CN107022624B - LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds - Google Patents
LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds Download PDFInfo
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Abstract
The invention discloses a LAMP specific primer composition for a rapid detection method of rice bakanae disease germs, which consists of four primers, namely an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP and a downstream inner primer BIP. The invention also discloses a loop-mediated isothermal amplification kit and a corresponding loop-mediated isothermal amplification method: firstly, adding dye hydroxy naphthol blue as a reaction indicator before amplification, and then carrying out LAMP amplification reaction; after the reaction is finished, the color is changed, and the judgment is positive, otherwise, the judgment is negative; and secondly, taking the amplification product, detecting by using 2% agarose gel electrophoresis, and judging as positive if a trapezoidal band appears, otherwise, judging as negative.
Description
Technical Field
The invention belongs to the technical field of plant fungus molecular biology detection, relates to a loop-mediated isothermal amplification primer group for detecting bakanae disease bacteria of rice by using a loop-mediated isothermal amplification (LAMP) technology and a using method thereof, and belongs to the technical field of early warning of plant disease detection, identification and prevention.
Background
The rice bakanae disease is a hereditary fungal disease, which occurs in each main rice production area, can occur from the seedling stage to the heading stage, and reduces the yield by 5-50% in the field with light incidence. The disease is mainly caused by infection of fusarium graminearum (fusarium. fujikuroi), and seeds carrying pathogenic bacteria are the main infection source of rice bakanae disease, for example, if seeds with bacteria and healthy seeds are soaked together, disease-free seeds can be polluted. When the seeds grow new buds, germs can invade the seedlings through the bud sheaths, hyphae gradually spread to the whole plants along with the growth of the seedlings with the germs, and a series of symptoms occur, such as internode extension, exposure of the nodes and the outside of the leaf sheaths, weakened tillering capability, yellow leaf color and abnormal root system development. The plants with light incidence degree are heading in advance, and the panicle shape is small but not solid. In the heading stage, diseases can also occur, and in severe cases, the ears become brown and cannot be fructified. The probability of rice infection with germs is highest within 21 days after ear emergence of rice plants, and seeds with germs become the initial source of infection in the next season during threshing. Therefore, the establishment of a rapid detection system for rice bakanae disease bacteria can quickly and accurately detect infected rice seeds in early stage, and has important theoretical and practical significance for predicting disease occurrence conditions, timely adopting effective control measures to control the spread and prevalence of pathogenic bacteria and reducing economic loss.
At present, most of the detection of rice bakanae disease continues to use the traditional tissue isolation culture and morphological identification method, but the identification method consumes long time, generally takes more than one week to complete detection once, has large workload, has low separation success rate and has great limitation. If the separated disease sample is not fresh, pathogenic bacteria are difficult to diagnose accurately, the actual requirement for diagnosing the bakanae disease of the rice is difficult to meet, and the detection, the prevention and the control of the pathogenic bacteria are very unfavorable. With the continuous development of nucleic acid-related identification methods, common PCR-based methods have been successfully used for detecting pathogenic bacteria, but the detection time consumption of PCR specificity and the like is long and needs 6-8 h, and meanwhile, the PCR method needs expensive instruments, namely a PCR instrument, and the detection process is complicated. Most PCR detection methods are carried out by using a culture of pathogenic bacteria, separation, culture and purification of the pathogenic bacteria are required in the early stage, field detection cannot be carried out, and the practical application significance is low.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology invented by Nippon grongy corporation, and is a novel nucleic acid amplification technology that can replace PCR because of the advantages of simple and rapid amplification operation, high specificity, low cost, and the like. It designs 4 pairs of specific primers aiming at 6 regions of a target gene, causes self-circulation strand displacement reaction under the action of Bst large-fragment polymerase, and can synthesize a large amount of target DNA within 60min to 65 ℃. The detection of the amplified product is generally performed by visual observation with a fluorescent dye, agarose gel electrophoresis, turbidity observation, and the like. The LAMP amplification process depends on 6 independent areas for identifying the target sequence, so the reaction specificity is very strong, the nucleic acid amplification process is carried out under the constant temperature condition, a common water bath pot or an isothermal thermos bottle can meet the reaction requirement, the detection cost is reduced, and the required time is short. The method has the characteristics of simple reaction, rapidness, high efficiency, economy and the like, so the method has extremely wide application prospect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a set of loop-mediated isothermal amplification primer composition for detecting rice bakanae disease, a kit containing the primer and an LAMP method for quickly detecting the rice bakanae disease from rice seeds.
