A LAMP primer for detecting highland barley smut, a kit and application thereof

Abstract

The invention discloses a LAMP primer for detecting highland barley smut, a kit and an application thereof, comprising an inner primer FIP / BIP, an outer primer F3 / B3 and a loop primer LF / LB, wherein the nucleotide sequence of the inner primer FIP / BIP is shown in SEQ ID No.1 and SEQ ID No.2, the nucleotide sequence of the outer primer F3 / B3 is shown in SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequence of the loop primer LF / LB is shown in SEQ ID No.5 and SEQ ID No.6. The primer set provided by the invention for detecting highland barley smut has the characteristics of simple operation, less time consumption and good accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology detection, and particularly relates to a LAMP primer for detecting highland barley smut, a kit and an application thereof. Background Art

[0002] The host range of fungi in the order Ustilagoales is extremely wide, covering almost all important food crops in the world. Ustilago hordei and Ustilago nuda, which infect highland barley, are members of the order Ustilagoales. They occur frequently in the main highland barley producing areas in my country and are also important diseases in the highland barley producing areas of Qinghai Province. Both are seed-borne diseases. The smut fungus that infects highland barley is a facultative parasitic fungus. Before the formation of winter spores, it is no different from healthy plants. Once the disease occurs in the field, it is meaningless to prevent and control it. The use of fungicidal seed treatment agents and the planting of resistant varieties in production can effectively control such diseases. Therefore, there have been few related studies in the past few decades. In recent years, with the increasing rise of organic agriculture, the use of chemical agents has received widespread attention from the society, and there is also a requirement to reduce the use of chemical agents in production, resulting in the renewed attention to seed-borne diseases.

[0003] After the pathogens of both infect plants, they will multiply and extend in large numbers between and within the tissue cells as the plants grow, especially in the meristems. After flowering, they will multiply in large numbers in the ovary, and eventually the inflorescence of the plants will be replaced by winter spores. The difference between Ustilago nuda and Ustilago hordei is that the former (Ustilagonuda) is significantly taller than healthy plants after infecting the plants, which facilitates the spread of winter spores. Soon after the bracts are exposed, the black powder capsule of the diseased ear breaks and releases winter spores. Some winter spores are blown by the wind to the opening floral organs of nearby plants, infecting the ovary, and lurking in the seeds in the form of dormant hyphae, becoming the source of infection for field diseases in the following year. The latter (Ustilago hordei) is significantly shorter than normal plants after infecting the plants. The hyphae always colonize in the meristem until the floral organs are formed. The hyphae of the pathogen invade the ovary and form winter spores wrapped in a thin film. During the harvesting and threshing process, the film breaks and releases winter spores, which are scattered in the field or gathered in the seed coat. The winter spores can germinate with the seeds and then infect the seed coleoptile for re-infection. Due to the difference between the two, the traditional detection methods are also different. U.nuda is mainly detected by microscopic observation after seed embryo staining, while U.hordei is detected by direct microscopic observation, microscopic observation of washed centrifuged seed coat materials, and frozen absorbent paper method.

[0004] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology reported by Notomi et al. in 2000. It can recognize four forward and reverse outer primers and forward and reverse inner primers in six specific regions on the target sequence, and use highly active strand displacement DNA polymerase to specifically, efficiently and quickly amplify the target gene under constant temperature conditions of 59℃ to 67℃. This specific, efficient and rapid molecular detection technology has been successfully applied to the detection of fungi, bacteria, viruses, etc., and the sensitivity of the detection system is 100 times that of ordinary PCR. The detection results of LAMP technology can be detected by electrophoresis, real-time PCR instrument and turbidity meter, and can also be identified by naked eyes after staining with SYBR Green I, calcein and hydroxynaphthol blue. The whole process is simple to operate, the reaction time is short, and the results are easy to judge. Summary of the invention

[0005] The invention is based on the ITS gene sequence design of highland barley smut, designs a primer set to establish a LAMP detection technology with real-time fluorescence amplification curve and SYBR Green I as dual judgment standards, in order to provide a scientific and convenient detection technology for the prevention and control of highland barley smut and resistance breeding.

