CN103589800B - LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group - Google Patents
LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group Download PDFInfo
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Abstract
The invention relates to an LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat. The LAMP primer group consists of LAMP primers detected to produce DON toxins and NIV toxins, wherein the LAMP primers detected to produce the DON toxins comprise a forward inner primer expressed by SEQ ID No.1, a reverse inner primer expressed by SEQ ID No.2, a forward outer primer expressed by SEQ ID No.3 and a reverse outer primer expressed by SEQ ID No.4; the LAMP primers detected to produce the NIV toxins comprise a forward inner primer expressed by SEQ ID No.5, a reverse inner primer expressed by SEQ ID No.6, a forward outer primer expressed by SEQID No.7 and a reverse outer primer expressed by SEQID No.8. In the application, a kit A containing the LAMP primers detected to produce the DON toxins and a kit B containing the LAMP primers detected to produce the NIV toxins are respectively used for synchronously implementing parallel LAMP amplification for DNA of extracted fusarium graminearum; the types of the toxins produced by the gibberellic disease infected wheat are directly distinguished according to amplification results.
Description
Technical field
The invention belongs to and genetically engineered field, relate to a kind of head blight of distinguishing by LAMP primer group and the application thereof of feeling wheat toxin producing type.
Background technology
By the important disease of one that the microbial head blight of reaping hook (Fusarium head blight or scab) is the multiple cereal crops such as harm wheat, barley, oat, corn, especially serious on wheat, mainly concentrate on flowering stage of wheat to infect, cause a disease at seed grouting parameter, not only cause the output underproduction, the toxin that same sickle-like bacteria produces retains and is accumulated in crop kernel, affects the seed output and quality of cereal.In China, Fusarium graminearum (Fusariumgraminearum Schwabe) is the main pathogenic fungi of head blight, it produces Type B Trichothecenes toxin, mainly comprise deoxynivalenol (Deoxynivalenol, be called for short DON) and nivalenol (Nivalenol is called for short NIV).And these two kinds of toxin have height hazardness to people and animals, cause humans and animals to occur vomiting, diarrhoea, the symptom such as dizzy, thus accurately differentiate that fusarium graminearum produces the management in the type of DON or NIV toxin and postpartum antenatal to wheat and quality evaluation has directive function fast.
The detection method of fusarium graminearum toxin producing type has three kinds, and one is chemical assay, and the method needs the Sample Purification on Single preparation procedure of expensive plant and instrument and complexity, required time length, technology content requires also higher; Two is immunizations, and the method is simple to operate, and expense is lower, but because normal containing some other derivatives in the toxin detected, so the result measured is often higher; Three is PCR methods, and the method needs specific PCR instrument device, reaction times relatively long, and result needs agarose electrophoresis-EB to detect, and easily causes the problems such as EB environmental pollution.
And ring type isothermal duplication (LAMP:loop-mediated isothermal amplification) technology is highly sensitive method of carrying further on the basis of detection of nucleic acids, advantages such as there is isothermal duplication, do not need specific PCR instrument device, the reaction times is short, to the detection of amplification, only need to add fluorescence dye, changed by visual color and carry out result of determination, thus realize the nucleic acid detection method of rapid amplifying, easy detection; The method is easy, reliable results, can be widely used in Plant Quarantine, food safety detection analysis.But use it for Toxin Classification not appear in the newspapers.
Summary of the invention
The object of the invention is, provide and distinguish head blight produces DON or NIV toxin LAMP primer group and application thereof by sense wheat.
The object of the invention is to be achieved through the following technical solutions: a kind of head blight of distinguishing is by the LAMP primer group feeling wheat toxin producing type, it is characterized in that: LAMP primer group forms by detecting the LAMP primer of producing DON toxin and detecting the LAMP primer of producing NIV toxin, wherein: detect the LAMP primer of producing DON toxin for the reverse outer primer DON-B3 shown in forward outer primer DON-F3, SEQ ID No.4 shown in reverse inner primer DON-BIP, SEQ ID No.3 shown in forward inner primer DON-FIP, SEQ ID No.2 shown in SEQ ID No.1; Detect the LAMP primer of producing NIV toxin for: just guide that the reverse outer primer NIV-B3 shown in forward outer primer NIV-F3, SEQ ID No.8 shown in reverse inner primer NIV-BIP, SEQ ID No.7 shown in interior thing NIV-FIP, SEQ ID No.6 forms into shown in SEQ ID No.5.
