CN102251055B - Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology - Google Patents
Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology Download PDFInfo
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- CN102251055B CN102251055B CN2011102409813A CN201110240981A CN102251055B CN 102251055 B CN102251055 B CN 102251055B CN 2011102409813 A CN2011102409813 A CN 2011102409813A CN 201110240981 A CN201110240981 A CN 201110240981A CN 102251055 B CN102251055 B CN 102251055B
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- isothermal amplification
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Abstract
The invention relates to a loop-mediated isothermal amplification kit and method for detecting fusarium. The kit comprises loop-mediated isothermal amplification reaction liquid, UNG (uracil-DNA-glycosylase), Bst DNA polymerase, developer and fusarium positive DNA, wherein the reaction liquid contains forward/reverse inner and outer primers, of which the sequences are respectively SEQ ID NO: 1-4. The detection method comprises the steps of fungus DNA extraction, loop-mediated isothermal amplification and development detection. The loop-mediated isothermal amplification primers are designed according to the fusarium gene conserved region sequences, and the fusarium in the sample is detected by loop-mediated isothermal amplification technology. The invention has the advantages of high speed, high sensitivity, high specificity, low cost and the like; and the whole process is not related to toxic reagents, thereby ensuring the safety of operating personnel and environment. Meanwhile, the UNG adopted in the invention can thoroughly eliminate false positive caused by nucleic acid pollution in multiple detection processes.
Description
Technical field
The invention belongs to encountered pathogenic fungi rapid detection technical field, be specifically related to a kind of primer and test kit that detects sickle-like bacteria based on loop-mediated isothermal amplification technique.
Background technology
Sickle-like bacteria (Fusarium) is distributed widely in natural soil, water and the organism, is a kind of important phytopathogen, can infect various plants (food crop, cash crop, medicinal plant and ornamental plant), kind surplus known host plant reaches 100.Fusarium infection host plant fibrovascular system; The root-rot, stem rot, stem foot that can cause plant be rotten, spend multiple diseases such as corruption and fringe corruption; And in the metabolic process of growing, produce the toxin damage to crops; It is dead to cause crop to wilt, and has a strong impact on the yield and quality of crop, is one of important disease of the most difficult control on producing.In addition, sickle-like bacteria also can infect fresh-water fishes such as perch, causes inflammation, festers, if untimely treatment can be sent out mass mortality.Minority reaping hook bacterial classification also can cause human diseases, comprises shallow table infection, local infection and disseminated infections approach, depends primarily on patient's immune state and infects the invasion approach.For the immunizing power normal patient, it is the main path of fusarium infection that wound or external foreign matter cause skin or mucous membrane breakage, wherein the most common with keratitis and onychomycosis.In the ophthalmic diseases that sickle-like bacteria causes, the vegetalitas wound is the principal element that causes corneal infection.And sickle-like bacteria produces proteolytic enzyme, collagenase and multiple toxin disorganize, often causes keratohelcosis, in fungal keratitis, occupy the first.
Discover that the sickle-like bacteria that causes keratitis mainly is Fusarinm solani, Fusarium oxysporum and fusarium moniliforme.To the immunodeficient crowd, the fusaridiosis more than 70% shows as disseminated infections, the most often involves skin, secondly is lung and nasal sinus.For the infection, particularly keratitis of sickle-like bacteria initiation, if untimely treatment just has the danger of blinding, so early stage efficient diagnosis is particularly important for instructing correct medication treatment.
The detection method of existing sickle-like bacteria has pathology sample fungi isolated culture, pathology sample microscope inspection method, serology experiment detection method, confocal microscopy sem observation direct observational method and PCR detection method etc.Wherein the pathology sample fungi separation and Culture cycle longer, process is loaded down with trivial details, is unfavorable for early diagnosis fast; Though the direct microscopy of pathology sample is simple fast, positive rate is not high; Serological method is relatively poor for shallow table and local INFECTION IN DETECTION validity, and is unfavorable for the detection of other biological; It is a kind of very directly detection method that confocal microscope direct viewing affected area has or not sickle-like bacteria mycelia or spore, but confocal microscope is not very universal detecting instrument, has limited it and has applied.Though the common PCR detection method is sensitive special, the PCR appearance that cost is higher and needs are special.And ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) technology not only has the sensitive specificity of regular-PCR detection method, can also be under isothermal condition amplified target sequence fast, efficiently, specifically; And easy and simple to handle, do not need expensive instrument and reagent, cost is low, demonstrates vast potential for future development at the pathogenic micro-organism detection range, but does not see the report that detects sickle-like bacteria with loop-mediated isothermal amplification method at present as yet.
