CN102230016B - Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba - Google Patents
Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba Download PDFInfo
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- CN102230016B CN102230016B CN201110168915A CN201110168915A CN102230016B CN 102230016 B CN102230016 B CN 102230016B CN 201110168915 A CN201110168915 A CN 201110168915A CN 201110168915 A CN201110168915 A CN 201110168915A CN 102230016 B CN102230016 B CN 102230016B
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Abstract
The invention discloses loop-mediated isothermal amplification primers and kit for detecting acanthamoeba. The sequences of the primers are represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. The loop-mediated isothermal amplification primers designed according to the sequence of the conserved region of the gene of the acanthamoeba can flexibly, quickly and safely detect acanthamoeba; meanwhile, in the kit disclosed by the invention, false positive caused by nucleic acid pollution in a multiple detection process can be radically eliminated by using uracil-DNA glycosylase (UNG), so the problem of susceptibility to pollution and interference, which limits the wide application of a loop-mediated isothermal amplification technique, is solved. When the kit disclosed by the invention is used, the acanthamoeba causing amebic keratitis can be detected within 3 hours, and instead of expensive instrument and equipment, only a water bath pot or metal bath and a centrifuge are needed in a detection process; the detection result is very easy to determine; and compared with the conventional detection technique, the kit is low in cost, the kit is very safe for operators and the environment for the whole process is free from toxic reagents, and the detection sensitivity of the kit is very high.
Description
Technical field
The invention belongs to encountered pathogenic microbial rapid detection technical field, specifically relate to a kind of loop-mediated isothermal amplification (LAMP) primer and test kit that is used to detect thorn ameba protozoon.
Background technology
Thorn ameba protozoon is a kind of mikrobe that extensively is present in the free living of occurring in nature, phase when two of trophonts, packing are arranged.Trophont is generally perched in fresh water, sewage, seawater or earth, is converted into packing when environment is unfavorable, and packing can be present in the air.Under certain condition, thorn ameba protozoon can get into human body from the eye conjunctiva of skin wound, penetrance cornea wound, damage or through respiratory tract, reproductive tract etc., and majority parasitizes positions such as eye, skin, if immunity of organisms weakens also and can further cause encephalitis.Acanthamoeba property keratitis is a kind of carrying out property keratohelcosis of stubbornness, and the patient has foreign body sensation, blurred vision, sheds tears, photophobia, and serious pain is often arranged, but blinding when serious.Its generation is relevant with certain Hazard Factor, mainly comprises water source and cornea wound of wearing of contact lens, contact stain etc.Because the wearing of contact lens, Acanthamoeba property keratitis has obvious ascendant trend in recent years.Acanthamoeba property keratitis performance is complicated various, is infecting early stage because the corneal infection that its clinical symptom and herpes simplex virus, fungi etc. cause is closely similar, so often by mistaken diagnosis and delay treatment.And the encephalitis that Acanthamoeba causes is sent out well in immunosuppressant patient, and head or eye wound were arranged before the infection, and its diagnosis also is quite difficult.Nervous system signs shows focal one-sided infringement, and serious focal necrosis and oedema are arranged.Patient headache, heating vomiting, neck rigidity, dizzy, drowsiness, psychiatric disorder, ataxia are until stupor and dead.
For thorn ameba protozoon laboratory diagnostic method commonly used at present the burnt microscopic examination of the inspection of lesion tissue scraping blade, separation and Culture and copolymerization is arranged.The microscopic examination of pathological tissues scraping blade has simple fast, do not need advantages such as specific apparatus, but positive rate is not high, and need knows a thing or two for detecting of amebic cyst.Separation and Culture to ameba in pathological tissues or the scraping blade is a kind of very definite method of inspection, but culturing process is loaded down with trivial details, is essential to want the long period, and this has also restricted the widespread use of this method.It is a kind of very directly detection method that confocal microscope direct viewing affected area has or not amebic cyst, be because the motion of the low light horizontal reflection of cornea tissue and eyeball has limited the generation of image, and confocal microscope neither very universal detecting instrument.Loop-mediated isothermal amplification method can remedy these deficiencies, does not see the report that detects thorn ameba protozoon with loop-mediated isothermal amplification method at present as yet.
Summary of the invention
The objective of the invention is to provide a kind of ring mediated isothermal amplification (LAMP) primer and test kit that is used to detect thorn ameba protozoon; So that the accurate fast thorn ameba protozoon that detects; Simultaneously detection reagent is concentrated in the test kit of standard, the detection that makes thorn ameba protozoon more accurately, sensitive, fast, safety.
The loop-mediated isothermal amplification (LAMP) primer of detection thorn ameba protozoon of the present invention, its primer sequence are respectively SEQ ID NO:1～4.
