CN105713960A - Specific LAMP primer composition used for detection of phomopsis longicolla and application thereof - Google Patents
Specific LAMP primer composition used for detection of phomopsis longicolla and application thereof Download PDFInfo
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Abstract
The present invention belongs to the field of genetic engineering, and discloses a specific LAMP primer composition used for detection of phomopsis longicolla and application thereof. A nucleotide sequence of phomopsis longicolla detection target sequence Tef-alpha is as shown in SEQ ID NO. 1, forward inner primer FIP of the target sequence-specific LAMP primer composition is as shown in SEQ ID NO. 2, reverse inner primer BIP is as shown in SEQ ID NO. 3, forward outer primer F3 is as shown in SEQ ID NO. 4, reverse outer primer B3 is as shown in SEQ ID NO. 5, and forward loop primer LF is as shown in SEQ ID NO.6. The detection system can be fast, efficient, high-specificity and high-sensitivity in detection of the phomopsis longicolla under LAMP amplification conditions, can be used for on-site detection of field quarantine inspection, import and export quarantine inspection, and the like, and is easy to promote and use in a large range.
Description
Technical field
The invention belongs to genetic engineering field, relate to a kind of specificity LAMP primer composition thing for detecting soybean Phomopsis seed decay pathogen and application thereof.
Background technology
Soybean Phomopsis seed decay pathogen (PhomopsislongicollaHobbs) infects Semen sojae atricolor, causes soybean root rot and sprout term disease, and can cause that soybean seed quality reduces[1],.Soybean Phomopsis seed decay pathogen found in the U.S. as far back as 1976, and hereafter, Canada, Argentina, Korea S, Italy, Yugoslavia all had been reported that[2,3].According to statistics, in 1994, the whole world is because this disease causes that soybean yield loss is up to 18.6 ten thousand tons[4,5].Occur abroad in recent years because of it general, economic impact is big, introduction risk is high and the report that not yet has generation in China, is classified as on Semen sojae atricolor one of 7 kinds of dangerous fungal diseases including soybean phytophthora by relevant expert[6].In order to stop the continuous expansion of soybean Phomopsis seed decay pathogen spread scope, soybean Phomopsis seed decay pathogen disease is made to be controlled, it is necessary to it is detected quickly and accurately.
Ring mediated isothermal amplification method (loop-mediatedisothermalamplification, LAMP), it is that Notomi of Rong Yan Co., Ltd. having Japan et al. is in a kind of novel nucleic acid amplification method [7] of exploitation in 2000, it is characterized in 4 specific primers of 6 region designs for target gene, under the effect of strand displacement DNA enzymatic (BstDNApolymerase), 60～65 DEG C of constant-temperature amplifications, 15～60min gets final product observed result, and efficiency is up to 109～1010The individual order of magnitude, has the features such as simple to operate, high specificity, product easily detection.When DNA synthesizes, the pyrophosphate ion precipitated out from desoxyribose triphosphoric acid substrate (dNTPs) reacts with the magnesium ion in reaction solution, produces a large amount of magnesium pyrophosphate precipitation, presents white.Therefore, it can using turbidity as the index reacted, the white opacity that only detects by an unaided eye precipitates, and just can identify whether expanding, without loaded down with trivial details electrophoresis and ultraviolet visualization.Due to LAMP reaction need not PCR instrument and expensive reagent, product can by whether the color change after forming white precipitate (muddiness) or SYBRGREEN I dyeing judges whether to react.Along with constantly improving and development of technology, can have a wide range of applications in fields such as food inspection clinical disease diagnosis[8,9]。
Summary of the invention
It is an object of the invention to provide a kind of specificity LAMP primer composition thing for detecting soybean Phomopsis seed decay pathogen.
It is a further object of the present invention to provide the application of this specificity LAMP primer composition thing.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of specificity LAMP primer composition thing for detecting soybean Phomopsis seed decay pathogen, it is made up of following primer: forward inner primer FIP is such as shown in SEQIDNO.2, reverse inner primer BIP is such as shown in SEQIDNO.3, forward outer primer F3 is such as shown in SEQIDNO.4, reverse outer primer B3 is such as shown in SEQIDNO.5, and forward ring primer LF is such as shown in SEQIDNO.6.
