CN116694737A - LAMP-based detection method for pathogenic bacteria of black spot disease of celery cabbage - Google Patents
LAMP-based detection method for pathogenic bacteria of black spot disease of celery cabbage Download PDFInfo
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Abstract
The invention relates to the technical field of plant pathogen detection, in particular to a LAMP-based detection method for pathogenic bacteria of black spot disease of celery cabbage. In addition, the method is carried out under isothermal conditions, special instruments and equipment are not needed, the reaction time is short, and the method is more suitable for real-time detection in the field or the field; the sample preparation and DNA extraction operations of steps S1 and S2 can ensure that high quality DNA samples are extracted, thereby improving the accuracy and reliability of the reaction. The specific preparation of the LAMP reaction system in S3 can ensure the stability and consistency of the reaction, and the amplification reaction is more reliable. In the aspect of result analysis, the accuracy and the reliability of the result can be ensured by using two methods of direct visual observation and thermophoresis electrophoresis.
Description
Technical Field
The invention relates to the technical field of plant pathogen detection, in particular to a detection method for pathogenic bacteria of black spot disease of celery cabbage based on LAMP.
Background
The black spot disease of chinese cabbage is caused by infection with fungi of the genus Alternaria (Alternaria). Black or dark brown spots or plaques are formed on the leaves after the cabbage is infected with germs, and the spots start to be small yellow spots and gradually expand to black or dark brown spots with the diameter of 2-6 mm, the shapes of the spots are irregular, and the edges are blurred. Later infected leaves develop withering, drying and dying. In the case of severe infections, the entire leaf may die. Under the high humidity condition, pathogenic bacteria may generate spores and form a black mold layer, and the black mold layer covers the leaves; the black spot bacteria may invade the roots and stems of plants, leading to root rot and stem lesions.
The black spot disease of the Chinese cabbage occurs all over the country and is a common leaf disease of the Chinese cabbage. The photosynthesis of the Chinese cabbage is weakened after the damage, the plant aging is accelerated, the stem and leaf taste is bitter, the quality is poor, the yield is obviously reduced, and the yield is reduced by 20-30% in the epidemic year in part of the area, so that the serious loss is caused. Research work for controlling cabbage black spot has been carried out both at home and abroad, and comprehensive control of this disease has been carried out mainly by seed treatment, biological control and plant antibacterial actives and chemicals (leibert et al 1992;Terras et al, 1993; daya et al 1997;Pedras et al, 1997;Shrestha et al, 2000;Tylkowska et al, 2001; xiaochang et al 2002; cui Xiurong et al 2005; wang Gang et al 2006). The implementation of the above measures requires that the pathogenic bacteria can be "symptomatically administered" only if they are clear, since different control measures are required for different pathogenic bacteria. The identification method of pathogenic bacteria of cabbage black spot has been systematically studied abroad (King et al, 1994;Hansen et al, 1997;Doullah et al, 2006), and mainly includes the following types: plant or isolated organ identification, toxin activity identification, cytology identification, tissue culture technology identification, biochemical identification screening and molecular identification. Molecular characterization is currently one of the more popular methods.
The LAMP (Loop-mediated isothermal amplification) method is a novel nucleic acid amplification technology, has the advantages of rapidness, simplicity, high efficiency, sensitivity, strong specificity and the like, and has been successfully applied to detection of various pathogens. Aiming at the detection of pathogenic bacteria of cabbage black spot, methods such as PCR, nested PCR and the like exist at present, but the methods have the following defects:
1. PCR and Nested PCR require multiple steps, such as DNA extraction, debugging of PCR reaction conditions, and the like, are cumbersome to operate, take long time, and are susceptible to environmental factors to produce false positive or false negative results.
