CN102643924A - Primer group and kit for rapidly detecting Listeria monocytogenes by utilizing LAMP (Loop-mediated Isothermal Amplification) technology - Google Patents
Primer group and kit for rapidly detecting Listeria monocytogenes by utilizing LAMP (Loop-mediated Isothermal Amplification) technology Download PDFInfo
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Abstract
The invention relates to a biological detection reagent, in particular to a primer and a kit for rapidly detecting Listeria monocytogenes by utilizing an LAMP (Loop-mediated Isothermal Amplification) technology. The primer for rapidly detecting the Listeria monocytogenes by utilizing the LAMP technology is a characteristic primer group of the Listeria monocytogenes, one primer group consists of two pairs of primers, one pair of primers is external primers, and the other pair of primers is internal primers. The kit provided by the invention consists of one primer group, reactant liquor, BstDNA (Bacillus Stearothermophilus Deoxyribonucleic Acid) polymerase, a sample pretreatment solution, a developer, a positive control solution and the like. The detection method comprises the following steps of: extraction of bacterium DNA, LAMP of nucleic acid, development detection and the like. The Listeria monocytogenes in a detection sample can be rapidly detected through simply treating the sample by utilizing the kit; and the kit has the advantages of high sensitivity, strong specificity, simplicity in operation, result determination based on naked eyes, and the like.
Description
Application of the present invention is that the patent No. is: 200810121029.X, patent name is: the monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection is with primer, test kit and detection method, and the applying date is: the dividing an application of the application for a patent for invention of 2008-09-16.
Technical field
The present invention relates to biological detection reagent, be specifically related to a kind of monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection with primer, test kit and detection method.
Background technology
Listeria monocytogenes (
Listeria monocytogenes, Lm) (hereinafter to be referred as " Listeria monocytogenes ") is a kind of important zoonosis pathogenic bacterium, extensively is present in soil, animal and fishery products etc., mainly passes through food transmissions such as milk and milk preparation, vegetables, fishery products, meat product.This bacterium has been classified as one of larger food pathogenic bacterium nineties 4, becomes the essential items for inspection of many state food health.
Traditional Listeria monocytogenes biochemistry detection method whole process needs 7～10 d at least, and detection limit is lower, wastes time and energy.Immunization is than very fast, but Monoclonal Antibody is relatively more difficult, is prone to produce cross reaction, poor specificity.And PCR or fluorescence quantifying PCR method are quick, and be special, sensitivity is very high, but need expensive PCR appearance.Once had a small amount of bibliographical information to utilize the mRNA template to detect the live body list to the iap gene with RT-PCR abroad and increase listeria spp, and showed detected result but need hybridize with film, time length, technical sophistication, expense height are difficult for applying.The fluorescent dye technology can detect viable bacteria, but technical sophistication needs high end instrument.Therefore it is necessary to set up a kind of easy, sensitive, quick, special method.
Ring mediated isothermal amplification gene engineering (loop-mediated isothermal amplication; Be called for short LAMP) (International Patent Publication No. WO 00/28082) be a kind of nucleic acid amplification new technology that Notomi in 2000 etc. develop; Two pairs of special primers of 6 zone design, one cover to target-gene sequence to be measured; Utilize strand displacement archaeal dna polymerase (Bst DNA polymerase) can specificity under 65 ℃ of left and right sides isothermal conditions, carry out nucleic acid amplification efficiently, fast; Its turbidity can directly judged or detect to amplification through naked eyes to amplification by product magnesium pyrophosphate deposition; The also available preferred SYBR Green of the optical dye I dyeing that combines two strands can be judged through naked eyes.Because two pairs of primers of LAMP technology amplification are 6 sections to target gene; Thereby have a specificity higher than PCR; Be promptly not need specific apparatus such as PCR appearance under the isothermal condition simultaneously; And the pre-treatment of sample is very simple, amplification efficiency advantages of higher more in the unit time, has caused people's attention.
(application number is Chinese invention patent: 200710030435.0,200710030437.X, 200710132320.2,200710026389.7,200810052321.0,200810015001.8,200810093986.6) disclose the method that adopts ring mediated isothermal amplification gene engineering tested for pathogens respectively.But ring mediated isothermal amplification gene engineering also of no use at present detects the report of the test kit of Listeria monocytogenes.
