CN113481310B - LAMP primer group and LAMP kit for detecting ginger rot pathogen and application of LAMP primer group and LAMP kit - Google Patents
LAMP primer group and LAMP kit for detecting ginger rot pathogen and application of LAMP primer group and LAMP kit Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention relates to the technical field of pathogen detection, and particularly discloses an LAMP primer group for detecting pathogen of ginger rot, an LAMP kit and application thereof. The LAMP primer group for detecting ginger rot pathogen comprises a pair of outer primers F3 and B3, wherein the sequence of the pair of inner primers FIP and BIP is shown as SEQ ID NO. 1, the sequence of B3 is shown as SEQ ID NO. 2, the sequence of FIP is shown as SEQ ID NO. 3, and the sequence of BIP is shown as SEQ ID NO. 4. The LAMP primer group can accurately detect the pathogen of ginger rot (enterobacter cloacae, enterobacter albomarginatum and citrobacter freundii), has strong specificity and high sensitivity, has the minimum detection limit of 10 fg/mu L on the pathogen of ginger rot, and has important significance in controlling the occurrence of ginger rot diseases, avoiding epidemic risks of diseases and improving the yield and economic benefit of ginger.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to an LAMP primer group for detecting pathogen of ginger rot, an LAMP kit and application thereof.
Background
The fresh ginger rot is a new fresh ginger disease discovered in recent years, causes rot and necrosis of the root and stem of the fresh ginger, has the characteristics of quick onset, strong infectivity, great harm and the like, further causes yield reduction and even harvest failure of the fresh ginger, and seriously influences the yield and quality of the fresh ginger. The symptoms of ginger rot are similar to those of ginger blast, but the pathogenic bacteria are completely different, and the pathogenic bacteria of ginger rot are identified as enterobacteria (enterobacter cloacae, enterobacter albosis, citrobacter freundii).
If the new disease of ginger rot is continuously controlled by a control method similar to ginger distemper, the treatment effect is poor, so that the method can accurately detect the rot pathogen of ginger in time at the early stage of seed ginger screening or disease infection, and has important significance for controlling disease occurrence, avoiding disease epidemic risk and improving the yield and economic benefit of ginger.
Disclosure of Invention
In view of the above, the application provides an LAMP primer group and an LAMP kit for detecting the pathogen of ginger rot and application thereof, which can simply and efficiently detect the pathogen of Jiang Fulan at birth, and have good specificity and high sensitivity.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
the LAMP primer group for detecting ginger rot pathogen comprises a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP, wherein the sequences of the outer primers are as follows:
F3：5'-CAGGCCGTTCCAACTCTG-3'；
B3：5'-AACGGGTGTATTTGGTCAGG-3'；
the sequence of the inner primer is as follows:
FIP：5'-GTAACACCGGAGTCAACGGCAA-GCTGATAAGCCGCTGGTTG-3'；
BIP：5'-ACGTGGCGGTACTGTTCAGTAC-TTCGCCGGATACATCTCGC-3'。
compared with the prior art, the LAMP primer group for detecting the ginger rot pathogen has the following advantages:
the method is based on a specific primer ropB sequence of enterobacter, a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP are specifically designed, so that a to-be-detected object containing a ginger rot pathogen DNA fragment can be greatly amplified by using the primers, and further the ginger rot pathogen (enterobacter cloacae, enterobacter arvensis and citrobacter freundii) can be accurately detected, and the method has no cross amplification reaction with ginger fever pathogen such as bacterial wilt, cabbage soft rot pathogen such as European fungus, bacillus, rice bacterial leaf blight pathogen such as xanthomonas, potato black shank pathogen such as pectobacterium nigrum, cucumber angular spot pathogen such as pseudomonas syringae, escherichia coli, ginger flower skin pathogen such as Alternaria, ginger flower skin pathogen such as Fusarium oxysporum and wheat root rot such as navel and child, and has strong specificity; the LAMP primer group has high sensitivity, the minimum detection limit of the pathogen of the ginger rot is 10 fg/mu L, the pathogen of the ginger rot can be accurately detected in time at the early stage of seed ginger screening or disease infection, meanwhile, the detection accuracy is high, special instruments and special operators are not needed, the aim of rapidly detecting the pathogen of the ginger rot in any occasion is fulfilled, and the LAMP primer group has important significance in controlling the occurrence of the pathogen of the ginger rot, avoiding epidemic risks of the disease and improving the yield and economic benefit of the ginger.
The invention also provides application of the LAMP primer group for detecting the pathogenic bacteria of the ginger rot in non-diagnostic detection of the pathogenic enterobacteria of the ginger rot.
Preferably, the enterobacteria is at least one of enterobacter cloacae, enterobacter albopictus or citrobacter freundii.
