CN106086211A - LAMP primer group, test kit and the detection method of detection Flos Nelumbinis corruption pathogenic bacteria - Google Patents
LAMP primer group, test kit and the detection method of detection Flos Nelumbinis corruption pathogenic bacteria Download PDFInfo
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Abstract
The present invention relates to biological technical field, specifically disclose LAMP primer group, test kit and the detection method thereof of a kind of quick detection Flos Nelumbinis corruption pathogenic bacteria.The present invention is directed to the design of Flos Nelumbinis corruption pathogenic bacteria translation elongation factor (TEF 1 α) gene order and obtain one group of primer sets F3/B3 and FIP/BIP, Flos Nelumbinis corruption pathogenic bacteria is detected by ring mediated isothermal amplification (LAMP) by this primer sets or the test kit containing this primer sets, there is low cost, easily operation, simply, quickly, testing result is directly perceived, naked eyes can determine whether, highly sensitive, the advantages such as high specificity, the present invention is applicable to department of basic unit Flos Nelumbinis (Rhizoma Nelumbinis) and introduces a fine variety quarantine, plant seed Rhizoma Nelumbinis quality testing and Flos Nelumbinis corruption pathogenic bacteria field early diagnosis etc., there is extensive and actual using value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of quick detection Flos Nelumbinis corruption pathogenic bacteria LAMP primer group,
Test kit and detection method thereof.
Background technology
Flos Nelumbinis is important economic plants, its subterraneous stem Rhizoma Nelumbinis, Semen Nelumbinis, is exactly always vegetable and the nourishing that Chinese often eat
Product.Along with the prosperity of China market economy, Flos Nelumbinis forms key product in some areas, becomes development local economy, culture
Pillar industry.In recent years, Pearl River Delta Flos Nelumbinis cultivation concentration zones, as wet in the three water Flos Nelumbinis worlds, Fanyu Lianhua Shan Mountain, Dongguan end of the bridge etc.
, there is rot in ground, have impact on the normal industry that produces and go sightseeing on a large scale.By Fusariumsp (Fusarium commune)
Causing rot is that Flos Nelumbinis growing area occurs the most generally, cause harm one of serious disease, in Flos Nelumbinis trophophase and Rhizoma Nelumbinis storage period
All can fall ill, be distributed wide, all there is generation in national each Flos Nelumbinis producing region.Flos Nelumbinis rot belongs to soil-borne disease, and pathogenic bacteria often results in Rhizoma Nelumbinis
Whip, Nodus Nelumbinis Rhizomatis, Rhizoma Nelumbinis root rot, simultaneously the leaf of upper part, flower also can be wilted yellow withered, and serious whole strain of falling ill is withered.
In recent years, have been reported that confirming that the method for PCR-based has been used successfully to detects lotus (Flos Nelumbinis) corrupt Pathogen detection (inspection
Survey Rhizoma Nelumbinis kind and carry method, test kit and the primer of lotus fungoid rot pathogen, the patent No.: 201310715070.0), PCR
Method on the operating time and detection specificity and sensitivity in terms of more conventional method improve a lot, but, PCR method
Must there is a temperature cycling device of costliness and precision, and its detection time the most long (3～6 hours), it is necessary to borrow
Helping electrophresis apparatus could differentiate the positive or negative of amplified production, cost is high, be difficult to operation, and course of reaction is easy to contaminated
The impact of thing, so that this method can not meet actually detected needs.Accordingly, it would be desirable to exploitation is a kind of highly sensitive and
It is swift in response, the detection method of PCR can be replaced to a certain extent.
