CN114752700A - LAMP primer for visual detection of black spot pathogen of kiwi fruit and detection method thereof - Google Patents
LAMP primer for visual detection of black spot pathogen of kiwi fruit and detection method thereof Download PDFInfo
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Abstract
The invention provides LAMP primers for visual detection of black spot pathogen of kiwi fruit, which comprise a forward primer FIP, a reverse primer BIP, a forward primer F3, a reverse primer B3, a forward primer Loop F and a reverse primer Loop B, wherein the nucleotide sequences are sequentially shown as SEQ ID NO. 1-6. The detection method is also provided, DNA of the fruit peel of the kiwi fruit is extracted, an LAMP reaction system is established, after the liquid level of the LAMP reaction system is sealed by mineral oil, isothermal reaction is carried out for 45min at the temperature of 65 ℃, amplification is carried out, and 10 XSYPR Green I dye is added after the reaction is finished; the reaction solution is fluorescent green to indicate that the black spot pathogen of the kiwi fruit is contained, and brown to indicate that the black spot pathogen of the kiwi fruit is not contained. The kit has the advantages of strong specificity, high sensitivity, simple operation process and low detection cost, and can effectively detect and prevent the black spot pathogen of the kiwi fruit.
Description
Technical Field
The invention belongs to the technical field of kiwi fruit black spot pathogen detection, and particularly relates to LAMP primers for visual detection of kiwi fruit black spot pathogen and a detection method thereof.
Background
The green kiwi fruit belongs to delicious kiwi fruit strain, and gradually becomes a main cultivation variety of kiwi fruit in Shaanxi province in recent years due to the advantages of excellent quality, sweet taste, early maturity, high quality, early marketing, high income and the like. From 2013, the green and fragrant kiwi fruit orchard from Shaanxi to county has black spot disease in successive years, the average orchard disease rate reaches 85.8%, and the fruit disease rate reaches more than 48.5%. According to investigation results of ill fruits in fields from weeks to counties and Mei counties of main kiwi fruit cultivation areas of the province of the authors in 2018 and 2019 in 8 months, the average disease garden rate of the Cuixiang variety reaches 100%, the average disease plant rate reaches 85.7%, the average disease fruit rate reaches 25.65%, and the average disease fruit rate reaches more than 80% in serious gardens. The pathogen of the disease is Diaporthe phaseolorum which belongs to epiphyte (with sexual state), is a major fruit disease which is newly appeared in Shaanxi kiwi fruit production in recent years, the disease is generated in each main production area of Shaanxi kiwi fruit, the damage is serious in successive years, the variety of the disease is most serious in emerald fragrance, and the disease is mild in slow fragrance variety; the field disease period begins to occur in the first 7 th of each year, the disease peak reaches from the last seventy to the last august, the fruits are mainly damaged, and the disease symptoms are shown as follows: black brown scabs are formed on the surface of the pericarp, small black brown spots appear on the epidermis part of the lower part of the fruit at the initial stage of the disease attack, then the scabs are gradually enlarged, and the scabs are connected into a scab shape when the disease attack is serious, the epidermis is hardened, and the epidermis villi is necrotic; the disease spots are mainly concentrated on the lower half part of the fruit, and anatomical observation shows that the disease spots only harm the fruit skin, the fruit flesh does not have obvious change, the diseased fruit is easy to fall off, becomes soft and rot, is not resistant to storage and transportation, and the commodity, the edibility and the economic value are obviously reduced, so that huge economic loss is caused to fruit growers, and the disease spots become important diseases which are concerned about and seriously harm the kiwi fruit industry.
The pathogenic bacteria of the kiwifruit black spot disease are weak parasitic fungi which are favored by high temperature and high humidity, can live through the winter in latent on soil, plant bodies and disease residues, can be rapidly propagated and diseased when the environmental conditions are proper, and are the key for prevention and treatment in the initial infection stage, the prevention and treatment cost is low, the prevention and treatment effect is optimal, but because the pathogenic bacteria have unobvious disease manifestation in the latent period and are difficult to observe, whether infection exists is difficult to judge, so that the optimal prevention and treatment period is missed, and the prevention and treatment measures and the prevention and treatment effect are influenced; meanwhile, by field sampling and determination, the field bacterial source quantity, the current year disease occurrence dynamic state and pathogen identification are determined, and a basis can be provided for prediction and prediction of the disease and timely formulation of a control strategy. Therefore, early diagnosis of disease field asymptomatic germ-carrying state is necessary, is an important basis for mastering the disease development stage, and is also the key for preventing and treating the kiwifruit black spot. However, research data or patent reports about a rapid detection technical method aiming at the black spot pathogen of the kiwi fruit are not found at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing the LAMP primer for visual detection of the kiwifruit black spot pathogen and the detection method thereof aiming at the defects of the prior art, and the method has the advantages of strong specificity, high sensitivity, simple operation process and low detection cost, and can effectively detect and prevent the kiwifruit black spot pathogen.