Summary of the invention
The purpose of this invention is to provide a kind of primer and test kit, so that detect sickle-like bacteria fast and accurately based on loop-mediated isothermal amplification technique detection sickle-like bacteria.
The loop-mediated isothermal amplification (LAMP) primer of detection sickle-like bacteria of the present invention; Be LAMP primer: comprise and to discern six not homotactic two inner primers of target DNA (forward inner primer and reverse inner primer) and two outer primers (forward outer primer and reverse outer primer) according to homology zone particular design between the kind of sickle-like bacteria 18s rRNA; Inner primer comprises the positive-sense strand and the antisense strand of target DNA, and its primer sequence is respectively SEQ ID NO:1～4.
Forward inner primer Fu-FIP: its nucleotide sequence is shown in SEQ ID NO:1;
Reverse inner primer Fu-BIP: its nucleotide sequence is shown in SEQ ID NO:2;
Forward outer primer Fu-F3: its nucleotide sequence is shown in SEQ ID NO:3;
Reverse outer primer Fu-B3: its nucleotide sequence is shown in SEQ ID NO:4.
Sickle-like bacteria nucleic acid isothermal amplification detection kit of the present invention comprises:
1) LAMP reaction solution, the LAMP reaction solution is made up of following component: 10 * Thermopol reaction buffer, each 1.0～2.0mM of dATP, dGTP and dCTP, dTTP, each 0.5～1.5mM of dUTP, MgSO
44～12mM, Betaine 0.8～1.6M; Each 1.2～1.8 μ M of forward and reverse inner primer and each 0.2～0.3 μ M of forward and reverse outer primer;
The Tris-HCl, 100mmol/L KCl, the 100mmol/L (NH that contain 200mmol pH8.8 in wherein said 10 * Thermopol reaction buffer
4)
2SO
4, 20mmol/L MgSO
4With 1%Triton X-100;
2) UNG enzyme is 1U/ μ L uracil dna glycosylase;
3) Bst archaeal dna polymerase, the Bst archaeal dna polymerase of interior dress 8U/ μ L;
4) developer, interior dress nucleic acid dye GeneFinder
TM
5) positive control nucleic acid is for containing the plasmid of sickle-like bacteria DNA.
Use the mentioned reagent box to detect sickle-like bacteria, its concrete grammar comprises the following steps (1)-(3) successively:
(1) extraction of sample to be checked or fungal DNA:
Utilize commercial kit or traditional method for extracting sample nucleic acid to be checked, the sample DNA OD that wherein extracts
260/ OD
280In the 1.6-2.0 scope, concentration is in 10-100ng/ μ L scope.
(2) carry out the loop-mediated isothermal amplification of sickle-like bacteria:
A. in the reaction tubes that 22 μ LLAMP reaction solutions are housed, add the UNG enzyme of 2 μ L template DNA to be checked and 0.5 μ L, in water bath with thermostatic control, place 3min for 50 ℃, place 3-5min, place 1-3min on ice immediately for 95 ℃;
B. in reaction tubes, add 1 μ L Bst archaeal dna polymerase, and in water-bath 60-65 ℃ of amplified reaction 45-90min in the water bath with thermostatic control;
C. 80-85 ℃ of termination reaction is transferred in water-bath, taken out to be checked behind the 5-10min;
(3) color developing detection:
In each reaction tubes to be checked, add 1 μ L developer, the colour-change that directly detects by an unaided eye, the reaction solution color becomes green, explains that sample to be checked contains or is sickle-like bacteria.
The present invention according to the LAMP primer of sickle-like bacteria gene conserved regions sequences Design can be sensitive, quick, safe the bacterial strain of detection Fusarium; And detection specificity is good; Adopt the UNG enzyme can thoroughly eliminate repeated detection process amplifying nucleic acid simultaneously in the test kit of the present invention and pollute the false positive that causes, solved easy the to be contaminated interferential problem of restriction LAMP technology widespread use.Use test kit of the present invention 2 hours with the interior detection that just can accomplish sickle-like bacteria in the sample, testing process need not expensive plant and instrument such as PCR appearance and gel imaging system, only need have water-bath or metal bath and whizzer to get final product; And detected result is very easy to differentiate; Compare with existing detection technique, test kit cost of the present invention is low, and whole process does not relate to toxic reagent, and is all as safe as a house to operator and environment, and detection sensitivity is high.
