CN103695542A - Detection target of Diaporthephaseolorum var.caulivora and LAMP (loop-mediated isothermal amplification) primer compositions and application thereof - Google Patents
Detection target of Diaporthephaseolorum var.caulivora and LAMP (loop-mediated isothermal amplification) primer compositions and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of gene engineering, and discloses a detection target of Diaporthephaseolorum var.caulivora and specific LAMP (loop-mediated isothermal amplification) primer compositions and application thereof. A nucleotide sequence of a detection target sequence Tef of Diaporthephaseolorum var.caulivora is shown in SEQ ID NO.1; in the specific LAMP primer compositions of the target sequence, a forward inner primer FIP is shown in SEQ ID NO.2, a reverse inner primer BIP is shown in SEQ ID NO.3, a forward outer primer F3 is shown in SEQ ID NO.4 and a reverse outer primer B3 is shown in SEQ ID NO.5. By adopting the detection system provided by the invention, the Diaporthephaseolorum var.caulivora can be quickly, efficiently, specifically and sensitively detected under the LAMP amplification condition, the crying needs on detection of the Diaporthephaseolorum var.caulivora at present can be well met, and the detection system is applied to field detection such as field inspection and quarantine, import and export inspection and quarantine and the like, and can be easily put into large-scale popularization and application.
Description
Technical field
The invention belongs to genetically engineered field, relate to detection target and LAMP primer sets compound and the application of soybean north stem canker.
Background technology
Soybean north stem canker (Diaporthephaseolorum var.caulivora) is one of most important destructive disease on north American soybean.After this infection process plant, develop rapidly and form the damage of cane ring-type, and can cause the plant that growing dead.The serious field of falling ill has 80% plant to be infected.The production loss causing is thus up to 50%
[1-4].Rapidly, except the U.S., there is the report of causing harm 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 it occurs generally in recent years abroad, economic impact is large, import the report that not yet there is generation in risk Gao Qie China into, China is classified as one of 13 kinds of quarantine pathogenic bacterias on the soybean of paying close attention to
[9].In order to stop the continuous expansion of soybean Phomopsis seed decay pathogen spread scope, make soybean south stem canker controlled, need to detect quickly and accurately it.
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) is that the people such as Notomi of Japanese Rong Yan Co., Ltd. are in a kind of novel nucleic acid amplification method of exploitation in 2000
[10], be characterized in 4 Auele Specific Primers of 6 zone design for target gene, under the effect of strand displacement DNA enzyme (Bst DNA polymerase), 60～65 ℃ of constant-temperature amplifications, 15～60min gets final product observations, and efficiency can reach 10
9～10
10the individual order of magnitude, has the easily feature such as detection of simple to operate, high specificity, product.When DNA is synthetic, the pyrophosphate ion of separating out from picodna triphosphoric acid matrix (dNTPs) reacts with the magnesium ion in reaction soln, produces a large amount of magnesium pyrophosphate precipitations, presents white.Therefore, can be using opacity as the index of reacting, the white opacity that only detects by an unaided eye precipitation, 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 can judge whether reaction by the colour-change whether forming after white precipitate (muddiness) or the dyeing of SYBR GREEN I.Along with constantly improving and development of technology, can have a wide range of applications in fields such as food inspection clinical disease diagnosis [11,12].
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 sets compound 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 sets compound thereof.
Object of the present invention can be achieved through the following technical solutions:
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 detecting or is identifying the application in the stem canker of the soybean north.
Specificity LAMP primer sets compound for the detection target sequence Tef design of described soybean north stem canker, comprise that forward inner primer FIP is as shown in SEQ ID NO.2, oppositely inner primer BIP is as shown in SEQ ID NO.3, forward outer primer F3 is as shown in SEQ ID NO.4, and oppositely outer primer B3 is as shown in SEQ ID NO.5.
Described specific LAMP primer sets compound preferably also comprises forward ring primer LF as shown in SEQ ID NO.6, oppositely encircles primer LB as shown in SEQ ID NO.7.
The specificity LAMP primer sets compound of the described detection target sequence Tef design for described soybean north stem canker is detecting or is identifying the application in the stem canker of the soybean north.
A LAMP detection kit for detection of soybean north stem canker, comprises described specific LAMP primer sets compound.
