CN114045358B - Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method - Google Patents

Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method Download PDF

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CN114045358B
CN114045358B CN202111203360.8A CN202111203360A CN114045358B CN 114045358 B CN114045358 B CN 114045358B CN 202111203360 A CN202111203360 A CN 202111203360A CN 114045358 B CN114045358 B CN 114045358B
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董莎萌
张欣杰
王帅帅
陈汉
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Nanjing Agricultural University
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Abstract

The invention discloses a primer composition for detecting twelve potato disease pathogenic bacteria based on a loop-mediated isothermal amplification technology and a detection method. The method is a molecular detection method for phytophthora infestans, alternaria alternata, fusarium oxysporum, fusarium equisetum, fusarium graminearum, elder fusarium, rhizoctonia solani, ralstonia solanacearum, pectobacterium nigeldanum, streptomycete and eschar. The method of the invention does not need complex instruments, can better meet the field detection of 12 potato disease pathogenic bacteria, provides a new technical platform for the detection of quarantine disease pathogenic bacteria, can better meet the urgent need of the field detection of potato diseases at present, is used for the field detection of import and export quarantine, field quarantine and the like, and is easy to popularize and apply in a large scale.

Description

Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method
Technical Field
The invention particularly relates to a primer composition for detecting twelve potato disease pathogenic bacteria by a loop-mediated isothermal amplification (LAMP) technology based on color judgment and a detection method, belonging to the technical field of biology. The special application is used for important potato disease pathogenic bacteria in the field: high-sensitivity rapid detection of phytophthora infestans (Phytophthora infestans), alternaria alternata (Alternaria solani), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (Fusarium equiseti), fusarium graminearum (Fusarium graminearum), fusarium layering (Fusarium proliferatum), fusarium sambucus (Fusarium sambucinum), rhizoctonia solani (Rhizoctonia solani), ralstonia solanacearum (Ralstonia solanacearum), pectobacter nigrum (Pectobacterium atrosepticum), streptomycete (Streptomyces scabies) and eschar silverum (Spongospora subterranea) can be used for early diagnosis of field potato diseases and monitoring of pathogenic bacteria.
Background
Potato disease is caused by infection of potatoes with a variety of pathogenic fungi and oomycetes, severely jeopardizing potato production. Pathogenic bacteria causing potato diseases are mainly phytophthora infestans (Phytophthora infestans), alternaria alternata (Alternaria solani), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (Fusarium equiseti), fusarium graminearum (Fusarium graminearum), fusarium graminearum (Fusarium proliferatum), fusarium sambucus (Fusarium sambucinum), rhizoctonia solani (Rhizoctonia solani), ralstonia solanacearum (Ralstonia solanacearum), pectobacterium nigrum (Pectobacterium atrosepticum), streptomycete (Streptomyces scabies), eschar farinacea (Spongospora subterranea) and the like. Potato diseases caused by the above pathogens include: late blight of potato, early blight of potato, wilt of potato, dry rot of potato, black mole of potato, bacterial wilt of potato, black shank of potato, scab of potato, and scab of potato. The diseases are widely distributed and seriously damaged, and often cause serious loss of agricultural production. The accurate detection of the pathogenic bacteria in time is beneficial to the rapid diagnosis of diseases and the timely guidance of prevention and control.
The traditional method for detecting various pathogenic bacteria is to separate and purify the pathogenic bacteria. The traditional method plays an important role in pathogen detection, but is time-consuming and labor-consuming and requires operators to have professional pathogen separation, morphological identification knowledge and abundant experience. With the development of nucleic acid related identification methods, PCR-based methods have been successfully used for detecting pathogenic bacteria, and although PCR methods have a great improvement in specificity and sensitivity, the detection time is still relatively long, approximately 4 to 5 hours, while PCR methods rely on precise temperature cycling devices. The detection sensitivity is relatively high, but the detection process is complex, and the requirement of rapid detection cannot be met.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology invented by Rongsheng corporation of Japan, and is a novel nucleic acid amplification technology capable of replacing PCR because of the advantages of simple operation, rapidness, high specificity, low cost and the like. The method designs 4 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 generates white magnesium pyrophosphate precipitation which is a byproduct when a large amount of target DNA is synthesized within 60min at 60-65 ℃. Because the LAMP amplification process relies on identifying 6 independent areas of a target sequence, the reaction specificity is strong, the nucleic acid amplification process is carried out under the constant temperature condition, the common water bath or equipment with stable heat source can meet the reaction requirement, and the detection cost is greatly reduced.
The choice of target gene is one of the important factors for LAMP detection. According to the invention, the sequence difference of target genes among different species and different species in different species of phytophthora infestans, alternaria alternata, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium sambucus, rhizoctonia solani, ralstonia solanacearum, pectobacterium nikoense, streptomycete and eschar are analyzed, specific LAMP primers are designed, and an LAMP system for detecting phytophthora infestans is established on the basis.
Disclosure of Invention
The invention aims to solve the problems that the biological detection method for common potato disease pathogenic bacteria in the prior art is long in period, time-consuming, labor-consuming, tedious and poor in specificity, and the technical problems that the PCR detection technology needs a thermal cycling instrument and can not detect each potato disease pathogenic bacteria rapidly, and provides an LAMP primer composition, a kit and a visual detection method for detecting twelve potato disease pathogenic bacteria. The method is a novel molecular detection method for phytophthora infestans, alternaria alternata, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium layering, fusarium sambucus, rhizoctonia solani, ralstonia solanaceae, pectobacterium nigeldanum, streptomycete and eschar, and carries out LAMP detection on the twelve pathogenic bacteria, and has the advantages of short detection period (only 1h is needed), high accuracy, high sensitivity and visual observation of detection results.
