CN103695542B - The soybean north detection target of stem canker and LAMP primer composition thing thereof and application - Google Patents
The soybean north detection target of stem canker and LAMP primer composition thing thereof and application Download PDFInfo
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Abstract
The invention belongs to genetically engineered field, disclose the soybean north detection target of stem canker and specificity LAMP primer composition thing thereof and application.The detection target sequence Tef of soybean north stem canker, nucleotide sequence is as shown in SEQ ID NO.1, the specific LAMP primer composition thing of this target sequence, forward inner primer FIP 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, and reverse outer primer B3 is as shown in SEQ ID NO.5.Detection system of the present invention is under LAMP amplification condition, energy fast, efficient, height is special, soybean north stem canker detected with sensitivity, can better meet at present to the detection of soybean north stem canker in the urgent need to, for field quarantine, the Site Detection importing and exporting quarantine etc., be easy to apply on a large scale.
Description
Technical field
The invention belongs to genetically engineered field, relate to the soybean north detection target of stem canker and LAMP primer composition thing thereof and application.
Background technology
Soybean north stem canker (Diaporthephaseolorum var.caulivora) is one of most important destructive disease on north American soybean.Develop rapidly after this infection process plant and form cane loop-like lesions, and the plant that growing can be caused dead.Serious field of falling ill has the plant of 80% to be infected.The production loss caused thus is up to 50%
[1-4].Rapidly, except the U.S., there is the report of occurring and damage in the Some European countries such as Argentina, Brazil, Canada, Paraguay and Italy, Former Yugoslavia, Croatia and area in this disease development in recent years
[5-8].
Because of its occur abroad in recent years general, economic impact is large, introduction risk is high and not yet have the report of generation in China, China is classified as one of 13 kinds of quarantine pathogenic bacterias on the soybean paid close attention to
[9].In order to stop the continuous expansion of soybean Phomopsis seed decay pathogen spread scope, soybean south stem canker being controlled, needs to detect quickly and accurately it.
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) is that the people such as the Notomi of Rong Yan Co., Ltd. of Japan are in a kind of novel nucleic acid amplification method of exploitation in 2000
[10], be characterized in 6 zone design, 4 Auele Specific Primers for target gene, under the effect of strand displacement DNA enzymatic (Bst DNA polymerase), 60 ~ 65 DEG C of constant-temperature amplifications, 15 ~ 60min gets final product observations, and efficiency can reach 10
9~ 10
10the individual order of magnitude, has the features such as simple to operate, high specificity, product easily detect.When DNA synthesizes, the magnesium ion in pyrophosphate ion and the reaction soln of separating out from picodna triphosphoric acid matrix (dNTPs) reacts, and produces a large amount of magnesium pyrophosphate and precipitates, present white.Therefore, can using the index of opacity as reaction, the white opacity that only detects by an unaided eye precipitates, and just can identify and whether increase, and not need loaded down with trivial details electrophoresis and ultraviolet visualization.Because LAMP reaction does not need PCR instrument and expensive reagent, reaction product judges whether to react by the colour-change after whether forming white precipitate (muddiness) or SYBR GREEN I and dyeing.Along with constantly improving and development of technology, [11,12] can be had a wide range of applications in fields such as food inspection clinical disease diagnosis.
Summary of the invention
The object of the present invention is to provide the detection target sequence Tef of a kind of soybean north stem canker.
Another object of the present invention is to provide its specificity LAMP primer composition thing of the detection target sequence Tef of this soybean north stem canker.
Another object of the present invention is to provide this soybean north detection target sequence Tef of stem canker and application of specificity LAMP primer composition thing thereof.
Object of the present invention realizes by following technical scheme:
The detection target sequence Tef of soybean north stem canker, nucleotide sequence is as shown in SEQ ID NO.1.
The detection target sequence Tef of described soybean north stem canker is in the application detected or in the stem canker of the qualification soybean north.