Ring mediated isothermal amplification method (Loop-mediated isothermal amplification, LAMP) is Japanology
The technology that a kind of novel in vitro isothermal duplication specific nucleic acid that person Notomi etc. (2000) invent judges, along with sending out of LAMP technology
Exhibition, LAMP technology be successfully applied to the cause of diseases such as virus, antibacterial, fungus and parasite detection (Liu Yongsheng, Ding Yaozhong,
Outstanding.The applied research [J] of ring mediated isothermal amplification (LAMP) technology, 2010,26 (8): 87-89).Have not yet to see application LAMP
Technology is for detecting the correlation technique report of Flos Nelumbinis rot.Therefore, the newest fruits of this biotech development is applied to lotus
The quick detection of flower rot, significant.
Summary of the invention
In order to solve the problems referred to above, an object of the present invention there are provided a kind of quick detection Flos Nelumbinis corruption pathogenic bacteria
LAMP primer group；The two of the purpose of the present invention there is provided the detection kit containing LAMP primer group；The purpose of the present invention it
Three there is provided the method utilizing LAMP primer group and test kit to detect Flos Nelumbinis corruption pathogenic bacteria.
The present invention is achieved through the following technical solutions:
A kind of LAMP primer group for detecting Flos Nelumbinis corruption pathogenic bacteria, described primer sets comprises a pair outer primer pair
F3/B3 and a pair inner primer are to FIP/BIP, and primer sequence is as follows:
F3:5 '-TTCCACAACCTCAATGAGC-3 '
B3:5 '-TGTCAGTACGAAGAGAAGTAGA-3 '
FIP:5 '-CCAGGCGTACTTGAAGGAACCGTCAAGCAGTCACTAACCAT-3 '
BIP:5 '-AGCGTGAGCGTGGTATCACCCAATGACGGTGACATAGTAG-3 '.
A kind of LAMP kit for detecting Flos Nelumbinis corruption pathogenic bacteria, described test kit contains above-mentioned LAMP primer group.
Described test kit is possibly together with extracting genome DNA liquid, LAMP reactant liquor, positive reference substance, negative controls, stable
Liquid and nitrite ion；Described LAMP reactant liquor in addition to containing above-mentioned primer sets possibly together with Bst archaeal dna polymerase and reaction buffer.
Wherein, described extracting genome DNA liquid is extract with CTAB liquid.The ultimate density of described reaction buffer be 1.3～
1.5mmol/L dNTPs, 0.6～0.9mmol/L glycine betaine and 7～9mmol/L MgSO4.Described positive reference substance is that Flos Nelumbinis is rotten
Lose pathogenic bacteria genomic DNA；Described negative controls is sterilized water.Described stabilizing solution is paraffin oil；Described nitrite ion is dilution
The SYBR Green I of 1000 times.
It is preferred that the reaction system of described test kit is: Bst archaeal dna polymerase 1.0 μ of genomic DNA 2.0 μ L, 8U/ μ L
L, reaction buffer 12.5 μ L, each 1.0 μ L of F3/B3 primer of 0.2 μm ol/L, each 1 μ L of FIP/BIP primer of 1.6 μm ol/L, use
Ultra-pure water polishing is to 25 μ l.
Utilize above-mentioned LAMP primer group or the method for mentioned reagent box detection Flos Nelumbinis corruption pathogenic bacteria, comprise the following steps:
S1. extracting genome DNA: if detection sample is Flos Nelumbinis corruption pathogenic bacteria mycelium or Flos Nelumbinis plant group in spite of illness
Knit, then utilize CTAB method to extract the DNA of the pathogen in Flos Nelumbinis corruption pathogenic bacteria or Flos Nelumbinis plant tissue；If detection sample is soil
Earth, then use soil genomic DNA rapid extraction test kit to extract and doubt the microorganism total DNA infecting soil；
S2. prepare reaction system: in described reaction system, add stabilizing solution, reaction tube lid adds nitrite ion；
The addition of described stabilizing solution is 20～25 μ L, and the addition of nitrite ion is 1.5～2 μ L；
S3. ring mediated isothermal amplification: with genome DNA as template, utilizes primer sets to obtain amplified production through amplification；Institute
Stating primer sets and comprise a pair outer primer to F3/B3 and a pair inner primer to FIP/BIP, the program of described amplified reaction is 63～65
DEG C 1h, 80～82 DEG C of 5min, 4 DEG C of preservations；
S4. amplified production detection: amplified reaction is complete, by mixed with amplified production concussion for the nitrite ion in reaction tube lid
Even, utilize development process that amplified production is analyzed, observe color change, product color becomes green, illustrates to treat test sample
Product contain Flos Nelumbinis corruption pathogenic bacteria；Product becomes orange, then do not contain Flos Nelumbinis corruption pathogenic bacteria.