In order to solve the technical problems, the invention adopts the technical scheme that: the LAMP primer for visual detection of the black spot pathogen of the kiwi fruit comprises a forward primer FIP, a reverse primer BIP, a forward primer F3, a reverse primer B3, a forward primer loopF and a reverse primer Loop B; the nucleotide sequence of the forward primer FIP is shown as SEQ ID NO. 1, the nucleotide sequence of the reverse primer BIP is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F3 is shown as SEQ ID NO. 3, the nucleotide sequence of the reverse primer B3 is shown as SEQ ID NO. 4, the nucleotide sequence of the forward primer loopF is shown as SEQ ID NO. 5, and the nucleotide sequence of the reverse primer loopB is shown as SEQ ID NO. 6.
The invention relates to a LAMP primer sequence designed aiming at EF1-a gene (OL702788) of D.phaseolorum (i.e. Diaporthe phaseolorum); the nucleotide sequence of the EF1-a gene of the D.phaseolorum is shown as SEQ ID NO. 7;
the invention also provides a method for detecting the kiwifruit black spot germs by using the LAMP primer for visual detection of the kiwifruit black spot germs, which comprises the following steps:
s1, extracting DNA of the fruit peel of the kiwi fruit to obtain a to-be-detected genome DNA;
s2, establishing a LAMP reaction system by using the genomic DNA to be detected obtained in S1, sealing the liquid level of the LAMP reaction system by using mineral oil, carrying out isothermal reaction for 45min at the temperature of 65 ℃ for amplification, and adding 10 × SYPR Green I dye after the reaction is finished; the reaction liquid is fluorescent green to indicate that the kiwi fruit black spot germ is contained, and brown to indicate that the kiwi fruit black spot germ is not contained;
the LAMP reaction system is as follows: 10 × Isothermal Amplification Buffer 2.5 μ L, 100 mmol.L-1MgSO (2) of4Solution 0.5. mu.L, 10 mmol. multidot.L-1dNTPs solution (3.5. mu.L, 10. mu. mol. L)-1The forward inner primer FIP of (1) 4.0. mu.L, 10. mu. mol. L-1Reverse inner primer BIP 4.0. mu.L, 10. mu. mol. L-130.5. mu.L, 10. mu. mol. L of forward outer primer F-1The reverse outer primer B of (2) 30.5. mu.L, 10. mu. mol. L-1The forward primer Loop F of (1.0. mu.L, 10. mu. mol. L)-1The reverse Loop primer Loop B of (1.0. mu.L, 8U. mu.L)-1Bst DNA polymerase solution (1.0. mu.L, 4 mol. L)-11. mu.L of betaine (D), 1. mu.L of genomic DNA to be detected, ddH2Make up to 25. mu.L of O.
Preferably, the mineral oil is used in an amount of 30. mu.L, and the 10 XSSYPR Green I dye is used in an amount of 1. mu.L.
Compared with the prior art, the invention has the following advantages:
the invention takes the EF1-a gene (OL702788) of D.phaseolorum as a target gene, designs LAMP specific primers, establishes a simple, rapid and sensitive detection method based on color judgment, and performs specificity and sensitivity tests and plant disease residue detection. The LAMP detection method has the advantages of strong specificity, high sensitivity, simple operation process, low detection cost, visual detection result and the like, can provide technical support for rapidly detecting field pathogenic bacteria, can provide scientific basis for early prediction of the disease, timely formulation of a disease control strategy and effective control, and has important guiding significance, wide application prospect and economic benefit.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 shows the Kiwi fruit Ustilago virens assay results of field harvested Kiwi fruit parts of example 1 of the present invention.
Detailed Description
Example 1
The LAMP primer for visual detection of Actinidia chinensis planch (Diaporthe phaseolorum) of the embodiment comprises a forward primer FIP, a reverse primer BIP, a forward primer F3, a reverse primer B3, a forward primer loopF and a reverse primer loopB; the nucleotide sequence of the forward primer FIP is shown as SEQ ID NO. 1, the nucleotide sequence of the reverse primer BIP is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F3 is shown as SEQ ID NO. 3, the nucleotide sequence of the reverse primer B3 is shown as SEQ ID NO. 4, the nucleotide sequence of the forward primer loopF is shown as SEQ ID NO. 5, and the nucleotide sequence of the reverse primer loopB is shown as SEQ ID NO. 6.