2. The specificity of PCR and Nested PCR is low and is susceptible to contamination by exogenous DNA.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a detection method for pathogenic bacteria of cabbage black spot based on LAMP, which introduces various specific nucleic acid primers and reaction control elements in the sample preparation and reaction processes so as to improve the specificity and sensitivity of detection. In addition, the reaction process of the LAMP method is carried out under isothermal conditions, special instruments and equipment are not needed, the reaction time is short, and the LAMP method is more suitable for real-time detection in fields or wild.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
a detection method of pathogenic bacteria of cabbage black spot based on LAMP comprises the following steps:
sample treatment, sampling the parts of the leaf, the leaf stalk, the root, the stem and the like of the celery cabbage to be detected, taking out the sample by using sterilized scissors and pliers, selecting the part to be detected, cutting off the sample with the length of about 1cm multiplied by 1cm, putting the sample into 1.5ml deionized water, centrifugally mixing the sample with Vortex, centrifugally mixing the sample at the speed of 12000g for 5min, and filtering the obtained supernatant by using an ultrafiltration tube after centrifuging to screen out germs. It should be noted that during sample processing, good sterility must be maintained to avoid sample contamination.
DNA extraction, culturing the bacteria obtained by screening at 60 ℃ for 12 hours, collecting bacterial liquid, and extracting total DNA of bacterial cells; the DNA extraction can be carried out by adopting a common DNA extraction kit, such as Omega DNA extraction kit, TIANamp Bacterial DNA Kit and the like, and the selection of the DNA extraction method is selected according to the specific requirements and experimental conditions of detection.
Preparing an LAMP reaction system, wherein the components of the LAMP reaction system are configured according to the following table:
wherein, the inner primer and the outer primer respectively contain two specific primers, and the annular primers consist of FIP and BIP to accelerate the amplification rate; the enhancer is useful for improving the amplification efficiency of the high GC region.
LAMP reaction, adding the extracted DNA into an LAMP reaction system, and carrying out DNA amplification in a reactor;
result analysis, the product after LAMP reaction is subjected to result analysis, and the direct observation of naked eyes is carried out: the amplified products were stained using 10. Mu.L SYBR Green I (1:10,000). If the color is changed from light green to fluorescent green, the positive result is judged, otherwise, the negative result is judged.
Thermophoresis electrophoresis: the reaction products were electrophoresed on a 1.5% agarose gel and observed by ultraviolet light, if the target band appeared, indicating the presence of the target sequence in the sample. It should be noted that the LAMP reaction is carried out without contamination, including reaction solutions, reagents and samples.
The method is based on the LAMP technology, has the characteristics of high sensitivity, rapidness, simplicity and the like, and is suitable for rapid detection of pathogenic bacteria of black spot disease of cabbages.
Further, the DNA extraction includes the steps of:
1. pretreatment of samples
The bacterial solution in PDA medium was centrifuged, the supernatant was discarded, the precipitated cells were washed with PBS, after centrifugation again, PBS was removed, and finally the precipitated cells were suspended with 1mLTE buffer (10 mM Tris-HCl,1mM EDTA,pH 8.0).
2. Cell disruption and lysis
Proteinase K and Lysozyme were added to the cell suspension and incubated at 60℃for 12 min.
3. Removal of proteins and other impurities
Adding Nuclei Lysis Solution to release nucleic acid from the cells; further, protein Precipitation Solution was added to precipitate proteins and other impurities, the reaction mixture was centrifuged, and the supernatant obtained by centrifugation was transferred to a new tube.
4. Washing DNA
The supernatant obtained above was transferred to DNA Binding Column, and impurities were removed by washing twice with Wash Buffer1 and Wash Buffer 2. Finally, DNABinding Column was subjected to the last Wash using Wash Buffer 3.
5. Extraction of DNA
DNABinding Column the DNA was washed out of Binding Column by adding an addition Buffer to a new centrifuge tube, and finally washing the Binding Column with Wash Buffer3 to Wash out all DNA. The extracted DNA may be stored using any method.
The whole process needs to pay attention to aseptic operation, and the operation is performed according to the instruction of the kit.
