CN111560450A - LAMP detection method for potato scab germs - Google Patents
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Abstract
The invention discloses an LAMP detection method of eschar potato germs, which comprises the steps of designing primers according to genes txtA, txtB and other sequences of a toxin synthesis gene cluster specific to eschar potato germs, and obtaining a group of LAMP detection primers through on-line screening and amplification effect screening; and optimizing the reaction system and verifying the specificity to establish the LAMP detection method of the scab potato germs and determine the sensitivity of the detection method. The LAMP detection primer improves the sensitivity and detection efficiency of pathogen detection, lays a foundation for the subsequent prevention of potato scab, and has high practical application value in the prevention of potato scab.
Description
Technical Field
The invention relates to the technical field of potato scab germ detection, and particularly relates to an LAMP detection method of potato scab germ.
Background
Potato scab (Potato common scab) is an important soil-borne and seed-borne disease in Potato production, is commonly generated in various Potato planting areas in the world at present, pathogenic bacteria causing the scab comprise various plant pathogenic streptomyces, can live in soil, is high in concealment, is mainly transmitted through field mechanical operation and diseased Potato seeds, is difficult to find in the early stage due to the fact that the scab mainly damages underground parts of Potato plants, and is difficult to prevent and control when the diseased potatoes have obvious symptom expression, so that a large amount of loss is caused, and in order to effectively and quickly detect the existence of the scab pathogenic bacteria, a quick detection method for the scab pathogenic bacteria is necessary to be established through screening general specific detection primers and a reaction system.
Intensive studies on the genome of the scab potato bacterium show that pathogenic genes of synthetic toxins are gathered in a large chromosome region, exist in streptomyces as a pathogenic island, and comprise multiple genes such as txtA, txtB, txtC and txtD. The pathogenicity of scab bacteria is closely related to the pathogenicity island. The pathogenic island contains a relatively large chromosomal region (10-200kb) and is only present in the chromosome of pathogenic strains, but not in non-pathogenic strains ]. Biosynthesis of thaxtomin A, which causes scab, is at a single locus (txt) of scab-causing species, txtA and txtB encode synthetic 4-nitroindole and L-phenylalanine that function to methylate and cyclize dipeptide synthetases.
Disclosure of Invention
The invention aims to provide a LAMP detection method for potato scab germs, which utilizes the LAMP detection method to screen stable amplification results and can be used as B3B3/B3F3, B3FIP/B3BIP and B3LF/B3LB of LAMP detection primers, thereby improving the sensitivity and detection efficiency of pathogen detection, laying a foundation for the subsequent work of preventing the potato scab germs, having higher practical application value for the prevention of the potato scab germs and solving the problems provided in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
an LAMP detection method for scab potato germs comprises the following steps:
s1: designing primers according to the sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to the common scab pathogen of potato, and obtaining a group of LAMP detection primers through on-line screening and amplification effect screening;
s2: optimizing a reaction system, verifying specificity, and determining LAMP detection sensitivity of the scab potato germs;
s3: and establishing stability verification of LAMP detection on the extracted potato scab tissues and soil DNA with scab germs.
Further, the specific method in S1 is as follows:
s101: designing primers for the sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to eschar potato using PrimeExplorer online LAMP primer design software;
s102: each group of designed primers comprises six primers which are respectively two inner primers, two outer primers and two loop primers, and then the primers are synthesized;
s103: according to the kit specification, the DNA of CPS-1 is taken as a template, the synthesized primer is screened under an initial reaction system, isothermal amplification is carried out for 30-60min under the condition of 65 ℃, and inactivation is carried out for 10min at 80 ℃;
s104: and after the amplification is finished, taking 5 mu L of amplification product, carrying out electrophoresis detection on the amplification product by using 1.6% agarose gel 110V for 35min, simultaneously adding 0.15 mu L of fluorescent dye into the rest 20 mu L of amplification product to observe color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, and otherwise, the reaction tube is in the orange color of the dye.
Further, LAMP detection primers in S1 are B3B3/B3F3, B3FIP/B3BIP, B3LF/B3 LB.
Further, the optimization of the reaction system in S2 includes Mg2+And (3) optimizing unidirectional factors of concentration, reaction temperature, concentration ratio of inner primers and outer primers and reaction time.