The LAMP primer of particular design comprises can 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), and inner primer comprises the positive-sense strand and the antisense strand of target DNA.
Thorn ameba protozoon nucleic acid isothermal amplification detection kit of the present invention comprises:
1) DNA extraction lysate;
Extracting lysate is the STEN lysate, and the STEN lysate is made up of following component: 10% sodium lauryl sulphate, 1～10 part; 100mM pH value is 8.0 Tutofusin tris, 1～10 part; The EDTA Disodium of 10mM, 1～10 part; 1M sodium-chlor, 1～10 part; Distilled water constant volume to 100 part;
2) Proteinase K, interior dress 20mg/mL Proteinase K;
3) albumen extract is the phenol of 25:24:1 for volume ratio: chloroform: primary isoamyl alcohol;
4) nucleic acid precipitated liquid is absolute ethyl alcohol;
5) nucleic acid washings is 70% ethanol;
6) pure water is the sterilization pure water;
7) UNG enzyme is uracil dna glycosylase;
8) LAMP reaction solution, the LAMP reaction solution is made up of following component: each 1.0～2.0mM of dATP, dGTP and dCTP, dTTP, each 0.5～1.5mM of dUTP, Tris-HCl 10～40mM, KCl 5～20mM, (NH
4)
2SO
45～15mM, Triton X-100 0.05%～1.0%, MgSO
44～12mM, Betaine 0.8～1.6M;
9) primer mixed solution, interior dress sequence are respectively the forward and reverse inner primer of LAMP and the forward and reverse outer primer of SEQ ID NO:1-4;
10) Bst archaeal dna polymerase, the Bst archaeal dna polymerase of interior dress 8U/ μ l;
11) developer, interior dress nucleic acid dye GeneFinder
TM
Also has positive control nucleic acid in the test kit of the present invention, the positive DNA of interior dress thorn ameba protozoon.
The present invention according to the LAMP primer of Acanthamoeba gene conserved regions sequences Design can be sensitive, quick, safe the detection thorn ameba protozoon; 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 3 hours with the interior detection that just can accomplish ameba in the amebic keratitis, 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
The present invention has designed 4 special primers and has utilized a kind of active archaeal dna polymerase of strand displacement that has, and at a certain temperature nucleic acid is carried out isothermal duplication.Primer of the present invention can effectively detect all worm strains of thorn ameba protozoon, and detection specificity is good.Test kit of the present invention in addition adopts the uridylic glycosylase to eliminate the pollution of LAMP amplified production; Avoided because the product amplification amount of LAMP detection method is very big; Test sample is polluted, causes false-positive result, and big limitations its widespread use in detection; Three of core is technical parameters of having optimized the entire operation process, and with it stdn, supportingization, forms detection kit, so that rig-site utilization.
Face primer of the present invention and test kit down and carry out concrete description.
The preparation of thorn ameba protozoon nucleic acid isothermal amplification detection kit:
Test kit of the present invention is by following the composition (5 kinds of parts):
(1) DNA extraction lysate, 1 the pipe, in adorn 500 μ l STEN lysates, the STEN lysate is made up of following component: 10 parts of 10% sodium lauryl sulphate (SDS); The Tutofusin tris of 100mM (TrisHCl, pH8.0) 10 parts; 10 parts of the EDTA Disodiums of 10mM (EDTA); 10 parts in 1M sodium-chlor (NaCl); Distilled water constant volume to 100 part;
(2) Proteinase K, 1 the pipe, in adorn 5 μ l Proteinase Ks (20mg/mL);
(3) nucleic acid extract, 1 pipe, in adorn 500 μ l phenol: chloroform: primary isoamyl alcohol (25:24:1);
(4) nucleic acid extraction liquid, 1 the pipe, in adorn 1000 μ l absolute ethyl alcohols;
(5) nucleic acid washings, 1 pipe, in adorn the ethanol of 1000 μ l70%;
(6) sterilization pure water, 1 pipe, in adorn 200 μ l sterilization pure water;
(7) UNG enzyme, 1 pipe, in adorn the uracil dna glycosylase of 25 U;
(8) LAMP reaction solution, 1 the pipe, in adorn 80 μ l LAMP reaction solutions, the LAMP reaction solution is made up of following component: each 1.4mM of dATP, dGTP and dCTP, dTTP, each 0.7mM of dUTP, Tris-HCl 20mM, KCl 10mM, (NH
4)
2SO
410mM, Triton X-100 0.1%, MgSO
48mM, Betaine 1M;
(9) primer mixed solution, 1 pipe, in adorn each 10 μ M of the forward and reverse inner primer of 20 μ L LAMP and each 1.25 μ M of forward and reverse outer primer;
(10) Bst archaeal dna polymerase, 1 pipe, in adorn the Bst archaeal dna polymerase of 5 μ L 8U/ μ l;
(11) developer, 1 pipe, in adorn 5 μ l10 and doubly dilute nucleic acid dye GeneFinder
TM
(12) positive control nucleic acid, 1 pipe, in adorn the positive DNA of 10 μ l thorn ameba protozoons;
The dna sequence dna of the LAMP primer described in the test kit is respectively SEQ ID NO:1～4.