The application in the reagent of preparation detection or qualification soybean Phomopsis seed decay pathogen of the described specificity LAMP primer composition thing.
A kind of LAMP detection kit for detecting soybean Phomopsis seed decay pathogen, comprises described specific LAMP primer composition thing.
The described LAMP detection kit for detecting soybean Phomopsis seed decay pathogen, comprises: 2.5 μ L10 × ThermoPolBuffer, 8mmol L-1MgSO4, 1.2mmol L-1The each 1.6 μm of ol L of dNTPs, inner primer FIP and BIP-1, each 0.4 μm of ol L of outer primer F3 and B3-1, LF0.8 μm of ol L of ring primer-1, 0.8mol L-1Glycine betaine, 0.1%Trion-X, 20mmol L-1Tris-HCl (pH8.8), 10mmol L-1KCl, 10mmol L-1(NH4)SO4, 8U μ L-1The detection solution of BstDNA polymerase and ultra-pure water composition and SYBRGREEN I dyestuff.
A kind of method detecting soybean Phomopsis seed decay pathogen, extracts the DNA of microorganism to be checked, with the DNA of extraction for template, utilizes described specific LAMP primer composition thing to carry out LAMP reaction；Amplified production adds SYBRGREEN I dyestuff, testing result under ordinary light, if product becomes displaing yellow under ordinary light, then it represents that there is soybean Phomopsis seed decay pathogen, shows orange, represent without this pathogenic bacteria under ordinary light.
The method of described detection soybean Phomopsis seed decay pathogen preferably extracts the DNA of microorganism to be checked, takes 3 μ lDNA solution, adds the detection solution described in 20.5 μ l and 1.5 μ l sterilizing deionized waters carry out LAMP reaction, and LAMP response procedures is: 64 DEG C of 60min.
Beneficial effect:
The present invention is on the basis of the detection target sequence Tef1-α and other allied species pathogenic bacteria differences in sequence that analyze soybean Phomopsis seed decay pathogen, 5 specific LAMP primer are designed and have screened in multiple potential sites, it is currently used for the PrimerExplorer that software is the exploitation of EikenChemical company of design LAMP primer, although utilizing software to can be designed that some the primers meeting design parameter, but still there is following defect in this software: (1) lacks the step of primer specificity detection, although LAMP primer is made up of a plurality of primer, specificity can improve a lot relative to PCR, but it is the absence of specific detection, there will be erroneous judgement, cause experimental result mistake；(2) even if specificity meets the requirements, actual experimental verification also there will be some primer and produce false-positive phenomenon, be probably primer can not form dumbbell shaped reacting precursor although producing these false-positive reasons, but amplification that can be linear.In order to overcome the defect of software design, carrying out artificial screening for meeting specific primer needs, this needs very abundant experiment experience and theoretical basis, and this is also the part that this patent is most crucial, and the location drawing of primer place target is shown in Fig. 1.Establish the LAMP reaction system of detection soybean Phomopsis seed decay pathogen on this basis.The detection system of the present invention is under LAMP amplification condition, energy is quickly, efficient, height is special, soybean Phomopsis seed decay pathogen detected with sensitivity, there is specificity between the kind of excellence, plant interior versatility and sensitivity, can meet very well current Site Detection to soybean Phomopsis seed decay pathogen in the urgent need to, for field quarantine, the Site Detection importing and exporting quarantine etc., it is easy to popularization and application on a large scale.
Accompanying drawing explanation
Fig. 1 LAMP specific primer target site, when to select the reason in this site be later stage artificial screening, the high specificity of primer, will not produce false positive, highly sensitive.
Fig. 2 embodiment 2 soybean Phomopsis seed decay pathogen LAMP primer specific test ordinary light light irradiates figure
Wherein, No. 1, No. 2 is soybean Phomopsis seed decay pathogen different strains, and No. 3 is southern stem canker of soybean, No. 4 is Semen sojae atricolor north stem canker, and No. 5 is tack anthrax, and No. 6 is rice blast fungus, No. 7 is alternaric bacteria, and No. 8 is ball Tuber Melanosporum, and No. 9 is soyabean phytophthora, No. 10 is Kidney bean shell ball spore bacterium, and No. 11 is aspergillus oryzae, and No. 12 is Kikuchi tail spore bacterium, No. 13 is oil bottle mycete, No. 14 is Flos Caryophylli phytophthora, and No. 15 is ramie mould, and No. 16 is negative control.