The aim of the invention can be achieved by the following technical scheme:
a LAMP primer composition for detecting twelve potato disease pathogens, the primer composition comprising at least one of the following primer sets (1) - (12):
(1) A phytophthora infestans detection primer set (F3, B3, FIP, BIP, LB);
(2) A Alternaria alternata detection primer set (F3, B3, FIP, BIP, LF);
(3) Fusarium oxysporum detection primer set (F3, B3, FIP, BIP, LF);
(4) Fusarium equisetum detection primer set (F3, B3, FIP, BIP, LF, LB);
(5) Fusarium graminearum detection primer set (F3, B3, FIP, BIP, LF, LB);
(6) Fusarium detection primer set (F3, B3, FIP, BIP, LF, LB);
(7) Fusarium sambucinum detection primer group (F3, B3, FIP, BIP, LF);
(8) A rhizoctonia solani detection primer set (F3, B3, FIP, BIP, LB);
(9) A primer set for detecting Ralstonia solanacearum (F3, B3, FIP, BIP, LF, LB);
(10) Pectobacterium nigrum detection primer set (F3, B3, FIP, BIP, LF, LB);
(11) Streptomyces detection primer set (F3, B3, FIP, BIP, LB);
(12) A eschar detecting primer set (F3, B3, FIP, BIP, LF, LB);
wherein,,
(1) The phytophthora infestans LAMP molecular detection primer group consists of five specific primers FIP, BIP, F, B3 and LB:
(2) The Alternaria alternata LAMP molecular detection primer group consists of five specific primers FIP, BIP, F, B3 and LF:
(3) Fusarium oxysporum LAMP molecular detection primer group consists of five specific primers FIP, BIP, F, B3 and LF:
(4) The fusarium equiseti LAMP molecular detection primer group consists of six specific primers FIP, BIP, F, B3, LF and LB:
(5) Fusarium graminearum LAMP molecular detection primer group consists of six specific primers FIP, BIP, F3, B3, LF and LB:
(6) The fusarium LAMP molecular detection primer group consists of six specific primers FIP, BIP, F, B3, LF and LB:
(7) Fusarium sambucinum LAMP molecular detection primer group consists of five specific primers FIP, BIP, F, B3 and LF:
(8) The rhizoctonia solani LAMP molecular detection primer group consists of five specific primers FIP, BIP, F, B3 and LB:
(9) The LAMP molecular detection primer group for the Ralstonia solanaceae consists of six specific primers FIP, BIP, F, B3, LF and LB:
(10) The LAMP molecular detection primer group for pectobacterium nigrum comprises six specific primers FIP, BIP, F, B3, LF and LB:
(11) The streptomycete LAMP molecular detection primer group consists of five specific primers FIP, BIP, F3, B3 and LB:
(12) The LAMP molecular detection primer group for the eschar consists of six specific primers FIP, BIP, F, B3, LF and LB:
according to the invention, a Ypt1 sequence of phytophthora infestans, an Alt a1 sequence of Alternaria alternata, a TEF-1 alpha sequence of Fusarium oxysporum, a TEF-1 alpha sequence of Fusarium equisetum, a TEF-1 alpha sequence of Fusarium graminearum, an RED1 sequence of Fusarium layering, a TEF-1 alpha sequence of Fusarium sambucus, an ITS sequence of Rhizoctonia solani, a 16S rRNA sequence of Ralstonia solanacearum, a gryB sequence of pectobacter nigrum, a Nec1 sequence of Streptomyces and an ITS sequence of Fusarium verrucosum are selected as target genes to design LAMP molecular detection primer groups of twelve potato pathogenic bacteria (the sequences of target genes of twelve potato pathogenic bacteria are disclosed);
the design of specific primers FIP, BIP, F3, B3 and LB aiming at the Ypt1 sequence of phytophthora infestans is shown in figure 3;
specific primers FIP, BIP, F, B3 and LF of Alt a1 sequences aiming at Alternaria alternata are designed in figure 4;
the design of specific primers FIP, BIP, F, B3 and LF aiming at the TEF-1 alpha sequence of fusarium oxysporum is shown in figure 5;
specific primers FIP, BIP, F, B3, LF and LB of TEF-1 alpha sequence aiming at Fusarium equisetum are designed in figure 6;
the design of specific primers FIP, BIP, F, B3, LF and LB aiming at TEF-1 alpha sequence of fusarium graminearum is shown in figure 7;
the specific primers FIP, BIP, F3, B3, LF and LB aiming at RED1 sequences of Fusarium layering are designed in the figure 8;
the design of specific primers FIP, BIP, F, B3 and LF aiming at TEF-1 alpha sequence of Fusarium sambucinum is shown in figure 9;
specific primers FIP, BIP, F, B3 and LB aiming at ITS sequences of rhizoctonia solani are designed as shown in figure 10;
specific primers FIP, BIP, F, B3, LF and LB of the 16S rRNA sequence aiming at Solanaceae Ralstonia are designed in FIG. 11;
specific primers FIP, BIP, F3, B3, LF and LB aiming at the gryB sequence of pectobacterium nigrum are designed in figure 12;
specific primers FIP, BIP, F, B3 and LB for the Nec1 sequence of streptomyces are shown in FIG. 13;
the specific primers FIP, BIP, F, B3, LF, LB for the ITS sequences of eschar are shown in FIG. 14.
The LAMP primer composition is applied to detection of twelve potato disease pathogenic bacteria.
The LAMP primer composition is applied to preparation of a kit for detecting twelve potato disease pathogenic bacteria.
A LAMP kit for detecting twelve potato disease pathogens, which comprises the LAMP primer composition.
As a preferable technical scheme, the reaction system of the kit is as follows:
1mL of the detection solution includes: 20. Mu.M forward inner primer FIP, 20. Mu.M reverse inner primer BIP, 10. Mu.M forward outer primer F3, 10. Mu.M reverse outer primer B3, 10. Mu.M forward loop primer LF, 10. Mu.M reverse loop primer LB, 10 XBuffer, 10mM dNTPs, 50mM MgSO 4 5M betaine, bst DNA polymerase U/. Mu.L, and ultrapure water were added to prepare 1mL of a detection solution. The shelf life was 1 year.
The LAMP kit is applied to detection of twelve potato disease pathogenic bacteria.
The LAMP detection method of twelve potato disease pathogens adopts the LAMP kit to carry out LAMP reaction, and the LAMP reaction comprises the following procedures: 70min at 62 ℃; detection of amplified products: SYBR Green I (fluorescent dye) is added into the product after isothermal amplification to serve as a reaction indicator, and the color change of the fluorescent dye is used as a result judgment standard; the yellow-green color indicates that the detection is positive, the target pathogenic bacteria exist, and the orange-yellow color indicates that the detection result is negative.