An application for above-mentioned LAMP primer group, is characterized in that:
A) LAMP primer detecting toxin DON is set up test kit A, the LAMP primer detecting toxin NIV is set up test kit B;
B) be separated the Fusarium graminearum of head blight by sense wheat, extract the DNA of this Fusarium graminearum, with the DNA extracted for template, utilize test kit A and test kit B synchronously to carry out parallel LAMP amplification respectively;
C) parallel LAMP amplified production carries out agarose gel electrophoresis respectively, detected result under ultraviolet light, there is gradient dispersivity band for positive; Or in amplified production, add SYBR Green respectively, color becomes green for positive, color keep orange constant be feminine gender;
D) intuitively distinguish according to the detected result of step c, the head blight that test kit A is positive produces DON toxin by sense wheat, the head blight that test kit B is positive produces NIV toxin by sense wheat, and namely the head blight that test kit A and test kit B is all negative is not produced DON toxin by sense wheat and do not produced NIV toxin yet.
In the application of above-mentioned LAMP primer group: described test kit A comprises: (NH4) of KCl, 10mM of Tris-HCl, 10mM of betaine, 20mM of dNTPs, 1M of the SEQ ID No.1 of 1.6 μMs, the SEQ ID No.2 of 1.6 μMs, the SEQ ID No.3 of 0.2 μM, SEQ ID No.4,1.6mM of 0.2 μM
2sO
4, 4mM MgSO
4, 0.1% Triton X-100,8U Bst DNA polymerase 500 unit, be prepared into detection solution, wherein the pH of Tris-HCl is 8.8; Described test kit B comprises: the SEQ ID No.5 of 1.6 μMs, 1.6 μMs, (NH4) of KCl, 10mM of Tris-HCl, 10mM of betaine, 20mM of dNTPs, 1M of SEQ ID No.6, the SEQ ID No.7 of 0.2 μM, SEQ ID No.8,1.6mM of 0.2 μM
2sO
4, 4mM MgSO
4, 0.1% Triton X-100,8U Bst DNA polymerase 500 unit, be prepared into detection solution, wherein the pH of Tris-HCl is 8.8.
In the application of above-mentioned LAMP primer group: the DNA extracting microorganism to be detected, with the DNA extracted for template, utilize test kit A and test kit B synchronously to carry out parallel LAMP amplification respectively to refer to: the DNA extracting microorganism to be checked, get 1 μ l DNA solution simultaneously, add the test kit A of 24 μ l or test kit B respectively to detect in solution and synchronously carry out LAMP, LAMP reaction conditions is: 60-65 DEG C, 60-90min; 80 DEG C, 10min.
In the application of above-mentioned LAMP primer group: the LAMP reaction conditions of test kit A preferably 64 DEG C, 60min; 80 DEG C, 10min; The LAMP reaction conditions of test kit B preferably 62 DEG C, 60min; 80 DEG C, 10min.
The invention has the advantages that: adopt ring type isothermal duplication (LAMP) technology to being put forward highly sensitive method further on the basis of detection of nucleic acids, advantages such as there is isothermal duplication, do not need specific PCR instrument device, the reaction times is short, to the detection of amplification, only need to add fluorescence dye, changed by visual color and carry out result of determination, thus realize the nucleic acid detection method of rapid amplifying, easy detection, realize producing DON or NIV Toxin Classification to by sense wheat.