The invention also provides an LAMP kit for detecting the pathogen of ginger rot, which comprises the LAMP primer group for detecting the pathogen of ginger rot.
Preferably, the kit further comprises buffer solution, magnesium sulfate, dNTPs, DNA polymerase and deionized water.
The invention also provides a method for detecting the pathogen of ginger rot by using the kit, which comprises the following specific operations: extracting genome DNA of the to-be-detected object as a template, carrying out loop-mediated isothermal amplification by using the LAMP kit, adding fluorescent dye after amplification is completed, and observing the result.
After the loop-mediated isothermal amplification is finished, fluorescent dye is added into the amplification product, and then the color change is observed, if the color change occurs, the fact that the object to be detected contains ginger rot pathogen enterobacteria DNA fragments is indicated; if the color is not changed, the fluorescent dye is also provided, which indicates that the sample does not contain DNA fragments of pathogenic enterobacteria of ginger rot or other pathogenic bacteria, and does not undergo amplification reaction.
The method for extracting genomic DNA of the test substance is a conventional DNA extraction method in the art.
The method for detecting the pathogen of the ginger rot has the advantages of simple operation, short reaction time and visual and accurate reaction result, and can be used for rapidly detecting the pathogen of the ginger rot.
Preferably, the reaction system for loop-mediated isothermal amplification comprises the following reagents and the dosage: 10 XThermopol buffer 2.5U L,100mM magnesium sulfate 1.5U L,10mM dNTP 3.5U L, 5U MF3, 5U M B3, 40U M FIP and 40U M BIP each 1U L,8000U/mL BstDNA polymerase 1U L, DNA template 1U L ~ 2U L, deionized water make up to 25U L.
The final concentration of each base in the dNTPs (deoxyribonucleoside triphosphate mixture) described above was 1.4mM.
The concentration of the DNA template in the reaction system of the loop-mediated isothermal amplification is more than 10 copies.
The above preferred reaction conditions may further improve the efficiency of detection of the pathogen of ginger rot.
Preferably, the reaction temperature of the loop-mediated isothermal amplification is 60 ℃, and the reaction time is 60min.
Preferably, the fluorescent dye is SYBR Green I nucleic acid dye, and the amount of the fluorescent dye is 0.5 mu L.
After the loop-mediated isothermal amplification is finished, adding 0.5 mu L of SYBR Green I nucleic acid dye into the amplified product, and observing color change, wherein if the color is fluorescent Green, the sample contains ginger rot pathogen enterobacteria DNA fragments; if the color is not changed, the fluorescent dye is orange, which indicates that the sample does not contain DNA fragments of pathogenic enterobacteria of ginger rot or other pathogenic bacteria, and does not undergo amplification reaction.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the result of a specific test for detecting the pathogen of ginger rot in example 1 of the present invention;
FIG. 2 is a graph showing the result of a sensitivity test of the method for detecting the pathogen of ginger rot according to example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The main reagents and instrumentation used in the following examples: bstDNA polymerase (NEB), buffer (NEB), magnesium sulfate (NEB), dNTP (NEB), SYBR Gree n I nucleic acid dye (Cool Lai Bo Co.), deionized water (Kangas reagent Co.), fungus genome DNA quick extraction kit (polymeric Mey Co.), PCR instrument (BIO-RAD Co., amplimer model T100 Th ermalCycler).
The preparation method comprises the steps of carrying out clinical detection, separation and identification on pathogenic enterobacter cloacae, pathogenic enterobacter albosis, pathogenic citrobacter freundii, pathogenic bacterial wilt and Ralstonia solanacearum, cabbage soft rot pathogen such as Euler's bacteria, bacillus, bacterial leaf blight pathogen such as xanthomonas, potato black shank pathogen such as pectobacterium nigrum, cucumber angular leaf pathogen such as pseudomonas syringae, escherichia coli, pathogenic Alternaria zingiberensis, pathogenic fusarium oxysporum, and wheat root rot such as Fusarium oxysporum.
Example 1
The LAMP primer group for detecting ginger rot pathogen comprises a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP, wherein the sequences of the outer primers are as follows:
F3：5'-CAGGCCGTTCCAACTCTG-3'；
B3：5'-AACGGGTGTATTTGGTCAGG-3'；
the sequence of the inner primer is as follows:
FIP：5'-GTAACACCGGAGTCAACGGCAA-GCTGATAAGCCGCTGGTTG-3'；
BIP：5'-ACGTGGCGGTACTGTTCAGTAC-TTCGCCGGATACATCTCGC-3'。
the LAMP kit for detecting ginger rot pathogen comprises the LAMP primer group, 10X thermo pol buffer solution, magnesium sulfate, dNTP, DNA polymerase and deionized water.