Embodiment
Face test kit of the present invention and detection method down and carry out concrete description.
The preparation of sickle-like bacteria nucleic acid isothermal amplification detection kit:
(1) LAMP reaction solution:
Totally 22 μ L/ parts; Every part contains 2.5 μ L, 10 * Thermopol reaction buffer, 3.5 μ LdNTP (each 10mM of dATP, dGTP and dCTP, dTTP, each 5mM of dUTP), 1 μ L, 40 μ M upper reaches inner primers, 1 μ L, 40 μ M downstream inner primers, 1 μ L, 5 μ M upper reaches outer primers, 1 μ L, 5 μ M downstream outer primers, 3 μ L 100mM MgSO4,5 μ L 5M trimethyl-glycines (betaine) and 4 μ L sterilization distilled water (ddH2O).
Wherein said primer is forward and reverse inner primer and forward and reverse outer primer, and sequence is respectively SEQ ID NO:1～4.
(2) UNG enzyme: 1U/ μ L;
(3) Bst archaeal dna polymerase: 8U/ μ L;
(4) developer: the optical dye GeneFinder that is 10 times of dilutions
TM
(5) positive control nucleic acid: for comprising the plasmid of sickle-like bacteria DNA.
Detection method is carried out according to following (1)-(3) program:
(1) extraction of sample DNA:
Use business-like fungal DNA to extract test kit and extract fungi sample DNA, DNA OD
260/ OD
280Can reach 1.8, concentration reaches 20ng/ μ L.
(2) carry out the loop-mediated isothermal amplification of sickle-like bacteria:
A. in the reaction tubes that 22 μ LLAMP reaction solutions are housed, add the UNG enzyme of 2 μ L template DNA to be checked and 0.5 μ L, in water bath with thermostatic control, place 3min for 50 ℃, place 5min, place 1min on ice immediately for 95 ℃;
B. in each reaction tubes, add 1 μ L Bst archaeal dna polymerase, and in water bath with thermostatic control 65 ℃ of amplified reactions 1 hour;
C. 80 ℃ of termination reactions are transferred in water-bath, are taken out to be checked behind the 10min;
(3) color developing detection:
In each reaction tubes to be checked, add 1 μ L developer, the colour-change that directly detects by an unaided eye, the reaction solution color becomes green, explains that sample to be checked is a sickle-like bacteria.
The LAMP detection method of sickle-like bacteria of the present invention can be used for the detection of sickle-like bacteria in the fungal keratitis, also can be used for the detection of sickle-like bacteria in the plant fusaridiosis, but also can detect whether to pollute in soil, the water body sickle-like bacteria is arranged.Following specific embodiment further specifies the present invention, but the restriction of the present invention of should not opposing.
Embodiment 1: the sensitivity Detection of test kit of the present invention
Positive nucleic acid samples in this test kit is the DNA plasmid that comprises sickle-like bacteria nucleic acid.The concrete operations of its preparation are behind pcr amplification sickle-like bacteria conserved region gene group DNA; Reclaim the purpose fragment with test kit; Be connected on the cloned plasmids, change among the intestinal bacteria E.coli, through ammonia benzyl culture medium flat plate screening and culturing; Picking positive colony sequence verification, the plasmid that extracts positive colony promptly can be used as positive nucleus acid.
With positive nucleic acid gradient dilution is 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0Copies/uL adopts directly that the LAMP reaction solution carries out the LAMP sensitivity Detection in LAMP primer of the present invention and the test kit, and concrete grammar is following:
1) LAMP reaction solution 22 μ L, the positive nucleic acid 2 μ L of each gradient Acanthamoeba add UNG enzyme 0.5 μ L, 50 ℃ of reaction 3min, the pollution template that enzymolysis possibly exist;
2) 95 ℃ of insulation 5min place rubble ice 1min rapidly, deactivation UNG enzyme then;
3) add 1 μ L Bst archaeal dna polymerase in the reaction tubes of each concentration gradient, 65 ℃ of insulation 60min are then in 80 ℃ of insulation 10min;
4) after the LAMP reaction finishes, in each concentration gradient reaction tubes, add 1 μ L GeneFinder
TM, the LAMP reaction product of observing sample then with eyes.
