CN105368918A - LAMP primer for detecting Asperillus Parasiticus, and kit including primer - Google Patents
LAMP primer for detecting Asperillus Parasiticus, and kit including primer Download PDFInfo
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- CN105368918A CN105368918A CN201410413463.0A CN201410413463A CN105368918A CN 105368918 A CN105368918 A CN 105368918A CN 201410413463 A CN201410413463 A CN 201410413463A CN 105368918 A CN105368918 A CN 105368918A
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Abstract
The invention relates to an LAMP primer for detecting Asperillus Parasiticus, and a kit including the primer. The LAMP primer comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; and the kit comprises the LAMP primer, a Bst DNA polymerase, a reaction buffer solution containing dNTPs, and deionized water. A loop-mediated isothermal amplification technology is used to rapidly detect Asperillus Parasiticus in the invention, so the Asperillus Parasiticus can be rapidly, accurately and conveniently detected, and the LAMP primer and the kit are of great significance to prevent and detect Asperillus Parasiticus pollution. A detection method also disclosed in the invention has the advantages of strong specificity, high sensitivity, lower detection limit reaching 1*10<-12>g/[mu]L, short reaction time, strong operability, no need of expensive apparatuses, convenient product detection, realization through macroscopic observation, and very high practical values.
Description
Technical field
The present invention relates to and a kind ofly detect the LAMP primer of Aspergillus parasiticus bacterium and comprise the test kit of this primer, relate to a kind of method that use comprises the test kit rapid detection Aspergillus parasiticus bacterium of this primer simultaneously.Belong to food safety detection and technical field of microbial detection.
Background technology
Aspergillus parasiticus bacterium is saprophytic microorganism common in aspergillus fungi.Be more common on the organism of mouldy grain, grain goods and other mould corruption.Aflatoxin is mycetogenetic poisonous secondary metabolite, produces primarily of mould metabolism such as flavus, Aspergillus parasiticus and Aspergillus tamariis, especially maximum with the research report of Aspergillus flavus and Aspergillus parasiticus bacterium.
Aflatoxin is found in 1961, and Britain there occurs the sudden death event of hundreds thousand of turkeys at that time, finally determines that this toxic substance is the secondary metabolite that Aspergillus flavus produces, therefore named aflatoxin.Research shows, the AFB1 toxicity in aflatoxin is 65 times of arsenic, 10 times of potassium cyanide, larger than the toxicity of highly toxic pesticide about 30 times, belongs to pole poisonous substance according to toxicity grading standard, within 1993, is classified as a class carcinogens by the World Health Organization.Aflatoxin can cause acute poisoning, also has the chronic hazards such as teratogenesis, carcinogenic, mutagenesis, belongs to strong carcinogen, particularly induced hepatocellular carcinoma.The toxin that aspergillus fungi produces can not only cause the mould quality losing the loss of rotten, nutritive substance and product of agricultural-food to reduce, import this toxin on a small quantity by natural way and can also cause the acute or chronic disease of vertebrates, by suppressing the synthesis of DNA, RNA in humans and animals body, protein and various enzyme and cause mycotoxins poisoning to cyto-architectural destruction.
Current China is all carry out classification and identification according to thalli morphology and features under microscope to the detection of Toxigenic fungi, but the method length qualification cycle, poor in timeliness, experienced expert or experimenter is needed accurately to differentiate, and easily there is false positive or false negative result, the food and feed that can not meet high speed development far away imports and exports detection demand.In the last few years, home and overseas had started to use the method such as biological chemistry and molecular biology to start to detect Toxigenic fungi.Ring mediated isothermal amplification method (the Loop-mediatedisothermalamplification that PCR basis develops, LAMP), be a kind of novel nucleic acids amplification method developed in 2000 by people such as the Notomi of Japanese Rong Yan Co., Ltd..It depends on 4 Auele Specific Primers and a kind of archaeal dna polymerase with strand displacement characteristic, under isothermal conditions can efficiently, fast, high amplified target sequence specifically.The archaeal dna polymerase of this strand displacement type, makes the primer junction at template two ends circulate and occurs cyclic single strand structure, ensures that primer is combined with template smoothly, carries out strand displacement amplification reaction.This technology more overcomes the feature needing repeatedly the PCR of thermally denature to react, and effectively prevent shortcoming consuming time in the middle of the process of heating and cooling, achieves the continuous rapid amplifying under constant temperature, can increase 10 within 15-60min
9-10
10times target sequence copy.Amplified production detects simple fast, and method is various, mainly contains the visual inspection precipitator method, detection of fluorescent dyes method, agarose gel electrophoresis method and turbidimeter detection method.It is high that LAMP has amplification efficiency, sensitivity is strong, the advantage that specificity is good, and do not need expensive PCR instrument and reagent, only need a constant temperature heating device, cost is low, is applicable to use in basic unit, in common laboratory, there is good application prospect, be applicable to the detection of Aspergillus parasiticus in Aspergillus Toxigenic fungi.