The method for detecting the pathogen of ginger rot by using the LAMP kit comprises the following specific operations:
extracting genome DNA of an object to be detected, wherein the object to be detected is respectively ginger rot pathogen enterobacter cloacae, ginger rot pathogen enterobacter albopictus, ginger rot pathogen citrobacter freundii, negative control of ginger rot pathogen is not contained, ginger fever pathogen bacterial wilt is not contained, cabbage soft rot pathogen is Eurotium, bacillus, rice bacterial leaf blight pathogen is xanthomonas, potato black shank pathogen is pectobacterium, cucumber angular spot pathogen pseudomonas syringae, escherichia coli, ginger flower skin pathogen alternaria, ginger flower skin pathogen fusarium oxysporum and wheat root rot navel child spore.
Step two, using the genome as a template, and respectively carrying out loop-mediated isothermal amplification by using the LAMP kit, wherein the isothermal amplification conditions are as follows: amplification reaction at 60℃for 60min; the reaction system for amplification comprises the following reagents and the dosage: 10 XThermopol buffer 2.5. Mu.L, 100mM magnesium sulfate 1.5. Mu.L, 10mM dNTP 3.5. Mu.L, 5. Mu.M outer primer F3, 5. Mu.M outer primer B3, 40. Mu.M inner primer FIP and 40. Mu.M inner primer BIP each 1. Mu.L, 8000U/mL BstDNA polymerase 1. Mu.L, DNA template 1. Mu.L, deionized water make up to 25. Mu.L, where the concentration in the DNA template is > 10 copies.
And step three, after the loop-mediated isothermal amplification reaction is finished, adding 0.5 mu L SYBR Green I nucleic acid dye into the amplified product, and then observing the color change. As a result, as shown in FIG. 1, the reaction tube of the specimen containing the DNA fragment of pathogenic enterobacter (Enterobacter cloacae, enterobacter alfasin, citrobacter freundii) of ginger rot was fluorescent green, and the reaction tube of the specimen containing other pathogenic bacteria was unchanged, and was orange in color of the dye itself.
The results show that the detection results of other pathogens on ginger and other plant fungi bacteria are negative, and the results of the ginger rot pathogens enterobacteria (enterobacter cloacae, enterobacter albomarginatus and citrobacter freundii) are positive, so that the LAMP primer group and the LAMP kit for detecting the ginger rot pathogens provided by the invention can clearly detect the ginger rot pathogens, have good specificity, have the advantage of no false positive results, and have no cross amplification reaction with the ginger blast pathogens such as bacterial wilt, cabbage soft rot pathogen such as European bacteria, bacillus, rice bacterial leaf blight pathogen such as xanthomonas, potato black shank pathogen such as pectobacterium nigrum, cucumber angular leaf pathogen such as pseudomonas syringae, escherichia coli, ginger flower skin pathogen such as Alternaria, ginger flower skin pathogen such as Fusarium oxysporum and wheat root rot such as Fusarium gracille.
Example 2
The LAMP primer group for detecting ginger rot pathogen comprises a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP, wherein the sequences of the outer primers are as follows:
F3：5'-CAGGCCGTTCCAACTCTG-3'；
B3：5'-AACGGGTGTATTTGGTCAGG-3'；
the sequence of the inner primer is as follows:
FIP：5'-GTAACACCGGAGTCAACGGCAA-GCTGATAAGCCGCTGGTTG-3'；
BIP：5'-ACGTGGCGGTACTGTTCAGTAC-TTCGCCGGATACATCTCGC-3'。
the LAMP kit for detecting ginger rot pathogen comprises the LAMP primer group, 10X thermo pol buffer solution, magnesium sulfate, dNTP, DNA polymerase and deionized water.
The method for detecting the pathogen of ginger rot by using the LAMP kit comprises the following specific operations:
step one, extracting DNA of pathogenic genome of ginger rot (Enterobacter cloacae, enterobacter alboldii and Citrobacter freundii) respectively, and diluting the DNA solution with the gradient of 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L and 1 fg/. Mu.L.
Step two, using the genome as a template, and respectively carrying out loop-mediated isothermal amplification by using the LAMP kit, wherein the isothermal amplification conditions are as follows: amplification reaction at 60℃for 60min; the reaction system for amplification comprises the following reagents and the dosage: 10 XThermopol buffer 2.5. Mu.L, 100mM magnesium sulfate 1.5. Mu.L, 10mM dNTP 3.5. Mu.L, 5. Mu.M outer primer F3, 5. Mu.M outer primer B3, 40. Mu.M inner primer FIP and 40. Mu.M inner primer BIP each 1. Mu.L, 8000U/mL BstDNA polymerase 1. Mu.L, DNA template, deionized water make up to 25. Mu.L, where the concentration in DNA template is > 10 copies.