The method for detecting twelve potato disease pathogenic bacteria by using the LAMP detection kit specifically comprises the following steps:
(1) LAMP detection of twelve potato disease pathogens: taking 4 mu L of DNA solution, adding 18 mu L of kit solution, 3 mu L of sterilized deionized water, and the total volume is 25 mu L;
(2) The reaction procedure is: 70min at 62 ℃;
(3) Detection of amplified products: after amplification, a fluorescent dye (SYBR Green I) was added as a reaction indicator, and the color change of the product was used as a result judgment standard. Yellow green indicates positive detection, the presence of such pathogenic bacteria. Orange-yellow indicates that the test result is negative.
The invention discloses a detection kit for detecting phytophthora infestans (Phytophthora infestans), alternaria alternata (Alternaria solani), fusarium oxysporum (Fusarium oxysporum), fusarium equisetum (Fusarium equiseti), fusarium graminearum (Fusarium graminearum), fusarium graminearum (Fusarium proliferatum), fusarium sambucus (Fusarium sambucinum), rhizoctonia solani (Rhizoctonia solani), ralstonia solanaceae (Ralstonia solanacearum), pectobacterium nigrum (Pectobacterium atrosepticum), streptomyces (Streptomyces scabies) and eschar ruber (Spongospora subterranea) based on a loop-mediated isothermal amplification (LAMP) technology of color judgment, and belongs to the fields of crop disease prevention and treatment and plant quarantine. The detection proves that the kit has good specificity sensitivity, rapid and efficient amplification and simple and convenient identification. The detection system can rapidly, conveniently, efficiently, highly specifically and highly sensitively detect phytophthora infestans, alternaria alternata, fusarium oxysporum, fusarium equisetum, fusarium graminearum, elder fusarium, rhizoctonia solani, solanaceae ralstonia, pectobacterium nigrum, streptomyces and eschar under the isothermal condition of 62 ℃, does not need a complex instrument, can better meet the field detection of 12 potato disease pathogenic bacteria, provides a new technical platform for the detection of quarantine disease pathogenic bacteria, can better meet the urgent need of the field detection of potato diseases at present, is used for the field detection of import and export quarantine, field quarantine and the like, and is easy to popularize and apply in a large scale.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) The practicability is good. Gel electrophoresis of products by common PCR reactions is easy to cause product diffusion, which is a major source of laboratory pollution; ethidium Bromide (EB) has huge toxicity and can accumulate and cause cancer; long-term observation of ultraviolet lamps also causes a degree of injury to the experimenters. The LAMP reaction is only carried out in a constant-temperature water bath kettle, and the result can be directly judged through the color change of the product after the reaction is finished, so that the application value of the LAMP reaction in the field is increased.
(2) And (5) amplifying at constant temperature. The isothermal amplification is realized, unlike the PCR method which needs thermal cycle, the dependence on a thermal cycle instrument is eliminated, the LAMP reaction can occur only by a stable heat source, the application range of the LAMP is greatly expanded, and the LAMP can react under a constant heat source because betaine is added into the LAMP reaction liquid, so that double-stranded DNA is in the dynamic balance of melting, and the amplification is realized under the action of Bst DNA polymerase.
(3) The accuracy is high. Because the traditional pathogen detection technology only determines quarantine objects according to morphological characteristics, interference of human factors cannot be eliminated, similar morphological species are difficult to distinguish, and the detection accuracy is only 60-80%; according to the invention, 12 specific or universal target genes of potato disease pathogenic bacteria are selected, the target genes not only show high specificity among other common potato pathogenic bacteria, but also show high conservation in homologous pathogenic bacteria, so that the Ypt1 sequence of phytophthora infestans, the Alt a1 sequence of Alternaria alternata, the TEF-1 alpha sequence of Fusarium oxysporum, the TEF-1 alpha sequence of Fusarium equisetum, the TEF-1 alpha sequence of Fusarium graminearum, the RED1 sequence of Fusarium sambucinum, the TEF-1 alpha sequence of Fusarium sambucus, the ITS sequence of rhizoctonia solani, the 16S rRNA sequence of Ralstonia solanaceae, the gryB sequence of pectobacterium nigrum, the Nec1 sequence of Streptomyces and the ITS sequence of Fusarium as target genes are finally selected. And comparing sequences of different pathogenic bacteria, other species and sequences of other pathogenic bacteria by using Bioedit software, and selecting a specific segment of sequence of each pathogenic bacteria to design a specific LAMP primer. LAMP primer design is carried out by PrimerExpore V5 software, a group of primers is screened according to indexes such as primer length, GC content, gibbs free energy and the like, the LAMP reaction specifically recognizes 6 independent areas on a target sequence through 4 primers, and compared with 2 independent areas of the target sequence recognized by PCR primers, the specificity and the sensitivity are high.
Drawings
FIG. 1 shows the specificity of LAMP for detecting twelve potato disease pathogens.
LAMP amplification was performed on a total of 12 strains of 12 pathogenic bacteria causing potato diseases. (1: negative control H 2 O;2: phytophthora infestans strain Pi;3: fusarium equisetum Fe;4: fusarium oxysporum Fo;5: fusarium graminearum Fg;6: rhizoctonia solani Rs;7: alternaria alternata As;8: layering fusarium Fp;9: pectobacterium nigrum Pba;10: ralstonia solanacearum Rs-2;11: streptomyces Ss;12: eschar Ss-2;13: fusarium sambucinum Fsam). And (3) checking the specificity of the LAMP method, and judging the result according to the color change in the reaction tube after reacting for 70 min.