[0006] In order to achieve the above technical objectives, the present invention is specifically implemented through the following technical solutions:

[0007] A LAMP primer for detecting highland barley smut comprises an inner primer FIP / BIP, an outer primer F3 / B3 and a loop primer LF / LB, wherein the nucleotide sequence of the inner primer FIP / BIP is shown in SEQ ID No.1 and SEQ ID No.2, the nucleotide sequence of the outer primer F3 / B3 is shown in SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequence of the loop primer LF / LB is shown in SEQ ID No.5 and SEQ ID No.6.

[0008] Preferably, the primers also include DNA molecules having the same functions as the above sequences after one or several nucleotides are substituted and / or deleted and / or added to the FIP / BIP, F3 / B3 and LF / LB sequences.

[0009] In another aspect of the present invention, a kit for detecting highland barley smut is provided, the kit comprising inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB.

[0010] The molar concentration ratio of the inner primer, the outer primer and the loop primer in the kit is 8:1:2.

[0011] In another aspect of the present invention, a method for detecting highland barley smut is provided, comprising the following steps:

[0012] 1) Extracting DNA from the sample to be tested;

[0013] 2) using the obtained DNA as a template, performing an amplification reaction using the above-mentioned inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB;

[0014] 3) After the reaction is completed, the detection results are double-judged based on the amplification curve and the addition of SYBR Green I dye (positive is green, negative is orange).

[0015] Further, the reaction system was: 20 ng / μL DNA template 2 μL, SYBR Green I 0.6×, dNTPs 1.8 mM, betaine 0.8 mM, magnesium ion 10 mM, DNA polymerase 0.32 U / L, 40 μmol / L inner primer FIP / BIP, 5 μmol / L outer primer F3 / B3, 10 μmol / L loop primer, and sterile deionized water was added to make up to 25 μL.

[0016] Furthermore, the reaction conditions were 65°C for 60 min, and the fluorescence was read at 1 min intervals.

[0017] In another aspect of the present invention, the application of the inner primers FIP / BIP, the outer primers F3 / B3 and the loop primers LF / LB in detecting highland barley smut is also provided.

[0018] The beneficial effects of the present invention are:

[0019] Seed infection is the primary infection source of U.hordei and U.nuda in the field. The rapid detection system established by the present invention is specific to U.hordei and U.nuda, and the detection sensitivity reaches 200fg / μL. The real-time fluorescence amplification curve and SYBR Green I colorimetric method are used for double determination, without the need for agarose electrophoresis, and the operation is simple, the cost is low, the sensitivity is high, and the false positive probability is small. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the LAMP primer amplification curve and analysis of the present invention;

[0021] Figure 2It is the sensitivity detection of the present invention; wherein, N: negative control; a: real-time fluorescence amplification curve of U.hordei; b: real-time fluorescence amplification curve of U.nuda; c: color change of SYBR Green I; 1-6: respectively represent the color change of DNA template of U.nuda at the concentration of 20fg / μL, 200f g / μL, 2pg / μL, 20pg / μL, 200pg / μL, 2ng / μL; 7-12: respectively represent the color change of DNA template of U.hordei at the concentration of 20fg / μL, 200fg / μL, 2pg / μL, 20pg / μL, 200pg / μL, 2ng / μL;

[0022] Figure 3 It is a specific detection of the present invention; wherein, N: negative control; a: real-time fluorescence amplification curve; b: SYBR GreenI color change; 1-6: respectively represent the color change when DNA is 6 common diseases of highland barley, U.hordei, U.nuda, Dactylobotrysgraminicola, Pyreno phora graminea, Pyrenophora teres, and Fusarium graminearum. DETAILED DESCRIPTION

[0023] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] 1 Materials, reagents and instruments

[0026] The pathogens of highland barley smut were collected from diseased ears with typical symptoms of hard smut and loose smut in the main highland barley producing areas of Qinghai. They were identified as hard barley smut (Ustilago hordei) and naked smut (Ustilago nuda) by laboratory identification. The pathogens of common diseases on highland barley were selected as comparison strains (see Table 1 for details). The test pathogens were all provided by the Key Laboratory of Integrated Management of Agricultural Pests of Qinghai Province.

