CN106893762A - A kind of LAMP detection primer combination of fumonisin and its LAMP detection kit and LAMP method - Google Patents
A kind of LAMP detection primer combination of fumonisin and its LAMP detection kit and LAMP method Download PDFInfo
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Abstract
The invention discloses a kind of LAMP primer composition and LAMP detection kit and LAMP detection method for detecting fumonisin.The LAMP primer composition is made up of positive outer primer F3, reverse outer primer B3, positive inner primer FIP, reverse inner primer BIP, positive ring primer LF and reverse ring primer LB.Detection method high specificity of the invention, accuracy are high, easy to operate, practicality is good, realize isothermal duplication, can be used for the high sensitivity quick detection of fumonisin, it is easy to large-scale promotion.
Description
Technical field
The invention belongs to biological technical field, and in particular to one kind volt horse (Fumonisin) LAMP detection primer combination and its
LAMP detection kit and LAMP method.
Background technology
Maize kernel rot is distributed all over the world, and its cause of disease is typically propagated by air-flow, and seed is invaded through filigree, causes seed
Kernel rot is rotten.Pathogen of maize ear rot bacterium huge number, but it is that sickle-like bacteria wherein takes turns a sickle to be distributed most extensively, to Influence of production maximum
Knife bacterium Fusarium verticillioides and fusarium prolifertum Fusarium proliferatum are dominant bacteria.Germ also can be through seed
Carry disease germs and enter milpa and fruit ear (Murillo et al., 2008 by systemic infection；Wilke et al., 2007)；Also can be through insect
The wound for causing turns pest-resistant into fruit ear or by the direct band of polypide is to the seed wound of insect moth food and causes serious production loss
Gene corn is effectively reduced generation (Song Liqiu etc., 2009 of ear rot；Munkvold et al., 1997,1999), in Corn Seeds
Grain harvests storage, and the germ of latent infection can also trigger seed to go mouldy (Ma Bingyuan etc., 1995).
In addition to production loss, sickle-like bacteria also produces various mycotoxins, and wherein fumonisin is a kind of most important of which.Volt horse
Toxin is a class by different many hydrogen alcohol diester compound similar with the structure that tricarballylic acid is constituted., Gelderblom in 1988
Deng isolating fumonisin from fusarium moniliforme nutrient solution first.Then, Laurent etc. separates ending of the dog days horse from fumonisin again
Toxin B1 (FB1) and fumonisin B2 (FB2).Up to the present, the fumonisin of discovery have FA1, FA2, FB1,
Totally 11 kinds of FB2, FB3, FB4, FC1, FC2, FC3, FC4 and FP1, wherein 60% above is FB1, its toxicity
It is most strong.Cause the main pathogen wheel branch sickle-like bacteria Fusarium verticillioides and fusarium prolifertum Fusarium of ear rot
Proliferatum bacterium produce substantial amounts of fumonisin.Effect of the fumonisin in people and animals' body is more complicated, it and sphingosine
The structure of (Sphinngosine, SO) and dihydrosphingosine (Shpinganine, SA) is very much like, then both of which
It is the chain backbone long of sphingolipid.Fumonisin is synthesized by suppressing N- fatty acyl group sphingosine synzyme, blocking SO,
The metabolism of sphingolipid or the function of influence sphingolipid are destroyed, this suppression can cause SA to raise, so as to cause tissue, blood, urine
Middle SA/SO ratios are raised.Research finds that the FB1 of high concentration can cause various domestic animals and experimental animal the urgency of species specificity occur
Toxication shape, such as horse white matter of brain soften syndrome, pig pulmonary edema and sheep liver nephropathy, it has further been discovered that FB1 may be with the food of people
Pipe cancer is relevant with liver cancer generation.
At present, the assay method of mycotoxin mainly has bioassary method, chemical assay and Radioimmunoassay of vascular endothelial growth.Biologicall test
Method is a lack of unified standardizing standard, and easily effected by environmental factors, is generally simply possible to use in qualitative analysis.Chemistry
Determination method is main assay method, with it is accurate, reproducible the characteristics of, but to depend on costliness scientific research apparatus, and
And the extraction purification complex steps of early stage, technical requirements are high, and time-consuming, it is difficult to meet the requirement of rapid field detection.Immunology
Determination method is simple to operate compared with chemical assay, quickly, but depends on specific antibody, is only capable of detecting a small number of toxin, and
And easily by the interference of some derivatives, false positive rate is high, and measurement result is not accurate enough.