Summary of the invention
The detection method that has Listeria monocytogenes now is not easy, sensitive low, the time is long in order to solve, the technological deficiency of poor specificity.An object of the present invention is to provide a kind of monotonic increasing Listeria hymenial veil mediated isothermality amplification (loop-mediatedisothermal amplication, LAMP) technological rapid detection is used primer; Second purpose of the present invention provides uses the test kit of above-mentioned detection with primer; The 3rd purpose of the present invention provides uses the kit test method of above-mentioned detection with primer.Test kit of the present invention and detection method have easy, sensitive, quick, special good characteristics, can be extensively with being widely used in fields such as food, fishery products, makeup and health care.
In order to realize first above-mentioned purpose, the present invention adopts following technical scheme:
The monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection is used primer, and described primer is a cover Listeria monocytogenes characteristic primer sets, and a cover primer sets is made up of two pairs of primers; A pair of primer is an outer primer; A pair of is inner primer, has six cover primers, and sequence is respectively:
Primer sets one:
Outer primer 1:CAAGCACTGTAGTAGTCGA
Outer primer 2:CGTAATAATACTGTTATCAACACC
Inner primer 1:GTTTGCTTTTTTAATTGCGTCAACATTTTAGCTGGTGATACTCTTTGG
Inner primer 2:ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG
Or primer sets two:
Outer primer 1:GGTGATACTCTTTGGGGTAT
Outer primer 2:CGTAATAATACTGTTATCAACACC
Inner primer 1:GACCTGGTACGATTTTATCTGTTGTTTTTCGCACAAAGTAAAGGGAC
Inner primer 2:ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG
Or primer sets three:
Outer primer 1:TTAAACGTCCGTACTGGC
Outer primer 2:CACTTCTTGTGTTGGTGC
Inner primer 1:CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA
Inner primer 2:GCTGGCACAAAATTACTTACAACGATTTTTGGAGTGCTTACTGCTTTG
Or primer sets four:
Outer primer 1:ACTGGTTTCGTTAACGGTAA
Outer primer 2:TGTCACCGCTTTTGACAG
Inner primer 1:AGGTGCAGCTTGTTGAGTAGTAGTTTTGCAGTAAGCACTCCAGTTG
Inner primer 2:CCTGCGCCTAAAGTAGCAGAATTTTGTGTGTAGTAGCATTTTGATCT
Or primer sets five:
Outer primer 1:TTAAACGTCCGTACTGGC
Outer primer 2:CACTTCTTGTGTTGGTGC
Inner primer 1:CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA
Inner primer 2:CTGGCACAAAATTACTTACAACGATTTTTTGGAGTGCTTACTGCTTTG
Or primer sets six:
Outer primer 1:GGTAACGGACCAACTACA
Outer primer 2:TCATTTGACCATTACCAACAT
Inner primer 1:TGTACGTGGAAGGGAGATACCTTTTTGATTGCTCTGGTTACACT
Inner primer 2:TCTGAATCTCAAGCAAAACCTGGTTTTTCGTGAGAAATTCCGCTACC.
In order to realize second above-mentioned purpose, the present invention adopts following technical scheme:
The monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit is characterized in that: this test kit comprises any one group of primer sets, reaction solution, Bst archaeal dna polymerase, sample pretreatment liquid and the positive control solution in the technique scheme.
As preferably, described primer sets is that 20-30 μ M outer primer 1, outer primer 2, the inner primer 1 of 1:1:4:4 formed with inner primer 2 by volume ratio.
As preferably, described reaction solution is to be 10 * Thermopol reaction buffer, the 7.5～12.5mM dNTP of 5:2:1:2 by volume ratio, 100～200mM MgSO
4Form with 25～37.5M trimethyl-glycine.Preferred as again, it is 1% triton x-100 that described 10 * Thermopol reaction buffer contains 200 mM pH8.8 trihydroxy methyl aminomethane hydrochlorides, 100 mM Repone K, 100 mM ammonium sulfate, 20 mM sal epsom and concentration of volume percent.
As preferably, the every microlitre of described Bst archaeal dna polymerase contains 8～16 activity units.
As preferably, described sample pretreatment liquid is by 20mM pH 8.0 Tris-HCl, and 2mM EDTA, concentration of volume percent are 1.2% triton x-100.
As preferably, described this test kit also comprises developer, and developer is an optical dye.Preferred optical dye is SYBR Green I;
As preferably, described positive control solution is a Listeria monocytogenes reference culture genomic dna.