The LAMP detection method of thorn ameba protozoon of the present invention can be used for the detection of ameba in the Acanthamoeba property keratitis, also can be used for detecting whether have ameba in water body, the food.Concrete steps are following:
(1) get testing sample and place miniature centrifuge tube, add 100 μ l and organize lysate and 1 μ l Proteinase K, 60 ℃ of effect 30～60min blow and beat for several times therebetween repeatedly;
(2) after the extracting of adding equal-volume nucleic acid extract, the centrifugal 5～10min of 12000rpm/min draws the upper strata and changes new miniature centrifuge tube over to;
(3) 2 times of volume nucleic acid extraction liquid thorough mixing of-20 ℃ of precoolings of adding with the centrifugal 5～10min of 12000r/min, are abandoned supernatant and are kept throw out;
(4) wash above-mentioned throw out 2 times with the nucleic acid washings, then with the centrifugal 2～5min of 10000～15000r/min, abandoning supernatant also keeps throw out;
(5) throw out with above-mentioned centrifuge tube bottom dries in room temperature, adds sterilization pure water dissolution precipitation thing, obtains sample nucleic acid;
(6) get positive control nucleic acid in an amount of above-mentioned sample nucleic acid and the test kit; Add 15 μ l LAMP reaction solutions, 4 μ l LAMP primer mixed solutions; And then adding UNG enzyme 0.5 μ l, 50 ℃ of reaction 3 min are as polluting the pollution template that the template removing method wherein possibly exist with enzymolysis;
(7) with the sample nucleic acid behind the above-mentioned enzymolysis and positive control nucleic acid reaction pipe in 95 ℃ of insulation 3～6min, and then place rubble ice 2～10min rapidly, deactivation UNG enzyme;
(8) sample nucleic acid after the above-mentioned processing and positive control nucleic acid reaction pipe are added 1 μ l Bst archaeal dna polymerase respectively;
(9) with above-mentioned tubule in 60～65 ℃ of insulation 40～70min, carry out LAMP reaction in 80 ℃ of insulation 5～10min then;
(10) after the LAMP reaction finishes, in each tubule, add GeneFinder
TM, observe the LAMP reaction product of sample then with eyes, if show green then represent that the thorn ameba protozoon detected result is positive in this sample, orange-yellowly represent that then thorn ameba protozoon detects negative in this sample if show.
Embodiment 1: primer of the present invention and test kit are to the detection of positive
Positive nucleic acid samples in this test kit is the DNA plasmid that comprises thorn ameba protozoon nucleic acid.The concrete operations of its preparation are behind pcr amplification Acanthamoeba genomic 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 15 μ l, LAMP primer mixed solution 4 μ l, the positive nucleic acid 1 μ l of each gradient Acanthamoeba adds UNG enzyme 0.5 μ l, sterilization pure water 4 μ l, 50 ℃ of reaction 3 min, the pollution template that enzymolysis possibly exist;
2) 95 ℃ of insulation 3～6min place rubble ice 2～10min rapidly, deactivation UNG enzyme then;
3) add 1 μ l Bst archaeal dna polymerase in the reaction tubes of each concentration gradient, 60～65 ℃ of insulation 40～70min are then in 80 ℃ of insulation 5～10min;
4) after the LAMP reaction finishes, in each concentration gradient reaction tubes, add 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 Acanthamoeba nucleic acid of trace, and sensitivity can reach 10 positive nucleic acid copy numbers (10
1), when use primer of the present invention detects in actually operating false negative can not appear.