Accompanying drawing explanation
Fig. 1 (a) and Fig. 1 (b) are the susceptibilitys of by the LAMP augmentation detection of test kit A, fusarium graminearum being produced to DON Type of toxin; Agarose electrophoresis figure (M:DL2000 DNA Marker [the DNA molecular amount standard of Fig. 1 (a) LAMP amplified production, maximum band is 2000bp], 1-7 is followed successively by the amplification respectively containing 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg DNA in the reaction system of 25 μ l); (reaction tubes respectively containing 100ng, 10ng, 1ng, 100pg, 10pg fusarium graminearum DNA in the reaction system of 25 μ l is aobvious green, is positive for the color development figure of Fig. 1 (b) LAMP amplified production; Reaction tubes respectively containing 0 [water], 1pg, 100fg fusarium graminearum DNA in the reaction system of 25 μ l shows orange, and be negative reaction).
Fig. 2 (a) and Fig. 2 (b) are the susceptibilitys of by the LAMP augmentation detection of test kit B, fusarium graminearum being produced to NIV Type of toxin; Agarose electrophoresis figure (M:DL2000 DNA Marker [the DNA molecular amount standard of Fig. 2 (a) LAMP amplified production, maximum band is 2000bp], 1-7 is followed successively by the amplification respectively containing 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg DNA in the reaction system of 25 μ l); (reaction tubes respectively containing 100ng, 10ng, 1ng, 100pg, 10pg fusarium graminearum DNA in the reaction system of 25 μ l is aobvious green, is positive for the color development figure of Fig. 2 (b) LAMP amplified production; Reaction tubes respectively containing 0 [water], 1pg, 100fg fusarium graminearum DNA in the reaction system of 25 μ l shows orange, and be negative reaction).
Fig. 3 is that the LAMP amplification checking of test kit A is to the reliability (1-7 is followed successively by JS18, JS1217, A17, JS08,68,127 and 117) of fusarium graminearum product DON Type of toxin.
Fig. 4 is that LAMP verifies that test kit B produces the reliability (1-7 is followed successively by JS18, JS1217, A17, JS08,68,127 and 117) of NIV Type of toxin to fusarium graminearum.
Embodiment
The invention will be further described in conjunction with specific embodiments.
embodiment 1
Test kit A detects the LAMP that fusarium graminearum produces DON Type of toxin, and it comprises:
SEQ ID No.1, forward inner primer DON-FIP:
5'-TCAACTACCGGCTGCTAGTAGATAGTGGATACGTCTTCCAGGA-3'
SEQ ID No.2, reverse inner primer DON-BIP:
5'-CCTGCCATGTAGAGGTGAGGAGCAGGGGAAGCACAATTGAGATC-3'
SEQ ID No.3, forward outer primer DON-F3:
5'-ACAGTTATTCCAAACAGTTCG-3'
SEQ ID No.4, reverse outer primer DON-B3:
5'-TGACAAGTCCGGTCGCAC-3'
Preparation detection solution in: 1.6 μMs of forward inner primer DON-FIP, 1.6 μMs of reverse inner primer DON-BIP, 0.2 μM of forward outer primer DON-F3,0.2 μM of reverse outer primer DON-B3,1.6mM dNTPs, 1M betaine, 20mM Tris-HCl(pH8.8), 10mM KCl, 10mM (NH4)
2sO
4, 4mM MgSO
4, 0.1%Triton X-100,8U Bst DNA polymerase 500 unit.
embodiment 2
Test kit B detects the LAMP that fusarium graminearum produces NIV Type of toxin, and it comprises:
SEQ ID No.5, forward inner primer NIV-FIP:
5'-CTATCTGCAGAACTCGAATTGCTCCATATCACTCACATATCCAT-3'
SEQ ID No.6, reverse inner primer NIV-BIP:
5'-ACATCGTCCAGGATGTAGTCTAGCAGGCTCAAGGTAAAGCG-3'
SEQ ID No.7, forward outer primer NIV-F3:
5'-CACCTTGATGTGGTTGCC-3'
SEQ ID No.8, reverse outer primer NIV-B3:
5'-GCATACATAGTACTGGCGAT-3'
Preparation detection solution in: 1.6 μMs of forward inner primer NIV-FIP, 1.6 μMs of reverse inner primer NIV-BIP, 0.2 μM of forward outer primer NIV-F3,0.2 μM of reverse outer primer NIV-B3,1.6mMdNTPs, 1M betaine, 20mM Tris-HCl(pH8.8), 10mM KCl, 10mM (NH4)
2sO
4, 4mM MgSO
4, 0.1%Triton X-100,8U Bst DNA polymerase 500 unit.
embodiment 3in LAMP reaction, verify that test kit A produces the susceptibility of DON Type of toxin to fusarium graminearum.