In order to solve the technical problems, the invention provides the following LAMP primer composition for detecting the rice bakanae disease, which consists of an upstream outer primer and a downstream inner primer, wherein the sequences of the primers are as follows:
upstream outer primer (F3): 5 '-CGGCCCTTATACCCCATTC-3';
downstream outer primer (B3): 5 '-GCGCCACTATTGCTGTCT-3';
inner upstream primer (FIP): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA-3';
downstream inner primer (BIP): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA-3'.
The invention also provides a loop-mediated isothermal amplification kit for detecting the rice bakanae disease bacteria, which comprises the LAMP specific primer composition.
As an improvement of the kit of the present invention: comprises 1.6 mu M of forward inner primer FIP, 1.6 mu M of reverse inner primer BIP, 0.2 mu M of forward outer primer F3 and 0.2 mu M of reverse outer primer B3; further comprising: 10 XThermoPol Buffer, 1mmol/L dNTPs, 4mmol/L MgCl20.6mmol/L betaine, 150mmol/L hydroxynaphthol blue (HNB), 8U/. mu.L Bst DNA polymerase, ddH2O。
Namely, the LAMP kit comprises a detection solution consisting of a loop-mediated isothermal amplification primer mixed solution and a loop-mediated isothermal amplification reaction premixed solution, and the concentration of the primer combined mixed solution is as follows: 1.6 μ M forward inner primer FIP, 1.6 μ M reverse inner primer BIP, 0.2 μ M forward outer primer F3, 0.2 μ M reverse outer primer B3; loop-mediated isothermal amplification reactionMixing liquid: 10 XThermoPol Buffer, 1mM dNTPs, 4mM MgCl20.6M betaine, 150. mu.M hydroxynaphthol blue (HNB), 8U/. mu.L Bst DNA polymerase, ddH2O。
The invention also provides application of the primer combination or the kit in detecting the bacteria-carrying condition of rice seeds.
The invention also provides a loop-mediated isothermal amplification method for detecting the bakanae disease bacteria of rice, wherein the total amount of detection solution is 24 mu L, and 1 mu L of DNA template to be detected is added to form a 25 mu L detection reaction system;
the 25. mu.L reaction system contained 8U/. mu.L of Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M of FIP primer 2.0. mu.L, 20. mu.M of BIP primer 2.0. mu.L, 10. mu.M of F3 primer 0.5. mu.L, 10. mu.M of B3 primer 0.5. mu.L, 25mM of MgCl24.0 μ L, 2.5 μ L of 10mM dNTPs, 3.0 μ L of 5M betaine, 1 μ L of 3.75mM hydroxynaphthol blue (HNB)1 μ L, DNA template, and making up to 25 μ L with double distilled water;
performing LAMP amplification reaction by using the 25-microliter detection reaction system; the LAMP amplification reaction program is as follows: 60min at 63 ℃; 80 ℃ for 10 min.
As an improvement of the loop-mediated isothermal amplification method of the present invention:
performing LAMP amplification reaction by using the 25. mu.L detection reaction system, and selecting any one of the following methods:
adding dye Hydroxy Naphthol Blue (HNB) as a reaction indicator before amplification, taking the color change of HNB as a result judgment standard, and then carrying out LAMP amplification reaction; after the reaction is finished, the color is changed, namely, the sky blue is observed as a color development result and is judged to be positive, namely, the rice to be detected contains rice bakanae disease germs; the color is not changed, the color development result is bluish purple, and the judgment is negative, namely the rice to be detected has no bakanae disease germs, or the content of the bakanae disease germs contained in the rice to be detected does not reach the lowest detection concentration of 0.01 ng/muL;
taking 5-10 mu L of amplification product, detecting by using 2% agarose gel electrophoresis, and if a trapezoidal strip appears, judging that the strip is positive, judging that the rice to be detected contains bakanae disease germs of rice; if the amplified band does not exist, the rice to be detected is judged to be negative, namely the rice to be detected does not have the bakanae disease germs, or the lowest detection concentration of the bakanae disease germs contained in the rice to be detected is 10 fg/muL.