Fig. 3 embodiment 2 soybean Phomopsis seed decay pathogen LAMP primer specific test electrophoretogram
Wherein, M is ML5000Marker, and No. 1, No. 2 is soybean Phomopsis seed decay pathogen different strains, No. 3 is southern stem canker of soybean, and No. 4 is Semen sojae atricolor north stem canker, and No. 5 is tack anthrax, No. 6 is rice blast fungus, and No. 7 is alternaric bacteria, and No. 8 is ball Tuber Melanosporum, No. 9 is soyabean phytophthora, and No. 10 is Kidney bean shell ball spore bacterium, and No. 11 is aspergillus oryzae, No. 12 is Kikuchi tail spore bacterium, and No. 13 is oil bottle mycete, and No. 14 is Flos Caryophylli phytophthora, No. 15 is ramie mould, and No. 16 is negative control.
Fig. 4 embodiment 3 soybean Phomopsis seed decay pathogen LAMP primer sensitivity test ordinary light irradiates figure
LAMP expands variable concentrations soybean Phomopsis seed decay pathogen genomic DNA；The reaction system of respectively 25 μ L from left to right contains the LAMP amplification of 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg soybean Phomopsis seed decay pathogen DNA respectively.LAMP amplified reaction can identify that soybean Phomopsis seed decay pathogen, picture represent that sensitivity can reach 1pg/ μ L specifically.
Fig. 5 susceptible soybean plant strain LAMP detection test ordinary light irradiates figure
Wherein 1 is soybean Phomopsis seed decay pathogen reference culture genome, 2-7 is inoculation soybean Phomopsis seed decay pathogen bacterial strain soybean plant strain genome after 6 days, 8 is the blank PDA culture medium soybean plant strain genome after 6 days of inoculation, and 9 is healthy soybean plant strain genome, and 10 is negative control.
Detailed description of the invention
Embodiment 1
A kind of LAMP detection kit for detecting soybean Phomopsis seed decay pathogen, comprises: 2.5 μ L10 × ThermoPolBuffer, 8mmol L-1MgSO4, 1.2mmol L-1The each 1.6 μm of ol L of dNTPs, inner primer FIP and BIP-1, each 0.4 μm of ol L of outer primer F3 and B3-1, LF0.8 μm of ol L of ring primer-1, 0.8mol L-1Glycine betaine, 8U μ L-1BstDNA polymerase, adds ultra-pure water and prepares into detection solution.The concentration of each component represents its final concentration in detection solution.
Embodiment 2 soybean Phomopsis seed decay pathogen LAMP primer specific test
In order to verify the specificity of LAMP method, selection soybean Phomopsis seed decay pathogen reference culture (46562TM) with the southern stem canker of soybean of the close kind of soybean Phomopsis seed decay pathogen, Semen sojae atricolor north stem canker, bacterium (the tack anthrax not belonged to together with soybean Phomopsis seed decay pathogen；Rice blast fungus；Alternaric bacteria；Ball Tuber Melanosporum；Soyabean phytophthora；Kidney bean shell ball spore bacterium；Aspergillus oryzae；Kikuchi tail spore bacterium；Oil bottle mycete；Flos Caryophylli phytophthora；Ramie mould) DNA as template, take 3 μ lDNA solution, add the detection solution described in 20.5 μ l embodiments 1 and 1.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 60min；Amplified production adds SYBRGREEN I dyestuff, detects under ordinary light, and result is as shown in Figure 2.Result display specificity LAMP primer composition thing can identify soybean Phomopsis seed decay pathogen specifically, sample (No. 1 and No. 2 pipe) containing soybean Phomopsis seed decay pathogen becomes yellow green under ordinary light, and other kinds (3～No. 15 pipes) are orange under ordinary light, negative control (No. 16) is also orange under ordinary light, product is through 2 agarose gel electrophoresiies, observe the band of amplification, result is shown in Fig. 3, only No. 1 and No. 2 pipes typical trapezoid-shaped strips of appearance, show there is soybean Phomopsis seed decay pathogen into No. 1 and No. 2 pipes, result shows discrimination standard that can be positive and negative as detection using SYBRgreen I chromogenic reaction being dyestuff, and the time that detection is required can be shortened.
