CN111088393A - LAMP (loop-mediated isothermal amplification) detection primer group for rhizoctonia cerealis and application of LAMP detection primer group - Google Patents
LAMP (loop-mediated isothermal amplification) detection primer group for rhizoctonia cerealis and application of LAMP detection primer group Download PDFInfo
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Abstract
The invention relates to a LAMP detection primer group for Rhizoctonia cerealis and application thereof, belonging to the technical field of genetic engineering, wherein the ITS of pathogenic bacteria is used as a target sequence to establish the LAMP detection technology for Rhizoctonia cerealis, the adopted primers have strong specificity and the sensitivity of a reaction system is high; through the detection of the diseased tissue, the reaction system can accurately and quickly detect the wheat sharp eyespot from the diseased wheat tissue. The detection method is simple, rapid and sensitive, does not need expensive instruments, is favorable for large-area popularization and use in a basic plant protection station, and provides technical support for early and accurate prevention and control of the wheat sharp eyespot.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a Rhizoctonia cerealis LAMP detection primer group and application thereof.
Background
Wheat sheath blight is one of the serious wheat soil-borne diseases in the world, and is also a main disease in the middle and lower reaches of Yangtze river and Huang-Huai-Mai area in China. Mainly prepared from rhizoctonia cerealis (Rhizoctonia cerealis) The typical symptom of infection is the formation of striae-like lesions on the leaf sheath. In recent years, the damage degree of banded sclerotial blight is increased year by year due to factors such as improvement of water and fertilizer conditions, dwarfing of varieties and the like, and the method becomes a great obstacle for influencing high yield and stable yield of wheat. At present, wheat sharp eyespot, wheat take-all and wheat root rot are the main soil-borne diseases of wheat, but the symptoms of the 3 diseases at the early stage of the disease are easy to be confused, so that the accurate diagnosis of the wheat soil-borne diseases at the early stage of the disease is necessary for the early prevention and control of the wheat soil-borne diseases.
Loop-mediated isothermal amplification (LAMP) is a constant temperature in vitro nucleic acid amplification technique. By designing a group of 4 specific primers aiming at 6 regions of a target sequence, and utilizing Bst DNA polymerase with strand displacement activity, 10 can be amplified out by 60 min at the constant temperature of 60-65 DEG C9And (c) a target sequence. Compared with the conventional PCR detection technology, the technology has the characteristics of strong specificity, high sensitivity, rapidness, convenience and low cost, and is widely applied to the detection of pathogenic microorganisms.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an LAMP detection primer group for Rhizoctonia cerealis, and aims to provide application of the LAMP detection primer group in the detection of Rhizoctonia cerealis.
In order to achieve the purpose, the invention adopts the specific scheme that:
the LAMP detection primer group for the rhizoctonia cerealis comprises four specific primers, and specifically comprises the following steps:
WK-FIP：GGATGAAAGAGAAGGTGTGCACATGTAGAGTCGGTTGTAGC；
WK-BIP：CCTGTTTAGACGGTCGAAGGAGATTTTATGTATTGGACGGGAA；
WK-F3：TAGGTGAACCTGCGGAAG；
WK-B3：TGCGTTTACATCGATTACATTC；
the target genes of the four specific primers are ITS of Rhizoctonia cerealis.
The invention also provides application of the LAMP detection primer group in the detection of rhizoctonia cerealis.
Has the advantages that:
the LAMP detection technology of the rhizoctonia cerealis is established based on the ITS of the pathogenic bacteria of the sheath blight of wheat as a target sequence and the four specific primers, the specificity is very strong, and the detection sensitivity is only 1 pg. Through detection of diseased tissues, the reaction system can still accurately detect the rhizoctonia cerealis under the condition of wheat DNA interference. Can accurately and quickly distinguish and detect the infection of rhizoctonia cerealis or other pathogenic bacteria. The technology of the invention provides technical support for early and accurate prevention and control of the wheat sharp eyespot.