In recent years, with the development of molecular biology, the particularly announcement of sickle-like bacteria whole genome sequence, domestic and international scientist exists
The synthesis of fumonisin achieves impressive progress with regulation and control aspect, has parsed each gene and multiple correlations in toxin synthetic gene cluster
The function of controlling gene.Sequence information based on toxin synthesis and controlling gene, develop quick and with low cost detection method by
Gradually turn into the focus of scientist's research.Such as：Baird etc. (2008) develops detection volt horse poison using Fum1 gene sequence informations
The PCR detection method of element, achieves good effect.But its detection needs carries out completing polymerase chain reaction in PCR instrument
Should, it is higher to instrument requirements, and Standard PCR reaction and time are long, and sensitivity is relatively low.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is that a kind of new isothermal expands
Increasing technology, the features such as because of its quick, specificity simple to operate, sensitivity high, low cost, being increasingly becoming can substitute normal PCR
New nucleic acid amplification detection technique.4 special primers are designed in 6 regions that it is directed to target gene, poly- using Bst DNA
The constant temperature high efficiency amplification in same reaction system of the strand-displacement activity of synthase, while a large amount of synthesis target DNAs, produces by-product
Thing-white magnesium pyrophosphate precipitation.6 independent regions of target DNA are depended on due to amplified reaction, therefore specificity is very high,
Reaction is carried out under isothermal conditions, then do not need the expensive devices such as PCR instrument, is only able to maintain that setting for stabilization constant temperature with water-bath etc.
Standby to complete, testing cost is substantially reduced, and range of application is significantly expanded, and can carry out real-time monitoring in field.Due to LAMP
The many advantages of technology, are widely used at aspects such as cause of disease analyte detections in recent years, but there is no correlation in fumonisin context of detection
Report for work.
The content of the invention
Goal of the invention：For the problem present on, it is an object of the invention to provide the LAMP primer group that one group of detection lies prostrate Ma Su
Close, fumonisin can be detected.It is a further object of the present invention to provide the LAMP detection kit of above-mentioned fumonisin.This hair
Bright another object is to provide the LAMP detection method of above-mentioned fumonisin.
Technical scheme：In order to realize foregoing invention purpose, the technical solution adopted by the present invention is：
A kind of LAMP primer composition for fumonisin, by positive outer primer F3, reverse outer primer B3, positive inner primer
FIP, reverse inner primer BIP, positive ring primer LF and reverse ring primer LB are constituted, and each primer sequence is as follows：
FIP：5’-AGTCTGTCCGCGACTCGACATAGAGAGCTTGAGGGCATCG-3’；
BIP：5’-ATCGACTTCTTCGCCACTGCTGGGTGCCAGCACCAATCTC-3’；
F3：5’-GTTGACCACTGCGAGGAAAT-3’；
B3：5’-GGATAACTTGAGCTCCGCC-3’；
LF：5’-CCATCTTCTGCCTGAAGAAGTT-3’；
LB：5’-GGCCCACGCTTCGTGTT-3’；
Application of the primer combination in detection volt Malaysia and China.
A kind of LAMP kit for detecting fumonisin, comprising following component：
(1) comprising the primer mixed liquor of above-mentioned primer combination, concentration is respectively：1 μM of positive outer primer F3, reverse outer primer B3 1
μM, positive 8 μM of inner primer FIP, 8 μM of reverse inner primer FIP, positive ring primer 2 μM, reverse ring primer 2 μM；
(2) reaction solution, comprising：7mM dNTPs, 100mM Tris-HCl, 50mM KCl, 50mM (NH4)2SO4, 40mM
MgSO4, 0.5%Triton X-100,5M Betain, 40U Bst archaeal dna polymerases, 25mM hydroxynaphthol blues；
(3) positive control is the genomic DNA for taking turns branch sickle-like bacteria CBS218.76, and negative control is that the reaction without genes of interest is mixed
Close liquid.
Application of the LAMP kit of described detection fumonisin in fumonisin is detected
A kind of method for detecting fumonisin, including the STb gene of detected materials is extracted, it is template with the DNA for extracting, utilize
LAMP primer group or LAMP kit carry out LAMP amplifications, and the color change of observing response solution, display purple represents inspection
It is feminine gender to survey, and in the absence of fumonisin, and shows that sapphire is the positive, there is fumonisin in sample.