(a) Color determination LAMP detection specificity color development diagram of phytophthora infestans. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(b) Color determination LAMP detection specificity color map of alternaria alternata. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(c) Color determination specific color development of LAMP detection of Fusarium oxysporum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(d) Color determination specific color development diagram for LAMP detection of Fusarium equisetum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(e) Color determination specific color development diagram for LAMP detection of Fusarium graminearum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(f) And (5) judging the color, and obtaining a specificity color development chart of fusarium by the LAMP detection layer. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(g) Color judgment specific color development chart for LAMP detection of Fusarium sambucinum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(h) Color determination LAMP detection of rhizoctonia solani specific color development. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(i) Color determination LAMP detection of specific color profile of ralstonia solanacearum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(j) Color judgment specific color development diagram for LAMP detection of pectobacterium nigrum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(k) Color determination LAMP detection streptomyces specific color map. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
(l) Color determination LAMP detection eschar specificity color chart. The positive reaction appeared yellow-green and the negative control appeared orange-yellow.
FIG. 2 shows the sensitivity of LAMP detection of Phytophthora infestans, alternaria alternata, fusarium oxysporum, fusarium equisetum, fusarium graminearum, fusarium sambucinum, rhizoctonia solani, ralstonia solanacearum, pectobacterium nigeldanum, streptomyces and Fusarium verrucosum.
Wherein, LAMP amplifies genome DNA with different concentrations; the reaction systems from left to right each contained 1ng, 100pg, 10pg, and 1pg of DNA as amplification results.
(a) Color determination sensitivity color chart of LAMP detection of Phytophthora infestans. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(b) Color determination sensitivity color chart of LAMP detection of Alternaria alternata. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(c) Color determination sensitivity color chart of LAMP detection of Fusarium oxysporum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(d) Color determination sensitivity color development diagram for LAMP detection of Fusarium equisetum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 100pg.
(e) Color determination sensitivity color chart of LAMP detection of Fusarium graminearum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(f) And (5) judging the color, and obtaining a sensitivity color development chart of fusarium by the LAMP detection layer. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(g) Color determination sensitivity color development diagram for LAMP detection of Fusarium sambucinum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1ng.
(h) Color determination sensitivity color development diagram for LAMP detection of Rhizoctonia solani. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(i) Color determination sensitivity color chart of LAMP detection of Ralstonia solanacearum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 1pg.
(j) Color determination sensitivity color chart of LAMP detection of pectobacterium nigrum. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 100pg.
(k) Color determination sensitivity color chart of LAMP detection Streptomyces. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 100pg.
(l) Color determination sensitivity color chart of LAMP detection of eschar. The positive reaction appeared yellow-green and the negative control appeared orange-yellow. The color development showed that the sensitivity of the LAMP reaction reached 10pg.
FIG. 3 is a diagram showing the design of specific primers FIP, BIP, F, B3 and LB of the Phytophthora infestans LAMP molecular detection primer set.
FIG. 4 is a design diagram of specific primers FIP, BIP, F, B3 and LF of Alternaria alternata LAMP molecular detection primer set.
FIG. 5 is a design diagram of specific primers FIP, BIP, F, B3 and LF of fusarium oxysporum LAMP molecular detection primer sets.
FIG. 6 is a design diagram of specific primers FIP, BIP, F, B3, LF and LB of the fusarium equiseti LAMP molecular detection primer set.
FIG. 7 is a schematic diagram of specific primers FIP, BIP, F, B3, LF and LB of fusarium graminearum LAMP molecular detection primer set.
FIG. 8 is a design drawing of specific primers FIP, BIP, F, B3, LF and LB of the fusarium LAMP molecular detection primer group.
Fig. 9 is a design diagram of specific primers FIP, BIP, F, B3 and LF of a fusarium sambucus LAMP molecular detection primer set.
FIG. 10 is a diagram showing the design of specific primers FIP, BIP, F, B3 and LB of the LAMP molecular detection primer set for Rhizoctonia solani.
FIG. 11 is a diagram showing the design of specific primers FIP, BIP, F, B3, LF and LB of the LAMP molecular detection primer group for Ralstonia solanaceae.
FIG. 12 is a design diagram of specific primers FIP, BIP, F, B3, LF and LB of the pectobacterium nigrum LAMP molecular detection primer set.
FIG. 13 is a diagram showing the design of specific primers FIP, BIP, F, B3 and LB of the Streptomyces LAMP molecular detection primer set.
FIG. 14 shows the design of specific primers FIP, BIP, F, B3, LF and LB of the LAMP molecular detection primer set for eschar.
FIG. 15 shows a sample I of detection of the presence of twelve pathogenic bacteria in field-affected potato leaves in Du-city, guizhou, by the LAMP system.
(1: phytophthora infestans strain Pi;2: alternaria alternata As;3: fusarium equisetum Fe;4: fusarium graminearum Fg;5: fusarium oxysporum Fo;6: fusarium layering Fp;7: rhizoctonia solani Rs;8: ralstonia solanaceae Rs-2;9: pectobacter nigrum Pba;10: streptomyces Ss;11: fusarium roseum Ss-2;12: fusarium sambucus Ffamum). And detecting a field sample by using the LAMP system, and judging the result according to the color change in the reaction tube after reacting for 70 min.
FIG. 16 is a graph showing the detection of the presence of twelve pathogenic bacteria in field-affected potato leaves in Du-city, guizhou, using the LAMP system-sample two.
(1: phytophthora infestans strain Pi;2: alternaria alternata As;3: fusarium equisetum Fe;4: fusarium graminearum Fg;5: fusarium oxysporum Fo;6: fusarium layering Fp;7: rhizoctonia solani Rs;8: ralstonia solanaceae Rs-2;9: pectobacter nigrum Pba;10: streptomyces Ss;11: fusarium roseum Ss-2;12: fusarium sambucus Ffamum). And detecting a field sample by using the LAMP system, and judging the result according to the color change in the reaction tube after reacting for 70 min.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described with reference to preferred embodiments, and the described embodiments are only some embodiments of the present invention, but not all, and the present invention is not limited by the following preferred embodiments.
The reagents used in the examples of the present invention are commercially available, and the test methods used are conventional methods unless otherwise specified.