Embodiment 3 soybean Phomopsis seed decay pathogen LAMP primer sensitivity test
In order to determine the sensitivity of LAMP detection method, carrying out 10 doubling dilutions with DEPC water after the standard soybean extracted is intended stem point seed rot disease bacteria strain DNA spectrophotometric determination concentration (1 μ g/ μ l) ,-70 DEG C preserve as template.Taking each concentration DNA diluent 3 μ l after 10 doubling dilutions respectively as template, add the detection solution described in 20.5 μ l embodiments 1 and 1.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 60min；Amplified production adds SYBRGREEN I dyestuff, detects under ordinary light, and as shown in Figure 4,1～6 pipe becomes yellow green to result under ordinary light, it was shown that the sensitivity of the inventive method is 1pg/ μ L.
The susceptible soybean plant strain LAMP detection test of embodiment 4
The DNA of the soybean plant strain after soybean Phomopsis seed decay pathogen 6 days is inoculated with NaOH alkaline lysis method of extracting 6 strain, template is it can be used as to expand for LAMP, soybean Phomopsis seed decay pathogen reference culture DNA is replaced DNA cloning as negative control as positive control, healthy plant DNA and aquesterilisa.Taking 3uLDNA solution, add the detection solution described in 20.5 μ l embodiments 1 and 1.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 60min.Amplified production adds SYBRGREEN I dyestuff, detect under ordinary light, result is as shown in Figure 5, display this method can become yellow green from the soybean plant strain (2～No. 7 pipes) of inoculation soybean Phomopsis seed decay pathogen under ordinary light, soybean Phomopsis seed decay pathogen can be detected specifically, effect and directly detection southern stem canker of soybean DNA (No. 1 pipe) do not have difference, and inoculate blank PDA culture medium soybean plant strain (No. 8 pipes) after 6 days, healthy plant (No. 9 pipes) and negative control aquesterilisa (No. 10 pipes) then become orange under ordinary light, visible this method may be used for Fields detection (Fig. 5).
List of references
1.SatoT,DeViedmaLQ,AlvarezE,RomeroM(1993)FirstoccurrenceofsoybeansouthernstemcankerinParaguay.JapanAgriculturalResearchQuarterly27:20-20.
2.RiccioniL.IdentificationbyPCR-RFLPofPhomopsis/Diaporth especiesonItaliansoybeanseeds；2003.
3.ZhangA,RiccioniL,PedersenW,KolliparaK,HartmanG(1998)MolecularidentificationandphylogeneticgroupingofDiaporthephaseolorumandPhomopsislongicollaisolatesfromsoybean.Phytopathology88:1306-1314.
4.HepperlyP,SinclairJ(1978)QualitylossesinPhomopsis-infectedsoybeanseeds.Urbana51:61801.
5.KmetzK,SchmitthennerA,EllettC(1978)Soybeanseeddecay:PrevalenceofinfectionandsymptomexpressioncausedbyPhomopsissp.,Diaporthephaseolorumvar.sojae,andD.phaseolorumvar.caulivora.Phytopathology68:836-840.
6. Wu Pin coral, sternly enters the Semen sojae atricolor new expression that (2003) merit attention. plant quarantine 17:226-228.
7.NotomiT,OkayamaH,MasubuchiH,YonekawaT,WatanabeK,etal.(2000)Loop-mediatedisothermalamplificationofDNA.NucleicAcidsRes28:E63.
8.MoriY,NagamineK,TomitaN,NotomiT(2001)Detectionofloop-mediatedisothermalamplificationreactionbyturbidityderivedfrommagnesiumpyrophosphateformation.BiochemBiophysResCommun289:150-154.
9. the easy sensitive ring mediated isothermal amplification gene diagnostic new technology of Xiao Bin, Zhu Yonghong, Zou Quanming (2005). China laboratory medicine magazine 28:761-763.