In order to verify the susceptibility of LAMP method, select the bacterial strain JS05 of known product DON toxin, after (1 μ g/ul) after spectrophotometric measurement concentration, carry out as template after 10 doubling dilutions, get each concentration DNA 1 μ l solution after dilution respectively, the detection solution adding 24 μ l embodiments 1 preparations carries out LAMP reaction, reaction conditions is 64 DEG C, 60min, 80 DEG C, 10min; After reaction terminates, LAMP reaction solution 4 μ l carries out electrophoresis detection, result shows: 1-5 swimming lane is the amplification containing 100ng, 10ng, 1ng, 100pg, 10pg DNA, they have obvious dispersivity band, 6-7 swimming lane is the amplification containing 1pg, 100fgDNA, they and negative control water do not have band, and electrophoresis result shows that the sensitivity that LMAP reacts reaches 10ng; After the completion of reaction, colour-change is observed after adding dyestuff SYBR Green, wherein No. 1-5 pipe is the amplification containing 100ng, 10ng, 1ng, 100pg, 10pg DNA, their color becomes green, No. 6-7 pipe is the amplification containing 1pg, 100fgDNA, they and negative control water come to the same thing, for orange; Proved by colour-change result of determination, test kit A also reaches 10pg(Fig. 1 (a) and Fig. 1 (b) to the detection sensitivity of producing DON toxin bacterium).
embodiment 4in LAMP reaction, verify that test kit B produces the susceptibility of NIV Type of toxin to fusarium graminearum.
In order to verify the susceptibility of LAMP method, select the bacterial strain JS70 of known product NIV toxin, after (1 μ g/ul) after spectrophotometric measurement concentration, carry out as template after 10 doubling dilutions, get each concentration DNA 1 μ l solution after dilution respectively, the detection solution adding 24 μ l embodiments 2 preparations carries out LAMP reaction, reaction conditions is 62 DEG C, 60min, 80 DEG C, 10min; Get LAMP reaction solution 4 μ l after reaction terminates and carry out electrophoresis detection, result shows, 1-4 swimming lane is the amplification containing 100ng, 10ng, 1ng, 100pg DNA, they have obvious dispersivity band, No. 5 swimming lanes are the amplification containing 10pg DNA, and its band is weak, and 6-7 is the amplification containing 1pg, 100fgDNA, they and negative control water do not have band, and electrophoresis result shows that the sensitivity that LMAP reacts reaches 10ng.After the completion of reaction, colour-change is observed after adding dyestuff SYBR Green, wherein No. 1-4 pipe is the amplification containing 100ng, 10ng, 1ng, 100pg DNA, their colors become green, No. 5 is the amplification containing 10pg DNA, and its color is pale green, and 6-7 is the amplification containing 1pg, 100fgDNA, they and negative control water come to the same thing, for orange; Shown by colour-change result of determination, test kit B also reaches 10pg (Fig. 2 (a) and Fig. 2 (b)) to the detection sensitivity of producing NIV toxin bacterium.
embodiment 5in LAMP reaction, verify that test kit A produces the reliability of DON Type of toxin to fusarium graminearum
In order to verify the reliability of test kit A in LAMP method, select the bacterial strain JS18 of known product DON toxin, JS1217, it is different Fusarium graminearum bacterial strains that A17(is separated what obtain from sick wheat) and one do not produce the bacterial strain JS08 of DON and NIV toxin and produce the bacterial strain 68 of NIV toxin, 127, it is different Fusarium graminearum bacterial strains that 117(is separated what obtain from sick wheat), extract the DNA of microorganism respectively as template, get 1 μ l DNA solution respectively, the test kit A adding 24 μ l embodiment 1 preparations carries out LAMP reaction, reaction conditions is 64 DEG C, 60min, 80 DEG C, 10min, after reaction terminates, often pipe adds 2 μ l SYBR Green, observe colour-change, result display 1-3 pipe corresponding toxin producing bacterial strain JS18, JS1217, A17, their color is green, confirm that they produce DON toxin, experimental result conforms to practical situation, and the corresponding toxin producing bacterial strain of 4-7 pipe is JS08,68,127 and 117, and they and water color are orange, confirm not produce DON toxin, also conform to practical situation (accompanying drawing 3).
