenic organisms are returned to parasitic plants, the pathogenic organisms are further identified indoors through morphological and physiological characteristics, the method is long in time consumption and low in detection rate, and in addition, the identification is interfered by other factors, such as the obligately parasitic pathogenic bacteria can not be cultured, the similar species are difficult to identify according to the form and biological characteristics, and the like, so that the early rapid diagnosis method for the black spot of the apocarya is developed and monitored in time, and is very important for ensuring the health production and food safety of the apocarya.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide LAMP detection primers for pecan alternaria alternata as well as a method for establishing a detection system and application of the LAMP detection primers.
The invention is realized by the following technical scheme:
the LAMP detection primer for the pecan alternaria alternata is characterized by comprising a primer group 1 and/or a primer group 2, wherein the primer group 1 comprises the following sequences:
F3：CATTAGTATTCTAGTGGGCATG，
B3:GGTATTCCTACCTGATCCGA；
FIP:GCCGTTGTATTTCAGGAACTACAACGAGCGTCATTTCAACCCT,
BIP:TTCTCGCTTTTGTTAGGTGCTATGGTCAACCACAAAAAATTGG；
F2:GAGCGTCATTTCAACCCT,
B2:GGTCAACCACAAAAAATTGG；
the primer set 2 comprises the following sequences:
F3：CATTAGTATTCTAGTGGGCATG，
B3:GGTCAACCACAAAAAATTGG；
FIP:AGGAACTACAACTCCTAAGAGAAGTGAGCGTATTTCAACCCTT,
BIP:AATACAACGGCGGATTTGTAGTCGGCTGGGAGTTATAGCA；
F2:GAGCGTCATTTCAACCCTT,
B2:CGGCTGGGAGTTATAGCA。
the method for establishing the detection system of the pecan alternaria solani by using the LAMP detection primer is characterized by comprising the following steps:
1) extracting the genomic DNA of a sample to be detected;
2) LAMP detection reaction system: amplifying by using the DNA extracted in the step 1) as a template and using F3/B3, FIP/BIP and F2/B2 primers of the primer group 1 and/or the primer group 2;
the reaction system is 25 mu L, comprises 2 xLamp Master Mix 12.5 mu L, FIP 2 mu L, BIP2 mu L, F30.5.5 mu L, B30.5 mu L, F21 mu L, B21 mu L, template DNA1 mu L, DNA Polymerase 0.5 mu L, Sterilized ddH2O 4 mu L, and is fully mixed;
3) LAMP reaction: preserving the temperature of the reaction system in the step 2) in a water bath kettle at 65 ℃ for 60min, and inactivating the reaction system at 80 ℃ for 10 min;
4) and (4) detecting a result: monitoring by agar gel electrophoresis and visual inspection of fluorescent dye;
after LAMP reaction, adding 0.5 mu L of SYBR Green I dye into the reaction solution, and determining that the reaction system is Green and positive and orange and negative;
the detection is carried out by utilizing an agar gel electrophoresis method, and the amplified band is positive in dispersibility and negative in strip shape.
The LAMP detection primer for the pecan alternaria alternata has the functions of diagnosis, monitoring and identification of the pecan alternata.
The LAMP detection primer of the pecan alternaria alternata performs LAMP reaction to detect the pecan alternata and plays a role in diagnosis, monitoring and identification of the pecan alternata.
The method is suitable for rapid and reliable monitoring and identification of the pecan alternaria alternata in diseased tissues. The designed LAMP primer is used for rapid detection of pecan alternaria alternata, LAMP nucleic acid amplification is carried out under the isothermal condition, only one water bath is needed, complex instruments and expensive molecular reagents are not needed, and the LAMP primer has the advantages of strong practicability and simplicity and quickness in operation. The method can also provide reliable technical support for early warning of the melasma caused by Pestalotiopsis microsporus and New Pestalotiopsis pseudosporidium, and has very important significance for determining the optimal period of disease control.