Summary of the invention
The invention provides a kind of LAMP primer detecting Aspergillus parasiticus bacterium, and a kind of test kit comprising this primer, and a kind of LAMP method using this test kit to detect Aspergillus parasiticus bacterium.Detection Aspergillus parasiticus bacterium that can be quick, special, easy, cheap, thus make up that prior art speed in correlation detection is slow, high in cost of production is not enough, farm crop, food, some medicine and feed safety are detected there is vital role.
For solving above technical problem, the present invention is realized by following technical proposals:
Detect a LAMP primer for Aspergillus parasiticus bacterium, it is characterized in that, comprising:
Outside forward primer F3:5 '-GCGGATTCTACGAGACGGA-3 ';
Outside reverse primer B3:5 '-AGTAGGGAGCATGCCAGA-3 '; And
Inner side forward primer FIP:5 '-TGGCCGATAGCCTATACTCCCA-CCGGACTCGGTCTTTCCAT-3 ';
Inner side reverse primer BIP:5 '-GACGGGCCCGGTAGTGTATTC-TGATTGAAACTCGCGCATCC-3 ';
And ring primer LF:5 '-TCCGCTAGGCTTTTGCAG-3 ';
Ring primer LB:5 '-TGGTTCACAGCTAAAATGGGG-3 '.
Comprise a test kit for the detection Aspergillus parasiticus bacterium of above-mentioned LAMP primer, comprise LAMP primer, BstDNA polysaccharase, containing the reaction buffer of dNTPs and deionized water, it is characterized in that, comprising LAMP primer be above-mentioned LAMP primer.
The test kit of above-mentioned detection Aspergillus parasiticus bacterium, is characterized in that, the described reaction buffer containing dNTPs comprises Tris-HCl, 20mmol/LKCl, 20mmol/L (NH of dNTPs2.8mmol/L, 40mmol/LpH8.8
4)
2sO
4, 16mmol/LMgSO
4, 0.2% polysorbas20 and 1.6mol/L trimethyl-glycine.
The test kit of above-mentioned detection Aspergillus parasiticus bacterium, it is characterized in that, described LAMP primer comprises 40pmol/ μ L primers F IP50 μ L, 40pmol/ μ L primer BIP50 μ L, 5pmol/ μ L primers F 350 μ L, 5pmol/ μ L primer B350 μ L, 20pmol/ μ L primer LF50 μ L, 20pmol/ μ L primer LB50 μ L.
The test kit of above-mentioned detection Aspergillus parasiticus bacterium, is characterized in that, in described test kit, BstDNA polymerase concentration is 8U/ μ L, and content is 50 μ L, and the reaction buffer content containing dNTPs is 1mL, and deionized water content is 1mL.
Utilize described test kit to detect a method for Aspergillus parasiticus bacterium, it is characterized in that, comprise the following steps:
(1) template DNA is extracted;
(2) with the DNA extracted in step (1) for template, adopt above-mentioned LAMP primer to carry out LAMP amplified reaction;
(3) LAMP amplification judges.
Above-mentioned detection method, is characterized in that, in step (2), Aspergillus parasiticus bacterium LAMP reaction system is 25 μ l, and each component concentration is as follows:
Above-mentioned detection method, is characterized in that, the temperature of the described LAMP amplified reaction of step (2) is 63 DEG C, and the time is 60min.
Above-mentioned detection method, is characterized in that, step (3) utilizes LA-320C turbidimeter to carry out LAMP amplified reaction, is identified the existence of specific amplification products in described step (2) by the opacity of programdisplay.
