CN113430286B - LAMP (loop-mediated isothermal amplification) detection primer group and kit for pathogenic klebsiella of mulberry bacterial wilt, and application of LAMP detection primer group and kit - Google Patents
LAMP (loop-mediated isothermal amplification) detection primer group and kit for pathogenic klebsiella of mulberry bacterial wilt, and application of LAMP detection primer group and kit Download PDFInfo
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Abstract
The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer group and kit for pathogenic klebsiella of mulberry bacterial wilt and application thereof. The invention provides an LAMP primer group for detecting Klebsiella multocida, which has a nucleotide sequence shown as SEQ ID NO: 1-4; the nucleotide sequence of SEQ ID NO: 1-2 are outer primers; the SEQ ID NO: 3-4 are inner primers. The LAMP primer group and the kit thereof can be used for detecting the mulberry bacterial wilt disease infected with the Klebsiella and rhizosphere soil samples thereof, have good detection specificity and sensitivity, and the reaction result can be identified by naked eyes, so that the LAMP primer group and the kit thereof have important significance for quickly detecting the Klebsiella which is a pathogen of the mulberry bacterial wilt disease in mulberry gardens, have good application value in quarantine and prevention and treatment of the mulberry bacterial wilt disease, and are worthy of popularization.
Description
Technical Field
The invention relates to the technical field of plant pathogen molecular detection, in particular to a LAMP detection primer group and a kit for detecting pathogen Klebsiella of mulberry bacterial wilt and application thereof.
Background
The development of the silkworm mulberry industry is deeply troubled by mulberry wilt, and the early identification of pathogenic bacteria of the mulberry wilt is an important means for preventing and controlling the mulberry wilt. The mulberry bacterial wilt disease is characterized by being a whole-plant disease, wherein in the early stage of the disease, branch strips on the upper part wither and brown, the outer edge of leaves firstly loses water and rolls over, and then the whole mulberry bacterial wilt disease begins to fall off from top to bottom. When the epidermis of the stem and root of the diseased plant is cut open, the xylem is yellowed and browned to different degrees, brown stripes are generated, and when the xylem is seriously browned and blacked all over the body, the root is rotten.
Bacterial blight pathogens of mulberry are diverse and include Enterobacter sp., Klebsiella sp., and Pantoea sp. At present, the prior art does not determine which main pathogenic bacteria of the mulberry bacterial wilt disease are, and different pathogenic bacteria need corresponding detection methods. At present, the detection method of mulberry bacterial wilt pathogens mainly focuses on enterobacter and Pantoea, and for Klebsiella, the patent CN111073988A only discloses a group of PCR detection primers of the Klebsiella which is a pathogen of mulberry bacterial wilt pathogens, but the detection efficiency of the PCR primers is low, and the detection limit can only detect 5 × 10-7The genome of the Klebsiella at ng/mul is not suitable for the rapid detection in the field, and the detection result can not be quickly interpreted, which seriously influences the quarantine and control of the mulberry bacterial wilt disease. In order to more conveniently and quickly carry out early prevention and control on mulberry bacterial wilt diseases, a method or a product which is suitable for field detection and is convenient and quick for detecting the Klebsiella multocida is urgently needed at present.
Disclosure of Invention
The invention aims to solve the problem of low detection efficiency of mulberry bacterial wilt disease caused by Klebsiella multocida in the prior art, and provides a LAMP detection primer group and a kit for the pathogen Klebsiella multocida disease, and application of the LAMP detection primer group and the kit.
The first purpose of the invention is to provide an LAMP primer group for detecting Klebsiella multocida.
The second purpose of the invention is to provide the application of the primer group in detecting the Klebsiella mori and/or the mulberry bacterial wilt.
The third purpose of the invention is to provide the application of the primer group in the preparation of a kit for detecting the bacterial wilt disease of the Klebsiella mori and/or the mulberry.
The fourth purpose of the invention is to provide a detection kit for the Klebsiella pneumoniae from mulberry and/or the bacterial wilt of mulberry
The fifth purpose of the invention is to provide the application of the kit in detecting the Klebsiella mori and/or the mulberry bacterial wilt.