Further, specific methods for specificity verification in S2 are as follows:
s201: selecting representative strains CPS-1, CPS-2, CPS-3, CPS-4, Sd-2 of 5 different scab pathogenic strains obtained from different regions, several nonpathogenic streptomycetes N8, shx101 and biocontrol streptomycete strains Men-myco-93-63, and fungal disease pathogenic strains including verticillium dahliae, rhizopus tritici and pythium, and DNA of bacterial strains bacillus amyloliquefaciens separated from soil as templates;
s202: determining an initial reaction system according to the kit specification, reacting for 60min at 65 ℃ for isothermal amplification, inactivating for 10min at 80 ℃, finally adding 0.15 mu L of fluorescent dye into an amplification product to observe color change, wherein if a target fragment is amplified in a large amount, a reaction tube is in fluorescent green, and otherwise, the dye is in orange color.
Further, the sensitivity detection method in S2 is: extracting potato scab bacteria DNA by 101、102、103、104、105、106、107、108Diluting the double gradient into 8 concentrations, using the concentrations as reaction templates to perform LAMP reaction by using optimized reaction systems and conditions, and determining the lowest DNA detection concentration of the reaction.
Further, the specific method for verifying the stability of the LAMP detection in S3 is as follows:
s301: carrying out conventional PCR detection and LAMP detection on DNA of tissue samples of different growth periods of the potato plants and DNA of tissue samples of different parts of diseased potato plants;
s302: and (3) conventional PCR detection: specific primers RTA1/RTA2 of the common scab bacteria are selected for detection, the detection sensitivity of the primers RTA1/RTA2 is known to be 20 pg/mu L, and the amplification reaction system is 25 mu L: nucleic-free Water: 10 mu L of the solution; 2 XGC buffer II: 12.5 mu L; template DNA: 0.5 mu L; primer: 0.5. mu.L each; dNTP (10 mM): 0.5 mu L; taq enzyme: 0.5 mu L; amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 50s, extension at 72 ℃ for 90s, 30 cycles, and final extension at 72 ℃ for 5 min; after the amplification is finished, adding 1.5 mu L of 6Loading Buffer into 7.5 mu L of amplification product, and carrying out electrophoresis detection on the amplification product by using 1.5% agarose gel at 110V for 30 min;
s303: LAMP detection: and (3) carrying out isothermal amplification for 30-60min by using an optimized reaction system at a proper temperature and with the sample DNA as a template, inactivating for 10min at 80 ℃, adding 0.15L of fluorescent dye after the amplification is finished, and observing color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, and otherwise, the reaction tube is in the dye color orange.
Compared with the prior art, the invention has the beneficial effects that:
according to the LAMP detection method for the potato scab, provided by the invention, the LAMP detection method is utilized to screen a stable amplification result, B3B3/B3F3, B3FIP/B3BIP and B3LF/B3LB which can be used as LAMP detection primers are adopted, the detection sensitivity and detection efficiency of pathogenic bacteria are improved, a foundation is laid for the subsequent work of preventing the potato scab, and the LAMP detection method has a high practical application value for preventing the potato scab.
Detailed Description
The following examples will explain the present invention in detail, however, the present invention is not limited thereto. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides an LAMP detection method for scab potato germs, which comprises the following steps:
the first step is as follows: designing primers according to the sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to the common scab pathogen of potato, and obtaining a group of LAMP detection primers through on-line screening and amplification effect screening;
the second step is that: optimizing a reaction system, verifying specificity, and determining LAMP detection sensitivity of the scab potato germs;
the third step: and establishing stability verification of LAMP detection on the extracted potato scab tissues and soil DNA with scab germs.
In the above embodiment, the specific method in the step one is as follows:
1. on-line LAMP primer design software for potato scab by using PrimeExplorerDesigning primers for sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to bacteria; wherein the PrimeExplorer on-line LAMP primer design software is referred to in (1)http://primerexplorer.jp/e/)；
2. Each group of designed primers comprises six primers which are respectively two inner primers, two outer primers and two loop primers, and the primers are synthesized in a synthetic mode which can be synthesized by Shanghai Biotechnology Limited company;
3. according to the kit specification, the DNA of CPS-1 is taken as a template, the synthesized primer is screened under an initial reaction system, isothermal amplification is carried out for 30-60min under the condition of 65 ℃, and inactivation is carried out for 10min at 80 ℃;
4. after the amplification is finished, carrying out electrophoresis detection on 5 mu L of amplification products by using 1.6% agarose gel 110V for 35min, simultaneously adding 0.15 mu L of fluorescent dye into the rest 20 mu L of amplification products to observe color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, otherwise, the color of the dye is orange; wherein BstDNA polymerase is added into the system at last, and the reaction tube cover is covered immediately after the BstDNA polymerase is added into the reaction equipment.
The sequences of the primers screened are shown in table 1 below:
table 1 primer sequences for study design
The primer sequences after screening are shown in table 2 below:
TABLE 2 LAMP detection primers
The LAMP detection primers obtained from Table 2 above are B3B3/B3F3, B3FIP/B3BIP, and B3LF/B3 LB.