embodiment 6in LAMP reaction, verify that test kit B produces the reliability of NIV Type of toxin to fusarium graminearum
Consistent with embodiment 5, bacterial strain JS18, JS1217, A17(of same selection known product DON toxin are separated the different Fusarium graminearum bacterial strain obtained from sick wheat) and a bacterial strain JS08 not producing DON with NIV toxin and product NIV toxin bacterial strain 68,127, from sick wheat, be separated what obtain be different Fusarium graminearum bacterial strains to 117(), extract the DNA of microorganism respectively as template, get 1 μ l DNA solution respectively, the test kit B adding 24 μ l embodiment 2 preparations carries out LAMP reaction, reaction conditions is 62 DEG C, 60min, 80 DEG C, 10min; After reaction terminates, often pipe adds 2 μ l SYBR Green, observe colour-change, the corresponding toxin producing bacterial strain 68,127,117 of result display 5-7 pipe, their color becomes green, NIV toxin is produced in confirmation, experimental result conforms to practical situation, and the corresponding toxin producing bacterial strain of 1-4 pipe is JS18, JS1217, A17 and JS08, and they and water color are orange, confirm not produce NIV toxin, also conform to practical situation (accompanying drawing 4).
embodiment 7utilize test kit A and test kit B from the susceptible wheat groat of head blight to the method that gibberellic hypha toxin producing type is classified
Produce the method for the malicious type of head blight susceptible wheat qualification product of malicious type from unknown gibberellic hypha, comprising:
The separation of Fusarium graminearum in susceptible wheat groat:
Sick for the wheat scab of adopting back tassel seed 70% ethanol surface sterilization, sterilizing filter paper dries, is put on PDA substratum in 25 DEG C of constant temperature culture.Cultivate after 2-3 days, treat that substratum grows red colonies, obtain the first isolate of pathogenic bacteria.Choose pin picking on a small quantity in sweet mung bean soup substratum by what PDA substratum is cultivated the first isolate sterilizing that obtains, under 25 DEG C of 175r/min, concussion is cultivated, and can produce conidium after 4-5 days.Draw 10 μ L spore suspensions, along straight line even setting-out on substratum of PDA flat plate bottom, 25 DEG C of constant temperature culture 24h, get single bacterium colony, and be transferred in new PDA flat board, 25 DEG C of constant temperature culture, namely obtain the bacterial strain of purifying.
DNA is extracted from Fusarium graminearum:
A) a small amount of with the gibberella saubinetii mycelia on sterilizing toothpick scraping PDA flat board, load in 1.5 ml sterile centrifugation tube;
B) add 150 μ l Lysis buffer [Lysis buffer (pH 8.0): Tris-HCl 200 mM, EDTA 50 mM, NaCl 20 mM, SDS 1%], grind 1 min with the electric drill with Glass drill head; Add 350 μ lLysisbuffer again and rinse drill bit, after vortex mixing, room temperature places 10 min;
C) 12000 rpm, 4 DEG C of centrifugal 10 min, Aspirate supernatant proceeds in new 1.5 ml centrifuge tubes;
D) add equal-volume dehydrated alcohol, put upside down mixing, 120 min placed by-20 ° of C refrigerators;
E) 12000 rpm, 4 DEG C of centrifugal 10 min;
F) precipitation 70 % washing with alcohol, 12000 rpm, 4 DEG C of centrifugal 1 min, outwell liquid, dry to alcohol-free taste;
G) 50 μ l ddH are added
2o dissolves, for the template of LAMP amplification.