For the specificity LAMP primer composition thing that the detection target sequence Tef of described soybean north stem canker designs, comprise forward inner primer FIP 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, and reverse outer primer B3 is as shown in SEQ ID NO.5.
Described specific LAMP primer composition thing preferably also comprises forward ring primer LF as shown in SEQ ID NO.6, and reverse ring primer LB is as shown in SEQ ID NO.7.
The described application of specificity LAMP primer composition thing in detection or qualification soybean north stem canker designed for the detection target sequence Tef of described soybean north stem canker.
For detecting a LAMP detection kit for soybean north stem canker, comprise described specific LAMP primer composition thing.
The described LAMP detection kit for detecting soybean north stem canker, comprise: by the reverse inner primer BIP of 20mM forward inner primer FIP, 20mM, the reverse outer primer B3 of 10mM forward outer primer F3,10mM, 10mM forward ring primer LF, 10mM reverse ring primer LR, 9.26%10xThermoPol Reaction Buffer, 50mM MgSO
4, 10mMdNTPMixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment and ultrapure water composition detection solution.
Detect a method for soybean north stem canker, extract the DNA of microorganism to be checked, with the DNA extracted for template, the specific LAMP primer composition thing described in utilization carries out LAMP reaction; Amplified production adds SYBR GREEN I dyestuff, detected result under ordinary light and/or UV-light, if reaction product becomes displaing yellow, produces intense fluorescence under ultraviolet light under ordinary light, then represent to there is soybean north stem canker, under ordinary light, under aobvious orange, UV-light, unstressed configuration produces and then represents not containing this germ.
The method of described detection soybean north stem canker preferably extracts the DNA of microorganism to be checked, gets 3 μ lDNA solution, adds detection solution described in 21.5 μ l and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, and LAMP response procedures is: 64 DEG C of 70min.
Beneficial effect:
The invention provides the detection target sequence Tef of a new soybean north stem canker, and analyze this target sequence Tef and the difference of other allied species germs in sequence, devise 6 specific LAMP primer, establish the LAMP reaction system detecting soybean north stem canker on this basis.Detection system of the present invention is under LAMP amplification condition, energy fast, efficient, height is special, soybean north stem canker detected with sensitivity, there is specificity between excellent kind, plant interior versatility and sensitivity, can meet very well at present to the Site Detection of soybean north stem canker in the urgent need to, for field quarantine, the Site Detection importing and exporting quarantine etc., be easy to apply on a large scale.
Accompanying drawing explanation
Between the stem canker kind of Fig. 1 embodiment 2 soybean north, specific test ordinary light irradiates figure
Wherein, No. 1, No. 2 is soybean north stem canker different strains, and No. 3, No. 4 is southern stem canker of soybean different strains, and 5 ~ No. 7 is plan stem point seed rot disease bacterium different strains, and No. 8 is negative control.
Specific test UV-irradiation figure between the stem canker kind of Fig. 2 embodiment 2 soybean north
Wherein, No. 1, No. 2 is soybean north stem canker different strains, and No. 3, No. 4 is southern stem canker of soybean different strains, and 5 ~ No. 7 is plan stem point seed rot disease bacterium different strains, and No. 8 is negative control.
Fig. 3 embodiment 3 soybean north stem canker specificity among genus test ordinary light rayed figure
Wherein, No. 1, No. 2 is soybean north stem canker different strains, and No. 3 is soybean rest fungus, No. 4 is glue born of the same parents anthrax-bacilus, and No. 5 is tack anthrax-bacilus, 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 mould, No. 14 is cloves phytophthora, and No. 15 is ramie mould, and No. 16 is negative control.
Fig. 4 embodiment 3 soybean north stem canker specificity among genus test UV-irradiation figure
Wherein, No. 1, No. 2 is soybean north stem canker different strains, and No. 3 is soybean rest fungus, No. 4 is glue born of the same parents anthrax-bacilus, and No. 5 is tack anthrax-bacilus, 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 mould, No. 14 is cloves phytophthora, and No. 15 is ramie mould, and No. 16 is negative control.