Compared with prior art, the method have the advantages that
(1) low cost, easily operation, simple: the maximum advantage of the present invention is to need not the PCR required for conventional PCR detection
The special instrument that instrument, electrophresis apparatus, gel imaging system etc. are expensive, it is only necessary to a cheap thermostatical instrument, such as water-bath；The present invention
Eliminate loaded down with trivial details operating procedure and code, simple.
(2) quick: application detection method, testing result can be obtained 1～1.5h, and conventional PCR or nido
PCR detection needs 3～6h just to can get testing result；The method of the invention is much shortens operating time, fast and easy.
(3) testing result is directly perceived, and naked eyes can determine whether: amplified production of the present invention can dye by adding developer, green
For the corruption pathogenic bacteria Han Flos Nelumbinis in the positive, i.e. testing sample, orange is negative, illustrates in testing sample without Flos Nelumbinis corruption pathogenic bacteria；
Naked eyes can determine whether testing result, it is not necessary to electrophoresis detection and gel imaging.
(4) false positive is reduced: nitrite ion of the present invention has been added in PCR pipe lid before isothermal duplication, logical after reaction
Crossing to be centrifuged or shake and mix with amplified production, uncapping after thus being avoided that amplification adds reactant liquor, causes Aerosol Pollution, can significantly subtract
Few false positive produces, it is achieved that without the sample quick visualization detection uncapped.
(5) sensitivity is high: lowest detection of the present invention limitation, can less than 1.25pg Flos Nelumbinis corruption pathogenic bacteria genomic DNA
It is directly used in Flos Nelumbinis plant and field in spite of illness to fall ill the detection of sample.
(6) high specificity: the primer sets of present invention design is the key of this technology, and this primer sets is for Flos Nelumbinis rot
4 specific primers of 6 region designs of bacterium TEF-1 α gene order, in 6 regions, any region is not mated with primer all
Nucleic acid amplification can not be carried out, therefore the detection method specificity of the present invention is high.
Accompanying drawing explanation
Fig. 1 is Flos Nelumbinis corruption pathogenic bacteria LAMP detection primer design diagram of the present invention.
The Flos Nelumbinis rhizome sample of Fig. 2 morbidity and control sample testing result figure；Wherein, 1～6: the Flos Nelumbinis Sect. Rhiziridium of morbidity
Tissue samples；7: negative control；8: positive control.
The specific detection of Fig. 3 LAMP kit；Wherein, 1～10: the Flos Nelumbinis corruption pathogenic bacteria of different geographic origin
(Fusarium commune) bacterial strain；11～15: non-Flos Nelumbinis rot bacterial strain；16: blank.
The susceptiveness detection of Fig. 4 LAMP kit；Wherein, 1～7:1.25 × 102ng·μL-1、1.25×101ng·μL-1、1.25×100ng·μL-1、1.25×102pg·μL-1、1.25×101pg·μL-1、1.25×100pg·μL-1、1.25×
102fg·μL-1；8: blank.
Specific embodiment
Below in conjunction with detailed description of the invention, the present invention is described in further detail, manages to help those skilled in the art
Solve the present invention.
Quickly detect LAMP primer group, test kit and the detection method thereof of Flos Nelumbinis corruption pathogenic bacteria.