The invention relates to a LAMP primer sequence designed aiming at EF1-a gene (OL702788) of D.phaseolorum (i.e. Diaporthe phaseolorum); the nucleotide sequence of the EF1-a gene of the D.phaseolorum is shown as SEQ ID NO. 7;
the embodiment also provides a method for detecting the kiwifruit black spot germs by using the LAMP primer for visual detection of the kiwifruit black spot germs, which comprises the following steps:
s1, extracting DNA of the fruit peel of the kiwi fruit to obtain a to-be-detected genome DNA;
s2, establishing a LAMP reaction system by using the genomic DNA to be detected obtained in S1, sealing the liquid level of the LAMP reaction system by using 30 mu L of mineral oil, carrying out isothermal reaction for 45min at the temperature of 65 ℃ for amplification, and adding 1 mu L of 10 xSYPR GreenI dye after the reaction is finished; the reaction solution is fluorescent green and shows that the reaction solution contains the kiwi fruit alternaria (positive reaction), and is brown and shows that the reaction solution does not contain the kiwi fruit alternaria (negative reaction);
the LAMP reverseThe system is as follows: 10 × Isothermal Amplification Buffer 2.5 μ L, 100 mmol.L-1MgSO (2) of4Solution 0.5. mu.L, 10 mmol. multidot.L-1dNTPs solution (3.5. mu.L, 10. mu. mol. L)-1The forward inner primer FIP of (1) 4.0. mu.L, 10. mu. mol. L-1Reverse inner primer BIP 4.0. mu.L, 10. mu. mol. L-130.5. mu.L, 10. mu. mol. L of forward outer primer F-1The reverse outer primer B of (3) 30.5. mu.L, 10. mu. mol. L-1The forward primer Loop F of (1.0. mu.L, 10. mu. mol. L)-1The reverse Loop primer Loop B of (1.0. mu.L, 8U. mu.L)-1Bst DNA polymerase solution (1.0. mu.L, 4 mol. L)-11. mu.L of betaine (D), 1. mu.L of genomic DNA to be detected, ddH2Make up to 25. mu.L of O.
Randomly sampling Tunxianguo fruits collected from Guangji town of Zhou to county, south town of Zhou to county, guan town of Zhou to county, Gaoyuwei town of Mei county and Wuquan town of Yangling, wherein the total number of the Tunxianguo fruits is 59, 49 diseased fruits are contained, 10 healthy fruits are contained, and DNA of the diseased fruits is extracted as fruit peels at the disease-health junction; healthy fruit sampling was random sampling. Taking healthy fruits as a reference, and extracting DNA of the fruit epidermis; and detecting according to an indoor pathogenic bacterium detection technology system. The results show that: diseased fruit was detected as fluorescent green (positive reaction) and healthy fruit and CK (sterilized double distilled water) control were brown (negative reaction) (fig. 1). The detection accuracy is 100%. The pathogenic bacteria detection technology system method is proved to be effective and feasible, and can be completely used for detecting the kiwi fruit black spot bacteria field samples.
The extraction method of the DNA comprises the following steps:
the method comprises the steps of adopting field-onset black spot disease green fragrant fruits and healthy materials, soaking and sterilizing the fruits and the healthy materials for 30min by using 0.06% sodium hypochlorite, cleaning the fruits and the healthy materials for 3 times by using sterile water, cutting peel at a disease-health junction of about 0.05g by using a sterile scalpel, and detecting and testing the processed peel by using an established LAMP method. Healthy fruit peel is used as a control, and sterile water is used as a negative control. The specific treatment method is referred to Kamel with slight modification: placing the tissue sample into a sterile 1.5mL centrifuge tube, adding 300. mu. LDNA extract (100mM Tris-HCl, pH 8.5,1M NaCl,50mM EDTA, 2% SDS, 2% PVP), grinding the sample to a fine powder using a grinding bar; adding 300 μ L of DNA extract and 10 μ L of LRNase A (preheated at 65 deg.C), and water-bathing at 65 deg.C for 15 min; adding 200. mu.L of 3M sodium acetate, centrifuging at 15000g for 8min, transferring 300. mu.L of the supernatant to a new 1.5mL centrifuge tube; adding isopropanol with the same volume, standing at room temperature for 10min, centrifuging at 8900g for 10min, and pouring off the supernatant; adding 50 mu L of 75% ethanol, centrifuging at 5700g for 2min, pouring out the supernatant, adding 20 mu L of sterile water after the ethanol is completely volatilized, and using the mixture as a DNA template for LAMP detection.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
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Claims (3)
1. The LAMP primer for visual detection of the black spot pathogen of the kiwi fruit is characterized by comprising a forward primer FIP, a reverse primer BIP, a forward primer F3, a reverse primer B3, a forward primer Loop F and a reverse primer Loop B; the nucleotide sequence of the forward primer FIP is shown as SEQ ID NO. 1, the nucleotide sequence of the reverse primer BIP is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer F3 is shown as SEQ ID NO. 3, the nucleotide sequence of the reverse primer B3 is shown as SEQ ID NO. 4, the nucleotide sequence of the forward primer Loop F is shown as SEQ ID NO. 5, and the nucleotide sequence of the reverse primer Loop B is shown as SEQ ID NO. 6.