Fig. 5 embodiment 4 soybean north stem canker sensitivity test ordinary light irradiates figure
LAMP amplification different concns soybean north stem canker genomic dna; From left to right be respectively the LAMP amplification respectively containing 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg soybean Phomopsis seed decay pathogen DNA in the reaction system of 25 μ L.LAMP amplified reaction can identify soybean north stem canker specifically, and picture represents that sensitivity can reach 10pg/ μ L.
Fig. 6 embodiment 4 soybean north stem canker sensitivity test UV-irradiation figure
LAMP amplification different concns soybean north stem canker genomic dna; From left to right be respectively the LAMP amplification respectively containing 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg soybean north stem canker DNA in the reaction system of 25 μ L.LAMP amplified reaction can identify soybean north stem canker specifically, and picture represents that sensitivity can reach 10pg/ μ L.
Fig. 7 susceptible soybean plant strain LAMP detection experiment ordinary light
Wherein 1 is soybean north stem canker reference culture genome, 2-7 is the soybean strain gene group of inoculation soybean north stem canker bacterial strain after 6 days, 8 is the soybean strain gene group of the blank PDA substratum of inoculation after 6 days, and 9 is healthy soybean plant strain genome, and 10 is negative control.
Fig. 8 susceptible soybean plant strain LAMP detection experiment UV-irradiation figure
Wherein 1 is soybean north stem canker reference culture genome, 2-7 is the soybean plant strain genome of inoculation soybean north stem canker bacterial strain after 6 days, 8 is the soybean plant strain genome of the blank PDA substratum of inoculation after 6 days, and 9 is healthy soybean plant strain genome, and 10 is negative control.
Embodiment
Embodiment 1
A kind of LAMP detection kit for detecting soybean north stem canker, comprise: the reverse inner primer BIP of 20mM forward inner primer FIP, 20mM, the reverse outer primer B3 of 10mM forward outer primer F3,10mM, 10mM forward ring primer LF, 10mM reverse ring primer LR, 9.26%10xThermoPol Reaction Buffer, 50mM MgSO
4, 10mMdNTPMixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment, add ultrapure water and be prepared into detection solution.Each constituent concentration all refers to detecting the final concentration in solution.
Specific test between the stem canker kind of the embodiment 2 soybean north
In order to verify the specificity of LAMP method, the DNA selecting soybean north stem canker reference culture (CBS177.55) and soybean north stem canker southern stem canker of soybean of the same race and belong to soybean Phomopsis seed decay pathogen not of the same race together is as template, get 3 μ lDNA solution, add detection solution described in 21.5 μ l embodiments 1 and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 70min; Amplified production adds SYBR GREEN I dyestuff, detects under ordinary light UV-light.As depicted in figs. 1 and 2, display Auele Specific Primer identifies soybean north stem canker to result specifically, and sample (No. 1 pipe and No. 2 pipes) the meeting yellowing under ordinary light containing soybean north stem canker, produces intense fluorescence under UV-light; And other kinds (3 ~ No. 7 pipes) become orange under ordinary light, also fluorescence is not produced under UV-light, negative control (No. 8 pipes) becomes orange under ordinary light, and also do not produce fluorescence under UV-light, this LAMP detection method showing that the present invention sets up has specificity between very high kind.