Drawings
FIG. 1 is a real-time fluorescence amplification curve of primer sets 1 and 5 of Pestalotiopsis pseudopeyrodis 2003-27;
FIG. 2 is a real-time fluorescence amplification curve of primer sets 2, 3, 4, 5 of Pestalotiopsis microsporus 2021-3;
FIG. 3 is a diagram showing the result of the specificity identification of the LAMP detection system for Pestalotiopsis microsporus and New Pestalotiopsis;
FIG. 4 is a diagram showing the results of practical identification of the optimal primers for Pestalotiopsis microphylla and New Pestalotiopsis neoformans.
Detailed Description
The present invention is further illustrated with reference to the following specific examples, which are not intended to limit the scope of the invention.
Example 1: pathogen DNA extraction and sequencing
Respectively culturing the pathogenic bacteria to be tested on a PDA culture medium, scraping hyphae, putting into a 1.5mL centrifugal tube filled with steel balls with the diameter of 2mm, freezing by liquid nitrogen, and then crushing the tissues for 6 min. The total DNA of the pathogenic bacteria is extracted by adopting a fungal genome DNA extraction kit, the specific operation is explained by referring to the kit, and the obtained DNA is diluted and stored at the temperature of minus 20 ℃ for later use.
The extracted DNA was amplified by tef1 sequence. The PCR amplification system is as follows: DNA 1. mu.L, Prime START Max Premix (2X) 12.5. mu.L, upstream and downstream primers 1. mu.L each, and sterile water to make up to 25. mu.L. PCR amplification procedure: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min. The amplification product was sent to the sequencing company for sequencing.
Example 2: primer design
After obtaining the ITS sequence, the MEGA6.0 software is used for comparison, the sections with low homology with other pathogenic bacteria of the pestalotiopsis microspora and the Neosporotrichia neostisolioschavialis are screened, the LAMP Primer is designed by the online software Primer Explore V5 (http:// Primer explorer.jp/lampv5e/index. html), the LAMP Primer is symbiotic to form a Primer sequence 5 group, the detailed table 1 is shown, and the Primer synthesis is completed by Shanghai biology GmbH.
TABLE 1
Example 3: screening of LAMP primer set
The optimal primer group is determined by a real-time fluorescent quantitative PCR method, the reaction system is 2 XSYBR Green dye method Mix 5. mu.L, the inner primer (FIP/BIP) 2. mu.L, the loop primer (F2/B2) 1. mu.L, the outer primer (F3/B3) 0.5. mu.L, DNA 1. mu.L and sterile deionized water for 10. mu.L. The real-time fluorescence LAMP reaction condition is 65 ℃ for 70min, and the fluorescence is read at intervals of 1 min. And after the reaction is finished, determining an optimal primer group according to the real-time amplification curve.
Example 4: establishment of LAMP detection method
2 × Lamp Master Mix 12.5 μ L, FIP 2 μ L and BIP2 μ L, F30.5.5 μ L and B30.5 μ L, F21 μ L and B21 μ L, template DNA1 μ L, DNA Polymerase 0.5 μ L, Sterilized ddH2O 4 μ L, mixed well for a total of 25 μ L. After closing the lid, the bottom was flicked to mix the solution thoroughly and centrifuged instantaneously, and finally 20ul of paraffin oil was added. The amplification reaction conditions are as follows: keeping the temperature of a water bath kettle at 65 ℃ for 60min, and inactivating the DNA Polymerase at 80 ℃ for 10min after the completion. After the reaction is finished, carrying out double judgment on the detection result according to gel electrophoresis and SYBR Green I dyeing, wherein the amplification strip is positive in a dispersion mode and negative in a strip mode; after adding 0.5. mu.L of SYBR Green I dye, the reaction system is positive when showing Green and negative when showing orange.
Example 5: LAMP detection
Detecting 2003-27 New Pestalotiopsis crassa and Pestalotiopsis microspora 2010-3 Chaetoceros crassa, 2070-1 Alternaria alternata and 2008-1 fruit producing anthrax Colletotrichum fructicola by using the screened optimal primer group and detection system; detection of 2010-3 Pestalotiopsis microspora and 2003-27 New Pestalotiopsis clavispora, 2070-1 Alternaria alternata and 2008-1 fruit producing Colletotrichum fructicola.