In order to realize the 3rd above-mentioned purpose, the present invention adopts following technical scheme:
The monotonic increasing Listeria hymenial veil mediated isothermality amplification technique method for quick adopts above-mentioned any described test kit of technical scheme, and this method comprises the steps:
1) extraction of Listeria monocytogenes DNA: A, get the 50ul incubated overnight enrichment liquid in the eppendorf pipe, the centrifugal 2min of 1000rpm abandons supernatant; B, adding 80ul sample pretreatment liquid mix with deposition gained thalline in centrifuge tube; Cool off 10min immediately after boiling 10min in C, the boiling water; D, the centrifugal 2min of 10000rpm, supernatant promptly can be used as template DNA to be checked;
2) reaction system is: 2.5 μ L primer sets, 5 μ L reaction solutions, 1 μ L Bst archaeal dna polymerase, 2 μ L template DNA to be checked or positive control solution and 14.5 μ L ddH2O;
3) ring mediated isothermal amplification of Listeria monocytogenes: the PCR for preparing is managed in 60～65 ℃ of reaction 1～1.5h, 80 ℃ of termination reactions;
4) analysis and judgement reaction product result: in reaction product, add the 2.5ul optical dye, mixing leaves standstill 5min, if shows green is then positive, orange then negative; Perhaps, identify, relatively show that detector tube occurs obviously muddy positive, do not see muddy negative with the negative control pipe through visual inspection.
The said loop-mediated isothermal amplification technique of the present invention (loop-mediated isothermal amplication; Be called for short LAMP); The method of rapid detection sample Listeria monocytogenes is the special inside and outside primer of two couples that utilizes the Bst archaeal dna polymerase and design according to target-gene sequence; Six isolated areas on the specific recognition target sequence; Start the endless chain replacement(metathesis)reaction, start complementary strand in target DNA district synthetic, and result's complementary sequence on same chain goes round and begins again and is formed with the very stem of polycyclic Cauliflower structure--circular DNA mixture.Carry out in loop-mediated isothermal amplification (the be called for short LAMP reaction) process pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product-----magnesium pyrophosphate milky white precipitate, can be through the visual inspection result of determination.The LAMP reaction is under constant temperature (about 65 ℃) condition, to accomplish in 45 to 90 minutes.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is oversimplified, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height.In addition, this detection method is lower to testing staff's technical quality requirement, and actually operating is very easy, does not need special reagent and plant and instrument, helps setting up rapid screening system with low cost.The LAMP method is a kind of gene amplification method of easy, quick, high degree of specificity.Constant temperature gene amplification technology and round pcr (comprising the fluorescence real-time quantitative PCR technology) are compared; Can find and technology on methodology indexs such as sensitivity, specificity and sensing range, to be equivalent to or to be superior to round pcr; And do not rely on any special plant and instrument and can realize on-the-spot high-throughput rapid detection, and detect cost far below fluorescent quantitative PCR technique.Domestic do not have the test kit of this respect to sell at present as yet.Be accredited as passing method main, that combine biochemical analysis and serological typing to identify with mikrobe separation and Culture and morphology in the national standard at present, preliminary evaluation needs 2-3 days, accomplishes probation report and needs 10 to 15 days; Adopt the gene diagnosis kit detection among the present invention only to need 2 hours.And, having added optical dye in the reaction solution of the present invention, qualification result is visual and clear more.
Advantage of the present invention is that advantage of the present invention is (1), does not need special reagent and equipment; (2), high specific: use six sections, four primers, whether the existence that just can judge target substance according to whether increasing to be, positive rate can reach greater than 99.9 ﹪, false positive rate is less than 0.1 ﹪; (3), efficient amplification fast: about 2 hours detection times; (4), highly sensitive: amplification template only needs 10 copies or still less, the lowest detection limit reaches 10CFU/ml; The recall rate of sample reaches 99 ﹪; (5), identify easy: identify through visual inspection, need not other any analytical procedures such as electrophoresis, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, producing by product-----magnesium pyrophosphate milky white precipitate, can identify through visual inspection; After adding optical dye, the positive findings colour developing is for green, and negative findings is orange, and is more obviously reliable; (6), purposes is wide: the fields such as food, fishery products, makeup and health care that can be widely used in are detected safely and fast.
Embodiment
Provide specific embodiment further to set forth technical scheme of the present invention below, but The Application of Technology of the present invention is not limited to embodiment.
The preparation of embodiment 1 test kit
Gene diagnosis kit provided by the present invention is by a cover primer sets, and a cover primer sets comprises two pairs of primers, Bst archaeal dna polymerase, reaction solution, sample pretreatment liquid, compositions such as developer and positive control solution.