The extracted DNA is subjected to purification treatment, and the DNA can be purified by using a phenol/chloroform method or a purification kit; finally, detecting the content and purity of the DNA by using an ultraviolet spectrophotometer, and ensuring the quality of the extracted DNA.
Further, the step of the LAMP reaction specifically includes:
1. preparation of the required reagents and tools, including Bst DNA polymerase, mgSO 4 dNTPs, LAMP inner primers, LAMP outer primers, LAMP loop primer FIP, LAMP loop primer BIP, LAMP enhancer, reaction buffer, etc.
2. According to the proportion in the table, accurately measuring each reagent and configuring an LAMP reaction system; the specific operation method is as follows:
the required volume of 1X Isothermal Amplification Buffer was added to the reaction tube.
Adding a corresponding amount of MgSO 4 And thoroughly mixing.
Adding dNTPs, and mixing thoroughly.
Adding an LAMP inner primer and an LAMP outer primer; the inner primer and the outer primer respectively contain two specific primers, and are fully and uniformly mixed after being added.
And adding the LAMP loop primers FIP and BIP, and fully and uniformly mixing.
Add LAMP enhancer (0.8M betaine) and mix well.
Finally Bst DNA polymerase was added. To avoid loss of enzyme activity, it is necessary to add Bst DNA polymerase to the bottom of the reaction tube and add it to the top of the reaction tube after thoroughly mixing.
3. Transferring the prepared LAMP reaction system into a reaction tube, adding a proper amount of DNA to be amplified, generally 10-50ng in volume and 10 concentration, according to the requirement -12 And (5) mol/L, and closing the cover of the reaction tube after fully and uniformly mixing.
4. The reaction tube was placed in a reactor, the reactor temperature was set to 65 ℃ (appropriate temperature range 62-68 ℃), and the reaction was started.
5. The reaction time is set as desired, and it is generally recommended that the reaction time be 40 to 60 minutes. After the reaction is completed, the result analysis can be directly performed.
During the operation, pollution is avoided, and aseptic technique is used as much as possible to ensure the accuracy and reliability of experimental results. In addition, the final concentrations of the individual components of the reaction system should be precisely formulated to ensure the accuracy and reliability of the reaction.
Further, the LAMP inner primer: comprises F3 and B3 primer, which are 20-24 base gene sequences;
LAMP outer primer: comprises FIP and BIP primers, which are both gene sequences of 30-40 bases;
F3：5'-CTGCTGGAGCTGGATAGC-3'；
B3：5'-GAACCTCGGACAGGAGAA-3'；
FIP：5'-GGGCGCGGTTCTAGAGTGAAACCGCATGGTTCCATGGT-3'；
BIP：5'-AAGTGAGCATGGCGGGTTCAGCAGTAGCGCGTCGCGG-3'。
the basis of these Primer designs is Primer design and verification using NCBI Primer-BLAST, a bioinformatics analysis tool for pathogenic ITS sequences.
The invention has the beneficial effects that:
the invention provides a LAMP-based detection method for pathogenic bacteria of cabbage black spot, which can improve the specificity and sensitivity of detection and reduce the occurrence of false positive and false negative results. In addition, the method is carried out under isothermal conditions, special instruments and equipment are not needed, the reaction time is short, and the method is more suitable for real-time detection in the field or the field. The sample preparation and DNA extraction operations of steps S1 and S2 can ensure that high quality DNA samples are extracted, thereby improving the accuracy and reliability of the reaction. The specific preparation of the LAMP reaction system in S3 can ensure the stability and consistency of the reaction, and the amplification reaction is more reliable. In the aspect of result analysis, the accuracy and the reliability of the result can be ensured by using two methods of direct visual observation and thermophoresis electrophoresis.