In the above examples, the optimization of the reaction system in step two includes Mg2+The optimization of one-way factors of concentration, reaction temperature, concentration ratio of inner primers and outer primers and reaction time is realized by using Mg which plays a key role in the reaction through a single-factor change test on the basis of an LAMP initial reaction system2+Optimizing conditions such as concentration, reaction temperature, concentration ratio of internal primer to external primer, reaction time and the like, and selecting the optimal reaction condition.
In the above examples, in the second step, in order to verify the specificity of the primers, including the specificity different from other disease pathogens and the specificity different from the nonpathogenic strain of eschar potato scaber, the specific verification method is as follows:
1. selecting representative strains CPS-1, CPS-2, CPS-3, CPS-4, Sd-2 of 5 different scab pathogenic strains obtained from different regions, several nonpathogenic streptomycetes N8, shx101 and biocontrol streptomycete strains Men-myco-93-63, and fungal disease pathogenic strains including verticillium dahliae, rhizopus tritici and pythium, and DNA of bacterial strains bacillus amyloliquefaciens separated from soil as templates;
2. determining an initial reaction system according to the kit specification, reacting for 60min at 65 ℃ for isothermal amplification, inactivating for 10min at 80 ℃, finally adding 0.15 mu L of fluorescent dye into an amplification product to observe color change, wherein if a target fragment is amplified in a large amount, a reaction tube is in fluorescent green, and otherwise, the dye is in orange color.
In the above embodiment, the sensitivity detection method in step two is as follows: extracting potato scab bacteria DNA by 101、102、103、104、105、106、107、108Diluting the double gradient into 8 concentrations, using the concentrations as reaction templates to perform LAMP reaction by using optimized reaction systems and conditions, and determining the lowest DNA detection concentration of the reaction.
In the above embodiment, the specific method for verifying the stability of the LAMP detection in step three is as follows:
1. carrying out conventional PCR detection and LAMP detection on DNA of tissue samples of different growth periods of the potato plants and DNA of tissue samples of different parts of diseased potato plants;
2. and (3) conventional PCR detection: specific primers RTA1/RTA2 of the common scab bacteria are selected for detection, and the detection sensitivity of the known primers RTA1/RTA2 is 20 pg/mu L; the optimized amplification reaction system is 25 mu L: nucleic-freeWater: 10 mu L of the solution; 2 XGC buffer II: 12.5 mu L; template DNA: 0.5 mu L; primer: 0.5. mu.L each; dNTP (10 mM): 0.5 mu L; taq enzyme: 0.5 mu L; amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 50s, extension at 72 ℃ for 90s, 30 cycles, and final extension at 72 ℃ for 5 min; after the amplification is finished, adding 1.5 mu L of 6Loading Buffer into 7.5 mu L of amplification product, and carrying out electrophoresis detection on the amplification product by using 1.5% agarose gel at 110V for 30 min;
3. LAMP detection: and (3) carrying out isothermal amplification for 30-60min by using an optimized reaction system at a proper temperature and with the sample DNA as a template, inactivating for 10min at 80 ℃, adding 0.15L of fluorescent dye after the amplification is finished, and observing color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, and otherwise, the reaction tube is in the dye color orange.
The design principle is as follows: the invention extracts spore genome DNA of five model strains existing in China, designs LAMP primers according to txtA and txtB conserved gene sequences in a toxin synthesis gene cluster specific to eschar potato scab bacteria registered in NCBI GenBank by using PrimerExplorer online LAMP primer design software, and obtains a group of LAMP detection primers by online screening and amplification effect screening: the LAMP detection method for the scab potato scab is established by optimizing a reaction system and verifying specificity, the detection sensitivity of the LAMP detection method is measured, and the method is applied to detect the extracted diseased tissues and the bacteria-carrying soil, so that the established method is strong in repeatability and high in usability.
In summary, the following steps: according to the LAMP detection method for the potato scab, provided by the invention, the LAMP detection method is utilized to screen a stable amplification result, B3B3/B3F3, B3FIP/B3BIP and B3LF/B3LB which can be used as LAMP detection primers are adopted, the detection sensitivity and detection efficiency of pathogenic bacteria are improved, a foundation is laid for the subsequent work of preventing the potato scab, and the LAMP detection method has a high practical application value for preventing the potato scab.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. The LAMP detection method for the scab potato germs is characterized by comprising the following steps:
s1: designing primers according to the sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to the common scab pathogen of potato, and obtaining a group of LAMP detection primers through on-line screening and amplification effect screening;
s2: optimizing a reaction system, verifying specificity, and determining LAMP detection sensitivity of the scab potato germs;
s3: and establishing stability verification of LAMP detection on the extracted potato scab tissues and soil DNA with scab germs.
2. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the specific method in S1 is as follows:
s101: designing primers for the sequences of genes txtA and txtB of a toxin synthesis gene cluster specific to eschar potato using PrimeExplorer online LAMP primer design software;
s102: each group of designed primers comprises six primers which are respectively two inner primers, two outer primers and two loop primers, and then the primers are synthesized;
s103: according to the kit specification, the DNA of CPS-1 is taken as a template, the synthesized primer is screened under an initial reaction system, isothermal amplification is carried out for 30-60min under the condition of 65 ℃, and inactivation is carried out for 10min at 80 ℃;
s104: and after the amplification is finished, taking 5 mu L of amplification product, carrying out electrophoresis detection on the amplification product by using 1.6% agarose gel 110V for 35min, simultaneously adding 0.15 mu L of fluorescent dye into the rest 20 mu L of amplification product to observe color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, and otherwise, the reaction tube is in the orange color of the dye.
3. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the LAMP detection primers in S1 are B3B3/B3F3, B3FIP/B3BIP, B3LF/B3 LB.
4. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the optimization of the reaction system in S2 comprises Mg2+And (3) optimizing unidirectional factors of concentration, reaction temperature, concentration ratio of inner primers and outer primers and reaction time.
5. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the specific method for specificity verification in S2 is as follows:
s201: selecting representative strains CPS-1, CPS-2, CPS-3, CPS-4, Sd-2 of 5 different scab pathogenic strains obtained from different regions, several nonpathogenic streptomycetes N8, shx101 and biocontrol streptomycete strains Men-myco-93-63, and fungal disease pathogenic strains including verticillium dahliae, rhizopus tritici and pythium, and DNA of bacterial strains bacillus amyloliquefaciens separated from soil as templates;
s202: determining an initial reaction system according to the kit specification, reacting for 60min at 65 ℃ for isothermal amplification, inactivating for 10min at 80 ℃, finally adding 0.15 mu L of fluorescent dye into an amplification product to observe color change, wherein if a target fragment is amplified in a large amount, a reaction tube is in fluorescent green, and otherwise, the dye is in orange color.
6. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the sensitivity detection method in S2 is: extracting potato scab bacteria DNA by 101、102、103、104、105、106、107、108Dilution in a multiple gradient to 8 concentrations toThe LAMP reaction is carried out by using the optimized reaction system and conditions for the reaction template, and the lowest DNA detection concentration of the reaction is determined.
7. The LAMP detection method of Sclerotinia solanacearum according to claim 1, wherein the stability verification of LAMP detection in S3 is as follows:
s301: carrying out conventional PCR detection and LAMP detection on DNA of tissue samples of different growth periods of the potato plants and DNA of tissue samples of different parts of diseased potato plants;
s302: and (3) conventional PCR detection: specific primers RTA1/RTA2 of the common scab bacteria are selected for detection, the detection sensitivity of the primers RTA1/RTA2 is known to be 20 pg/mu L, and the amplification reaction system is 25 mu L: nucleic-free Water: 10 mu L of the solution; 2 XGC buffer II: 12.5 mu L; template DNA: 0.5 mu L; primer: 0.5. mu.L each; dNTP (10 mM): 0.5 mu L; taq enzyme: 0.5 mu L; amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 50s, extension at 72 ℃ for 90s, 30 cycles, and final extension at 72 ℃ for 5 min; after the amplification is finished, adding 1.5 mu L of 6Loading Buffer into 7.5 mu L of amplification product, and carrying out electrophoresis detection on the amplification product by using 1.5% agarose gel at 110V for 30 min;
s303: LAMP detection: and (3) carrying out isothermal amplification for 30-60min by using an optimized reaction system at a proper temperature and with the sample DNA as a template, inactivating for 10min at 80 ℃, adding 0.15L of fluorescent dye after the amplification is finished, and observing color change, wherein if a target fragment is amplified in a large quantity, the reaction tube is in fluorescent green, and otherwise, the reaction tube is in the dye color orange.
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| CN114045358A (en) * | 2021-10-15 | 2022-02-15 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
| CN114045358B (en) * | 2021-10-15 | 2023-08-18 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
| CN114592079A (en) * | 2022-03-24 | 2022-06-07 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | LAMP detection primer group, reagent and detection method for detecting pathogenic bacteria of potato scab and application |
| CN114592079B (en) * | 2022-03-24 | 2024-03-29 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | LAMP detection primer group, reagent and detection method for detecting pathogenic bacteria of potato scab and application of LAMP detection primer group |
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