The described LAMP detection kit for detection of soybean north stem canker, comprise: by 20mM forward inner primer FIP, the reverse inner primer BIP of 20mM, 10mM forward outer primer F3, the reverse outer primer B3 of 10mM, 10mM forward ring primer LF, 10mM oppositely encircle primer LR, 9.26%10xThermoPol Reaction Buffer, 50mM MgSO
4, the detection solution that forms of 10mMdNTPMixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment and ultrapure water.
Detect a method for soybean north stem canker, extract the DNA of microorganism to be checked, the DNA extracting of take is template, utilizes described specific LAMP primer sets compound to carry 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 UV-light under ordinary light, represent to exist soybean north stem canker, under ordinary light, under aobvious orange, UV-light, without fluorescence, produce and represent containing this germ.
The method of described detection soybean north stem canker is preferably extracted the DNA of microorganism to be checked, gets 3 μ lDNA solution, adds detection solution and 2.5 μ l sterilizing deionized waters described in 21.5 μ l to carry out LAMP reaction, and LAMP response procedures is: 64 ℃ 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, design 6 specific LAMP primers, set up on this basis the LAMP reaction system that detects soybean north stem canker.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 be fine meet at present to the Site Detection of soybean north stem canker in the urgent need to, Site Detection for field quarantine, import and export quarantine etc., is easy to apply on a large scale.
Accompanying drawing explanation
Between Fig. 1 embodiment 2 soybean north stem canker kinds, specific test ordinary light irradiates figure
Wherein, be for No. 1, No. 2 soybean north stem canker different strains, No. 3, No. 4 is southern stem canker of soybean different strains, 5～No. 7 for intending stem dibbling maize ear rot bacterium different strains, No. 8 negative contrasts.
Specific test UV-irradiation figure between Fig. 2 embodiment 2 soybean north stem canker kinds
Wherein, be for No. 1, No. 2 soybean north stem canker different strains, No. 3, No. 4 is southern stem canker of soybean different strains, 5～No. 7 for intending stem dibbling maize ear rot bacterium different strains, No. 8 negative contrasts.
Fig. 3 embodiment 3 soybean north stem canker specificity among genus test ordinary light rayed figure
Wherein, be for No. 1, No. 2 soybean north stem canker different strains, be for No. 3 soybean rest fungus, being for No. 4 glue born of the same parents anthrax-bacilus, is for No. 5 tack anthrax-bacilus, is for No. 6 rice blast fungus, being for No. 7 alternaric bacteria, is for No. 8 ball Tuber Melanosporum, is for No. 9 soyabean phytophthora, being for No. 10 Kidney bean shell ball spore bacterium, is for No. 11 aspergillus oryzae, is for No. 12 Kikuchi tail spore bacterium, it is for No. 13 oil bottle mould, being for No. 14 cloves phytophthora, is for No. 15 ramie mould, No. 16 negative contrasts.
Fig. 4 embodiment 3 soybean north stem canker specificity among genus test UV-irradiation figure
Wherein, be for No. 1, No. 2 soybean north stem canker different strains, be for No. 3 soybean rest fungus, being for No. 4 glue born of the same parents anthrax-bacilus, is for No. 5 tack anthrax-bacilus, is for No. 6 rice blast fungus, being for No. 7 alternaric bacteria, is for No. 8 ball Tuber Melanosporum, is for No. 9 soyabean phytophthora, being for No. 10 Kidney bean shell ball spore bacterium, is for No. 11 aspergillus oryzae, is for No. 12 Kikuchi tail spore bacterium, it is for No. 13 oil bottle mould, being for No. 14 cloves phytophthora, is for No. 15 ramie mould, No. 16 negative contrasts.
Fig. 5 embodiment 4 soybean north stem canker sensitivity test ordinary lights irradiate figure
LAMP amplification different concns soybean north stem canker genomic dna; From left to right be respectively the LAMP amplification that contains respectively 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 be identified 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 that contains respectively 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 be identified soybean north stem canker specifically, and picture represents that sensitivity can reach 10pg/ μ L.
The susceptible soybean plant strain LAMP of Fig. 7 detects test 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, 10 negative contrasts.
The susceptible soybean plant strain LAMP of Fig. 8 detects test 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, 10 negative contrasts.
Embodiment
A kind of LAMP detection kit for detection of soybean north stem canker, comprise: 20mM forward inner primer FIP, the reverse inner primer BIP of 20mM, 10mM forward outer primer F3, the reverse outer primer B3 of 10mM, 10mM forward ring primer LF, 10mM oppositely encircle primer LR, 9.26%10xThermoPol Reaction Buffer, 50mM MgSO
4, 10mMdNTP Mixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment, add ultrapure water to be prepared into detection solution.Each constituent concentration all refers to the final concentration in detecting solution.