The invention introduces a plurality of specific nucleic acid primers and reaction control elements, and the design of the nucleic acid primers is based on the special genome sequence of the pathogenic bacteria of the black spot disease of the Chinese cabbage. These primers can specifically bind to the target DNA, thereby improving the specificity and sensitivity of detection. In addition, the introduction of the reaction control element can effectively reduce nonspecific reaction and can more accurately detect the pathogenic bacteria of the black spot disease of the Chinese cabbage.
The LAMP method of the invention has the advantages that the reaction process is carried out under isothermal conditions, compared with the PCR method (polymerase chain reaction), the LAMP method has the reaction process based on isothermal amplification reaction, does not need complex instruments and equipment, has shorter reaction time, and can carry out real-time detection in fields or outdoors. This approach can significantly reduce the time and cost of sample detection.
The result analysis of the invention adopts naked eye direct observation and thermophoresis electrophoresis, has simple operation and visual result, and can rapidly judge whether the sample is positive.
In conclusion, the LAMP-based detection method for the pathogenic bacteria of the black spot disease of the celery cabbage is efficient, accurate and convenient, early warning can be carried out in advance, and a corresponding prevention and control strategy can be adopted, so that the loss caused by the disease is reduced.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The LAMP-based detection method for the pathogenic bacteria of the black spot disease of the celery cabbage, disclosed by the embodiment, comprises the following steps:
s1: pretreating Chinese cabbage to obtain a sample to be detected, and screening out germs;
s2: culturing the bacteria obtained in the step S1, and extracting DNA;
s3: adding the DNA extracted in the step S2 into an LAMP reaction system for amplification;
s4: and (3) analyzing the result of the amplification product obtained in the step (S3).
In this embodiment, the step S1 specifically includes the following substeps:
s1.1: sampling plants at different positions such as leaf, root and stem of Chinese cabbage;
s1.2: taking out the sample by using sterilized scissors and pliers, and selecting a required detection part to cut a 1cm multiplied by 1cm sample;
s1.3: putting the sample obtained in the step S1.2 into 1.5ml of deionized water, centrifugally mixing with Vortex, wherein the centrifugal speed is 12000g, and the centrifugal time is 5min;
s1.4: after centrifugation, the resulting supernatants were each filtered through ultrafiltration tubes to screen out pathogens.
In this embodiment, the step S2 specifically includes the following substeps:
s2.1: after sample treatment, incubating the screened bacterial strain in a PDA culture medium to a logarithmic phase, wherein the concentration of bacterial liquid reaches about OD < 600 > = 0.5-0.8;
s2.2: centrifuging bacterial liquid in PDA culture medium, discarding supernatant, washing precipitated cells with PBS, centrifuging again, discarding PBS, and suspending the precipitated cells with 1mLTE buffer (10 mM Tris-HCl,1mM EDTA,pH 8.0);
s2.3: cell disruption and lysis, proteinase K and Lysozyme were added to the cell suspension and incubated at 60℃for 12 min;
s2.4: removing proteins and other impurities, adding Nuclei Lysis Solution, and releasing nucleic acid from the cells; adding Protein Precipitation Solution to precipitate protein and other impurities, centrifuging the reaction solution, and transferring the supernatant to a new tube;
s2.5: washing the DNA, transferring the supernatant obtained above to DNA Binding Column, and washing twice with Wash Buffer1 and Wash Buffer2 to remove impurities; finally, using Wash Buffer3 to Wash DNABinding Column for the last time;
s2.6: DNA was extracted, DNABinding Column was placed in a new centrifuge tube, and an addition Buffer was added to Wash the DNA from the Binding Column, and finally Wash the Binding Column with Wash Buffer3 to Wash out all DNA.
In this example, the LAMP reaction system in step S3 includes Bst DNA polymerase, mgSO 4 dNTPs, LAMP inner primer, LAMP outer primer, LAMP loop primer FIP, LAMP loop primer BIP, LAMP enhancer and reaction buffer, wherein the concentration of each component is configured according to the following table:
wherein, the inner primer and the outer primer respectively contain two specific primers; the circular primer consists of FIP and BIP, so that the amplification rate is increased; the enhancer is useful for improving the amplification efficiency of the high GC region.