The result shows that primer of the present invention and LAMP reaction system just can amplify positive findings to the sickle-like bacteria nucleic acid of trace, and sensitivity can reach 100 positive nucleic acid copy numbers (10
2), when use primer of the present invention detects in actually operating false negative can not appear.
Embodiment 2: the specific detection of test kit of the present invention
Utilize the common pathogenic micro-organism of ophthalmology to comprise the fungi Fusarinm solani; Fusarium oxysporum; Fusarium moniliforme, and bacterium Pseudomonas aeruginosa, the genomic dna of viral herpes simplex virus type 1 and thorn ameba protozoon carries out the LAMP specific detection to primer of the present invention and test kit.
Concrete operations are with the concrete grammar of embodiment 1; The result shows; Primer of the present invention and LAMP reaction system only all have amplification to the DNA of three kinds of sickle-like bacteria; Other ophthalmology common bacterias, virus and ameba itself are not had amplification, can not become green in the promptly above-mentioned amplified reaction pipe, explain that primer of the present invention false positive can not occur when detecting.
Embodiment 3: test kit of the present invention is to the detection of animal pathology tissue sample
Utilize the pathology cornea tissue highly suspect fungal keratitis clinically to scrape to get thing test kit of the present invention is carried out validation verification, concrete grammar is following:
(1) use the fungal DNA extraction test kit extraction pathology cornea of Omega Bio-tek company to scrape the nucleic acid of getting thing, OD
260/ OD
280Reach 1.8, concentration reaches 20ng/ μ L.
(2) get positive control nucleic acid in above-mentioned sample nucleic acid of 2 μ L and the 2 μ L test kits, add 22 μ L LAMP reaction solutions, and then add UNG enzyme 0.5 μ L, 50 ℃ of reaction 3min do the negative control that does not add template simultaneously;
(3) with above-mentioned enzymolysis afterreaction pipe in 95 ℃ of insulation 5min, and then place rubble ice 1min rapidly;
(4) above-mentioned processing afterreaction pipe is added 1 μ L Bst archaeal dna polymerase respectively, in 65 ℃ of insulation 60min; Carry out the LAMP reaction in 80 ℃ of insulation 10min then;
(5) after the LAMP reaction finishes, in each tubule, add 1 μ L GeneFinder
TM, the LAMP reaction product of observing sample with eyes.
Pathology cornea tissue sample nucleic acid and positive control nucleic acid reaction Guan Junxian green are positive as a result, and the negative control pipe is apparent orange.
Embodiment 4: test kit of the present invention is to the detection of pedotheque
Utilize and suspect that highly the pedotheque that pollutes sickle-like bacteria carries out validation verification to test kit of the present invention, concrete grammar is following:
Get the soil of the doubtful pollution of 250mg sickle-like bacteria, use
soil DNA of MO BIO company to extract organism nucleic acid in the test kit extraction soil; Surplus step is with (2) among the embodiment 3～(5), and the result shows that Contaminated soil and positive control nucleic acid all show green, and negative control is apparent orange.
Claims (4)
1. loop-mediated isothermal amplification (LAMP) primer group that is used to detect sickle-like bacteria, its primer sequence is respectively SEQ ID NO:1～4.
2. test kit that utilizes ring mediated isothermal amplification to detect sickle-like bacteria is characterized in that this test kit comprises the component of (1)-(5):
(1) loop-mediated isothermal amplification liquid: 10 * Thermopol reaction buffer, each 1.0～2.0mM of dATP, dGTP and dCTP, dTTP, each 0.5～1.5mM of dUTP, MgSO
44～12mM, Betaine 0.8～1.6M, each 1.2～1.8 μ M of forward and reverse inner primer and each 0.2～0.3 μ M of forward and reverse outer primer; The Tris-HCl, 100mmol/L KCl, the 100mmol/L (NH that wherein contain 200mmol pH8.8 in 10 * Thermopol reaction buffer
4)
2SO
4, 20mmol/L MgSO
4With 1%Triton X-100;
2) Bst archaeal dna polymerase, the Bst archaeal dna polymerase of interior dress 8U/ μ L;
3) UNG enzyme is 1U/ μ L uracil dna glycosylase;
4) developer, interior dress nucleic acid dye GeneFinder
TM
5) positive control nucleic acid, interior dress comprises the plasmid of sickle-like bacteria conserved regions DNA;
Wherein the sequence of forward and reverse inner primer is shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of forward and reverse outer primer is shown in SEQ ID NO:3 and SEQ ID NO:4.