The LAMP of fusarium graminearum toxin producing type detects:
The LAMP that fusarium graminearum produces DON Type of toxin detects: get 1 μ l DNA solution simultaneously, add the test kit A described in the example 1 and test kit B described in embodiment 2 respectively; Wherein:
The cumulative volume of test kit A is 25 μ l; Reaction conditions is: 64 DEG C, 60min; 80 DEG C, 10min; Amplification terminates rear interpolation 2 μ l SYBR Green, and color presents green, judges to produce DON toxin with this; Color does not have vicissitudinous orange, then judge that wheat scab does not produce DON toxin.
The cumulative volume of test kit B is 25ul; Reaction conditions is: 62 DEG C, 60min; 80 DEG C, 10min; Using SYBR Green as reaction indicator, the color that amplification terminates the LAMP reaction system of rear product NIV toxin presents green, judges that fusarium graminearum produces NIV toxin with this; Color does not have vicissitudinous orange, then judge that wheat scab does not produce DON toxin.
Utilize the method for the present embodiment, gather and come from Yancheng, Jiangsu Province, Nanjing, the susceptible wheat of head blight that the unknown gibberellic hypha being respectively 001,002,003,004,005,006,007,008,009,010,011,012,013,014,015,016,017,018,019,020 from numbering of Wuxi produces malicious type carry out laboratory classification, its outcome record in Table 1 ("-" represent do not produce DON and NIV toxin).
Gibberellic hypha toxin producing type statistics identified by table 1
| Numbering | 001 | 002 | 003 | 004 | 005 | 006 | 007 | 008 | 009 | 010 |
| Result | DON | NIV | DON | DON | NIV | DON | DON | NIV | DON | DON |
| Numbering | 011 | 012 | 013 | 014 | 015 | 016 | 017 | 018 | 019 | 020 |
| Result | DON | DON | - | NIV | DON | DON | DON | DON | NIV | DON |
From table, the sample producing DON toxin has 14, and the sample of product NIV toxin has 5, and namely not producing the sample that DON toxin do not produce NIV toxin yet has 1.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> distinguishes head blight by LAMP primer group and the application thereof of feeling wheat toxin producing type
<130> 2013-11-19
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> DNA
<213> artificial sequence
<400> 1
tcaactaccg gctgctagta gatagtggat acgtcttcca gga 43
<210> 2
<211> 44
<212> DNA
<213> artificial sequence
<400> 2
cctgccatgt agaggtgagg agcaggggaa gcacaattga gatc 44
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
acagttattc caaacagttc g 21
<210> 4
<211> 18
<212> DNA
<213> artificial sequence
<400> 4
tgacaagtcc ggtcgcac 18
<210> 5
<211> 44
<212> DNA
<213> artificial sequence
<400> 5
ctatctgcag aactcgaatt gctccatatc actcacatat ccat 44
<210> 6
<211> 41
<212> DNA
<213> artificial sequence
<400> 6
acatcgtcca ggatgtagtc tagcaggctc aaggtaaagc g 41
<210> 7
<211> 18
<212> DNA
<213> artificial sequence
<400> 7
caccttgatg tggttgcc 18
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
gcatacatag tactggcgat 20
Claims (5)
1. distinguish the LAMP primer group of head blight by sense wheat toxin producing type for one kind, it is characterized in that: LAMP primer group forms by detecting the LAMP primer of producing DON toxin and detecting the LAMP primer of producing NIV toxin, wherein: detect the LAMP primer of producing DON toxin for the forward outer primer DON-F3 shown in reverse inner primer DON-BIP, SEQ ID No.3 shown in forward inner primer DON-FIP, SEQ ID No.2 shown in SEQ ID No.1 and the reverse outer primer DON-B3 shown in SEQ ID No.4; Detect the LAMP primer of producing NIV toxin for: just guide the forward outer primer NIV-F3 shown in reverse inner primer NIV-BIP, SEQ ID No.7 shown in interior thing NIV-FIP, SEQ ID No.6 and the reverse outer primer NIV-B3 shown in SEQ ID No.8 into shown in SEQ ID No.5.