The stem canker specificity among genus test of the embodiment 3 soybean north
In order to verify the specificity of LAMP method, select bacterium (the soybean rest fungus that soybean north stem canker reference culture (CBS177.55) does not belong to together with soybean north stem canker; Glue born of the same parents anthrax-bacilus; Tack anthrax-bacilus; Rice blast fungus; Alternaric bacteria; Ball Tuber Melanosporum; Soyabean phytophthora; Kidney bean shell ball spore bacterium; Aspergillus oryzae; Kikuchi tail spore bacterium) DNA as template, get 3 μ lDNA solution, add detection solution described in 21.5 μ l embodiments 1 and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 70min; Amplified production adds SYBR GREEN I dyestuff, detects under ordinary light UV-light.As shown in Figure 3 and Figure 4, display Auele Specific Primer identifies soybean north stem canker to result specifically, and sample (No. 1 pipe and No. 2 pipes) the meeting yellowing under ordinary light containing soybean north stem canker, produces intense fluorescence under UV-light; And other kinds (3 ~ No. 15 pipes) become orange under ordinary light, also fluorescence is not produced under UV-light, negative control (No. 16 pipes) becomes orange under ordinary light, and also do not produce fluorescence under UV-light, this LAMP detection method showing that the present invention sets up has very high specificity among genus.
Embodiment 4 soybean north stem canker sensitivity test
In order to determine the sensitivity of LAMP detection method, the standard extracted north stem canker bacterial strain (CBS177.55) DNA spectrophotometric determination concentration (1 μ g/ μ l) being carried out 10 doubling dilutions with DEPC water afterwards, preserving as template for-70 DEG C.Get each concentration DNA diluent 3 μ l after 10 doubling dilutions respectively as template, add detection solution described in 21.5 μ l embodiments 1 and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 70min; Amplified production adds SYBRGREEN I dyestuff, detects under ordinary light and UV-light, and result as shown in Figure 5, Figure 6.Under ordinary light, detect soybean north stem canker sensitivity is as seen from Figure 5 10pg/ μ L, and to detect the stem canker sensitivity of the soybean north be under ultraviolet light 10pg/ μ L.
Embodiment 5 susceptible soybean plant strain LAMP detection experiment
With the DNA of NaOH alkaline lysis method of extracting 6 strain inoculation standard soybean north stem canker (CBS177.55) soybean plant strain after 6 days, template is it can be used as to increase for LAMP, using soybean north stem canker reference culture DNA as positive control, healthy plant DNA and aqua sterilisa replace DNA cloning as negative control.Get 3uLDNA solution, add detection solution described in 21.5 μ l embodiments 1 and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 70min.Amplified production adds SYBR GREEN I dyestuff, detects under ordinary light and UV-light.Result is as shown in Fig. 7 ~ 8, display present method can detect soybean north stem canker specifically from the soybean plant strain (2 ~ No. 7 pipes) of inoculation soybean north stem canker, effect and direct-detection soybean north stem canker (No. 1 pipe) DNA do not have difference, inoculate the soybean plant strain of blank PDA substratum after 6 days (No. 8 pipes) and then under ordinary light, become orange with healthy plant (managing for No. 9) and negative control aqua sterilisa (managing for No. 10), also do not produce fluorescence under UV-light, visible present method may be used for Fields detection.
Reference
1.Crall J(1950)Soybean disease in Iowa in1949.Plant Disease Reporter34:96-97.
2.Sinclair JB(1982)Compendium of soybean diseases:American Phytopathological Society andUniversity of Illinois.
3.McGee D,Biddle J(1987)Seedborne Diaporthe phaseolorum var.caulivora in Iowa and itsrelationship to soybean stem canker in the Southern United States.Plant Disease71:620-622.
4.Athow KL,Caldwell RM(1954)A comparative study of Diaporthe stem canker and pod and stemblight of Soy-bean.Phytopathology44:319-325.
5.Pioli RN,Morandi EN,Martínez MC,Lucca F,Tozzini A,et al.(2003)Morphologic,molecular,and pathogenic characterization of Diaporthe phaseolorum variability in the coresoybean-producing area of Argentina.Phytopathology93:136-146.
6.Fernández FA,Hanlin RT(1996)Morphological and RAPD analyses of Diaporthe phaseolorumfrom soybean.Mycologia:425-440.
7.Sato T,De Viedma LQ,Alvarez E,Romero M(1993)First occurrence of soybean southern stemcanker in Paraguay.Japan Agricultural Research Quarterly27:20-20.