The method of described detection fumonisin, extracts the STb gene of testing sample, takes 1 μ LDNA solution, adds 5 μ L
Reaction solution, 5 μ L primers liquid, 14 μ L sterilizing ultra-pure waters carry out LAMP reactions, and response procedures are 60~65 DEG C, and the time is 30~50
Min, amplified production carries out the observation of color change.
The present invention detection fumonisin method, including testing sample DNA extraction, with DNA as template, drawn using described
Thing combination carries out LAMP reactions.Hydroxynaphthol blue (Hydroxynaphthol blue, HNB) belongs to Metal ion indicator
One kind, is Mg2+Titration and agent, its color changes and changes with solution PH, therefore can be by monitoring Mg2+Concentration and
Color indicator is played a part of in the change of PH.HNB is added in reaction solution before reaction, solution is in purple, in course of reaction
It is a large amount of to form accessory substance magnesium pyrophosphate precipitation, make Mg2+Concentration declines, and PH changes, and solution colour is changed into day by purple
It is blue.Therefore, the color change after reaction terminates by reaction system judges the presence or absence of fumonisin.Display purple represents detection
It is feminine gender, in the absence of fumonisin, and shows that sapphire is the positive, there is fumonisin in sample.
One of key technology of the invention is the efficient specific amplification primer sequence and its amplification method of fumonisin.Draw to verify
The specificity of thing sequence, the present invention is with 3 plants of sickle-like bacteria not of the same race for producing fumonisin toxin and 21 plants of generation other types toxin
Or the sickle-like bacteria not of the same race of toxin producing is not material to be tested (table 1), and sickle-like bacteria genome is extracted using Fast DNA extraction method
DNA.Specific method is as follows：With sterilized toothpick or pipette tips on sample surfaces a small amount of mycelia of picking, be put into PCR pipe, plus
Enter 50 μ L BufferA solution (100mM NaOH, 2%Tween20), 95 DEG C of incubation 10min.Add 50 μ L BufferB
Solution (100mM Tris-HCl, 2mM EDTA), vibration is mixed, 12000rpm centrifugation 15s, is taken 1 μ L of supernatant liquid and is done
The template of LAMP amplifications.
When there is LAMP reactions, substantial amounts of magnesium pyrophosphate precipitation is produced to cause turbidity to rise.By HNB chromogenic reaction knots
Shown in fruit, sky blue is in the sickle-like bacteria example reaction pipe for producing fumonisin, it is positive findings, and produces other kind of poison
Element and not toxin producing sickle-like bacteria sample cell are in purple, are negative findings.Proving the LAMP primer composition of design has specificity.
Illustrate that primer combination can be used for the field Testing and appraisal fast and reliable with fumonisin in results cereal.When used for results cereal
During middle Trichothecenes Mycotoxin identification, STb gene is extracted using rapid cleavage method, detailed process is as follows：By grain sample to be measured
2mL centrifuge tubes are added, and adds diameter 4mm steel balls simultaneously, with Biospec beveller speed lapping 1min after liquid nitrogen flash freezer,
Add 200 μ L Buffer solution As (100mM NaOH, 2%Tween20), 95 DEG C of incubation 10min.Add 200 μ L
BufferB solution (100mM Tris-HCl, 2mM EDTA), vibration is mixed, 12000rpm centrifugation 30s, is taken supernatant and is done
The template of LAMP amplifications.
Beneficial effect：Prior art is compared, of the invention a little to be shown with good effect：
1) high specificity.The present invention devises 5 spies for 6 regions of sickle-like bacteria fumonisin synthesis key gene Fum1 sequences
Different primer, specific recognition is carried out to producing malicious sickle-like bacteria, and its high specificity, sensitivity are high, enable the invention to quick and precisely complete
Into the detection of fumonisin.
2) it is simple and efficient to handle.The square LAMP methods of the detection fumonisin that the present invention is provided, overcome existing biological and chemical detection
Method sample treatment is cumbersome, and detection cycle is long, the problems such as waste time and energy and need PCR thermal cyclers.The method is without PCR
Detection can be completed in annealing, the change of renaturation equitemperature in reaction, 30~50min, the efficiency of detection is substantially increased.