In this embodiment, the step S3 specifically includes the following substeps:
s3.1: 1X IsothermalAmplification Buffer of the required volume is added into the reaction tube; adding a corresponding amount of MgSO 4 And fully and uniformly mixing;
s3.2: adding dNTPs, and fully and uniformly mixing; adding an LAMP inner primer and an LAMP outer primer;
s3.3: adding LAMP loop primers FIP and BIP, and fully and uniformly mixing; adding LAMP enhancer (0.8 Mboetaine), and mixing completely;
s3.4: adding Bst DNA polymerase, wherein in order to avoid the loss of enzyme activity, the Bst DNA polymerase is added at the bottom of the reaction tube, and then added at the top of the reaction tube after fully and uniformly mixing;
s3.5: transferring the prepared LAMP reaction system into a reaction tube, adding the DNA obtained in the step S2, fully and uniformly mixing, and closing a cover of the reaction tube to prepare for amplification;
s3.6: the reaction tube is put into a reactor, and the temperature of the reactor is set to be 62-68 ℃ to start the reaction; the reaction time is 40-60 minutes, and the reaction system is 25 mu L;
s3.7: and analyzing the results of the LAMP reaction.
In this embodiment, the LAMP inner primers in step S3 include F3 and B3 primers, and the LAMP outer primers include FIP and BIP primers; the primer sequences of each group are as follows:
F3：5'-CTGCTGGAGCTGGATAGC-3'；
B3：5'-GAACCTCGGACAGGAGAA-3'；
FIP：5'-GGGCGCGGTTCTAGAGTGAAACCGCATGGTTCCATGGT-3'；
BIP：5'-AAGTGAGCATGGCGGGTTCAGCAGTAGCGCGTCGCGG-3'。
in this embodiment, the analysis of the result in step S3.7 specifically includes direct observation by naked eyes: the amplified product was stained using 10. Mu.L SYBR Green I (1:10,000); if the color is changed from light green to fluorescent green, the test result is positive, otherwise, the test result is negative;
thermophoresis electrophoresis: the reaction products were electrophoresed on a 1.5% agarose gel and observed by ultraviolet light, if the target band appeared, indicating the presence of the target sequence in the sample.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (7)
1. The LAMP-based detection method for the pathogenic bacteria of the black spot disease of the celery cabbage is characterized by comprising the following steps of:
s1: pretreating Chinese cabbage to obtain a sample to be detected, and screening out germs;
s2: culturing the bacteria obtained in the step S1, and extracting DNA;
s3: adding the DNA extracted in the step S2 into an LAMP reaction system for amplification;
s4: and (3) analyzing the result of the amplification product obtained in the step (S3).
2. The LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as claimed in claim 1, wherein: the step S1 specifically comprises the following substeps:
s1.1: sampling plants of different parts such as leaf, leaf stalk, root and stem of Chinese cabbage;
s1.2: taking out the sample by using sterilized scissors and pliers, and selecting a required detection part to cut a 1cm multiplied by 1cm sample;
s1.3: putting the sample obtained in the step S1.2 into 1.5ml of deionized water, centrifugally mixing with Vortex, wherein the centrifugal speed is 12000g, and the centrifugal time is 5min;
s1.4: after centrifugation, the resulting supernatants were each filtered through ultrafiltration tubes to screen out pathogens.