Technique scheme of the present invention has the following advantages compared to existing technology:
1. of the present inventionly a kind ofly detect the LAMP primer of Aspergillus parasiticus bacterium and comprise the test kit of this primer, described LAMP primer with the histidine kinase mRNA gene of Aspergillus parasiticus bacterium for target gene increases; This gene order has the specificity of height, thus can realize the specific detection of Aspergillus parasiticus bacterium; Describedly comprise detection reagent used in the test kit of this primer and be commercially available common chemical reagent, it is convenient to detect.
2. of the present inventionly a kind ofly detect the LAMP primer of Aspergillus parasiticus bacterium and comprise the test kit of this primer, when detecting Aspergillus Toxigenic fungi more fast and efficient, nucleic acid amplification can complete in 1h, starts amplified reaction occurs at 11min.The present invention is exclusively used in detect Aspergillus parasiticus bacterium, to the prevention of Aspergillus parasiticus fungi pollution with detect significant.
3. LAMP method of the present invention detects Aspergillus parasiticus bacterium, for 6 zone design, 4 species-specific primers of target sequence, in 6 regions, any one and primer do not mate and all can not carry out nucleic acid amplification, and amplified reaction do not occur with the bacterial classification of other kind, thus have high specific; Present method has highly sensitive simultaneously, and required template copy amount is few, and Monitoring lower-cut is about 1 × 10
-12g/ μ L.
4. LAMP reaction of the present invention is workable, avoids using expensive thermal cycler, only requires the reaction kit of simple constant temperature, and the heating in water bath only preparing constant temperature if desired can complete this operation; And product is easy to detect, just can be realized by visual inspection opacity.
Accompanying drawing explanation
Fig. 1 is the real-time Turbidity measurement amplification histogram that in the embodiment of the present invention 3, LAMP atopic is analyzed, and X-coordinate represents the reaction times (min), and ordinate zou represents turbidity.Left 1 is Aspergillus parasiticus; Left 2 is flavus; Left 3 is Aspergillus tamarii; Left 4 is Aspergillus fumigatus; Left 5 is aspergillus versicolor; ; Left 6 is Fusarium graminearum; Left 7 is eggplant fusarium; Left 8 is penicillium islandicum; Right 1 is penicillium viridicatum; Right 2 is streptococcus aureus; Right 3 is intestinal bacteria; Right 4 is negative control.
Fig. 2 is the real-time Turbidity measurement amplification curve diagram that in the embodiment of the present invention 3, LAMP atopic is analyzed, and X-coordinate represents the reaction times (min), and ordinate zou represents turbidity.In figure, curve is Aspergillus parasiticus amplification curve, and amplified reaction does not occur other bacterial classification.
Fig. 3 is the real-time Turbidity measurement amplification histogram that in the embodiment of the present invention 4, LAMP reaction sensitivity is analyzed.X-coordinate represents the reaction times (min), and ordinate zou represents turbidity.Left 1 is 10
-8; Left 2 is 10
-9; Left 3 is 10
-10; Left 4 is 10
-11; Left 5 is 10
-12; Left 6 is 10
-13; Left 7 is 10
-14; Left 8 is 10
-15; Right 1 is 10
-16; Right 2 is 10
-17; Right 3 negative controls.
Fig. 4 is the visualization result that in inventive embodiments 5, LAMP reacts water bath with thermostatic control experiment.1 is system positive control; 2 is Aspergillus parasiticus; 3 is negative control.
Embodiment
Embodiment 1 is for detecting the Design and synthesis of Aspergillus parasiticus bacterium LAMP primer
Choose the histidine kinase mRNA gene (AY430047.1) of Aspergillus parasiticus bacterium, according to NCBI comparison result, choose specific sequence zone design primer.Use LAMP method design of primers support software PrimerExplorer to design primer, Photographing On-line network address is: http://primerexplorer.jp/e/.By choosing a set of primer meeting LAMP primer design requirements.Described primer comprises 2 Outside primer (F3, B3), and 2 inner primer (FIP, BIP) and 2 ring primers (LF and LB), synthesized by Sangon Biotech (Shanghai) Co., Ltd..Histidine kinase mRNA gene can be used as the specific gene of specificity identification Aspergillus parasiticus bacterium.