Specific test between embodiment 2 soybean north stem canker kinds
In order to verify the specificity of LAMP method, the DNA that selects 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 and 2.5 μ l sterilizing deionized waters described in 21.5 μ l embodiment 1 to carry out LAMP reaction, response procedures is: 64 ℃ of 70min; Amplified production adds SYBR GREEN I dyestuff, under ordinary light UV-light, detects.As depicted in figs. 1 and 2, demonstration Auele Specific Primer is identified soybean north stem canker to result specifically, and the sample that contains soybean north stem canker (No. 1 pipe and No. 2 pipes) meeting yellowing under ordinary light, produces intense fluorescence under UV-light; And other kinds (3～No. 7 pipes) become orange under ordinary light, under UV-light, do not produce fluorescence yet, negative control (No. 8 pipes) becomes orange under ordinary light, does not also produce fluorescence under UV-light, and this LAMP detection method that shows that the present invention sets up has specificity between very high kind.
In order to verify the specificity of LAMP method, bacterium (the soybean rest fungus of selecting soybean north stem canker reference culture (CBS177.55) and soybean north stem canker not to belong to together; 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, gets 3 μ lDNA solution, adds detection solution and 2.5 μ l sterilizing deionized waters described in 21.5 μ l embodiment 1 to carry out LAMP reaction, and response procedures is: 64 ℃ of 70min; Amplified production adds SYBR GREEN I dyestuff, under ordinary light UV-light, detects.As shown in Figure 3 and Figure 4, demonstration Auele Specific Primer is identified soybean north stem canker to result specifically, and the sample that contains soybean north stem canker (No. 1 pipe and No. 2 pipes) meeting yellowing under ordinary light, produces intense fluorescence under UV-light; And other kinds (3～No. 15 pipes) become orange under ordinary light, under UV-light, do not produce fluorescence yet, negative control (No. 16 pipes) becomes orange under ordinary light, does not also produce fluorescence under UV-light, and this LAMP detection method that shows that the present invention sets up has very high specificity among genus.
In order to determine the sensitivity of LAMP detection method, the standard north stem canker bacterial strain (CBS177.55) extracting is carried out to 10 doubling dilutions with DEPC water after spectrophotometric determination concentration (1 μ g/ μ l) for DNA ,-70 ℃ of preservations are as template.Get respectively each concentration DNA diluent 3 μ l after 10 doubling dilutions as template, add detection solution and 2.5 μ l sterilizing deionized waters described in 21.5 μ l embodiment 1 to carry out LAMP reaction, response procedures is: 64 ℃ of 70min; Amplified production adds SYBR GREEN I dyestuff, under ordinary light and UV-light, detects, and result as shown in Figure 5, Figure 6.Under ordinary light, detecting as seen from Figure 5 the stem canker sensitivity of the soybean north is 10pg/ μ L, and detect the stem canker sensitivity of the soybean north under UV-light, is 10pg/ μ L.
The susceptible soybean plant strain LAMP of embodiment 5 detects test
DNA with the soybean plant strain of NaOH alkaline lysis method of extracting 6 strain inoculation standard soybean north stem cankers (CBS177.55) after 6 days, using it as template, for LAMP, increase, 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 and 2.5 μ l sterilizing deionized waters described in 21.5 μ l embodiment 1 to carry out LAMP reaction, response procedures is: 64 ℃ of 70min.Amplified production adds SYBR GREEN I dyestuff, under ordinary light and UV-light, detects.Result is as shown in Fig. 7～8, show that 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 (No. 8 pipe) of blank PDA substratum after 6 days becomes orange with healthy plant (No. 9 pipes) and negative control aqua sterilisa (No. 10 pipes) under ordinary light, under UV-light, also do not produce fluorescence, visible present method can be 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?and?University?of?Illinois.
3.McGee?D,Biddle?J(1987)Seedborne?Diaporthe?phaseolorum?var.caulivora?in?Iowa?and?its?relationship?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?stem?blight?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?core?soybean-producing?area?of?Argentina.Phytopathology93:136-146.
6.Fernández?FA,Hanlin?RT(1996)Morphological?and?RAPD?analyses?of?Diaporthe?phaseolorum?from?soybean.Mycologia:425-440.