The scheme of the invention comprises the following steps:
(1) designing a specific primer according to a DNA sequence of the NPRS31 gene of the rice bakanae disease bacteria;
(2) LAMP amplification and detection of amplification products;
(3) detecting the specificity of the LAMP primer;
(4) comparing LAMP amplification sensitivity with PCR amplification sensitivity;
(5) and (3) detecting the rice seeds carrying bakanae disease bacteria.
The scheme of the invention is as follows:
1. designing a primer:
the inventors firstly obtained the NPRS31 gene sequence of rice bakanae disease, designed multiple sets of primers using primereplorer V5(http:// primereplorer. jp/lampv5e/index. html), and further selected the primers according to the comprehensive factors such as the conservation of the sequence region where the primers are located, the hairpin structure of the primers, the dimer GC content and the Tm value. Finally, 4 primers including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP) are selected for the rice bakanae disease bacteria. The primer information is shown in Table 1.
TABLE 1 primer sequence information
2. And (3) specific detection of the primers:
to detect the specificity of the primer, using Fusarium graminearum (Fusarium fujikuroi) as a positive control, using Fusarium graminearum (f.fujikuroi), Fusarium oxysporum (f.oxysporum), Fusarium laminarinum (f.proliferum), Fusarium solani (f.solani), Fusarium graminearum (f.graminearum), penicillium sp (penicillium sp.), aspergillus oryzae (usagonoideae virens), Pyricularia oryzae (Pyricularia grisea), Alternaria alternata (Alternaria alternata), Rhizoctonia solani (Rhizoctonia solani) as a negative control, and using double distilled water as a blank control to test, the primer was found to have the specificity of detecting Fusarium graminearum (Fusarium fujikuroi) (fig. 1).
3. The LAMP reaction system is as follows:
the 25. mu.L system contained 8U/. mu.L Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20.0. mu.M FIP primer 2.0. mu.L, 20.0. mu.M BIP primer 2.0. mu.L, 10. mu.M F3 primer 0.5. mu.L, 10. mu.M B3 primer 0.5. mu.L, 25.0mM MgCl 4.0. mu.L, 10.0mM dNTPs 2.5. mu.L, 5.0M betaine 3.0. mu.L, 3.75mM hydroxynaphthol blue (HNB) 1. mu. L, DNA template 1. mu.L, and double distilled water to make up to 25. mu.L; the amplification conditions were 63 ℃ incubation for 60min and 80 ℃ reaction for 10 min.
After the reaction, the specificity of the primers was determined by agarose gel electrophoresis and HNB dye color development, respectively. Agarose gel electrophoresis: taking 5 mu L of LAMP amplification product, detecting by 2% agarose gel electrophoresis, EB dyeing, observing in a gel imaging system, wherein the characteristic ladder-shaped strip is positive, and the strip which does not appear is negative; HNB dye color development method: and (3) observing the color change of the LAMP reaction mixed solution by naked eyes, wherein sky blue is a positive reaction, and blue purple is a negative reaction.
4. Comparison of LAMP amplification and conventional PCR amplification sensitivity:
the extracted genomic DNA of the rice bakanae disease is diluted to 7 concentrations (10 ng/muL-1 ng/muL) according to a gradient of 10 times after the concentration is measured by the NanoDrop 1000, and the concentration is used for measuring the LAMP sensitivity. The LAMP reaction system and amplification conditions were as above.
Meanwhile, the diluted DNA was used as a template, and F3 and B3 were used as primers to perform conventional PCR amplification as a control. The reaction system is as follows: the PCR system was 25.0. mu.L, 2 XTaq MasterMix 12.5. mu.L, upstream and downstream primers 10. mu.M each 1.0. mu.L, DNA template 1.0. mu.L, double distilled water 9.5. mu.L; the PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 10 min.
After the amplification is finished, 5 mu L of LAMP amplification and PCR amplification gradient products are subjected to electrophoresis analysis on 2% agarose gel, and the results are observed by using a gel imaging system after ethidium bromide staining.
5. Optimization of the reaction program:
aims to establish the LAMP optimal reaction temperature capable of detecting the rice bakanae disease. The primer combination is utilized to form a detection solution with the reaction premix, 1 mu L of rice bakanae disease bacteria DNA template is added, the reaction temperature is respectively set to be 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and 66 ℃, after the amplification reaction is finished, the color change of the reaction tube is observed, and the detection is carried out by 2% agarose gel electrophoresis as the verification in the next step.