2. an application for LAMP primer group described in claim 1, is characterized in that:
A) LAMP primer detecting toxin DON is set up test kit A, the LAMP primer detecting toxin NIV is set up test kit B;
B) be separated the Fusarium graminearum of head blight by sense wheat, extract the DNA of this Fusarium graminearum, with the DNA extracted for template, utilize test kit A and test kit B synchronously to carry out parallel LAMP amplification respectively;
C) parallel LAMP amplified production carries out agarose gel electrophoresis respectively, detected result under ultraviolet light, there is gradient dispersivity band for positive; Or in amplified production, add SYBR Green respectively, color becomes green for positive, color keep orange constant be feminine gender;
D) intuitively distinguish according to the detected result of step c, the head blight that test kit A is positive produces DON toxin by sense wheat, the head blight that test kit B is positive produces NIV toxin by sense wheat, and namely the head blight that test kit A and test kit B is all negative is not produced DON toxin by sense wheat and do not produced NIV toxin yet.
3. the application of LAMP primer group described in claim 2, is characterized in that: described test kit A comprises: (NH4) of KCl, 10mM of Tris-HCl, 10mM of betaine, 20mM of dNTPs, 1M of the SEQ ID No.1 of 1.6 μMs, the SEQ ID No.2 of 1.6 μMs, the SEQ ID No.3 of 0.2 μM, SEQ ID No.4,1.6mM of 0.2 μM
2sO
4, 4mM MgSO
4, 0.1% Triton X-100,8U Bst DNA polymerase 500 unit, be prepared into detection solution, wherein the pH of Tris-HCl is 8.8; Described test kit B comprises: (NH4) of KCl, 10mM of Tris-HCl, 10mM of betaine, 20mM of dNTPs, 1M of the SEQ ID No.5 of 1.6 μMs, the SEQ ID No.6 of 1.6 μMs, the SEQ ID No.7 of 0.2 μM, SEQ ID No.8,1.6mM of 0.2 μM
2sO
4, 4mM MgSO
4, 0.1% Triton X-100,8U Bst DNA polymerase 500 unit, be prepared into detection solution, wherein the pH of Tris-HCl is 8.8.
4. the application of the LAMP primer group according to Claims 2 or 3, it is characterized in that: the DNA extracting microorganism to be detected, with the DNA extracted for template, utilize test kit A and test kit B synchronously to carry out parallel LAMP amplification respectively to refer to: the DNA extracting microorganism to be checked, get 1 μ l DNA solution simultaneously, add respectively in the test kit A of 24 μ l and the detection solution of test kit B and synchronously carry out LAMP amplification, their LAMP reaction conditions is: 60-65 DEG C, 60-90min; 80 DEG C, 10min.
5. the application of LAMP primer group according to claim 4, is characterized in that: the LAMP reaction conditions of test kit A preferably 64 DEG C, 60min; 80 DEG C, 10min; The LAMP reaction conditions of test kit B preferably 62 DEG C, 60min; 80 DEG C, 10min.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310580088.4A CN103589800B (en) | 2013-11-19 | 2013-11-19 | LAMP primer group for distinguishing types of toxins produced by gibberellic disease infected wheat and application of LAMP primer group |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1715915A (en) * | 2004-07-02 | 2006-01-04 | 华中农业大学 | Molecular detection method for fusarium toxin |
| CN102094080A (en) * | 2009-12-09 | 2011-06-15 | 中国农业科学院植物保护研究所 | Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof |
| CN102443590A (en) * | 2010-10-09 | 2012-05-09 | 华中农业大学 | Molecular identification method of trichothecene type-A toxins of fusarium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1715915A (en) * | 2004-07-02 | 2006-01-04 | 华中农业大学 | Molecular detection method for fusarium toxin |
| CN102094080A (en) * | 2009-12-09 | 2011-06-15 | 中国农业科学院植物保护研究所 | Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof |
| CN102443590A (en) * | 2010-10-09 | 2012-05-09 | 华中农业大学 | Molecular identification method of trichothecene type-A toxins of fusarium |
Non-Patent Citations (1)
| Title |
|---|
| LAMP(环介导等温扩增)的原理及在寄生虫检测上的应用;史亚东等;《中国人兽共患病学报》;20111231;第27卷(第10期);935-939 * |
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