The sixth purpose of the invention is to provide a method for detecting the Klebsiella mori.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides an LAMP primer group for detecting Klebsiella multocida, which has a nucleotide sequence shown as SEQ ID NO: 1-4; the nucleotide sequence of SEQ ID NO: 1-2 are outer primers; the nucleotide sequence of SEQ ID NO: 3-4 are inner primers.
The outer primers are 2F3 and 2B3, the inner primers are 2FIP and 2BIP, and specific nucleotide sequences are shown as follows:
the invention claims the following applications:
the primer group is applied to detection of Klebsiella mulberensis and/or mulberry bacterial wilt.
The primer group is applied to the preparation of a kit for detecting the bacterial wilt disease of the mulberry and/or the Klebsiella pneumoniae of the mulberry.
Preferably, the mulberry bacterial wilt disease is caused by Klebsiella multocida; see example 7.
The invention also claims a detection kit for the Klebsiella pneumoniae from mulberry and/or the bacterial wilt disease of mulberry, and the kit comprises the LAMP primer group.
Preferably, the LAMP reaction reagent kit further comprises 2 × LAMP reaction buffer, Bst DNA polymerase, ddH2O, a fluorescent dye STYO 9 and a chromogenic dye SYBR Green I; see example 4.
More preferably still, the first and second liquid crystal compositions are,the 2 × LAMP reaction buffer contained 20mM Tris-HCl, pH8.8, 10mM KCl, 2mM MgSO4；20mM(NH4)2SO40.1% Triton X-100, 2.8mM dNTPs, 1M betaine, 25mM MgCl2(ii) a See example 4.
The application of the kit in detecting the Klebsiella multocida and/or the mulberry bacterial wilt disease is also within the protection range of the invention.
The invention also claims a method for detecting the Klebsiella multocida, which comprises the steps of taking DNA of a sample to be detected as a template, carrying out LAMP reaction by using the LAMP primer group, and judging whether the amplification product has the Klebsiella multocida or not by a real-time fluorescence curve method, a color development method or gel electrophoresis after the reaction is finished.
Preferably, the judging method is: after LAMP is finished, judging that the fluorescence curve is positive by detecting the fluorescence curve through a real-time fluorescence curve method, and judging that the fluorescence curve is negative when the fluorescence curve is not detected; or adding SYBR Green I into the reaction solution by a color development method, and observing that the reaction solution is Green and is judged to be positive, and the reaction solution is brown and is judged to be negative; or detecting the amplification product by gel electrophoresis, judging that multiple bands appear as positive, and judging that the multiple bands do not appear as negative; see example 4.
Preferably, when the LAMP reaction is carried out, the final concentration ratio of the outer primer to the inner primer of the LAMP primer group is 1: (4-10); see example 3.
More preferably, when the LAMP reaction is carried out, the final concentration ratio of the outer primer to the inner primer of the LAMP primer group is 1: 8; see example 3.
Preferably, the system of the LAMP reaction is: 12.5 μ L of 2 × LAMP reaction buffer, 0.5 μ L of Bst DNA polymerase, 0.1 μ M of each outer primer, 0.8 μ M of each inner primer, 1 μ L of DNA template, 90.5 μ L of fluorescent dye STYO, ddH2And O is supplemented to 25 mu L.
Preferably, the LAMP reaction is carried out for 60min at the constant temperature of 61-65 ℃; see example 2.
More preferably, the LAMP reaction is carried out at the constant temperature of 65 ℃ for 60 min; see example 2.
Compared with the prior art, the invention has the following beneficial effects:
the LAMP primer group and the kit thereof with high specificity and sensitivity for the Klebsiella multocida are designed, can be used for detecting the Klebsiella multocida-infected bacterial wilt disease plants and rhizosphere soil samples thereof, can be distinguished from the Klebsiella anthropogonis, have good detection specificity and sensitivity, and can be visually identified by reaction results, so that the LAMP primer group and the kit thereof have important significance for quickly detecting the Klebsiella multocida bacterial wilt pathogen bacteria in mulberry diseases, have good application value in quarantine and prevention and control of the mulberry bacterial wilt diseases, and are worthy of popularization.