The reaction result shows (figure 2), when the reaction temperature is 63 ℃, the color change of the reaction tube is most obvious, and the electrophoresis LAMP band is clear.
Aims to establish the LAMP optimal reaction time capable of detecting the rice bakanae disease. The primer combination and the reaction premixed solution form a detection solution, 1 mu L of the DNA template of the rice bakanae disease is added, the reaction time is set to be 30min, 40min, 50min and 60min, and the color change of the reaction tube is observed after the amplification reaction is finished. Further detection was performed by 2% agarose gel electrophoresis as a confirmation.
The reaction results show (fig. 3), when the reaction time reaches 60min or longer, the color change of the reaction tube is obvious, the electrophoresis LAMP band is clear, and the detection time is saved, namely 60min is the amplification time.
According to the optimization result of the reaction program, the reaction program is 63 ℃ and 60 min.
6. Detecting the rice seeds carrying bakanae disease:
respectively taking the rice seeds inoculated with the bakanae disease bacteria and the healthy seeds without the bakanae disease bacteria to mix in different proportions, randomly taking the mixed seed samples, then cracking the seed samples by using a 5% Chelex-10 solution, extracting DNA, and carrying out LAMP detection as above.
The invention establishes a monitoring technology system which is rapid, simple, convenient, strong in specificity and high in sensitivity and can be observed by naked eyes, can detect the germ carrying rate of rice seeds before seed soaking and germination acceleration in agricultural production, and has very important significance for preventing and controlling the rice bakanae disease.
Compared with the prior art, the invention has the following technical advantages:
(1) the specificity is strong: the results of the two specific detection implementation methods show that positive results are only generated when the rice seeds carry bakanae bacteria.
(2) The sensitivity is high: by diluting the genomic DNA of the seed bakanae bacteria by a multiple ratio for sensitivity detection, the LAMP technology can detect that the lowest concentration of the bakanae bacteria DNA is 64 fg/muL, which is 100 times higher than that of the conventional PCR.
(3) After the rapid and convenient reaction is finished, the reaction result can be directly observed by naked eyes, the whole process only needs 60min, and the detection time is greatly shortened.
(4) The cost is low: the LAMP technology does not need expensive instruments such as a PCR instrument, gel electrophoresis and a gel electrophoresis imaging system, the reaction can be completed by using a water bath kettle and a common vacuum cup, and the LAMP technology is suitable for large-scale detection in common laboratories and fields.
(5) The technology can be used for directly detecting the bacteria-carrying condition of rice seeds, and can detect the bacteria-carrying rate of 0.06 percent or more without separation and culture of germs and complicated DNA extraction procedures such as centrifugation.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a comparison of primer specificity assays;
a: is an LAMP chromogenic change map;
b: is LAMP agarose gel electrophoresis picture;
m represents DL 2000DNA Marker, and the test samples were respectively prepared by using 9 pathogenic bacteria of 1. Fusarium cancearum (F.fujikurio), 2. Fusarium oxysporum (F.oxysporum), 3. Fusarium proliferatum (F.proliferum), 4. Fusarium solani (F.solani), 5. Fusarium graminearum (F.graminearum), 6. Penicillium (Penicillium sp.), 7. Rhizopus oryzae (Ustilaginoidea virens), 8. Pyricularia oryzae (Pyricularia grisea), 9. Alternaria alternata (Alternaria altate), 10. Rhizoctonia solani (Rhizoctonia solani) as negative controls, and using double distilled water (11) as a blank control.
FIG. 2 is a diagram of reaction temperature optimization;
a: is an LAMP chromogenic change map;
b: is LAMP agarose gel electrophoresis picture;
m represents DL 2000DNA Marker;
1. 2, 3, 4, 5, 6, 7, 8 represent 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C, 65 deg.C, 66 deg.C, respectively;
the color change is most obvious at 63 ℃, and the agarose gel electrophoresis band of the LAMP product is bright.
FIG. 3 is a graph of reaction time optimization;
a: is an LAMP chromogenic change map;
b: is LAMP agarose gel electrophoresis picture;
m represents DL 2000DNA Marker;
1. 2, 3 and 4 respectively represent 60min, 50min, 40min and 30 min;
all showed bright bands on LAMP agarose gel electrophoresis after 60 min.
Detailed Description
The invention will be further described by way of example only with reference to the accompanying drawings, which do not in any way limit the scope of the invention, but are given by way of illustration only.