Drawings
FIG. 1 is a diagram showing the results of a specificity test of LAMP primers; wherein, 1-cereal smut, 2-wheat take-all pathogen, 3-wheat root rot pathogen, 4-wheat scab pathogen, 5-wheat straw smut pathogen, 6-wheat leaf embroidery pathogen, 7-wheat embroidery disease pathogen, 8-wheat powdery mildew pathogen, 9-trim micronucleus, 10-rhizoctonia solani, 11-alternaria, 12-pythium admittedly, 13-fruit pythium, 14-soybean phytophthora, 15-tobacco phytophthora, 16-negative contrast;
FIG. 2 is a graph showing the results of a sensitivity test of LAMP primers; wherein N is a negative control;
FIG. 3 is a diagram showing the result of the LAMP detection of the pathogenic tissue of wheat; wherein N is a negative control and P is a positive control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
1 materials and methods
1.1 test strains
The total 15 plant pathogenic bacteria of the test strains are important pathogenic bacteria and common soil-borne pathogenic bacteria of wheat, wherein 8 strains of rhizoctonia cerealis and 1 strain of each pathogenic bacteria of other species are shown in Table 1.
Watch (A)
Information for LAMP detection of strains
1.2 test Medium
The test media were Potato Dextrose Agar (PDA) and V8 juice agar (V8 juice agar, V8A). PDA culture medium: 200 g of potatoes, 20 g of glucose, 15 g of agar powder and 1L of distilled water; V8A medium: 200 mL of V8 juice, 3 g of calcium carbonate, 15 g of agar powder and 800 mL of distilled water; when the strain is cultured in liquid, the culture medium is the corresponding liquid culture medium without agar powder.
1.3 design and Synthesis of primers
The ITS sequence (JQ 768036.1) of Rhizoctonia cerealis R.cerealis is downloaded from GenBank website, LAMP primers are designed by using PrimeExplore online software, 3 sets of primers are selected for synthesis by Venetian Biotechnology engineering (Shanghai) GmbH, 1 set of LAMP primers meeting the requirements are screened by a pre-test (Table 2), and the primers are stored in a refrigerator at 20 ℃ for later use. Wherein, the primer sequence of WK-FIP is shown as SEQ ID NO: 1 is shown in the specification; the primer sequence of WK-BIP is shown as SEQ ID NO: 2 is shown in the specification; the primer sequence of WK-F3 is shown in SEQ ID NO: 3 is shown in the specification; the primer sequence of WK-B3 is shown in SEQ ID NO: 4, respectively.
Watch (A)
LAMP primer sequence
1.4 extraction of genomic DNA
Liquid culture of pathogenic bacteria for 3d, filter paper filtration and collection of hyphae (obligate parasitic bacteria such as wheat straw smut bacteria, wheat stripe rust bacteria, wheat leaf rust bacteria, wheat powdery mildew and the like directly collect sporophyte from the diseased part of wheat), extraction of genome DNA is carried out by using a plant genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd.), and the concentration and purity of the DNA are measured by a NanoDrop 1000 ultramicro spectrophotometer.
1.5 LAMP reaction System and procedure
The LAMP reaction system is determined by a pre-test as follows: 2.5. mu.L of 10 × ThermoPol Buffer, 1.5. mu.L of 100 mM MgSO4, 3.5. mu.L of 10 mM dNTPs, 4. mu.L each of 10. mu.M WK-FIP and WK-BIP, 0.5. mu.L each of 10. mu.M WK-F3 and WK-B3, 1. mu.L of 8000U/mL Bst DNA Polymerase and 1. mu.L of template DNA, sterilized ultrapure water to make up 25. mu.L.
Reaction procedure: after the reagents are uniformly mixed, the mixture is placed in a water bath kettle with the constant temperature of 64 ℃ for reaction for 60 min, then 1 mu L of nucleic acid dye SYBR Green I is added into a reaction system, the color change of the reaction system is observed after the mixture is uniformly mixed, and the positive color is fluorescent yellow Green and the negative color is orange under the irradiation of a blue light gel cutting instrument (the wavelength is 440-485 nm).
1.6 detection of LAMP primer specificity and sensitivity
In order to detect the specificity of the LAMP primer, 14 important wheat pathogenic bacteria such as wheat take-all pathogen, wheat root rot pathogen, wheat scab pathogen, wheat straw smut pathogen, wheat leaf rust pathogen, wheat stripe rust pathogen, wheat powdery mildew, tidy micronucleus, rhizoctonia solani, alternaria alternate, pythium admittedly, pythium aphanidermatum, phytophthora sojae, phytophthora nicotianae and the like and common soil-borne pathogenic bacteria are used as reference strains, rhizoctonia cerealis is used as a target strain, and sterilized ultrapure water is used as negative control to carry out LAMP reaction.