Drawings
FIG. 1 shows the colony of Klebsiella as pathogen of Mulberry bacterial wilt in LB nutrient agar medium.
FIG. 2 is a real-time fluorescence plot of LAMP primer set screening; wherein the labels 1-4 are primer groups KL-1, KL-2, KL-3 and KL-4 respectively.
FIG. 3 is a real-time fluorescence plot for LAMP reaction optimal temperature screening.
FIG. 4 is a real-time fluorescence curve diagram of LAMP reaction primer ratio screening; wherein, the outer primers with the labels of 1-4: the proportion of the inner primers is 1: 2. 1: 4. 1: 8. 1: 10.
FIG. 5 is a real-time fluorescence curve diagram of LAMP reaction primer specificity experiment; wherein, the reference numerals 2 to 12 are Enterobacter cloacae (Enterobacter cloacae), Bacillus pumilus (Curtobacterium sp.), Azotobacter sp, Pseudomonas sp, Enterobacter pseudomonad (Pseudomonas sp), Pantoea agglomerans (Pantoea agglomerans), Laurella solanacearum (Ralstonia solanacearum), Enterobacter albuginella (Enterobacter albureae), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pantoea ananatis (Pantoea ananatis), and Paenibacillus polymyxa (Paenibacillus polymyxa), respectively, the reference numeral 1 is positive control Klebsiella pneumoniae (Klebsiella sp.) and the reference numeral 13 is negative control sterile water.
FIG. 6 is a real-time fluorescence curve diagram of the LAMP reaction primer sensitivity experiment; wherein the numbers 1 to 5 are respectively 2X 105cfu/mL、2×104cfu/mL、2×103cfu/mL、2×102cfu/mL, 20cfu/mL Klebsiella bacterial fluid.
FIG. 7 is a LAMP detection real-time fluorescence curve diagram of mulberry bacterial wilt disease samples in different regions;
FIG. 8 is a color development chart of LAMP detection results of mulberry bacterial wilt disease samples in different regions;
FIG. 9 is a LAMP detection result electrophoretogram of mulberry bacterial wilt disease sample in different areas;
in fig. 7 to 9, reference numeral 1 is positive control klebsiella mori, reference numeral 2 is total DNA of mulberry wilt branches in chemical city in Guangdong province, reference numeral 3 is total DNA of rhizosphere soil of mulberry wilt sample in chemical city in Guangdong province, reference numeral 4 is total DNA of ramulus mori wilt branches in Guangxi willow, reference numeral 5 is total DNA of ramulus mori rhizosphere soil of ramulus mori wilt disease in Guangxi willow, reference numeral 6 is total DNA of ramulus mori wilt disease branches in Guangdong Yangshan, reference numeral 7 is total DNA of rhizosphere soil of mulberry wilt sample in Guangdong Yangshan silkworm breeding farm, reference numeral 8 is total DNA of roots of mulberry wilt disease in Guangdong English silkworm breeding farm, reference numerals 9 to 12 are total DNA of roots of klebsiella tieback experiment wilt nursery stock, reference numeral 13 is total DNA of healthy mulberry sample, and reference numeral 14 is negative control sterile water.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 LAMP primer set primer design and screening
In the invention, the specific gene sequence of the Klebsiella mulberensis is obtained by comparison and analysis of the whole genome in the previous research, the gene codes a hydrolytic protease domain gene (hdp), a specific LAMP detection primer is designed based on the target gene,
first, experiment method
1. Extraction of Klebsiella genome DNA
The isolation of Klebsiella from the mulberry bacterial wilt disease strain is shown in FIG. 1, and its colony on LB nutrient agar medium. The extraction method of the Ezup columnar bacteria genome DNA extraction kit (Shanghai's worker) is adopted to extract the DNA of the Klebsiella mulberella, and the extraction method refers to the kit specification.