The main reagents and instruments used in the following examples Bst DNA polymerase (New England Biolabs, MgCl)2(Sigma), dNTPs, betaine, DEPC water, molecular mass standard DNA Maker (TaKaRa bioengineering company genome), a fungus genome DNA rapid extraction kit (bioengineering company, Inc.), an eppendorf common PCR amplification instrument, and a DK-8D type electric heating constant-temperature water bath of Shanghai sperm macroexperiment instrument factory.
Example 1:
sensitive detection of primers
The DNA of the genome of Fusarium fujikuroi (Fusarium fujikuroi) was extracted and the concentration of the DNA solution was measured to be 200 ng/. mu.L. The DNA solution was diluted to have a gradient of 0.0001 ng/. mu.L, 0.001 ng/. mu.L, 0.01 ng/. mu.L, 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, or 100 ng/. mu.L. The reaction system was prepared in 25. mu.L of the following: 8U/. mu.L Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M FIP 2.0. mu.L, 20. mu.M BIP 2.0. mu.L, 10. mu. M F30.5.5. mu.L, 10. mu. M B30.5.5. mu.L, 25mM MgCl2mu.L of 4.0. mu.L, 2.5. mu.L of 10mM dNTPs, 3.0. mu.L of 5M betaine, 1. mu.L of 3.75mM hydroxynaphthol blue (HNB), 1. mu.L of the DNA template of each concentration gradient described above (1. mu.L of double distilled water as a blank), and finally, each amount was made up to 25. mu.L with double distilled water.
The following primer combinations were used for the experiments:
upstream outer primer (F3): 5 '-CGGCCCTTATACCCCATTC-3';
downstream outer primer (B3): 5 '-GCGCCACTATTGCTGTCT-3';
inner upstream primer (FIP): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA-3';
downstream inner primer (FIP): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA-3'.
The LAMP amplification reaction program used was: amplifying at 63 ℃ for 60 min; inactivating at 80 deg.C for 10 min.
After the reaction, the reaction results were positive at DNA template concentrations of 0.01 ng/. mu.L, 0.1 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L, and 100 ng/. mu.L, the color of the reaction tube changed from bluish purple to sky blue before and after the reaction, and specific bands were detected by 2% agarose gel electrophoresis. When the concentration is lower than 0.01 ng/. mu.L, the reaction tube becomes negative when no color change occurs, and no specific band appears by using 2% agarose gel electrophoresis.
The results showed that the lowest concentration of DNA detected for rice bakanae disease bacteria (F. fujikuroi) was 0.01 ng/. mu.L.
Example 2: analysis of primer specificity
The method comprises the following steps of extracting 9 pathogenic bacteria of fusarium granatum (F.fujikurio), fusarium oxysporum (F.oxysporum), fusarium solani (F.solani), fusarium graminearum (F.graminearum), penicillium sp (Penicillium sp.), Ustilaginoides, Pyricularia oryzae (Pyricularia grisea), Alternaria alternata (Alternaria alternata) and Rhizoctonia solani (Rhizoctonia solani) as negative controls, and using double distilled water as blank controls.
A25-microliter reaction system is used and comprises the following components in proportion: 8U/. mu.L Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M FIP 2.0. mu.L, 20. mu.M BIP 2.0. mu.L, 10. mu. M F30.5.5. mu.L, 10. mu. M B30.5.5. mu.L, 25mM MgCl2mu.L of 4.0. mu.L, 2.5. mu.L of 10mM dNTPs, 3.0. mu.L of 5M betaine and 1. mu. L, DNA solution of 3.75mM hydroxynaphthol blue (HNB) as a template, and adding 1. mu.L of the template solution to make up to 25. mu.L of the template solution by double distilled water.
The following primer combinations were used for the experiments:
upstream outer primer (F3): 5 '-CGGCCCTTATACCCCATTC-3';
downstream outer primer (B3): 5 '-GCGCCACTATTGCTGTCT-3';
inner upstream primer (FIP): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA-3';
downstream inner primer (FIP): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA-3'.
The LAMP amplification reaction program used was: amplifying at 63 ℃ for 60 min; inactivating at 80 deg.C for 10 min.
The test results show that the templates except for the rice bakanae disease pathogen (F. fujikuroi) are negative, the color of the reaction solution in the reaction tube is not changed, and no specific band appears after 2% agarose gel electrophoresis detection. Shows that the loop-mediated isothermal amplification system formed by the primer combination can detect the rice bakanae disease germ (F.fujikuroi) and has specificity.