(1) wherein said primer has 6 covers, is respectively:
Primer sets one:
Outer primer 1:CAAGCACTGTAGTAGTCGA
Outer primer 2:CGTAATAATACTGTTATCAACACC
Inner primer 1:GTTTGCTTTTTTAATTGCGTCAACATTTTAGCTGGTGATACTCTTTGG
Inner primer 2:ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG
Or primer sets two:
Outer primer 1:GGTGATACTCTTTGGGGTAT
Outer primer 2:CGTAATAATACTGTTATCAACACC
Inner primer 1:GACCTGGTACGATTTTATCTGTTGTTTTTCGCACAAAGTAAAGGGAC
Inner primer 2:ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG
Or primer sets three:
Outer primer 1:TTAAACGTCCGTACTGGC
Outer primer 2:CACTTCTTGTGTTGGTGC
Inner primer 1:CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA
Inner primer 2:GCTGGCACAAAATTACTTACAACGATTTTTGGAGTGCTTACTGCTTTG
Or primer sets four:
Outer primer 1:ACTGGTTTCGTTAACGGTAA
Outer primer 2:TGTCACCGCTTTTGACAG
Inner primer 1:AGGTGCAGCTTGTTGAGTAGTAGTTTTGCAGTAAGCACTCCAGTTG
Inner primer 2:CCTGCGCCTAAAGTAGCAGAATTTTGTGTGTAGTAGCATTTTGATCT
Or primer sets five:
Outer primer 1:TTAAACGTCCGTACTGGC
Outer primer 2:CACTTCTTGTGTTGGTGC
Inner primer 1:CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA
Inner primer 2:CTGGCACAAAATTACTTACAACGATTTTTTGGAGTGCTTACTGCTTTG
Or primer sets six:
Outer primer 1:GGTAACGGACCAACTACA
Outer primer 2:TCATTTGACCATTACCAACAT
Inner primer 1:TGTACGTGGAAGGGAGATACCTTTTTGATTGCTCTGGTTACACT
Inner primer 2:TCTGAATCTCAAGCAAAACCTGGTTTTTCGTGAGAAATTCCGCTACC
Primer is made up of with inner primer 2 20-30 μ M outer primer 1, outer primer 2, the inner primer 1 of 1:1:4:4
(2) reaction solution is 10 * Thermopol reaction buffer, the 7.5-12.5mM dNTP by 5:2:1:2, and 100-200mM MgSO4 and 25-37.5M trimethyl-glycine are formed; Wherein, to contain 200 mM pH8.8 trihydroxy methyl aminomethane hydrochlorides, 100 mM Repone K, 100 mM ammonium sulfate, 20 mM sal epsom and concentration of volume percent be 1% triton x-100 to 10 * Thermopol reaction buffer;
(3) the Bst archaeal dna polymerase is Bst DNA polymerase, and every microlitre contains 8 activity units;
(4) sample pretreatment liquid is 20mM pH 8.0 Tris-HCl, and 2mM EDTA, concentration of volume percent are that 1.2% triton x-100 is formed;
(5) developer is an optical dye, preferred SYBR Green I;
(6) positive control solution is the Listeria monocytogenes genomic dna.
Embodiment 2 detection methods
Test sample: test samples such as a little food or body fluid are placed 37 ℃ of cultivations of enrichment liquid.
(1), to the pre-treatment of test sample: extract the DNA gene by ordinary method:
A, get the 50ul incubated overnight enrichment liquid in the eppendorf pipe, centrifugal 2 minutes of 1000rpm abandons supernatant;
B, adding 80ul sample pretreatment liquid mix with deposition gained thalline in centrifuge tube;
Boil in C, the boiling water after 10 minutes and cooled off immediately 10 minutes;
Centrifugal 2 minutes of D, 10000rpm, supernatant promptly can be used as template DNA for use.
(2), loop-mediated isothermal amplification technique reaction process:
A, in 200ulPCR pipe preparation reaction system: primer mixture 2.5ul, reaction solution 5.0 ul, Bst polymerase Large Fragment 1ul (8U), ready template DNA 2ul adds 14.5ul;
B, with the PCR for preparing manage in 65 ℃ the reaction 1-1.5h, 80 ℃ of termination reactions.
(3), analysis and judgement reaction product result: in reaction product, add 2.5ul optical dye (SYBRGREENI), mixing leaves standstill 5min, if shows green is then positive, orange then negative.