The preparation of embodiment 2 Aspergillus parasiticus bacterium genomic DNA template
Improved method of CTAB is used to extract Aspergillus parasiticus bacterium genomic dna.Aspergillus parasiticus bacterium (40365) reference culture is provided by Liaoning Entry-Exit Inspection and Quarantine Bureau.Detailed step is: (1) gets a certain amount of mycelium in mortar, and liquid nitrogen grinding is powdered to be transferred to rapidly in 1.5mL centrifuge tube afterwards; (2) the CTAB solution (2%CTAB of 600 μ L preheatings is added, 200mmol/LTris-HCl, 20mmol/LEDTApH=8.0,1.4mmol/LNaCl, 1%PVP-40,0.2% beta-mercaptoethanol), cover tightly rapidly pipe lid, abundant concussion, 65 DEG C of water-bath 30min, period constantly shakes; (3) add isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1) after room temperature cooling, repeatedly put upside down the centrifugal 10min of mixing 1min, 13000r/min; (4) draw supernatant in another centrifuge tube, add isopyknic chloroform-isoamyl alcohol (24: 1), repeatedly put upside down the centrifugal 10min of mixing 1min, 13000r/min; (5) draw supernatant in another centrifuge tube, add the RNaseA solution of 5 μ L10mg/L, 37 DEG C of water-bath 1h; (6) repeating step (3), (4); (7) draw supernatant in another centrifuge tube, add the NaCl solution of the Virahol of 0.7 times of volume and the 5mol/L of 1/5 times of volume, put upside down mixing, 4 DEG C of precipitations latter 4 DEG C, the centrifugal 10min of 13000r/min; (8) remove supernatant, add 500 μ L70% ethanol purge precipitations, 4 DEG C, the centrifugal 10min of 13000r/min, after removing supernatant, back-off is on filter paper, the solution that the absorption mouth of pipe and tube wall remain, and place dry DNA, time of drying is about 15min; (9) 50 μ LTE buffer solution genomic dnas are added, in-20 DEG C of Refrigerator stores.
Embodiment 3 utilizes LAMP primer to detect the specificity analyses of Aspergillus parasiticus bacterium
Using the Aspergillus flavus in Aspergillus, Aspergillus tamarii bacterium, aspergillus fumigatus, Fusarium graminearum between aspergillus versicolor and genus, eggplant fusarium, penicillium islandicum, penicillium viridicatum and streptococcus aureus and intestinal bacteria etc. as sample, carry out specific assay, increase according to LAMP reaction system, the specificity of checking Aspergillus parasiticus combination of primers.Analyze appearance time and the peak value of LA-320C program, as depicted in figs. 1 and 2.As can be seen from Fig. 1 and Fig. 2, primer is only positive with Aspergillus parasiticus bacterium, with the Aspergillus flavus in genus, and Aspergillus tamarii bacterium, aspergillus fumigatus, aspergillus versicolor; Fusarium graminearum between genus, eggplant fusarium, penicillium islandicum; And streptococcus aureus and intestinal bacteria all occur without amplified reaction, prove that this Aspergillus parasiticus bacterium primer has good specificity.
Embodiment 4 utilizes LAMP primer to detect the sensitivity analysis of Aspergillus parasiticus bacterium
By Aspergillus parasiticus template DNA by 10
nratio is diluted to 10 respectively
-8, 10
-9, 10
-10, 10
-11, 10
-12, 10
-13, 10
-14, 10
-15, 10
-16, 10
-17ten gradients doubly, unit is g/ μ L.Get each dilution 2 μ L respectively and carry out LAMP experiment, the sensitivity of checking LAMP method, determines that LAMP method detects the minimal detectable concentration of Aspergillus parasiticus.Analyze the peak value of LA-320C program, as shown in Figure 3.As can be seen from Figure 3, Aspergillus parasiticus genomic DNA template is diluted to 1 × 10
-12also can detect after g/ μ L that amplified reaction occurs, sensitivity reaches pik (pg) rank.Although produce turbidity on the right side of Fig. 3 left side 5, be considered as non-specific amplification compared with negative control.
Embodiment 5 utilizes LAMP primer to detect the water bath with thermostatic control experiment of Aspergillus parasiticus bacterium
Water bath with thermostatic control experiment is carried out to the detection of Aspergillus parasiticus bacterium, adds LAMP primer of the present invention according to reaction system, carry out water bath with thermostatic control experiment.Reaction conditions is constant temperature water bath 63 DEG C, reaction 60min.Judge whether that amplification occurs by observing opacity.As shown in Figure 4.Found by water bath with thermostatic control experiment, when 63 DEG C of waters bath with thermostatic control, through certain reaction times, reaction tubes becomes turbid, and illustrates that the specificity LAMP primer of Aspergillus parasiticus in the present invention under the condition of water bath with thermostatic control, amplified reaction can occur.Water bath with thermostatic control experiment demonstrates LAMP reaction does not need special instrument and easy to operate feature.Detected result directly with the naked eye just can be observed, and does not need large-scale instrument to carry out experimental result inspection.