Comparative examples 1-1,
The following primer combinations were used for the experiments:
upstream outer primer (F3-1): 5 '-CGGCCCTTATACCCCATTT-3';
downstream outer primer (B3-1): 5 '-GCGCCACTATTGCTGTCA-3';
inner upstream primer (FIP): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA-3';
downstream inner primer (FIP): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA-3'.
Note: the last bases C and T at the 3' ends of the two outer primers are respectively replaced by T and A so as to verify the specificity of the primers.
A25-microliter reaction system is used and comprises the following components in proportion: 8U/. mu.L Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M FIP 2.0. mu.L, 20. mu.M BIP 2.0. mu.L, 10. mu. M F3-10.5. mu.L, 10. mu. M B3-10.5. mu.L, 25mM MgCl2mu.L of 4.0. mu.L, 2.5. mu.L of 10mM dNTPs, 3.0. mu.L of 5M betaine, 1. mu.L of 3.75mM hydroxynaphthol blue (HNB), 1. mu.L of DNA solutions with concentrations of 1 ng/. mu.L, 10 ng/. mu.L and 100 ng/. mu.L, respectively, as a template, and made up to 25. mu.L with double distilled water.
The test results show that the test results are negative, the color of the reaction solution in the reaction tube is not changed, and no specific band appears after 2% agarose gel electrophoresis detection. Shows that the loop-mediated isothermal amplification system formed by the primer combination cannot effectively detect the rice bakanae disease germ (F.fujikuroi), and cannot be effectively distinguished even if the concentration of the DNA template is increased to 100 ng/. mu.L.
Comparative examples 1 to 2,
The following primer combinations were used for the experiments:
upstream outer primer (F3): 5 '-CGGCCCTTATACCCCATTC-3';
downstream outer primer (B3): 5 '-GCGCCACTATTGCTGTCT-3';
inner upstream primer (FIP-1): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGT-3';
downstream inner primer (FIP-1): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTC-3'.
Note: the last bases A and A at the 3' ends of the two outer primers are respectively replaced by T and C to verify the specificity of the primers.
A25-microliter reaction system is used and comprises the following components in proportion: 8U/. mu.L Bst DNA polymerase 0.5. mu.L (4U), 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M FIP 2.0. mu.L, 20. mu.M BIP 2.0. mu.L, 10. mu. M F30.5.5. mu.L, 10. mu. M B30.5.5. mu.L, 25mM MgCl24.0. mu.L, 2.5. mu.L of 10mM dNTPs, 3.0. mu.L of 5M betaine, 1. mu.L of 3.75mM hydroxynaphthol blue (HNB), at concentrations of 1 ng/. mu.L and 10 ng/. mu.L, respectivelymu.L of DNA solution at a concentration of 100 ng/. mu.L was added as a template, and the mixture was made up to 25. mu.L with double distilled water.
The test results show that the test results are negative, the color of the reaction solution in the reaction tube is not changed, and no specific band appears after 2% agarose gel electrophoresis detection. Shows that the loop-mediated isothermal amplification system formed by the primer combination cannot effectively detect the rice bakanae disease germ (F.fujikuroi), and cannot be effectively distinguished even if the concentration of the DNA template is increased to 100 ng/. mu.L.
Example 3 detection of Rice seeds carrying Miao disease bacteria
(1) Preparation of Rice seed samples
Beating 0.5cm on the bakanae disease strain plate cultured for 5 days2The fungus cake of (1) is mixed with 10g of healthy rice seeds without bakanae bacteria, and then cultured for 5 days at 25 ℃. Then 1 rice seed with bakanae disease obtained as above is mixed with 0, 200, 400, 800, 1600 and 3200 healthy seeds without bakanae disease respectively, and then is shaken for 60min at 300 rpm.
(2) Rapid extraction of bakanae disease DNA of rice seeds
Randomly taking one seed from the seed samples obtained by mixing in different proportions, respectively placing the seeds in 200 mu L of 5% Chelex-100, shaking on a vortex shaking instrument for 10s, boiling for 5min, further shaking for 10s, boiling for 5min, naturally cooling, and storing for later use.
(3) The extracted sample genomes were subjected to LAMP amplification, and the reaction results were observed by agarose gel electrophoresis and HNB dye method, and the detection method was the same as in example 1.