And step three, after the loop-mediated isothermal amplification reaction is finished, adding 0.5 mu L SYBR Green I nucleic acid dye into the amplified product, and then observing the color change.
As a result, as shown in FIG. 2, when the DNA concentrations of Enterobacter cloacae, enterobacter alboldii and Citrobacter freundii were 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L and 10 fg/. Mu.L, respectively, the reaction results showed positive, and the reaction tube containing the test substance of the DNA fragment of Enterobacter canker of ginger rot was fluorescent green. Therefore, the LAMP primer group and the LAMP kit for detecting the ginger rot pathogen provided by the invention can clearly detect the ginger rot pathogen, and have high sensitivity, and the detection minimum of the ginger rot pathogen DNA is 10 fg/. Mu.L.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
SEQUENCE LISTING
<110> university of agriculture in Hebei
<120> LAMP primer group for detecting ginger rot pathogen, LAMP kit and application thereof
<130> 20210624
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Synthesis
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caggccgttc caactctg 18
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<212> DNA
<213> Synthesis
<400> 2
aacgggtgta tttggtcagg 20
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<211> 41
<212> DNA
<213> Synthesis
<400> 3
gtaacaccgg agtcaacggc aagctgataa gccgctggtt g 41
<210> 4
<211> 41
<212> DNA
<213> Synthesis
<400> 4
acgtggcggt actgttcagt acttcgccgg atacatctcg c 41
Claims (8)
1. An LAMP primer group for detecting ginger rot pathogen, which is characterized in that: the kit comprises a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP, wherein the sequences of the outer primers are as follows:
F3：5'-CAGGCCGTTCCAACTCTG-3'；
B3：5'-AACGGGTGTATTTGGTCAGG-3'；
the sequence of the inner primer is as follows:
FIP：5'-GTAACACCGGAGTCAACGGCAA-GCTGATAAGCCGCTGGTTG -3'；
BIP：5'-ACGTGGCGGTACTGTTCAGTAC-TTCGCCGGATACATCTCGC-3'。
2. the use of a LAMP primer set for detecting ginger rot pathogen as claimed in claim 1, characterized in that: the LAMP primer group for detecting the pathogenic bacterial infection of the ginger rot is applied to non-diagnostic detection of the pathogenic enterobacter of the ginger rot, wherein the enterobacter is at least one of enterobacter cloacae, enterobacter albopictus or citrobacter freundii.
3. The LAMP kit for detecting the pathogen of ginger rot is characterized by comprising the following components: the kit comprises the LAMP primer group for detecting ginger rot pathogen according to claim 1.
4. The LAMP kit for detecting ginger rot pathogen as set forth in claim 3, wherein: the LAMP kit also comprises buffer solution, magnesium sulfate, dNTPs, DNA polymerase and deionized water.
5. The method for detecting ginger rot pathogen using the LAMP kit of claim 4, characterized in that: the specific operation is as follows: extracting genome DNA of the to-be-detected object as a template, carrying out loop-mediated isothermal amplification by using the LAMP kit, adding fluorescent dye after amplification is completed, and observing the result.
6. The method for detecting the pathogen of ginger rot as set forth in claim 5, wherein: the reaction system for loop-mediated isothermal amplification comprises the following reagents and the dosage: 10 XThermopol buffer 2.5U L,100mM magnesium sulfate 1.5U L,10mM dNTP 3.5U L, 5U M F3, 5U M B3, 40U M FIP and 40U M BIP each 1U L,8000U/mL BstDNA polymerase 1U L, DNA template 1U L ~ 2U L, deionized water to 25U L.
7. The method for detecting the pathogen of ginger rot as set forth in claim 6, wherein: the reaction temperature of the loop-mediated isothermal amplification is 60 ℃, and the reaction time is 60min.
8. The method for detecting the pathogen of ginger rot as set forth in claim 6, wherein: the fluorescent dye is SYBR Green I nucleic acid dye, and the dosage of the fluorescent dye is 0.5 mu L.
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| US5994066A (en) * | 1995-09-11 | 1999-11-30 | Infectio Diagnostic, Inc. | Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
| CN108441569B (en) * | 2018-04-12 | 2021-10-29 | 华南农业大学 | Specific sequence and primer set Yt4 of mulberry source enterobacter cloacae and application of specific sequence and primer set Yt4 in detection of enterobacter cloacae |
| CN108531646A (en) * | 2018-07-10 | 2018-09-14 | 重庆文理学院 | The LAMP detection primer and detection method of curing ginger stalk rot P. myriotylum |
| CN110241239B (en) * | 2019-06-24 | 2020-04-10 | 浙江大学 | Kit for detecting enterobacter cloacae |
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