Obviously, above-described embodiment is only for example of the present invention is clearly described, and the restriction not to embodiment.To those of ordinary skill in the art, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (9)
1. detect a LAMP primer for Aspergillus parasiticus bacterium, it is characterized in that, comprising:
Outside forward primer F3:5 '-GCGGATTCTACGAGACGGA-3 ';
Outside reverse primer B3:5 '-AGTAGGGAGCATGCCAGA-3 '; And
Inner side forward primer FIP:5 '-TGGCCGATAGCCTATACTCCCA-CCGGACTCGGTCTTTCCAT-3 ';
Inner side reverse primer BIP:5 '-GACGGGCCCGGTAGTGTATTC-TGATTGAAACTCGCGCATCC-3 ';
And ring primer LF:5 '-TCCGCTAGGCTTTTGCAG-3 ';
Ring primer LB:5 '-TGGTTCACAGCTAAAATGGGG-3 '.
2. one kind comprises the test kit of the detection Aspergillus parasiticus bacterium of the LAMP primer described in claim 1, comprise LAMP primer, BstDNA polysaccharase, containing the reaction buffer of dNTPs and deionized water, it is characterized in that, comprising LAMP primer be LAMP primer according to claim 1.
3. the test kit of detection Aspergillus parasiticus bacterium according to claim 2, is characterized in that, the described reaction buffer containing dNTPs comprises Tris-HCl, 20mmol/LKCl, 20mmol/L (NH of dNTPs2.8mmol/L, 40mmol/LpH8.8
4)
2sO
4, 16mmol/LMgSO
4, 0.2% polysorbas20 and 1.6mol/L trimethyl-glycine.
4. the test kit of detection Aspergillus parasiticus bacterium according to claim 2, it is characterized in that, described LAMP primer comprises 40pmol/ μ L primers F IP50 μ L, 40pmol/ μ L primer BIP50 μ L, 5pmol/ μ L primers F 350 μ L, 5pmol/ μ L primer B350 μ L, 20pmol/ μ L primer LF50 μ L, 20pmol/ μ L primer LB50 μ L.
5. the test kit of detection Aspergillus parasiticus bacterium according to claim 2, is characterized in that, in described test kit, BstDNA polymerase concentration is 8U/ μ L, and content is 50 μ L; Reaction buffer content containing dNTPs is 1mL; Deionized water content is 1mL.
6. utilize the test kit described in claim 2 to detect a method for Aspergillus parasiticus bacterium, it is characterized in that, comprise the following steps:
(1) template DNA is extracted;
(2) with the genomic dna extracted in step (1) for template, adopt the test kit of the detection Aspergillus parasiticus bacterium described in claim 2 to carry out LAMP amplified reaction;
(3) LAMP amplification judges.
7. detection method according to claim 4, is characterized in that, in step (2), Aspergillus parasiticus bacterium LAMP amplification reaction system is 25 μ l, and each component concentration is as follows:
8. detection method according to claim 4, is characterized in that, the temperature of the described LAMP amplified reaction of step (2) is 63 DEG C, and the time is 60min.
9. detection method according to claim 4, is characterized in that, step (3) utilizes LA-320C turbidimeter to carry out LAMP amplified reaction, is identified the existence of specific amplification products in described step (2) by the opacity of programdisplay.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117144053A (en) * | 2023-10-27 | 2023-12-01 | 江苏美克医学技术有限公司 | Aspergillus fumigatus detection primer set, kit and application thereof |
-
2014
- 2014-08-21 CN CN201410413463.0A patent/CN105368918A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117144053A (en) * | 2023-10-27 | 2023-12-01 | 江苏美克医学技术有限公司 | Aspergillus fumigatus detection primer set, kit and application thereof |
| CN117144053B (en) * | 2023-10-27 | 2024-01-19 | 江苏美克医学技术有限公司 | Aspergillus fumigatus detection primer set, kit and application thereof |
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Application publication date: 20160302 |