3) low cost.This method is without expensive, accurate test apparatus equipment, it is only necessary to which thermostat water bath can complete detection, and institute
Conventional reagent is with DNA rapid extractions and PCR processes, it is cheap.
Therefore this method is practical, can meet field and harvest fumonisin quick detection in cereal, for food security is provided
Ensure.
Table 1 is used for the specific reaping hook bacteria strain for producing not isotoxin of detection primer
* bacterial strain produces toxin to confirm through LC-MS
(4) illustrate
Fig. 1：The specificity verification result of LAMP primer composition
Strain number is identical with table 1 in figure, shows that 1~3 should manage to produce fumonisin sickle-like bacteria in figure, and testing result is positive, its
As a result he show feminine gender to produce other toxin or not producing malicious sickle-like bacteria.
Fig. 2：The sensitivity test result of LAMP primer composition
1.1000ng；2.100ng；3.10ng；4.1ng；5.100pg；6.10pg；7.1pg.25 μ L reactants are shown in figure
System is contained within 10pg-100ng DNA and shows the positive, and contains 1pg DNA and be negative, and illustrates that reaction sensitivity is 10pg.
Fig. 3：Corn second of the three ten-day periods of the hot season horse Mycotoxin identification result
1. positive control (CBS218.76)；2~5. inoculation corns；6~7. are not inoculated with corn；8. negative control (water).Figure
The corn of middle display inoculation sickle-like bacteria contains fumonisin, and remaining sample is negative.
(5) specific embodiment
It is described further with specific embodiment below, but the present invention is not limited by the following examples.
Embodiment 1：The specificity experiments of fumonisin LAMP reactions
In order to verify the specificity of LAMP method, the sickle-like bacteria with 24 kinds is object of participating in the experiment, wherein 3 kinds produce volt horse
Toxin, remaining 17 kind produce other toxin, 4 kinds not toxin producing (table 1).
Template DNA is extracted：With sterilized toothpick or pipette tips on sample surfaces a small amount of mycelia of picking, be put into PCR pipe, plus
Enter 50 μ L BufferA solution (100mM NaOH, 2%Tween20), 95 DEG C of incubation 10min.Add 50 μ L BufferB
Solution (100mM Tris-HCl, 2mM EDTA), vibration is mixed, 12000rpm centrifugation 15s, is taken 1 μ L of supernatant liquid and is done
The template of LAMP amplifications.
LAMP reacts：1 μ L DNA solutions are taken, adds 5 μ L reaction solutions, 5 μ L primer sets mixed liquors, 14 μ L sterilizings super
Pure water carries out LAMP reactions, and response procedures are 65 DEG C, and the time is 45min, and amplified production carries out the observation of color change.
LAMP testing results as shown in figure 1,2 plants produce fumonisin bacterial strain reaction tubes show sapphire positive reaction,
And other 23 plants of bacterial strains are in the negative reaction of purple.
Embodiment 2：The sensitivity experiment of fumonisin LAMP reactions
Divide to verify the sensitivity of LAMP method, the positive reaping hook bacteria strain CBS218.76 of extraction fumonisin, and using
Light photometric determination DNA concentration (1000ng/ μ L), 10 times of gradient dilutions are carried out with ultra-pure water, and template is done in -20 DEG C of preservations.
The μ L of DNA solution 1 for taking each concentration respectively do template, add 5 μ L reaction solutions, 5 μ L primer sets mixed liquors, 14 μ L sterilizings
Ultra-pure water carries out LAMP reactions, and response procedures are 65 DEG C, and the time is 45min, and amplified production carries out the observation of color change.
Result shows (Fig. 2) that the reaction tube for adding 1000ng, 100ng, 10ng, 1ng, 100pg, 10pg DNA is in obvious
Sky blue, and the reaction tube of 1pg DNA is added in purple, show that the sensitivity of LAMP reactions reaches 10pg genomic DNAs.
Embodiment 3：Fumonisin is detected in corn
Template DNA is extracted：Institute of Plant Protection of testing sample the Chinese Academy of Agricultural Sciences Langfang Experimental Base sample, 2~No. 6 samples are inoculation wheel branch
Sickle-like bacteria corn, No. 7 samples are not to be inoculated with corn.Corn sample to be measured is added into 2mL centrifuge tubes, and adds diameter 4mm simultaneously
Steel ball, with Biospec beveller speed lapping 1min after liquid nitrogen flash freezer, 200 μ L BufferA solution of addition (100mM NaOH,
2%Tween20), 95 DEG C of incubation 10min.Add 200 μ L BufferB solution (100mM Tris-HCl, 2mM
EDTA), vibration is mixed, 12000rpm centrifugation 30s, takes the template that supernatant is cooked LAMP amplifications.