One, design of primers
The present invention, using translation elongation factor (TEF-1 α) gene of Flos Nelumbinis corruption pathogenic bacteria as detection target, is divided by comparison
After analysis, utilize Primer Explorer Version 4.0 (http://primerexplorer.jp/e/) Photographing On-line 4
LAMP specific primer, wherein F3 (5 '-TTCCACAACCTCAATGAGC-3 ') and B3 (5 '-TGTCAGTACGAAGAGAAGTAG
A-3 ') it is Outside primer, FIP (5 '-CCAGGCGTACTTGAAGGAACCGTCAAGCAGTCACTAACCAT-3 ') and BIP (5 '-
AGCGTGAGCGTGGTATCACCCAATGACGGTGACATAGTAG-3 ') it is inner primer, its detection primer design diagram is such as
Shown in Fig. 1.
Wherein, translation elongation factor (TEF-1 α) the gene complete sequence of Flos Nelumbinis corruption pathogenic bacteria is as follows.
GTCGACCACTGTGAGTACTCCCCTTGGACGATGAGCTTATCTGCCATCGTTAATCCCGACCAAGACCTG
GCGGGGTATTTCTCAAAGGCAATATGCTGATATCGTTTCACAGACCGGTCACTTGATCTACCAGTGCGGTGGTATCG
ACAAGCGAACCATCGAGAAGTTCGAGAAGGTTAGTCACTTTCCCTTCGATCGCGCGTCCTCTGCCCATCGATTTCCC
CTACGACTCGAAACCTGCCCGCTACCCCGCTCGAGACCAAAAATTTTGCGATATGACCGTAATTTTTTTGGTGGGGC
ATTTACCCCGCCACTCGAGCGACGGGC-GCGTTTGCCCTCCCATTTCCACAACCTCAATGAGCGCATCGTCACGTGT
CAAGCAGTCACTAACCATTCAATAATAGGAAGCCGCTGAGCTCGGTAAGGGTTCCTTCAAGTACGCCTGGGTTCTTG
ACAAGCTCAAGGCCGAGCGTGAGCGTGGTATCACCATCGATATTGCTCTCTGGAAGTTCGAGACTCCTCGCTACTAT
GTCACCGTCATTGGTATGTTGTCGCTCATGCTTCATTCTACTTCTCTTCGTACTGACATATCACTCAGACGCTCCCG
GTCACCGTG
Two, the Field information of LAMP detection
S1. extracting genome DNA: the Flos Nelumbinis rhizome sample that in June, 2015, field collection was fallen ill (has Flos Nelumbinis rot
Early symptom, blade is withered, and subterraneous stem vascular tissue browning rots), use CTAB method that Flos Nelumbinis rhizome tissue carries out Flos Nelumbinis rotten
Lose pathogenic bacteria genome DNA to extract, obtain sample and number consecutively is sample 1～6；
S2. reaction system is prepared: total system 25 μ L, wherein, the Bst archaeal dna polymerase of genomic DNA 2.0 μ L, 8U/ μ L
1.0 μ L, reaction buffer 12.5 μ L, each 1.0 μ L of F3/B3 primer of 0.2 μm ol/L, each 1 μ of FIP/BIP primer of 1.6 μm ol/L
L, with ultra-pure water polishing to 25 μ l.Additionally adding stabilizing solution paraffin oil 25 μ L on every PCR pipe reaction system, PCR pipe lid adds
Nitrite ion SYBR Green I 2 μ L；
S3. ring mediated isothermal amplification: respectively with sample 1～6 and Flos Nelumbinis corruption pathogenic bacteria genomic DNA (positive control, labelling
Be No. 8) and ddH2O (negative control is labeled as No. 7) be template, use Flos Nelumbinis corruption pathogenic bacteria translation elongation factor (TEF-1 α)
Inside and outside two couples of special primers F3, B3 (outer primer to) of gene order design and FIP, BIP (inner primer to) obtain through amplification
Amplified production；
The program of amplified reaction is: 63 DEG C of 1h, 80 DEG C of 5min, 4 DEG C of preservations；
Wherein, described extracting genome DNA liquid is extract with CTAB liquid, and its formula is: 2%CTAB, 20mmol/L EDTA,
100mmol/L Tris-HCl (pH is 8.0), 1.4mol/L NaCl；
Wherein, reaction buffer is dNTPs, MgSO4And glycine betaine, its formula is that final concentration is respectively 1.4mmol/L
DNTPs, 0.8mmol/L glycine betaine and 8mmol/L MgSO4；
S4. amplified production detection: after amplified reaction terminates, is inverted PCR pipe and vibrates gently, makes showing on PCR pipe lid
Color liquid and amplified production are sufficiently mixed dyeing, and Fig. 2 is coloration result.