To detect the sensitivity of the LAMP primer, the Rhizoctonia cerealis DNA was diluted to 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L and 1 fg/. mu.L, and 1. mu.L was added to the system to perform the LAMP reaction. Sterilized ultrapure water was used as a negative control.
1.7 detection of diseased tissue and field samples
Culturing Rhizoctonia cerealis 3d on a flat plate, cutting hypha, inoculating the hypha to the base part of a wheat stem, preserving moisture for 3 h, 6 h, 12h, 24h and 48h, then intercepting the tissue of the inoculated part of the wheat, extracting the DNA of the wheat tissue by using a plant genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd.), using the DNA as a DNA template for LAMP detection, using the DNA of the Rhizoctonia cerealis as a positive control, and using the DNA of the healthy wheat tissue as a negative control.
In 2019, in 3 months, 42 parts of suspected wheat sharp eyespot samples are collected from luoyang city, Henan province, root and stem bases are selected, a plant genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd.) is used for extracting sample DNA, the Rhizoctonia cerealis DNA is used as a positive control, the healthy wheat tissue DNA is used as a negative control, LAMP detection is carried out, and each sample is repeatedly detected for 3 times.
2 results and analysis
2.1 specificity of LAMP primers
LAMP primers (WK-FIP, WK-BIP, WK-F3 and WK-B3) are used for carrying out LAMP amplification on DNA of 15 pathogenic bacteria such as gramineous smut, wheat take-all pathogen, wheat root rot pathogen, wheat scab pathogen, wheat straw smut pathogen, wheat leaf rust pathogen, wheat stripe rust pathogen, wheat powdery mildew, tidy micronucleus, rhizoctonia solani, alternaria, pythium stuartifactum, pythium aphanidermatum, phytophthora sojae, phytophthora nicotianae and the like, and the result shows that only LAMP reaction of the gramineous sclerotium appears to be yellow-green positive reaction, and LAMP reaction of other pathogenic bacteria appears to be orange negative reaction (figure 1), which shows that the specificity of the reaction system is very strong.
2.2 sensitivity of LAMP primers
LAMP amplification was carried out using 10-fold gradient dilution of Rhizoctonia cerealis DNA as a template, and the results showed that the LAMP reaction was a yellowish green positive reaction when the amounts of the DNA templates were 10ng, 1 ng, 100 pg, 10 pg and 1pg, and the LAMP reaction was an orange negative reaction when the amounts of the DNA templates were 100 fg, 10 fg and 1 fg (FIG. 2), indicating that the sensitivity of the reaction system was 1 pg.
2.3 detection of diseased tissue
Extracting wheat tissue DNA inoculated with the Rhizoctonia cerealis for 3 h, 6 h, 12h, 24h and 48h, performing LAMP amplification, and showing that the LAMP reaction is orange negative reaction when the DNA template is inoculated with the wheat tissue DNA for 3 h and 6 h, and the LAMP reaction is yellow-green positive reaction when the DNA template is inoculated with the wheat tissue DNA for 12h, 24h and 48h (figure 3), so that the reaction system can accurately detect the Rhizoctonia cerealis under the wheat DNA interference condition, and the detection time is more than 12h of infection.
2.4 detection of field wheat samples
The LAMP primer is used for LAMP detection of 42 suspected wheat sharp eyespot samples collected from Loyang, and the result shows that 15 samples are in positive reaction, the detection rate is 35.71%, so that the symptoms of the wheat sharp eyespot at the seedling stage are easy to be confused with other diseases, most samples are not the wheat sharp eyespot, and the LAMP technology can accurately and quickly detect the wheat sharp eyespot.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
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<120> Rhizoctonia cerealis LAMP detection primer group and application thereof
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Claims (2)
1. The LAMP detection primer group for the rhizoctonia cerealis is characterized by comprising the following components in parts by weight: comprises four specific primers as follows:
WK-FIP：GGATGAAAGAGAAGGTGTGCACATGTAGAGTCGGTTGTAGC；
WK-BIP：CCTGTTTAGACGGTCGAAGGAGATTTTATGTATTGGACGGGAA；
WK-F3：TAGGTGAACCTGCGGAAG；
WK-B3：TGCGTTTACATCGATTACATTC；
the target genes of the four specific primers are ITS of the Rhizoctonia cerealis Gulley nucleus.
2. The LAMP detection primer group of claim 1 is applied to the detection of Rhizoctonia cerealis.
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