7.Sato?T,De?Viedma?LQ,Alvarez?E,Romero?M(1993)First?occurrence?of?soybean?southern?stem?canker?in?Paraguay.Japan?Agricultural?Research?Quarterly27:20-20.
8.Vrandecic?K,Cosic?J,Riccioni?L,Duvnjak?T,Jurkovic?D(2005)Isolation?of?Diaporthe?phaseolorum?var.caulivora?from?Abutilon?theophrasti?in?Croatia.Plant?Pathology54:576-576.
9. Wu Pin coral, sternly enters the new disease of soybean that (2003) merit attention. Plant Quarantine 17:226-228.
10.Notomi?T,Okayama?H,Masubuchi?H,Yonekawa?T,Watanabe?K,et?al.(2000)Loop-mediated?isothermal?amplification?of?DNA.Nucleic?Acids?Res28:E63.
11.Mori?Y,Nagamine?K,Tomita?N,Notomi?T(2001)Detection?of?loop-mediated?isothermal?amplification?reaction?by?turbidity?derived?from?magnesium?pyrophosphate?formation.Biochem?Biophys?Res?Commun289:150-154.
12. Xiao Bin, Zhu Yonghong, the ring mediated isothermal amplification gene diagnostic new technology of the easy sensitivity of Zou Quanming (2005). Chinese laboratory medicine magazine 28:761-763.
Claims (9)
1. the detection target sequence Tef of soybean north stem canker, is characterized in that nucleotide sequence is as shown in SEQ ID NO.1.
The detection target sequence Tef of the soybean north stem canker shown in 2.SEQ ID NO.1 is detecting or is identifying the application in the stem canker of the soybean north.
3. for the specific LAMP primer sets compound of the detection target sequence Tef design of soybean claimed in claim 1 north stem canker, it is characterized in that forward inner primer FIP is as shown in SEQ ID NO.2, oppositely inner primer BIP is as shown in SEQ ID NO.3, forward outer primer F3 is as shown in SEQ ID NO.4, and oppositely outer primer B3 is as shown in SEQ ID NO.5.
4. specific LAMP primer sets compound according to claim 3, is characterized in that also comprising forward ring primer LF as shown in SEQ ID NO.6, oppositely encircles primer LB as shown in SEQ ID NO.7.
5. the application in the reagent of preparation detection or evaluation soybean north stem canker for LAMP primer sets compound claimed in claim 1 described in claim 3 or 4.
6. for detection of a LAMP detection kit for soybean north stem canker, it is characterized in that comprising the specific LAMP primer sets compound described in claim 3 or 4.
7. the LAMP detection kit for detection of soybean north stem canker according to claim 5, it is characterized in that described test kit comprises: by 20mM forward inner primer FIP, the reverse inner primer BIP of 20mM, 10mM forward outer primer F3, the reverse outer primer B3 of 10mM, 10mM forward ring primer LF, 10mM oppositely encircle primer LR, 9.26%10xThermoPol Reaction Buffer, 50mM MgSO
4, the detection solution that forms of 10mM dNTP Mixture, 5M trimethyl-glycine, 8000U/mL Bst DNA Polymerase Large fragment and ultrapure water.
8. a method that detects soybean north stem canker, is characterized in that extracting the DNA of microorganism to be checked, and the DNA extracting of take is template, utilizes the specific LAMP primer sets compound described in claim 3 or 4 to carry out LAMP amplified reaction; Amplified production adds SYBR GREEN I dyestuff, under ordinary light and/or UV-light, detects, if reaction product becomes displaing yellow, produces intense fluorescence under UV-light under ordinary light, represents to exist soybean north stem canker; If reaction product produces without fluorescence under aobvious orange, UV-light under ordinary light, represent containing this germ.
9. the method for detection according to claim 8 soybean north stem canker, it is characterized in that extracting the DNA of microorganism to be checked, get 3 μ l DNA solutions, add 21.5 μ l detection solution claimed in claim 7 and 2.5 μ l sterilizing deionized waters to carry out LAMP reaction, response procedures is: 64 ℃ of 70min; Volume increase thing adds SYBR GREEN I dyestuff, and detected result under UV-light, if reaction product produces intense fluorescence, represents to exist soybean north stem canker, without fluorescence, produces and represents not containing this germ.
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| CN111041123A (en) * | 2020-01-08 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
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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)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111041123A (en) * | 2020-01-08 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
| CN111041123B (en) * | 2020-01-08 | 2022-07-08 | 北京林业大学 | LAMP primer and kit for detecting Botryosphaeria sinensia |
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