Judge that testing result, green are positive, i.e. containing Flos Nelumbinis rot in testing sample according to coloration result in Fig. 2
Bacterium, orange is negative, i.e. without Flos Nelumbinis corruption pathogenic bacteria in testing sample.In the present embodiment, 1-8 sample result shows and shows: 6
Individual sample and positive control are the positive (green), detect Flos Nelumbinis corruption pathogenic bacteria, and negative control is negative (orange).
Three, the specificity analyses of LAMP detection
According to the method in case study on implementation 2, be isolatable from different regions 10 Flos Nelumbinis rot bacterial strains as object of study,
5 non-Flos Nelumbinis rot bacterial strains are control material (sample number into spectrum and bacterial strain information are as shown in table 1), and sterilized water is blank,
The specificity of checking LAMP detection method.With the DNA of each bacterial strain as template, the reaction system optimized is used to test, knot
Fruit is analyzed by fluorescent dye determination.
Table 1 is for detection primer specific Flos Nelumbinis rot bacterial strain and control strain
Judge that testing result, green are positive, i.e. containing Flos Nelumbinis rot in testing sample according to coloration result in Fig. 3
Bacterium, orange is negative, i.e. without Flos Nelumbinis corruption pathogenic bacteria in testing sample.In the present embodiment, 1～No. 16 sample result shows and shows:
1～the 10 samples Flos Nelumbinis corruption pathogenic bacterias of geographic origin (different) are positive (green), detect Flos Nelumbinis corruption pathogenic bacteria, and 11～15 samples
Product and negative control are negative (orange).Testing result shows that Flos Nelumbinis corruption pathogenic bacteria is had well by this primer sets and test kit
Specificity.
Four, the sensitivity analysis of LAMP detection
With the diluent after the DNA stock solution of Flos Nelumbinis rot bacterial strain Hhgw-2-4 extracted and 10 times of gradient dilutions as template
Carry out LAMP detection, bacterial strain Hhgw-2-4 genomic DNA is diluted to 1.25 × 10 successively from 125ng/ μ L2fg·μL-1, with nothing
Bacterium water is negative control, and result shows, Flos Nelumbinis rot bacterial strain Hhgw-2-4 genome concentration is 125ng/ μ L-1.25pg/ μ L
All colour developings be green, concentration is 1.25 × 102fg·μL-1With negative control colour developing for orange, show the Monitoring lower-cut of LAMP
For 1.25pg/ μ L (see Fig. 4).
Claims (9)
1. the LAMP primer group being used for detecting Flos Nelumbinis corruption pathogenic bacteria, it is characterised in that described primer sets comprises and externally draws
Thing is to F3/B3 and a pair inner primer to FIP/BIP, and primer sequence is as follows:
F3:5 '-TTCCACAACCTCAATGAGC-3 '
B3:5 '-TGTCAGTACGAAGAGAAGTAGA-3 '
FIP:5 '-CCAGGCGTACTTGAAGGAACCGTCAAGCAGTCACTAACCAT-3 '
BIP:5 '-AGCGTGAGCGTGGTATCACCCAATGACGGTGACATAGTAG-3 '.
2. the LAMP kit being used for detecting Flos Nelumbinis corruption pathogenic bacteria, it is characterised in that described test kit contains claim 1
Described LAMP primer group.