3. The LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as claimed in claim 1, wherein: the step S2 specifically includes the following substeps:
s2.1: after sample treatment, incubating the screened bacterial strain in a PDA culture medium to a logarithmic phase, wherein the concentration of bacterial liquid reaches about OD < 600 > = 0.5-0.8;
s2.2: centrifuging the bacterial solution in the PDA culture medium, discarding the supernatant, washing the precipitated cells with PBS, centrifuging again, discarding the PBS, and finally suspending the precipitated cells with 1mLTE buffer (10 mM Tris-HCl,1mMEDTA,pH8.0);
s2.3: cell disruption and lysis, proteinase K and Lysozyme were added to the cell suspension and incubated at 60℃for 12 min;
s2.4: removing proteins and other impurities, and adding nucleic acids solution to release nucleic acids from the cells; adding protein and other impurities into the solution, centrifuging the reaction solution, and transferring the supernatant into a new tube;
s2.5: washing the DNA, transferring the supernatant obtained above to DNABinding Column, and washing with WashBuffer1 and WashBuffer2 twice to remove impurities; finally, washing DNABinding column for the last time by using WashBuffer 3;
s2.6: DNA was extracted, DNABinding column was placed in a new centrifuge tube, elutionBuffer was added to Wash out DNA from BindingColumn, and BindingColumn was washed out with Wash Buffer3 to Wash out all DNA.
4. The LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as claimed in claim 1, wherein: the LAMP reaction system in the step S3 comprises BstDNA polymerase and MgSO 4 dNTPs, LAMP inner primer, LAMP outer primer, LAMP loop primer FIP, LAMP loop primer BIP, LAMP enhancer and reaction buffer, wherein the concentration of each component is configured according to the following table:
wherein, the inner primer and the outer primer respectively contain two specific primers, and the reactants are amplified together; the circular primer consists of FIP and BIP, so that the amplification rate is increased; the enhancer is useful for improving the amplification efficiency of the high GC region.
5. The LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as claimed in claim 4, wherein: the step S3 specifically comprises the following substeps:
s3.1: adding 1×IsothermalAmplification buffer with required volume into a reaction tube; adding a corresponding amount of MgSO 4 And fully and uniformly mixing;
s3.2: adding dNTPs, and fully and uniformly mixing; adding an LAMP inner primer and an LAMP outer primer;
s3.3: adding LAMP loop primers FIP and BIP, and fully and uniformly mixing; adding LAMP enhancer (0.8 Mboetaine), and mixing completely;
s3.4: adding BstDNA polymerase, wherein in order to avoid the loss of enzyme activity, the BstDNA polymerase needs to be added into the bottom of the reaction tube, fully and uniformly mixed and then added into the top of the reaction tube;
s3.5: transferring the prepared LAMP reaction system into a reaction tube, adding the DNA obtained in the step S2, fully and uniformly mixing, and closing a cover of the reaction tube to prepare for amplification;
s3.6: the reaction tube is put into a reactor, and the temperature of the reactor is set to be 62-68 ℃ to start the reaction; the reaction time is 40-60 minutes, and the reaction system is 25 mu L;
s3.7: and analyzing the results of the LAMP reaction.
6. The LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as set forth in claim 5, wherein: the LAMP inner primer in the step S3 comprises F3 and B3 primers, and the LAMP outer primer comprises FIP and BIP primers; the primer sequences of each group are as follows:
F3：5'-CTGCTGGAGCTGGATAGC-3'；
B3：5'-GAACCTCGGACAGGAGAA-3'；
FIP：5'-GGGCGCGGTTCTAGAGTGAAACCGCATGGTTCCATG GT-3'；
BIP：5'-AAGTGAGCATGGCGGGTTCAGCAGTAGCGCGTCGC GG-3'。
7. the LAMP-based detection method of pathogenic bacteria of black spot disease of celery cabbage as set forth in claim 5, wherein: the analysis of the result in the step S3.7 specifically includes direct visual observation: the amplified product was stained with 10. Mu. LSYBRGrenI (1:10,000); if the color is changed from light green to fluorescent green, the test result is positive, otherwise, the test result is negative;
thermophoresis electrophoresis: the reaction products were electrophoresed on a 1.5% agarose gel and observed by ultraviolet light, if the target band appeared, indicating the presence of the target sequence in the sample.
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