Sequence table
< 110>China Measures Institute
<120>
The monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection is with primer sets and test kit
<160>24
<210>?1
<211>19
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?1
CAAGCACTGTAGTAGTCGA 19
<210>2
<211>24
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?2
CGTAATAATACTGTTATCAACACC 24
<210>3
<211>48
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?3
GTTTGCTTTTTTAATTGCGTCAACATTTTAGCTGGTGATACTCTTTGG 48
<210>4
<211>48
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?4
ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG 48
<210>5
<211>20
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?5
GGTGATACTCTTTGGGGTAT 20
<210>6
<211>24
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?6
CGTAATAATACTGTTATCAACACC 24
<210>7
<211>47
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?7
GACCTGGTACGATTTTATCTGTTGTTTTTCGCACAAAGTAAAGGGAC 47
<210>8
<211>45
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?8
ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG 48
<210>9
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>9
TTAAACGTCCGTACTGGC 18
<210>10
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>?10
CACTTCTTGTGTTGGTGC?18
<210>11
<211>51
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>11
CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA 51
<210>12
<211>48
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>12
GCTGGCACAAAATTACTTACAACGATTTTTGGAGTGCTTACTGCTTTG 48
<210>13
<211>20
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>13
ACTGGTTTCGTTAACGGTAA 20
<210>14
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>14
TGTCACCGCTTTTGACAG 18
<210>15
<211>46
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>15
AGGTGCAGCTTGTTGAGTAGTAGTTTTGCAGTAAGCACTCCAGTTG 46
<210>16
<211>47
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>16
CCTGCGCCTAAAGTAGCAGAATTTTGTGTGTAGTAGCATTTTGATCT 47
<210>17
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>17
TTAAACGTCCGTACTGGC 18
<210>18
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>18
CACTTCTTGTGTTGGTGC 18
<210>19
<211>51
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>19
CGTTAGATTCGGTTGTTTCAACAGTTTTAACAGTATTATTACGTCCATCAA 51
<210>20
<211>48
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>20
CTGGCACAAAATTACTTACAACGATTTTTTGGAGTGCTTACTGCTTTG 48
<210>21
<211>18
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>21
GGTAACGGACCAACTACA 18
<210>22
<211>21
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>22
TCATTTGACCATTACCAACAT 21
<210>23
<211>44
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>23
TGTACGTGGAAGGGAGATACCTTTTTGATTGCTCTGGTTACACT 44
<210>24
<211>47
<212>?DNA
< 213>artificial sequence
< 223>primer
<400>24
TCTGAATCTCAAGCAAAACCTGGTTTTTCGTGAGAAATTCCGCTACC 47
Claims (9)
1. the monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection is used primer; It is characterized in that: described primer is a cover Listeria monocytogenes characteristic primer sets, and a cover primer sets is made up of two pairs of primers, and a pair of primer is an outer primer; A pair of is inner primer, and sequence is respectively:
Outer primer 1:CAAGCACTGTAGTAGTCGA
Outer primer 2:CGTAATAATACTGTTATCAACACC
Inner primer 1:GTTTGCTTTTTTAATTGCGTCAACATTTTAGCTGGTGATACTCTTTGG
Inner primer 2:ACAAGTAAATAATGAGGTTGCTGCTTTTTCGGACGTTTAACCAAGTTG.
2. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit, it is characterized in that: this test kit comprises primer sets as claimed in claim 1, reaction solution, Bst archaeal dna polymerase, sample pretreatment liquid and positive control solution.
3. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: described primer sets is that the 20-30 μ M outer primer 1, outer primer 2, inner primer 1 of 1:1:4:4 formed with inner primer 2 by volume ratio.
4. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2; It is characterized in that: described reaction solution is to be 10 * Thermopol reaction buffer, the 7.5～12.5mM dNTP of 5:2:1:2 by volume ratio, 100-200mM MgSO
4Form with 25～37.5M trimethyl-glycine.
5. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 4 is characterized in that: it is 1% triton x-100 that described 10 * Thermopol reaction buffer contains 200 mM pH8.8 trihydroxy methyl aminomethane hydrochlorides, 100 mM Repone K, 100 mM ammonium sulfate, 20 mM sal epsom and concentration of volume percent.
6. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: the every microlitre of Bst archaeal dna polymerase contains 8～16 activity units.
7. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2; It is characterized in that: sample pretreatment liquid is by 20mM pH 8.0 Tris-HCl; 2mM EDTA, concentration of volume percent are that 1.2% triton x-100 is formed.
8. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: this test kit also comprises developer, and developer is an optical dye.
9. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: positive control solution is a Listeria monocytogenes reference culture genomic dna.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861744A (en) * | 2016-03-24 | 2016-08-17 | 天津出入境检验检疫局动植物与食品检测中心 | Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof |
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