Example 1 kit Components
Twelve potato disease pathogenic bacteria detection kits comprise the following components:
five specific primers FIP, BIP, F, B3 and LB for phytophthora infestans LAMP molecular detection:
f3 (forward outer primer): GCTAAGTGATGGACCGCTT;
b3 (reverse outer primer): AGCCATCATCATGAATGCCT;
FIP (forward inner primer) (f1c+f2): TTGGCCGTTAGATCGCTCTTGT-TGATTTGCAGATACGCCTGT;
BIP (reverse inner primer) (b1c+b2): CCGACGCCGCCAAGGAATTT-GTTCTTCGCACTGGTCTCC;
LB (reverse loop primer): AGCAGCTTGTTCACGTTCTC
Five specific primers FIP, BIP, F3, B3 and LF for detecting Alternaria alternata LAMP molecules:
f3 (forward outer primer): GCTTGAGGATCACAAGTGGT;
b3 (reverse outer primer): TGGGAAGAGTGGTGGTGG;
FIP (forward inner primer) (f1c+f2): CTTCTGCCTCAGGAGCAGGC-ACTCTTGCGGCGAGAACA;
BIP (reverse inner primer) (b1c+b2): CGACGAGTAAGTTGCCCTCGTG-CGACGTAGGTGATGCTGGA;
LF (forward loop primer): TCGAAAGAGAAGTCCATGAAGC.
Five specific primers FIP, BIP, F3, B3 and LF for fusarium oxysporum LAMP molecular detection:
f3 (forward outer primer): GCGTTTGCCCTCTTACCATT;
b3 (reverse outer primer): GCATGAGCGACAACATACCA;
FIP (forward inner primer) (f1c+f2): CGAGCTCAGCGGCTTCCTATT-CACAACCTCAATGAGTGCGT;
BIP (reverse inner primer) (b1c+b2): TTCTTGACAAGCTCAAGGCCGA-AGGAGTCTCGAACTTCCAGA;
LF (forward loop primer): GACTGCTTCACACGTGACG.
Six specific primers FIP, BIP, F3, B3, LF and LB for detecting fusarium equiseti LAMP molecules:
f3 (forward outer primer): TGCATAGACCGGTCACTTGA;
b3 (reverse outer primer): GCCCCACCAAAAAATTACGG;
FIP (forward inner primer) (f1c+f2): GCGTGCGATCGAGGAAAATGGA-TACCAGTGCGGTGGTATCG;
BIP (reverse inner primer) (b1c+b2): CCTCTGCCCATCGATCCAGC-TGTACTCGAGCGGGGTAAC;
LF (forward loop primer): ACTTCTCGATGGTTCGCTTGT;
LB (reverse loop primer): ACCCGAATCAGTCTCGACG.
Six specific primers FIP, BIP, F3, B3, LF and LB for detecting fusarium graminearum LAMP molecules:
f3 (forward outer primer): GAATCGCCCTCACACGAC;
b3 (reverse outer primer): AGGAACCCTTACCGAGCT;
FIP (forward inner primer) (f1c+f2): TGAGCCCCACCGGGAAAAAAAT-CGATACGCGCCTGTTACC;
BIP (reverse inner primer) (b1c+b2): GTCTGCCCTCTTCCCACAAACC-GACAGGTGGTTAGTGACTGG;
LF (forward loop primer): CAAAATTTTTGACCTCGAGCGG;
LB (reverse loop primer): CGCTCATCATCACGTGTCAA.
Six specific primers FIP, BIP, F3, B3, LF and LB for fusarium LAMP molecular detection are layered:
f3 (forward outer primer): CGTTCGAGAACCCGCATAT;
b3 (reverse outer primer): TCTCAAGGGCATAGCCGATT;
FIP (forward inner primer) (f1c+f2): GTGAGCTGGCAACTCCACTGTA-ACCTGGGATCATTGCGACA;
BIP (reverse inner primer) (b1c+b2): TGGCTTGCATCAGAGGAAGCT-CAGAAGCTCTTGGGCGTC;
LF (forward loop primer): ACTGAGGTCGGGATTGATTCC;
LB (reverse loop primer): GAGTTCTTAAAGGGCAAGTTCACC.
Five specific primers FIP, BIP, F3, B3 and LF for detecting fusarium sambucus LAMP molecules:
f3 (forward outer primer): ACCGGTCACTTGATCTACCA;
b3 (reverse outer primer): GCTTTAGAGGAAGGGCATGT;
FIP (forward inner primer) (f1c+f2): GAAAGTAGGGCGCGCGATCG-CAAGCGAACCATCGAGAAGT;
BIP (reverse inner primer) (b1c+b2): GACTCGACACACGCCTGCTAC-GGGTATGAGCCCCACCAA;
LF (forward loop primer): GAAAATGAGACCAACCTTCTCGA.
Five specific primers FIP, BIP, F3, B3 and LB for LAMP molecular detection of rhizoctonia solani:
f3 (forward outer primer): GGTATTGGAGGTCTTTTGCA;
b3 (reverse outer primer): AGATCAGATCATAAAGGTATTGTC;
FIP (forward inner primer) (f1c+f2): TTATCACGCCGAGTGGAACC-GCTCCTCTTTGTTCATTAGCT;
BIP (reverse inner primer) (b1c+b2): TCTATCGCTGAGGACACTGTAA-AAGTCAATGGACTATTAGAAGC;
LB (reverse loop primer): AAGGTGGCCAAGGTAAATGC.
Six specific primers FIP, BIP, F3, B3, LF and LB for LAMP molecular detection of Ralstonia solanaceae:
f3 (forward outer primer): CGACCTGAGGGTGAAAGTG;
b3 (reverse outer primer): TGTCCAAAATTCCCCACTGC;
FIP (forward inner primer) (f1c+f2): GGTAGGCCTTTACCCCACCAAC-CGCAAGGCCTCATGCTAT;
BIP (reverse inner primer) (b1c+b2): AAGGCGACGATCAGTAGCTGGT-TGCCTCCCGTAGGAGTCT;
LF (forward loop primer): ATCAGACATCGGCCGCTCC;
LB (reverse loop primer): CTGAGAGGACGATCAGCCA.