Claims (6)
1. the specificity LAMP primer composition thing being used for detecting soybean Phomopsis seed decay pathogen, it is characterized in that being made up of following primer: forward inner primer FIP is such as shown in SEQIDNO.2, reverse inner primer BIP is such as shown in SEQIDNO.3, forward outer primer F3 is such as shown in SEQIDNO.4, reverse outer primer B3 is such as shown in SEQIDNO.5, and forward ring primer LF is such as shown in SEQIDNO.6.
2. the application in the reagent of preparation detection or qualification soybean Phomopsis seed decay pathogen of the specificity LAMP primer composition thing described in claim 1.
3. the LAMP detection kit being used for detecting soybean Phomopsis seed decay pathogen, it is characterised in that comprise the specificity LAMP primer composition thing described in claim 1.
4. the LAMP detection kit for detecting soybean Phomopsis seed decay pathogen according to claim 3, it is characterised in that described test kit comprises: 2.5 μ L10 × ThermoPolBuffer, 8mmol L-1MgSO4, 1.2mmol L-1The each 1.6 μm of ol L of dNTPs, inner primer FIP and BIP-1, each 0.4 μm of ol L of outer primer F3 and B3-1, LF0.8 μm of ol L of ring primer-1, 0.8mol L-1Glycine betaine, 8U μ L-1BstDNA polymerase, and sterilizing deionized water composition detection solution and SYBRGREEN I dyestuff.
5. the method detecting soybean Phomopsis seed decay pathogen, it is characterised in that extract the DNA of microorganism to be checked, with the DNA of extraction for template, utilizes the specific LAMP primer composition thing described in claim 1 to carry out LAMP amplified reaction；Amplified production adds SYBRGREEN I dyestuff, testing result under ordinary light, if product becomes yellow green under ordinary light, then it represents that there is soybean Phomopsis seed decay pathogen, shows orange, represent without this pathogenic bacteria under ordinary light.
6. the method for detection soybean Phomopsis seed decay pathogen according to claim 5, it is characterized in that extracting the DNA of microorganism to be checked, take 3 μ lDNA solution, adding the detection solution described in 20.5 μ l claim 4 and 1.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 60min.
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Cited By (3)
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| CN105713962A (en) * | 2014-12-03 | 2016-06-29 | 南京农业大学 | LAMP primer composition for detection of phomopsis longicolla hobbs and application thereof |
| CN107245517A (en) * | 2017-06-02 | 2017-10-13 | 合肥市第六中学 | A kind of LAMP primer, kit and its application for being used to detect soybean Phomopsis seed decay pathogen |
| CN108330209A (en) * | 2018-05-15 | 2018-07-27 | 浙江省检验检疫科学技术研究院 | The RAA detection fluorescence methods and the primer probe and kit of southern stem canker of soybean |
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| CN101787390A (en) * | 2009-01-23 | 2010-07-28 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Kit used for detecting soybean diaporthe/phomopsis syndrome and detection method |
| CN103388026A (en) * | 2013-07-17 | 2013-11-13 | 南京农业大学 | Phomopsis longicolla Hobbs detection target, PCR primer composition thereof and applications of the detection target and the PCR primer composition |
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| CN101787390A (en) * | 2009-01-23 | 2010-07-28 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Kit used for detecting soybean diaporthe/phomopsis syndrome and detection method |
| CN103388026A (en) * | 2013-07-17 | 2013-11-13 | 南京农业大学 | Phomopsis longicolla Hobbs detection target, PCR primer composition thereof and applications of the detection target and the PCR primer composition |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105713962A (en) * | 2014-12-03 | 2016-06-29 | 南京农业大学 | LAMP primer composition for detection of phomopsis longicolla hobbs and application thereof |
| CN107245517A (en) * | 2017-06-02 | 2017-10-13 | 合肥市第六中学 | A kind of LAMP primer, kit and its application for being used to detect soybean Phomopsis seed decay pathogen |
| CN108330209A (en) * | 2018-05-15 | 2018-07-27 | 浙江省检验检疫科学技术研究院 | The RAA detection fluorescence methods and the primer probe and kit of southern stem canker of soybean |
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