2. LAMP primer set primer design and screening
Specific gene segments encoding the hydrolase were selected, and primer design was performed using the online software PrimeExplorer V5 (http:// PrimeExplorer. jp/elamp4.0.0/index. html) to obtain 4 sets of primers, respectively as follows:
taking the genome DNA of the Klebsiella to be detected as a template, and respectively adopting the four groups of primers to respectively carry out LAMP amplification reaction, wherein the LAMP reaction system is as follows: reaction buffer 12.5. mu.L, Bst DNA polymerase 0.5. mu.L, inner primers FIP/BIP 0.8. mu.M each, outer primers F3/B3 0.1. mu.M each: (outer primer: inner primer: 1: 8), DNA template 1. mu.L, fluorescent dye STYO 90.5. mu.L, ddH2And O is supplemented to 25 mu L. The results were determined by fluorescence curve method.
Second, experimental results
The results of the test screening are shown in figure 2, the four groups of primer groups can amplify the bands of the Klebsiella multocida, and the primer groups KL-2 and KL-3 are about 20min, so that the fastest and fluorescence curve appears at the same time, and therefore the primer group KL-2 is selected as the primer for detecting the Klebsiella multocida.
Example 2 screening of optimal temperature for LAMP primer set amplification reaction
First, experiment method
Different LAMP amplification reaction temperatures of 61 ℃, 62 ℃, 63 ℃, 64 ℃ and 65 ℃ were designed, LAMP reaction was carried out using the primer set KL-2 and the amplification reaction system of example 1, and the results were determined by the fluorescence curve method.
Second, experimental results
As a result of experimental screening, as shown in FIG. 3, curves were amplified at all 5 temperatures, and the fluorescence threshold of the curves gradually increased with the increase in temperature, so 65 ℃ was selected as the optimum temperature for LAMP reaction. .
Example 3 screening of the proportion of the final concentration of the primers in the LAMP primer set amplification reaction
First, experiment method
Optimizing the final concentration ratios of different inner primers and different outer primers in the LAMP reaction system, and designing the following different final concentration ratios of the primers, (1) the outer primers: inner primer 1: 2; (2) an outer primer: inner primer 1: 4; (3) an outer primer: inner primer 1: 8; (4) an outer primer: inner primer 1: 10. the result was determined by a fluorescence curve method using LAMP reaction performed using the primer set KL-2 of example 1 and the amplification reaction system.
Second, experimental results
Results are screened by experiments, the results are shown in fig. 4, 4 primers with different proportions can amplify curves, curves 2, 3 and 4 appear faster, but curve 3 appears fastest, and the threshold is highest, so that the inner primers are selected in the LAMP amplification reaction system finally: the concentration ratio of the outer primer is that: inner primer 1: 8.
example 4 detection kit for Morus pneumoniae
Composition of detection kit
A detection kit for Klebsiella pneumoniae of Morus origin comprises a primer set KL-2 of embodiment 1:
further comprises 2 × LAMP reaction buffer, Bst DNA polymerase, ddH2O, a fluorescent dye STYO 9 and a chromogenic dye SYBR Green I. Wherein the 2 × LAMP reaction buffer contains 20mM Tris-HCl, pH8.8, 10mM KCl, 2mM MgSO4；20mM(NH4)2SO40.1% Triton X-100, 2.8mM dNTPs, 1M betaine, 25mM MgCl2。
Second, the application method
Taking sample DNA as a template, and adopting a kit to perform LAMP reaction, wherein the amplification reaction system is as follows: reaction buffer 12.5. mu.L, DNA polymerase 0.5. mu.L, STYO 9 dye 0.5. mu.L, final concentrations of inner primers 2FIP/2BIP each 0.8. mu.M, outer primers 2F3/2B3 each 0.1. mu.M, DNA template 1. mu.L, ddH2Supplementing O to 25 mu L; the amplification reaction procedure was: reacting at constant temperature of 65 ℃ for 60 min.