Six specific primers FIP, BIP, F3, B3, LF and LB for LAMP molecular detection of pectobacterium nigrum:
f3 (forward outer primer): GGCGGTATCAAGGCATTCG;
b3 (reverse outer primer): GTATTCAGCGTACGGGTCAT;
FIP (forward inner primer) (f1c+f2): TCCACGCCGATGTCATCTTTCA-ACCTGAACCGTAACAAGACG;
BIP (reverse inner primer) (b1c+b2): TGCAGTGGAACGATGGTTTCCA-AAGTGTGTACCACCATCACG;
LF (forward loop primer): ACACGTTCGGGTGGATTGG;
LB (reverse loop primer): TACTGCTTTACCAACAATATTCCGC.
Five specific primers FIP, BIP, F, B3 and LB for LAMP molecular detection of streptomycete:
f3 (forward outer primer): TGACTCTCTCTTCGCTGACC;
b3 (reverse outer primer): TCGAAGGAGATCAGCACGA;
FIP (forward inner primer) (f1c+f2): TCAAGACGTTCGCTGACGCG-ATTCAGCATTGCAGAGGGCA;
BIP (reverse inner primer) (b1c+b2): CGCGCAGCAGATCGGGACT-TGACCGCATCCGACAGTC;
LB (reverse loop primer): GAGGTTGTCTTCGGCGAGGG.
Six specific primers FIP, BIP, F3, B3, LF and LB for LAMP molecular detection of eschar
F3 (forward outer primer): GGTTCCCACAACGATGAAGA;
b3 (reverse outer primer): CTTTCAAGCCATGGACCGA;
FIP (forward inner primer) (f1c+f2): CGAAAGCGCAACTTGCGTTCAA-GCAGCGAAATGCGATACGT;
BIP (reverse inner primer) (b1c+b2): AGCATGCCTCTTTGAGTGTCGG-CCAGAGCTCATAGTCCCCTT;
LF (forward loop primer): GATTCACTGAATTCTGCAATTCGC;
LB (reverse loop primer): TTTCTATTCTCCCGGAAACGCCTG.
Kit reaction system
1mL of the detection solution includes: 20. Mu.M forward inner primer FIP, 20. Mu.M reverse inner primer BIP, 10. Mu.M forward outer primer F3, 10. Mu.M reverse outer primer B3, 10. Mu.M forward loop primer LF, 10. Mu.M reverse loop primer LB, 10 XBuffer, 10mM dNTPs, 50mM MgSO 4 5M betaine, bst DNA polymerase U/. Mu.L, and ultrapure water were added to prepare 1mL of a detection solution. The shelf life was 1 year.
Example 2 Presence of twelve pathogenic bacteria in field-affected potato leaves in Du-even City, guizhou province
The method for detecting twelve pathogenic bacteria by using the twelve potato disease pathogenic bacteria detection kit comprises the following steps:
1) Collecting field samples:
the subject group randomly collects diseased potato leaves from the field of Du city in Guizhou province in 2021, adopts a five-point sampling method, randomly selects 5 points for sampling at each place 4-10 steps away from four sides of the field, and puts the potato leaves into a plastic package bag for independent storage.
2) Isolation of potato disease pathogens on diseased tissue:
washing fresh leaves with tap water, sucking water, placing a filter paper sheet at the bottom of a culture dish with the diameter of 90mm, spraying a little sterilized water, placing the back surface of the washed fresh leaves upwards on the wet filter paper sheet, and filling the gun head to suspend the tissues. Moisturizing the diseased potato leaf for about 24 hours until a large amount of white mold layer grows at the diseased spots, then picking hyphae, inoculating the hyphae on a rye selective culture medium (containing antibiotics ampicillin, rifampicin and pentachloronitrobenzene) plate and a PDA culture medium plate, and separating oomycetes and fungi.
Cutting diseased potato leaves, placing the crushed potato leaves into a 50mL centrifuge tube containing sterilized water, vibrating the potato leaves by using a vortex oscillator to obtain microorganism suspension, then performing a series of dilutions (such as 1:10, 1:100, 1:1000 and 1:10000), dipping a small amount of microorganism suspension to be separated by using an inoculating loop in a sterile operation mode, continuously scribing on the surface of a sterile flat plate, reducing the number of microorganism cells along with the increase of scribing times, and picking single colonies on the surface of the flat plate for separating bacteria.
3) LAMP detection of diseased potato leaf, comprising:
(1) Extraction of DNA from diseased potato leaves: extracting genome of potato leaf with disease spots by using a novel plant genome extraction kit (Tiangen DP320-100 times), wherein the method is in accordance with the specification;
(2) LAMP detection of different pathogens: taking 4 mu L of field sample DNA solution, adding 18 mu L of kit solution and 3 mu L of sterilized deionized water, and keeping the total volume to be 25 mu L;
(3) The reaction procedure is: 70min at 62 ℃;
(4) Detection of amplified products: detecting twelve potato disease pathogenic bacteria on the diseased potato leaves by using an LAMP system, adding SYBR Green I (fluorescent dye) into the LAMP amplified product as a reaction indicator, and taking the color change of the fluorescent dye as a result judgment standard. The yellow-green color indicates that the detection is positive, the target pathogenic bacteria exist, and the orange-yellow color indicates that the detection result is negative. By examining two diseased potato leaves in Duzhou city, guizhou province, it was found that Phytophthora infestans, alternaria alternata, fusarium graminearum, ralstonia solanacearum and pectobacterium nigrum could be detected (see FIGS. 15 and 16).
4) Sequencing and verifying isolated pathogenic bacteria
Extracting DNA from separated field pathogenic bacteria with different forms, designing corresponding primers according to fungus conserved ITS genes and bacterial conserved 16S rRNA genes, performing Blast comparison on sequenced sequence information on NCBI, and determining the types of the separated pathogenic bacteria. And comparing the LAMP detection result of the field sample with the separated pathogenic bacteria to verify the accuracy of the LAMP primer, and determining and separating phytophthora infestans, alternaria alternata and fusarium graminearum after sequencing and comparison. The LAMP detection system can detect the Ralstonia solanacearum and the pectobacterium nigrum, but the two pathogenic bacteria cannot be separated later, and the reasons are analyzed: the isolated material is diseased potato leaf, potato black shank is stem disease, potato bacterial wilt is systemic disease endangering potato stems and potato blocks, ralstonia solanaceae and pectobacterium nigrum are not main bacteria on diseased potato leaf, and are difficult to separate on leaf.