[0027] Table 1 Information on strains used for LAMP detection of highland barley pathogens

[0028]

[0029] Reagents: Fungal genomic DNA extraction kit, OMEGA; Prime STAR Max Premix (2×), TaKaRa; Bst 2.0 WarmStart DNA Polymerase (10×Isothermal Amplification Buffer, MgSO 4, Bst DNA polymerase), NEB, USA; betaine 5M, Sigma-Aldrich; 10mM dNTPs, Takara; SY BRGreen I, Solarbio.

[0030] Instruments: Monad MiniSpeed ​​3300 centrifuge, Beijing Biji Biotechnology Co., Ltd.; Powerpacuniversal universal electrophoresis instrument, BIO-RAD Corporation, USA; BBx24B cell tissue disruptor, BIO-RAD Corporation, USA; PCR instrument, BIO-RAD Corporation, USA; Thermo Fisher 7500 RT-PCR instrument, BIO-RAD Corporation, USA; No-Onec micro-nucleic acid protein analyzer, BIO-RAD Corporation, USA.

[0031] 2 Methods

[0032] 2.1 Pathogen DNA extraction and sequencing

[0033] For DNA extraction of smut fungus, winter spore powder was placed in a 1.5mL centrifuge tube with 0.2mm steel beads, and then the mixture was broken by a cell tissue disruptor for 10 minutes after adding buffer. Other control pathogens were cultured on PDA medium, and then the mycelium was scraped and placed in a 1.5mL centrifuge tube with 2mm steel beads, and then frozen in liquid nitrogen and then the tissue was broken for 6 minutes. The total DNA of the pathogen was extracted using a fungal genomic DNA extraction kit. The specific operation was referred to the instructions of the kit. The obtained DNA was diluted and stored at -20℃ for later use.

[0034] The extracted DNA was used for ITS sequence amplification. The selected ITS primers were ITS4 / ITS5 (5'-TCCTCCGCTTATTGATATGC-3' / 5'-GGAAGTAAAAGTCGTAACAAGG-3'). The PCR amplification system was: 2μl DNA, 12.5μl Prime STAR Max Premix (2×), 1μl each of 10μmol / L upstream and downstream primers, and sterile water was added to make up to 25μl.

[0035] Amplification program: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 58°C for 1 min, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0036] After the reaction was completed, the amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0037] 2.2 Primer design

[0038] After obtaining the ITS sequence, DNAMAN software was used for comparison, and the segments with low homology between U.hordei and U.nuda and other pathogens were screened, and LAMP primers for highland barley smut were designed. See Table 2 for details, which was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0039] Table 2 LAMP primer sets used in this study

[0040]

[0041] 2.3 Establishment of real-time fluorescence LAMP detection method

[0042] The established real-time fluorescence LAMP detection method reaction system: 20 ng / μL DNA template 2μL, SYBR Green I0.6×, dNTPs 1.8mM, betaine 0.8mM, magnesium ion 10mM, DNA polymerase 0.32U / L, 40μmol / L inner primer FIP / BIP, 5μmol / L outer primer F3 / B3, 10μmol / L loop primer LF / LB, add sterile deionized water to 25μL, close the lid and tap to mix the solution thoroughly and centrifuge it instantaneously, and finally add 20μL paraffin oil.

[0043] Amplification reaction conditions: 65°C, 60 min, read fluorescence every 1 min. After the reaction is completed, the detection results are double-judged based on the amplification curve ("s" for positive; "-" for negative) and the addition of 0.5 μL SYBR Green I dye (positive is green, negative is orange).

[0044] The results showed that the real-time fluorescence LAMP reaction was carried out at 65°C using the genomes of Ustilago sutchuenensis and Ustilago sutchuenensis as templates. Figure 1 ), the real-time fluorescence amplification curves of the two types of smut of the primer set designed by the present invention were "S" type, and the negative control was "-" type, which determined that the primers had the best effect and had no nonspecific amplification.

[0045] Example 2 Sensitivity Detection

[0046] According to the established detection method, the Ustilago farfara DNA adjusted to 20 ng / μL was diluted to 2 ng / μL, 200 pg / μL, 20 pg / μL, 2 pg / μL, 200 fg / μL, and 20 fg / μL, and 2 μL of the above different concentrations of Ustilago farfara DNA was used as a template for sensitivity detection. The sensitivity of LAMP detection was determined by two methods: real-time fluorescence amplification curve and SYBR Green I staining.