Embodiment 2: primer of the present invention and test kit are to the detection of negative sample
Utilize the common pathogenic micro-organism of ophthalmology to comprise the bacterium Pseudomonas aeruginosa, the fungi Fusarinm solani, viral herpes simplex virus type 1, thorn ameba protozoon and people's corneal epithelial cell genomic dna carry 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 have amplification to the DNA of thorn ameba protozoon; Other ophthalmology common bacterias, fungi, virus and cornea 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: primer of the present invention and test kit are to the detection of pathological tissues sample
Utilize the pathology cornea tissue highly suspect Acanthamoeba property keratitis clinically to scrape to get thing that primer of the present invention and test kit are carried out validity and test, concrete grammar is following:
(1) get the keratopathy tissue and scrape and get thing and place miniature centrifuge tube, add 100 μ L and organize lysate and 1 μ L Proteinase K, 60 ℃ of effect 60min, piping and druming is for several times repeatedly therebetween;
(2) add the extracting of equal-volume nucleic acid extract after, 12000rpm/min is centrifugal 5, draws the upper strata and changes new miniature centrifuge tube over to;
(3) add 2 times of volume nucleic acid extraction liquid thorough mixing of-20 ℃ of precoolings, centrifugal 5 with 12000r/min, abandon supernatant;
(4) wash above-mentioned throw out 2 times with the nucleic acid washings, then with the centrifugal 5min of 10000r/min, abandoning supernatant;
(5) throw out with above-mentioned centrifuge tube bottom dries in room temperature, adds 10 μ L TE damping fluid dissolution precipitation things, obtains sample nucleic acid;
(6) get positive control nucleic acid in above-mentioned sample nucleic acid of 5 μ L and the 1 μ L test kit, add 15 μ L LAMP reaction solutions, 4 μ L LAMP primer mixed solutions, and then add UNG enzyme 0.5 μ L, 50 ℃ of reaction 3 min do the negative control that does not add template simultaneously;
(7) with above-mentioned enzymolysis afterreaction pipe in 95 ℃ of insulation 5min, and then place rubble ice 2min rapidly;
(8) above-mentioned processing afterreaction pipe is added 1 μ L Bst archaeal dna polymerase respectively, in 63 ℃ of insulation 60min; Carry out the LAMP reaction in 80 ℃ of insulation 10min then;
(9) 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: primer of the present invention and test kit are to the detection of polluted-water sample
Utilize the contact lens soak solution of highly suspecting the pollution thorn ameba protozoon that primer of the present invention and test kit are carried out validity and test, concrete grammar is following:
Get the soak solution of 2ml contact lens, the centrifugal 10min of 10000r/min abandons supernatant and keeps deposition; In deposition, add 100 μ L and organize lysate and 1 μ L Proteinase K, 60 ℃ of effect 30min blow and beat for several times therebetween repeatedly; Surplus step is with (2) among the embodiment 3 ~ (9), and the result shows that the contact lens soak solution of pollution and positive control nucleic acid all show green, and negative control is apparent orange.
Claims (4)
1. loop-mediated isothermal amplification (LAMP) primer that is used to detect thorn ameba protozoon, its primer sequence is respectively SEQ ID NO:1～4.
2. thorn ameba protozoon loop-mediated isothermal amplification detection kit comprises following component:
1) DNA extraction lysate;
Extracting lysate is the STEN lysate, and the STEN lysate is made up of following component: 10% sodium lauryl sulphate, 1～10 part; 100mM pH value is 8.0 Tutofusin tris, 1～10 part; The EDTA Disodium of 10mM, 1～10 part; 1M sodium-chlor, 1～10 part; Distilled water constant volume to 100 part;
2) Proteinase K, interior dress 20mg/mL Proteinase K;
3) albumen extract is the phenol of 25:24:1 for volume ratio: chloroform: primary isoamyl alcohol;
4) nucleic acid precipitated liquid is absolute ethyl alcohol;
5) nucleic acid washings is 70% ethanol;
6) pure water is the sterilization pure water;
7) UNG enzyme is uracil dna glycosylase;
8) loop-mediated isothermal amplification liquid, reaction solution is made up of following component: each 1.0～2.0mM of dATP, dGTP and dCTP, dTTP, each 0.5～1.5mM of dUTP, Tris-HCl 10～40mM, KCl 5～20mM, (NH
4)
2SO
45～15mM, Triton X-100 0.05%～1.0%, MgSO
44～12mM, Betaine 0.8～1.6M;
9) primer mixed solution, interior dress sequence are respectively the forward and reverse inner primer of isothermal duplication and the forward and reverse outer primer of SEQ ID NO:1-4;
10) Bst archaeal dna polymerase, the Bst archaeal dna polymerase of interior dress 8U/ μ L;
11) developer, interior dress nucleic acid dye GeneFinder
TM
3. test kit as claimed in claim 2 is characterized in that above-mentioned test kit also includes positive control nucleic acid, and described positive control nucleic acid is the positive DNA of thorn ameba protozoon.
4. claim 2 or 3 described test kits are used for the detection of water body, food thorn ameba protozoon.
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| CN201110168915A CN102230016B (en) | 2011-06-22 | 2011-06-22 | Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba |
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| CN109593755A (en) * | 2018-12-29 | 2019-04-09 | 北京优迅医学检验实验室有限公司 | A method of for extracting genomic DNA in the animal sample saved |
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