And judging whether the amplification product has the Klebsiella through a real-time fluorescence curve method, a color development method or gel electrophoresis after the reaction is finished. The judging method comprises the following steps: the real-time fluorescence curve method detects that the fluorescence curve is judged to be positive, and the fluorescence curve which is not detected is judged to be negative; or adding SYBR Green I into the reaction solution by a color development method, and observing that the reaction solution is Green and is judged to be positive, and the reaction solution is brown and is judged to be negative; or detecting the amplification product by gel electrophoresis, judging the multiple bands to be positive if the multiple bands appear, and judging the multiple bands to be negative if the multiple bands do not appear.
Example 5 specific detection of LAMP primer set
First, experiment method
The method comprises the steps of using genome DNA of 12 strains of Klebsiella mori (Klebsiella sp.), Enterobacter cloacae (Enterobacter cloacae), Brevibacterium (Curtobacterium sp.), azotobacter (Kosakonia sp.), Pseudomonas sp, Pantoea agglomerans (Pantoea agglomerans), Lawsonia solani (Ralstonia solanacearum), Enterobacter albuginella (Enterobacter albureae), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pantoea ananatis (Pantoea ananatis), Paenibacillus polymyxa (Paenii bacillus polymyxa) as templates (1, 2-12), using genome DNA of Klebsiella (Klebsiella sp.) as a positive marker, using a PCR positive reference (LAMP 13) as a reference, and performing detection by using a comparison method (LAMP 13).
Second, experimental results
The results are shown in FIG. 5, except that the amplification curve of the genomic DNA of the positive control bacterium Klebsiella appears, no amplification curve of the other bacterial genomic DNAs appears, which indicates that the primer group KL-2 has good specificity and can be used for specifically detecting the Klebsiella mori.
Example 6 detection of sensitivity of LAMP primer set
First, experiment method
Diluting Klebsiella bacteria liquid to 2 × 10 respectively5cfu/mL、2×104cfu/mL、2×103cfu/mL、2×102cfu/mL and 20cfu/mL (1-5), heating and cracking at 100 ℃ for 10min, centrifuging and taking the supernatant, and performing LAMP detection by using the detection kit and the method in the embodiment 4.
Second, experimental results
The results are shown in FIG. 6, when the concentration of bacterial liquid reaches 2X 102When cfu/mL, the fluorescence curve appears after about 40min, and when the bacterial liquid concentration is diluted to 20cfu/mL, the fluorescence curve can not be detected, so that the lowest detection limit of KL-2 of the primer group is 2 multiplied by 102cfu/mL of Klebsiella bacterial liquid. The detection sensitivity of the LAMP primer group to the Klebsiella is higher.
Example 7 detection of Mulberry wilt disease Strain samples in different regions
First, experiment method
In order to rapidly detect the mulberry bacterial wilt disease caused by the Klebsiella, total DNA (deoxyribonucleic acid) of soil samples of xylem, root and rhizosphere of the mulberry wilt disease collected in different areas is extracted to be used as a sample template of a mulberry wilt disease plant (reference numerals 2-12, the extraction method refers to example 1), a healthy mulberry sample and sterile water are used as negative controls (reference numerals 13-14), the Klebsiella pneumoniae of Moraceae is used as a positive control (reference numeral 1), and LAMP detection is carried out by adopting the detection kit and the method of example 4. And judging the result by a real-time fluorescence curve method, a color development method and gel electrophoresis after the reaction is finished.
The number 2 is the total DNA of the mulberry wilt disease branches in the Huazhou city of Guangdong province, the number 3 is the total DNA of the rhizosphere soil of the mulberry wilt disease sample in the Huazhou city of Guangdong province, the number 4 is the total DNA of the Tuzhou Gongan mulberry wilt disease branches, the number 5 is the total DNA of the rhizosphere soil of the Guangxi Liuzhou Gongan mulberry wilt disease branches, the number 6 is the total DNA of the Guangdong Yanshan mulberry wilt disease branches, the number 7 is the total DNA of the rhizosphere soil of the Guangdong Yanshan mulberry wilt disease sample, the number 8 is the total DNA of the roots of the Guangdong Yangdong silkworm wilt disease branches, and the numbers 9-12 are the total DNA of the roots of the wilt nursery stock for the Klebsiella tieback test.