(4) And respectively taking 16 mixed seeds and inoculating the seeds on a PDA (personal digital assistant) plate with the length of 9cm, and counting the bacterial carrying rate of the bakanae disease of the seeds after 2 days.
The result shows that the LAMP reaction can not detect the bakanae disease in a healthy seed sample which is not polluted by the bakanae disease, the bakanae disease is detected in the seed with the bakanae disease and a mixed sample of the healthy seed after being polluted by the bakanae disease, the reaction solution in the tube is changed from purple to sky blue, ladder-shaped strips appear in 2 percent agarose gel electrophoresis, and the seed sample with the bakanae disease carrying rate of 0.0625 percent (namely, after 1 rice seed with the bakanae disease is mixed with 1600 healthy seeds without the bakanae disease) can still be rapidly diagnosed by the method, namely, the detection limit is 1/1600-0.0625 percent.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
<110> Zhejiang agriculture and forestry university
<120> LAMP method and kit for rapidly detecting bakanae disease bacteria of rice from rice seeds
<160> 4
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> upstream outer primer F3
<400> 1
cggcccttat accccattc 19
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> downstream outer primer B3
<400> 2
gcgccactat tgctgtct 18
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> inner upstream primer FIP
<400> 3
acttgcgctg gaaagaccag aacatccgcc acctcactga 40
<210> 4
<211> 42
<212> DNA
<213> Artificial sequence
<220>
<223> downstream inner primer BIP
<400> 4
ggtcaccaat cctcggctac agccctagga gatggtcgct ta 42
Claims (2)
1. A loop-mediated isothermal amplification method for detecting rice bakanae disease bacteria utilizes an LAMP specific primer composition for a rapid detection method of the rice bakanae disease bacteria, and is characterized in that:
the LAMP specific primer composition consists of four primers, namely an upstream and downstream outer primer and an upstream and downstream inner primer;
upstream outer primer (F3): 5 '-CGGCCCTTATACCCCATTC-3';
downstream outer primer (B3): 5 '-GCGCCACTATTGCTGTCT-3';
inner upstream primer (FIP): 5 '-ACTTGCGCTGGAAAGACCAGAACATCCGCCACCTCACTGA-3';
downstream inner primer (BIP): 5 '-GGTCACCAATCCTCGGCTACAGCCCTAGGAGATGGTCGCTTA-3';
the LAMP reaction system is 25 mu L and comprises: 8U/. mu.L Bst DNA polymerase 0.5. mu.L, 10 × ThermoPol Buffer 2.5. mu.L, 20. mu.M FIP 2.0. mu.L, 20. mu.M BIP 2.0. mu.L, 10. mu. M F30.5.5. mu.L, 10. mu. M B30.5.5. mu.L, 25mM MgCl24.0 mul, 2.5 mul of 10mM dNTPs, 3.0 mul of 5M betaine, 1 mul of 3.75mM hydroxynaphthol blue, 1 mul of DNA template of rice bakanae disease bacteria, and supplementing to 25 mul by double distilled water;
the amplification conditions were 63 ℃ incubation for 60min, 80 ℃ reaction for 10 min.
2. The loop-mediated isothermal amplification method according to claim 1, wherein:
performing LAMP amplification reaction by using the 25 mu L detection reaction system, and selecting any one of the following methods:
adding dye hydroxyl naphthol blue as a reaction indicator before amplification, taking the color change of HNB as a result judgment standard, and then carrying out LAMP amplification reaction; after the reaction is finished, the color is changed, namely, the sky blue is observed as a color development result and is judged to be positive, namely, the rice to be detected contains rice bakanae disease germs; the color is not changed, the color development result is bluish purple, and the judgment is negative, namely the rice to be detected has no bakanae disease germs, or the content of the bakanae disease germs contained in the rice to be detected does not reach the lowest detection concentration of 0.01 ng/muL;
taking 5-10 mu L of amplification product, detecting by using 2% agarose gel electrophoresis, and if a trapezoidal strip appears, judging that the strip is positive, judging that the rice to be detected contains bakanae disease germs of rice; if the amplified band does not exist, the rice to be detected is judged to be negative, namely the rice to be detected does not have the bakanae disease germs, or the lowest detection concentration of the bakanae disease germs contained in the rice to be detected is 10 fg/muL.
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