3. the described test kit of claim 2 is applied to the detection of sickle-like bacteria in water body and/or the soil.
4. detection as claimed in claim 3 is characterized in that comprising successively following (1)-(3) step:
(1) extraction of sample to be checked or fungal DNA:
Utilize commercial kit or traditional method for extracting sample nucleic acid to be checked, the sample DNA OD that wherein extracts
260/ OD
280In the 1.6-2.0 scope, concentration is in 10-100ng/ μ L scope;
(2) carry out the loop-mediated isothermal amplification of sickle-like bacteria:
A, in the reaction tubes that 22 μ LLAMP reaction solutions are housed, add the UNG enzyme of 2 μ L template DNA to be checked and 0.5 μ L, in water bath with thermostatic control, place 3min for 50 ℃, place 3-5min, place 1-3min on ice immediately for 95 ℃;
B, in reaction tubes, add 1 μ L Bst archaeal dna polymerase, and in water-bath 60-65 ℃ of amplified reaction 45-90min in the water bath with thermostatic control;
C, 80-85 ℃ of termination reaction is transferred in water-bath, taken out to be checked behind the 5-10min;
(3) color developing detection:
In each reaction tubes to be checked, add 1 μ L developer, the colour-change that directly detects by an unaided eye, the reaction solution color becomes green, explains that sample to be checked contains or is sickle-like bacteria.
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| CN2011102409813A CN102251055B (en) | 2011-08-22 | 2011-08-22 | Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology |
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| CN104263813B (en) * | 2013-07-18 | 2016-07-06 | 宁波市农业科学研究院 | For identifying Fusarinm solani or/and the primer sequence of Fusarium oxysporum, test kit and method thereof |
| CN105907885A (en) * | 2016-04-26 | 2016-08-31 | 张梅 | Detection kit and detection method for identifying fusarium sporotrichioides in food |
| CN106868114B (en) * | 2017-01-26 | 2020-01-07 | 中国疾病预防控制中心传染病预防控制所 | Fusarium disease pathogenic bacterium RT-PCR detection primer and probe combination and kit |
| CN107475396A (en) * | 2017-09-06 | 2017-12-15 | 范瑶飞 | A kind of environment measuring kit |
| PL237733B1 (en) * | 2017-10-23 | 2021-05-17 | Genomtec Spolka Akcyjna | Kit of starters for detection of Fusarium oxysporum, method for detection of Fusarium oxysporum, using the kit of starters and the kit for detection of Fusarium oxysporum |
| CN108315469B (en) * | 2018-04-09 | 2021-10-22 | 中国农业科学院农产品加工研究所 | Primer composition and kit for detecting pathogenic fusarium by loop-mediated isothermal amplification method and application of primer composition and kit |
| CN112852992A (en) * | 2021-02-18 | 2021-05-28 | 中国农业科学院农业质量标准与检测技术研究所 | Primer group, kit and method for identifying Pleurotus citrinopileatus based on loop-mediated isothermal amplification technology |
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| WO2006123423A1 (en) * | 2005-05-20 | 2006-11-23 | Sapporo Breweries Limited | Primer for detecting fungus of the genus fusarium |
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| JP2005245257A (en) * | 2004-03-02 | 2005-09-15 | Sapporo Breweries Ltd | Primer for detecting bacterium of genus fusarium |
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| WO2006123423A1 (en) * | 2005-05-20 | 2006-11-23 | Sapporo Breweries Limited | Primer for detecting fungus of the genus fusarium |
| CN101205558A (en) * | 2006-12-22 | 2008-06-25 | 福建省农业科学院植物保护研究所 | Banana wilt bacterium molecule detecting genes and detecting method thereof |
Non-Patent Citations (3)
| Title |
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