The result of the detection
The total DNA of the pestalotiopsis and pestalotiopsis minimali is taken as a template to carry out the screening of a primer group, and the amplification result shows that the real-time fluorescence amplification curves of the pestalotiopsis 2003-27 primer group 1 and 5 are S-shaped, and the real-time fluorescence amplification curves of the pestalotiopsis minimali 2021-3 primer group 2, 3, 4 and 5 are S-shaped. Therefore, the primer group 1 of the pestalotiopsis praecox 2003-27 is determined as an optimal primer group; the primer set 2 of Pestalotiopsis parvula 2021-3 is the best primer set, as shown in FIG. 1-FIG. 2. After adding SYBR Green I dye, the color of the 2003-27 detection system is changed into Green, the SYBR Green I color development results of other reference strains are negative, and the other reference strains do not generate amplification, which indicates that the established LAMP detection system can specifically detect the new pestalotiopsis crassifolia; similarly, the LAMP detection system is also suitable for detecting Pestalotiopsis microspora, which is shown in the figure 3-figure 4.
Claims (4)
1. The LAMP detection primer for the pecan alternaria alternata is characterized by comprising a primer group 1 and/or a primer group 2, wherein the primer group 1 comprises the following sequences:
F3：CATTAGTATTCTAGTGGGCATG，
B3:GGTATTCCTACCTGATCCGA；
FIP: GCCGTTGTATTTCAGGAACTACAACGAGCGTCATTTCAACCCT,
BIP: TTCTCGCTTTTGTTAGGTGCTATGGTCAACCACAAAAAATTGG；
F2: GAGCGTCATTTCAACCCT,
B2: GGTCAACCACAAAAAATTGG；
the primer set 2 comprises the following sequences:
F3：CATTAGTATTCTAGTGGGCATG，
B3:GGTCAACCACAAAAAATTGG；
FIP: AGGAACTACAACTCCTAAGAGAAGTGAGCGTATTTCAACCCTT,
BIP: AATACAACGGCGGATTTGTAGTCGGCTGGGAGTTATAGCA；
F2: GAGCGTCATTTCAACCCTT,
B2: CGGCTGGGAGTTATAGCA。
2. the method for establishing the pecan alternaria solani detection system by using the LAMP detection primer as claimed in claim 1, is characterized by comprising the following steps:
1) extracting the genomic DNA of a sample to be detected;
2) LAMP detection reaction system: amplifying by using the DNA extracted in the step 1) as a template and using F3/B3, FIP/BIP and F2/B2 primers of the primer group 1 and/or the primer group 2;
the reaction system is 25 mu L, comprises 2 xLamp Master Mix 12.5 mu L, FIP 2 mu L, BIP2 mu L, F30.5.5 mu L, B30.5 mu L, F21 mu L, B21 mu L, template DNA1 mu L, DNA Polymerase 0.5 mu L, Sterilized ddH2O 4 mu L, and is fully mixed;
3) LAMP reaction: preserving the temperature of the reaction system in the step 2) in a water bath kettle at 65 ℃ for 60min, and inactivating the reaction system at 80 ℃ for 10 min;
4) and (4) detecting a result: monitoring by agar gel electrophoresis and visual inspection of fluorescent dye;
after LAMP reaction, adding 0.5 mu L of SYBR Green I dye into the reaction solution, and determining that the reaction system is Green and positive and orange and negative;
the detection is carried out by utilizing an agar gel electrophoresis method, and the amplified band is positive in dispersibility and negative in strip shape.
3. The LAMP detection primer for pecan alternaria alternata as claimed in claim 1, and has effects in diagnosis, monitoring and identification of pecan alternata.
4. The method for detecting the pecan alternaria solani by the LAMP detection primer of the pecan alternaria solani of claim 2 through the LAMP reaction, and has the functions of diagnosis, monitoring and identification of the pecan alternaria solani.
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| CN114182034A (en) * | 2021-11-04 | 2022-03-15 | 中国林业科学研究院亚热带林业研究所 | SSR molecular marker of apocarya variety McMillian and application thereof |
| CN114182034B (en) * | 2021-11-04 | 2023-08-04 | 中国林业科学研究院亚热带林业研究所 | SSR molecular marker of apocarya variety McMillian and application thereof |
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Application publication date: 20210910 |