Sequence listing
<110> Nanjing agricultural university
<120> primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method
<160> 66
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gctaagtgat ggaccgctt 19
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
agccatcatc atgaatgcct 20
<210> 3
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ttggccgtta gatcgctctt gttgatttgc agatacgcct gt 42
<210> 4
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ccgacgccgc caaggaattt gttcttcgca ctggtctcc 39
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
agcagcttgt tcacgttctc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gcttgaggat cacaagtggt 20
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tgggaagagt ggtggtgg 18
<210> 8
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
cttctgcctc aggagcaggc actcttgcgg cgagaaca 38
<210> 9
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cgacgagtaa gttgccctcg tgcgacgtag gtgatgctgg a 41
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tcgaaagaga agtccatgaa gc 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gcgtttgccc tcttaccatt 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gcatgagcga caacatacca 20
<210> 13
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
cgagctcagc ggcttcctat tcacaacctc aatgagtgcg t 41
<210> 14
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ttcttgacaa gctcaaggcc gaaggagtct cgaacttcca ga 42
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gactgcttca cacgtgacg 19
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tgcatagacc ggtcacttga 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gccccaccaa aaaattacgg 20
<210> 18
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gcgtgcgatc gaggaaaatg gataccagtg cggtggtatc g 41
<210> 19
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
cctctgccca tcgatccagc tgtactcgag cggggtaac 39
<210> 20
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
acttctcgat ggttcgcttg t 21
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
acccgaatca gtctcgacg 19
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gaatcgccct cacacgac 18
<210> 23
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
aggaaccctt accgagct 18
<210> 24
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tgagccccac cgggaaaaaa atcgatacgc gcctgttacc 40
<210> 25
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gtctgccctc ttcccacaaa ccgacaggtg gttagtgact gg 42
<210> 26
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
caaaattttt gacctcgagc gg 22
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
cgctcatcat cacgtgtcaa 20
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
cgttcgagaa cccgcatat 19
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
tctcaagggc atagccgatt 20
<210> 30
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gtgagctggc aactccactg taacctggga tcattgcgac a 41
<210> 31
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tggcttgcat cagaggaagc tcagaagctc ttgggcgtc 39
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
actgaggtcg ggattgattc c 21
<210> 33
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gagttcttaa agggcaagtt cacc 24
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
accggtcact tgatctacca 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gctttagagg aagggcatgt 20
<210> 36
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gaaagtaggg cgcgcgatcg caagcgaacc atcgagaagt 40
<210> 37
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gactcgacac acgcctgcta cgggtatgag ccccaccaa 39
<210> 38
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gaaaatgaga ccaaccttct cga 23
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ggtattggag gtcttttgca 20
<210> 40
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
agatcagatc ataaaggtat tgtc 24
<210> 41
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ttatcacgcc gagtggaacc gctcctcttt gttcattagc t 41
<210> 42
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
tctatcgctg aggacactgt aaaagtcaat ggactattag aagc 44
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
aaggtggcca aggtaaatgc 20
<210> 44
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cgacctgagg gtgaaagtg 19
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
tgtccaaaat tccccactgc 20
<210> 46
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
ggtaggcctt taccccacca accgcaaggc ctcatgctat 40
<210> 47
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
aaggcgacga tcagtagctg gttgcctccc gtaggagtct 40
<210> 48
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
atcagacatc ggccgctcc 19
<210> 49
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
ctgagaggac gatcagcca 19
<210> 50
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
ggcggtatca aggcattcg 19
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
gtattcagcg tacgggtcat 20
<210> 52
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
tccacgccga tgtcatcttt caacctgaac cgtaacaaga cg 42
<210> 53
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
tgcagtggaa cgatggtttc caaagtgtgt accaccatca cg 42
<210> 54
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
acacgttcgg gtggattgg 19
<210> 55
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
tactgcttta ccaacaatat tccgc 25
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
tgactctctc ttcgctgacc 20
<210> 57
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
tcgaaggaga tcagcacga 19
<210> 58
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
tcaagacgtt cgctgacgcg attcagcatt gcagagggca 40
<210> 59
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
cgcgcagcag atcgggactt gaccgcatcc gacagtc 37
<210> 60
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
gaggttgtct tcggcgaggg 20
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ggttcccaca acgatgaaga 20
<210> 62