8.Vrandecic K,Cosic J,Riccioni L,Duvnjak T,Jurkovic D(2005)Isolation of Diaporthephaseolorum var.caulivora from Abutilon theophrasti in Croatia.Plant Pathology54:576-576.
9. Wu Pin coral, sternly enters the soybean new expression that (2003) merit attention. Plant Quarantine 17:226-228.
10.Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K,et al.(2000)Loop-mediatedisothermal amplification of DNA.Nucleic Acids Res28:E63.
11.Mori Y,Nagamine K,Tomita N,Notomi T(2001)Detection of loop-mediated isothermalamplification reaction by turbidity derived from magnesium pyrophosphate formation.Biochem Biophys Res Commun289:150-154.
12. Xiao Bin, the ring mediated isothermal amplification gene diagnostic new technology of Zhu Yonghong, Zou Quanming (2005) easy sensitivity. Chinese laboratory medicine magazine 28:761-763.
Claims (7)
1. for the specific LAMP primer composition thing that the detection target sequence Tef of the soybean north stem canker shown in SEQ ID NO.1 designs, it is characterized in that forward inner primer FIP 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, and reverse outer primer B3 is as shown in SEQ ID NO.5.
2. specific LAMP primer composition thing according to claim 1, it is characterized in that also comprising forward ring primer LF as shown in SEQ ID NO.6, reverse ring primer LB is as shown in SEQ ID NO.7.
3. the LAMP primer composition thing described in claim 1 or 2 detects in preparation or identifies the application in the reagent of soybean north stem canker.
4., for detecting a LAMP detection kit for soybean north stem canker, it is characterized in that the specific LAMP primer composition thing comprised described in claim 1 or 2.
5. the LAMP detection kit for detecting soybean north stem canker according to claim 4, it is characterized in that described test kit comprises: by 20 mM forward inner primer FIP, the reverse inner primer BIP of 20 mM, the reverse outer primer B3 of 10mM forward outer primer F3,10mM, 10mM forward ring primer LF, 10mM reverse ring primer LR, 9.26% 10xThermoPol Reaction Buffer, 50mM MgSO
4, 10mM dNTP Mixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment and ultrapure water composition detection solution.
6. detect a method for soybean north stem canker, it is characterized in that the DNA extracting microorganism to be checked, with the DNA extracted for template, utilize the specific LAMP primer composition thing described in claim 1 or 2 to carry out LAMP amplified reaction; Amplified production adds SYBR GREEN I dyestuff, day ordinary light and/or UV-light under detect, if reaction product under day ordinary light displaing yellow, produce intense fluorescence under ultraviolet light, then represent to there is soybean north stem canker; If reaction product under day ordinary light under aobvious orange, UV-light unstressed configuration produce; represent not containing this germ.
7. according to the method for the detection soybean north stem canker described in claim 6, it is characterized in that the DNA extracting microorganism to be checked, get 3 μ l DNA solutions, add 21.5 μ l detection solution according to claim 5 and 2.5 μ l sterilizing deionized waters carry out LAMP reaction, response procedures is: 64 DEG C of 70 min; Amplified production adds SYBR GREEN I dyestuff, detected result under ultraviolet light, if reaction product produces intense fluorescence, then represent to there is soybean north stem canker, unstressed configuration produces and then represents not containing this germ.
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| CN102154501A (en) * | 2011-04-15 | 2011-08-17 | 中华人民共和国连云港出入境检验检疫局 | PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs |
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| CN102154501A (en) * | 2011-04-15 | 2011-08-17 | 中华人民共和国连云港出入境检验检疫局 | PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs |
Non-Patent Citations (2)
| Title |
|---|
| Diaporthe phaseolorum var. caulivora, a Causal Agent for Both Stem Canker and Seed Decay on Soybean;suli sun等;《The plant pathology journal》;20120131;第28卷(第1期);第55-59页 * |
| JF461467.1;genbank;《Genbank》;20130123;全文 * |
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