[0047] Using primers, the concentration is 2ng / μL~2×10 -5 ng / μL of the pathogenic bacteria DNA of highland barley loose smut and hard smut were used for real-time fluorescence LAMP detection. The real-time amplification curve ( Figure 2 -a, b) and chromogenic method ( Figure 2 -c) all showed that the lowest detection concentration was 2×10 -4 ng / μL. At the same time, according to the amplification time of DNA at each concentration, the amplification reaction time was determined to be 60 min.

[0048] Example 3 Specificity Detection

[0049] The primer set and the optimal detection system were used to perform real-time fluorescence LAMP amplification on the total DNA of Ustilago farinae, Ustilago nuda, Botrytis cinerea, Pyricularia trichomoniasis, Diplocteosporium hordei, and Fusarium graminearum. After the reaction, the specificity of the detection method was evaluated by two methods: real-time fluorescence amplification curve and naked eye observation after adding SYBR Green I.

[0050] The total DNA of Ustilago sutchuenensis, Ustilago sutchuenensis, Botrytis cinerea, Pyricularia tataricola, Diplodia hordei, and Fusarium graminearum were used as templates for real-time fluorescence LAMP amplification. The results showed that the amplification curves of loose black stigma of highland barley and hard black stigma of highland barley were "S" shaped ( Figure 3 -a), after adding SYBR Green I, the color changes to green ( Figure 3 -b), while the amplification curves using DNA from barley streak pathogen, barley web spot pathogen, barley ear rot pathogen, and barley Fusarium graminearum as templates were straight lines ( Figure 3 -a), and the color becomes orange after adding SYBRGreen I ( Figure 3 –b), indicating that this primer set can specifically detect two types of smut in highland barley.

[0051] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents. Sequence Listing <110> Qinghai Academy of Agriculture and Forestry Sciences <120> A LAMP primer for detecting highland barley smut, a kit and application thereof <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 43 <212> DNA <213> Artificial Sequence <400> 1 agagcaaaat cgagctttcg tcttgaaata gattagcgca tcc 43 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <400> 2 gccgggtttt gatactatca ggagaagcac tccaaacagc a 41 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 ttttgccatt taccgtgg 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 taaaataaac tcggcatggg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 aatctatttc aagggagcca 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 ggagaggttg agatgggta 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 tcctccgctt attgatatgc 20 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <400> 8 ggaagtaaaa gtcgtaacaa gg 22

Claims

1. A method for detecting highland barley smut, characterized in that, The following steps are involved: 1) Extract DNA from the sample to be tested; 2) Using the obtained DNA as a template, perform an amplification reaction using LAMP primers; 3) After the reaction is completed, the detection results are double-judged based on the amplification curve and the addition of SYBR Green I dye; Wherein, the LAMP primers include inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB, the nucleotide sequences of the inner primers FIP / BIP are shown in SEQ ID No.1 and SEQ ID No.2, the nucleotide sequences of the outer primers F3 / B3 are shown in SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequences of the loop primers LF / LB are shown in SEQ ID No.5 and SEQ ID No.6; The reaction system of the amplification reaction is: 2 μL of 20 ng / μL DNA template, 0.6× SYBR Green I, 1.8 mM dNTPs, 0.8 mM betaine, 10 mM magnesium ion, 0.32 U / L DNA polymerase, 40 μmol / L inner primer FIP / BIP, 5 μmol / L outer primer F3 / B3, 10 μmol / L loop primer, and sterile deionized water is added to make up to 25 μL; the reaction conditions of the amplification reaction are 65° C., 60 min, and fluorescence is read at 1 min intervals; Wherein, the pathogenic bacteria of highland barley smut is Ustilago hordei Ustilago hordei Ustilago nudica Ustilago nuda .

2. The application of LAMP primers in detecting highland barley smut is characterized in that: The pathogenic bacteria of highland barley smut is Ustilago hordei Ustilago hordei Ustilago nudica Ustilago nuda ; Wherein, the LAMP primers include inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB, the nucleotide sequences of the inner primers FIP / BIP are shown in SEQ ID No.1 and SEQ ID No.2, the nucleotide sequences of the outer primers F3 / B3 are shown in SEQ ID No.3 and SEQ ID No.4, and the nucleotide sequences of the loop primers LF / LB are shown in SEQ ID No.5 and SEQ ID No.6.

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