LAMP reacts：1 μ L DNA solutions are taken, adds 5 μ L reaction solutions, 5 μ L primer sets mixed liquors, 14 μ L sterilizings super
Pure water carries out LAMP reactions, and response procedures are 65 DEG C, and the time is 45min, and amplified production carries out the observation of color change.
LAMP testing results as shown in figure 3, inoculation wheel branch sickle-like bacteria corn sample be positive, show containing fumonisin,
And corn sample is not inoculated with for feminine gender, do not contain fumonisin.
Sequence table：
Claims (6)
1. a kind of LAMP primer composition for fumonisin, it is characterised in that by positive outer primer F3, reverse outer primer B3, positive inner primer FIP, reverse inner primer BIP, positive ring primer LF and reverse ring primer LB are constituted, and each primer sequence is as follows：
FIP：5’-AGTCTGTCCGCGACTCGACATAGAGAGCTTGAGGGCATCG-3’；
BIP：5’-ATCGACTTCTTCGCCACTGCTGGGTGCCAGCACCAATCTC-3’；
F3：5’-GTTGACCACTGCGAGGAAAT-3’；
B3：5’-GGATAACTTGAGCTCCGCC-3’；
LF：5’-CCATCTTCTGCCTGAAGAAGTT-3’；
LB：5’-GGCCCACGCTTCGTGTT-3’.
2. application of the primer combination in fumonisin is detected described in claim 1.
3. it is a kind of detect fumonisin LAMP kit, it is characterised in that comprising following component：
(1) containing the primer sets mixed liquor described in claim 1, concentration is respectively：1 μM of positive outer primer F3,1 μM of reverse outer primer B3, positive 8 μM of inner primer FIP, 8 μM of reverse inner primer FIP, positive ring primer 2 μM, reverse ring primer 2 μM；
(2) reaction solution, comprising：7mM dNTPs, 100mM Tris-HCl, 50mM KCl, 50mM (NH4)2SO4, 40mM MgSO4, 0.5%Triton X-100,5M Betain, 40U Bst archaeal dna polymerases, 25mM hydroxynaphthol blues；
(3) positive control is the genomic DNA for taking turns branch sickle-like bacteria CBS218.76, and negative control is the reaction mixture without genes of interest.
4. application of the LAMP kit of the detection fumonisin described in claim 3 in fumonisin is detected.
5. it is a kind of detect fumonisin method, it is characterised in that：STb gene including extracting detected materials, it is template with the DNA for extracting, LAMP amplifications are carried out using LAMP primer group or LAMP kit, the color change of observing response solution, display purple is represented and is detected as feminine gender, in the absence of fumonisin, and show that sapphire is the positive, there is fumonisin in sample.
6. it is according to claim 5 detection fumonisin method, it is characterised in that：The STb gene of testing sample is extracted, 1 μ L DNA solutions are taken, adds 5 μ L reaction solutions, 5 μ L primer sets mixed liquors, 14 μ L sterilizing ultra-pure waters to carry out LAMP reactions, response procedures are 60~65 DEG C, and the time is 30~50min, and amplified production carries out the observation of color change.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456685A (en) * | 2018-04-27 | 2018-08-28 | 中国水稻研究所 | Three kinds of specific genes for generating fumonisin Races of Fusarium Oxysporum F. Sp and its application |
| CN114686617A (en) * | 2022-05-19 | 2022-07-01 | 国家食品安全风险评估中心 | Detection method of fumonisin synthetic gene in aspergillus niger group strain |
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2015
- 2015-12-17 CN CN201510940477.2A patent/CN106893762A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456685A (en) * | 2018-04-27 | 2018-08-28 | 中国水稻研究所 | Three kinds of specific genes for generating fumonisin Races of Fusarium Oxysporum F. Sp and its application |
| CN114686617A (en) * | 2022-05-19 | 2022-07-01 | 国家食品安全风险评估中心 | Detection method of fumonisin synthetic gene in aspergillus niger group strain |
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