Second, experimental results
The real-time fluorescence curve method judgment result is shown in figure 7, the positive control and mulberry wilt disease plant samples (labels 1-12) can detect the fluorescence curve, and the healthy mulberry sample and sterile water (labels 13-14) can not detect the fluorescence curve; the result of the color development method is shown in figure 8, the color development results of the positive control and mulberry wilt disease plant samples (1-12) show positive (green), while the color development results of the healthy mulberry sample and sterile water (13-14) show negative (brown), and the results are consistent with the fluorescence curve method; the results of the gel electrophoresis are shown in FIG. 9, where the positive control and the mulberry wilt disease plant sample (reference numerals 1-12) showed multiple bands, while the healthy mulberry sample and the sterile water (reference numerals 13-14) showed no bands. The LAMP primer group and the kit thereof can be used for detecting the mulberry bacterial wilt disease infected with the Klebsiella and rhizosphere soil samples thereof, have important significance for quickly detecting the pathogenic bacteria Klebsiella of the mulberry bacterial wilt disease in mulberry field, have good application value in quarantine and prevention and treatment of the mulberry bacterial wilt disease, and are worthy of popularization.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
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Claims (10)
1. An LAMP primer group for detecting Klebsiella multocida is characterized in that the nucleotide sequence is shown as SEQ ID NO: 1-4; the nucleotide sequence of SEQ ID NO: 1-2 are outer primers; the nucleotide sequence of SEQ ID NO: 3-4 are inner primers.
2. The use of the primer set according to claim 1 for detecting Klebsiella multocida or bacterial wilt of mulberry, wherein the sample to be detected is a mulberry sample.
3. The use of the primer set of claim 1 for the preparation of a kit for detecting Klebsiella mulberosa or bacterial wilt of mulberry.
4. A detection kit for Klebsiella mulberosa or Mulberry bacterial wilt disease, comprising the LAMP primer set of claim 1.
5. The detection kit according to claim 4, wherein the kit further comprises 2 × LAMP reaction buffer, Bst DNA polymerase, ddH2O, a fluorescent dye STYO 9 and a chromogenic dye SYBR Green I.
6. A method for detecting Klebsiella mulberberberella is characterized in that DNA of a sample to be detected is used as a template, the LAMP primer group of claim 1 is used for LAMP reaction, and then a real-time fluorescence curve method, a color development method or gel electrophoresis is used for judging whether the amplification product has the Klebsiella mulberella, wherein the sample to be detected is a mulberry source sample.
7. The method for detecting Klebsiella multocida according to claim 6, wherein when the LAMP reaction is performed, the final concentration ratio of the outer primer to the inner primer of the LAMP primer set according to claim 1 is 1: (4-10).
8. The method of claim 6, wherein the LAMP reaction system is: 12.5. mu.L of 2 XTLAMP reaction buffer, 0.5. mu.L of Bst DNA polymerase, final concentrations of the outer primers of claim 1 each of 0.1. mu.M, final concentrations of the inner primers of claim 1 each of 0.8. mu.M, 1. mu.L of DNA template, 90.5. mu.L of fluorescent dye STYO, ddH2And O is supplemented to 25 mu L.
9. The method for detecting Klebsiella multocida according to claim 8, wherein the determination method comprises: after LAMP is finished, judging that the fluorescence curve is positive by detecting the fluorescence curve through a real-time fluorescence curve method, and judging that the fluorescence curve is negative when the fluorescence curve is not detected; or adding SYBR Green I into the reaction solution by a color development method, and observing that the reaction solution is Green and is judged to be positive, and the reaction solution is brown and is judged to be negative; or detecting the amplification product by gel electrophoresis, judging the multiple bands to be positive if the multiple bands appear, and judging the multiple bands to be negative if the multiple bands do not appear.
10. The method according to claim 6, wherein the LAMP reaction is carried out at a constant temperature of 61-65 ℃ for 60 min.
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