LAMP kit the most according to claim 2, it is characterised in that described test kit is possibly together with extracting genome DNA
Liquid, LAMP reactant liquor, positive reference substance, negative controls, stabilizing solution and nitrite ion；Described LAMP reactant liquor is except wanting containing having the right
Ask primer sets described in 1 outer possibly together with Bst archaeal dna polymerase and reaction buffer.
LAMP kit the most according to claim 3, it is characterised in that described extracting genome DNA liquid is extract with CTAB
Liquid.
LAMP kit the most according to claim 3, it is characterised in that the ultimate density of described reaction buffer is 1.3
～1.5mmol/L dNTPs, 0.6～0.9mmol/L glycine betaine and 7～9mmol/L MgSO4。
LAMP kit the most according to claim 3, it is characterised in that described positive reference substance is Flos Nelumbinis corruption pathogenic bacteria base
Because of group DNA；Described negative controls is sterilized water.
LAMP kit the most according to claim 3, it is characterised in that described stabilizing solution is paraffin oil；Described nitrite ion
SYBR Green I for dilution 1000 times.
LAMP kit the most according to claim 3, it is characterised in that the reaction system of described test kit is: genome
The Bst archaeal dna polymerase 1.0 μ L of DNA2.0 μ L, 8U/ μ L, reaction buffer 12.5 μ L, the F3/B3 primer each 1.0 of 0.2 μm ol/L
μ L, each 1 μ L of FIP/BIP primer of 1.6 μm ol/L, with ultra-pure water polishing to 25 μ l.
9. utilize test kit detection Flos Nelumbinis rot described in LAMP primer group described in claim 1 or any one of claim 2 to 8
The method of bacterium, it is characterised in that comprise the following steps:
S1. extracting genome DNA: if detection sample is Flos Nelumbinis corruption pathogenic bacteria mycelium or Flos Nelumbinis plant tissue in spite of illness, then
CTAB method is utilized to extract the DNA of the pathogen in Flos Nelumbinis corruption pathogenic bacteria or Flos Nelumbinis plant tissue；If detection sample is soil, then
Use soil genomic DNA rapid extraction test kit to extract and doubt the microorganism total DNA infecting soil；
S2. prepare reaction system: in described reaction system, add stabilizing solution, reaction tube lid adds nitrite ion；Described
The addition of stabilizing solution is 20～25 μ L, and the addition of nitrite ion is 1.5～2 μ L；
S3. ring mediated isothermal amplification: with genome DNA as template, utilizes described primer sets to obtain amplified production through amplification；Institute
Stating primer sets and comprise a pair outer primer to F3/B3 and a pair inner primer to FIP/BIP, the program of described amplified reaction is 63～65
DEG C 1h, 80～82 DEG C of 5min, 4 DEG C of preservations；
S4. amplified production detection: amplified reaction is complete, by the nitrite ion in reaction tube lid and amplified production concussion mixing, profit
Being analyzed amplified production with development process, observe color change, product color becomes green, illustrates that testing sample contains
Flos Nelumbinis corruption pathogenic bacteria；Product becomes orange, then do not contain Flos Nelumbinis corruption pathogenic bacteria.
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| CN110423748A (en) * | 2019-09-12 | 2019-11-08 | 中国农业科学院麻类研究所 | A kind of LAMP primer and its application, detection method of quick detection javanese root knot nematode |
| CN113801950A (en) * | 2021-08-27 | 2021-12-17 | 贵州大学 | Primer group for detecting Pantoea agglomerans causing plum bacterial perforation disease and application |
| CN115161412A (en) * | 2022-08-04 | 2022-10-11 | 云南省烟草农业科学研究院 | Detection method of shared fusarium and application thereof |
| CN115161412B (en) * | 2022-08-04 | 2024-06-04 | 云南省烟草农业科学研究院 | Detection method for shared fusarium and application thereof |
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