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
ctttcaagcc atggaccga 19
<210> 63
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
cgaaagcgca acttgcgttc aagcagcgaa atgcgatacg t 41
<210> 64
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
agcatgcctc tttgagtgtc ggccagagct catagtcccc tt 42
<210> 65
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
gattcactga attctgcaat tcgc 24
<210> 66
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
tttctattct cccggaaacg cctg 24

Claims (6)

1. The LAMP primer composition for detecting twelve potato disease pathogens is characterized by comprising the following primer groups (1) - (12): (1) a phytophthora infestans detection primer set; (2) a set of Alternaria alternata detection primers; (3) fusarium oxysporum detection primer set; (4) a fusarium equiseti detection primer set; (5) Fusarium graminearum Gu Mu detection primer set; (6) layering out a fusarium detection primer group; (7) a fusarium sambucus detection primer set; (8) a rhizoctonia solani detection primer set; (9) a primer set for detecting Ralstonia solanaceae; (10) pectobacterium nigrum detection primer set; (11) a Streptomyces detection primer set; (12) a eschar detecting primer set;
the phytophthora infestans detection primer group consists of five specific primers F3, B3, FIP, BIP and LB:
F3:GCTAAGTGATGGACCGCTT;
B3:AGCCATCATCATGAATGCCT;
FIP:TTGGCCGTTAGATCGCTCTTGTTGATTTGCAGATACGCCTGT;
BIP:CCGACGCCGCCAAGGAATTTGTTCTTCGCACTGGTCTCC;
LB:AGCAGCTTGTTCACGTTCTC;
the Alternaria alternata detection primer group consists of five specific primers F3, B3, FIP, BIP and LF:
F3:GCTTGAGGATCACAAGTGGT;
B3:TGGGAAGAGTGGTGGTGG;
FIP:CTTCTGCCTCAGGAGCAGGCACTCTTGCGGCGAGAACA;
BIP:CGACGAGTAAGTTGCCCTCGTGCGACGTAGGTGATGCTGGA;
LF:TCGAAAGAGAAGTCCATGAAGC;
the fusarium oxysporum detection primer group consists of five specific primers F3, B3, FIP, BIP and LF:
F3:GCGTTTGCCCTCTTACCATT;
B3:GCATGAGCGACAACATACCA;
FIP:CGAGCTCAGCGGCTTCCTATTCACAACCTCAATGAGTGCGT;
BIP:TTCTTGACAAGCTCAAGGCCGAAGGAGTCTCGAACTTCCAGA;
LF:GACTGCTTCACACGTGACG;
the fusarium equiseti detection primer group consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:TGCATAGACCGGTCACTTGA;
B3:GCCCCACCAAAAAATTACGG;
FIP:GCGTGCGATCGAGGAAAATGGATACCAGTGCGGTGGTATCG;
BIP:CCTCTGCCCATCGATCCAGCTGTACTCGAGCGGGGTAAC;
LF:ACTTCTCGATGGTTCGCTTGT;
LB:ACCCGAATCAGTCTCGACG;
the fusarium graminearum Gu Mu detection primer group consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:GAATCGCCCTCACACGAC;
B3:AGGAACCCTTACCGAGCT;
FIP:TGAGCCCCACCGGGAAAAAAATCGATACGCGCCTGTTACC;
BIP:GTCTGCCCTCTTCCCACAAACCGACAGGTGGTTAGTGACTGG;
LF:CAAAATTTTTGACCTCGAGCGG;
LB:CGCTCATCATCACGTGTCAA;
the fusarium detecting primer group consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:CGTTCGAGAACCCGCATAT;
B3:TCTCAAGGGCATAGCCGATT;
FIP:GTGAGCTGGCAACTCCACTGTAACCTGGGATCATTGCGACA;
BIP:TGGCTTGCATCAGAGGAAGCTCAGAAGCTCTTGGGCGTC;
LF:ACTGAGGTCGGGATTGATTCC;
LB:GAGTTCTTAAAGGGCAAGTTCACC;
the fusarium sambucus detection primer group consists of five specific primers F3, B3, FIP, BIP and LF:
F3:ACCGGTCACTTGATCTACCA;
B3:GCTTTAGAGGAAGGGCATGT;
FIP:GAAAGTAGGGCGCGCGATCGCAAGCGAACCATCGAGAAGT;
BIP:GACTCGACACACGCCTGCTACGGGTATGAGCCCCACCAA;
LF:GAAAATGAGACCAACCTTCTCGA;
the rhizoctonia solani detection primer group consists of five specific primers F3, B3, FIP, BIP and LB:
F3:GGTATTGGAGGTCTTTTGCA;
B3:AGATCAGATCATAAAGGTATTGTC;
FIP:TTATCACGCCGAGTGGAACCGCTCCTCTTTGTTCATTAGCT;
BIP:TCTATCGCTGAGGACACTGTAAAAGTCAATGGACTATTAGAAGC;
LB:AAGGTGGCCAAGGTAAATGC;
the primer group for detecting the Ralstonia solanaceae consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:CGACCTGAGGGTGAAAGTG;
B3:TGTCCAAAATTCCCCACTGC;
FIP:GGTAGGCCTTTACCCCACCAACCGCAAGGCCTCATGCTAT;
BIP:AAGGCGACGATCAGTAGCTGGTTGCCTCCCGTAGGAGTCT;
LF:ATCAGACATCGGCCGCTCC;
LB:CTGAGAGGACGATCAGCCA;
the pectobacterium nigrum detection primer set consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:GGCGGTATCAAGGCATTCG;
B3:GTATTCAGCGTACGGGTCAT;
FIP:TCCACGCCGATGTCATCTTTCAACCTGAACCGTAACAAGACG;
BIP:TGCAGTGGAACGATGGTTTCCAAAGTGTGTACCACCATCACG;
LF:ACACGTTCGGGTGGATTGG;
LB:TACTGCTTTACCAACAATATTCCGC;
the streptomycete detection primer group consists of five specific primers F3, B3, FIP, BIP and LB:
F3:TGACTCTCTCTTCGCTGACC;
B3:TCGAAGGAGATCAGCACGA;
FIP:TCAAGACGTTCGCTGACGCGATTCAGCATTGCAGAGGGCA;
BIP:CGCGCAGCAGATCGGGACTTGACCGCATCCGACAGTC;
LB:GAGGTTGTCTTCGGCGAGGG;
the eschar detection primer group consists of six specific primers F3, B3, FIP, BIP, LF and LB:
F3:GGTTCCCACAACGATGAAGA;
B3:CTTTCAAGCCATGGACCGA;
FIP:CGAAAGCGCAACTTGCGTTCAAGCAGCGAAATGCGATACGT;
BIP:AGCATGCCTCTTTGAGTGTCGGCCAGAGCTCATAGTCCCCTT;
LF:GATTCACTGAATTCTGCAATTCGC;
LB:TTTCTATTCTCCCGGAAACGCCTG。
2. the use of the LAMP primer composition of claim 1 for detecting twelve potato disease pathogens.
3. The use of the LAMP primer composition of claim 1 in the preparation of a kit for detecting twelve potato disease pathogens.
4. A LAMP kit for detecting twelve potato disease pathogens, comprising the LAMP primer composition of claim 1.
5. The use of the LAMP kit of claim 4 for detecting twelve pathogenic bacteria of potato diseases.
6. The LAMP detection method for twelve potato disease pathogens is characterized by adopting the LAMP kit as set forth in claim 4 to carry out LAMP reaction, wherein the LAMP reaction comprises the following procedures: 70min at 62 ℃; detection of amplified products: SYBR Green I is added into the product after isothermal amplification as a reaction indicator, and the color change of fluorescent dye is used as a result judgment standard; the yellow-green color indicates that the detection is positive, the target pathogenic bacteria exist, and the orange-yellow color indicates that the detection result is negative.
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