2. A method for detecting the alternaria kiwii by using the LAMP primer for visual detection of the alternaria kiwii as claimed in claim 1, is characterized by comprising the following steps:
s1, extracting DNA of the fruit peel of the kiwi fruit to obtain a to-be-detected genome DNA;
s2, establishing an LAMP reaction system by using the genomic DNA to be detected obtained in S1, sealing the liquid level of the LAMP reaction system by using mineral oil, carrying out isothermal reaction for 45min at the temperature of 65 ℃ for amplification, and adding 10 × SYPR GreenI dye after the reaction is finished; the reaction liquid is fluorescent green to indicate that the kiwi fruit black spot germ is contained, and brown to indicate that the kiwi fruit black spot germ is not contained;
the LAMP reaction system is as follows: 10 × Isothermal Amplification Buffer 2.5 μ L, 100 mmol.L-1MgSO (2) of4Solution 0.5. mu.L, 10 mmol. multidot.L-1dNTPs solution (3.5. mu.L, 10. mu. mol. L)-1The forward inner primer FIP of (1) 4.0. mu.L, 10. mu. mol. L-1The reverse inner primer BIP of (4.0. mu.L, 10. mu. mol. L)-130.5. mu.L, 10. mu. mol. L of forward outer primer F-1The reverse outer primer B of (2) 30.5. mu.L, 10. mu. mol. L-1The forward primer Loop F of (1.0. mu.L, 10. mu. mol. L)-1The reverse Loop primer Loop B of (1.0. mu.L, 8U. mu.L)-1Bst DNA polymerase solution (1.0. mu.L, 4 mol. L)-11. mu.L of betaine, 1. mu.L of genomic DNA to be detected, ddH2Make up to 25. mu.L of O.
3. The method as claimed in claim 3, wherein the mineral oil is used in an amount of 30 μ L and the 10 XSSYPR GreenI dye is used in an amount of 1 μ L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210435826.5A CN114752700A (en) | 2022-04-24 | 2022-04-24 | LAMP primer for visual detection of black spot pathogen of kiwi fruit and detection method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045742A (en) * | 2012-12-27 | 2013-04-17 | 西北农林科技大学 | Method for detecting Pseudomonas syringae causing kiwi canker by loop-mediated isothermal amplification |
| CN103710440A (en) * | 2013-12-13 | 2014-04-09 | 南京农业大学 | Detection target of southern stem ulcer bacteria as well as loop-mediated isothermal amplification (LAMP) primer composition and application thereof |
| CN112646914A (en) * | 2020-12-25 | 2021-04-13 | 扬州大学 | LAMP primer group for rapidly detecting amycolatopsis persicae and rapid detection method and kit thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045742A (en) * | 2012-12-27 | 2013-04-17 | 西北农林科技大学 | Method for detecting Pseudomonas syringae causing kiwi canker by loop-mediated isothermal amplification |
| CN103710440A (en) * | 2013-12-13 | 2014-04-09 | 南京农业大学 | Detection target of southern stem ulcer bacteria as well as loop-mediated isothermal amplification (LAMP) primer composition and application thereof |
| CN112646914A (en) * | 2020-12-25 | 2021-04-13 | 扬州大学 | LAMP primer group for rapidly detecting amycolatopsis persicae and rapid detection method and kit thereof |
Non-Patent Citations (3)
| Title |
|---|
| WANG,L.: "OL702788.1 Diaporthe phaseolorum isolate HT1 beta-tubulin (TUB) gene, partial cds" * |
| YAMING YANG 等: "Pathogenic Fungi Diversity of ‘CuiXiang’ Kiwifruit Black Spot Disease during Storage" * |
| 沈浩 等: "基于环介导等温扩增技术检测大豆北方茎溃疡病菌" * |
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