CN101368203A - Primer, reagent kit and detection method for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique fast detection - Google Patents

Abstract

The invention relates to a biological detection reagent, in particular to a primer, a kit and a detection method which are used for the fast detection of the loop-mediated isothermal amplification technology of a monotonic increasing listeria . The primer used for the fast detection of the loop-mediated isothermal amplification technology of the monotonic increasing is a set of character primer group of the monotonic increasing; one set of primers consists of two pairs of primers; one pair of primers refers to outer primers and the other pair refers to inner primers; the invention has six sets of primers. The kit comprises a set of primers, a reaction liquid, BstDNA polymerase, a sample pre-processing liquid, a visualization reagent, a masculine contrast liquid; the detection method includes extracting the DNA of bacteria, the loop-mediated isothermal amplification reaction and coloration detection. The method has the advantages of fast speed, strong specificity, high sensitivity and low cost. The monotonic increasing in the sample can be fast detected by using the kit to carry out simple processing on the sample; thus having the advantages of high sensitivity, strong specificity, simple operation, and the like; besides, the result can be judged by sight.

Description

Monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection primer, test kit and detection method

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 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 be present in soil, animal and fishery products etc., mainly pass through food transmissions such as milk and milk preparation, vegetables, fishery products, meat product.This bacterium has been listed in 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 7～10d at least, and detection limit is lower, wastes time and energy.Immunization is than very fast, but Monoclonal Antibody is relatively more difficult, easily produces cross reaction, poor specificity.And PCR or fluorescence quantifying PCR method are quick, and be special, sensitivity is very high, but need expensive PCR instrument.Once had a small amount of bibliographical information to utilize the mRNA template to detect the live body list at 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 at target-gene sequence to be measured, utilize the strand displacement archaeal dna polymerase (Bst DNA polymerase) can specificity under 65 ℃ of left and right sides isothermal conditions, efficiently, carry out nucleic acid amplification fast, its turbidity can directly be judged or detect to amplification by naked eyes to amplification by product magnesium pyrophosphate precipitation, also available in conjunction with the double-stranded preferred SYBR Green of fluorescence dye I dyeing, can judge by naked eyes.Because two pairs of primers of LAMP technology amplification are 6 sections at target gene, thereby have a specificity higher than PCR, be promptly not need specific apparatus such as PCR instrument 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) technology rapid detection 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:

Monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection primer, described primer are 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.As preferred again, it is 1% triton x-100 that described 10 * Thermopol reaction buffer contains 200mM pH8.8 trihydroxy methyl aminomethane hydrochloride, 100mM Repone K, 100mM ammonium sulfate, 20mM 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 pH8.0 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 a fluorescence dye.Preferred fluorescence 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 the above-mentioned described test kit of any one 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 precipitation gained thalline in centrifuge tube; Cool off 10min immediately after boiling 10min in C, the boiling water; The centrifugal 2min of D, 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 LBst 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 pipe for preparing is reacted 1～1.5h, 80 ℃ of termination reactions in 60～65 ℃;

4) analysis and judgement reaction product result: add the 2.5ul fluorescence dye in reaction product, 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 by visual inspection.

The said loop-mediated isothermal amplification technique of the present invention (loop-mediated isothermalamplication, 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 by the visual inspection result of determination.The LAMP reaction is to finish in 45 to 90 minutes under constant temperature (about 65 ℃) condition.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 that this technology is equivalent to or is better than round pcr on methodology indexs such as sensitivity, specificity and sensing range, 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.At present in the national standard with microorganism separation and Culture and morphology be accredited as main, in conjunction with the passing method that biochemical analysis and serological typing are identified, preliminary evaluation needs 2-3 days, finishes 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 fluorescence dye in the reaction solution of the present invention, qualification result is more visual and clear.

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 by 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 by visual inspection; After adding fluorescence 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 the 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 7.5-12.5mMdNTP by 5:2:1:2, and 100-200mM MgSO4 and 25-37.5M trimethyl-glycine are formed; Wherein, to contain 200mM pH8.8 trihydroxy methyl aminomethane hydrochloride, 100mM Repone K, 100mM ammonium sulfate, 20mM 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 pH8.0Tris-HCl, and 2mM EDTA, concentration of volume percent are that 1.2% triton x-100 is formed;

(5) developer is a fluorescence 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 according to a conventional 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 precipitation 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 stand-by template DNA.

(2), loop-mediated isothermal amplification technique reaction process:

A, in 200ulPCR pipe preparation reaction system: primer mixture 2.5ul, reaction solution 5.0ul, Bstpolymerase Large Fragment1ul (8U), ready template DNA 2ul adds 14.5ul;

B, with the PCR pipe for preparing in 65 ℃ of reaction 1-1.5h, 80 ℃ of termination reactions.

(3), analysis and judgement reaction product result: add 2.5ul fluorescence dye (SYBRGREENI) in reaction product, mixing leaves standstill 5min, if shows green is then positive, orange then negative.

Sequence table

＜110〉China Measures Institute

＜120〉monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection primer, test kit and detection method

<160>24

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>1

<210>2

<211>24

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>2

<210>3

<211>48

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>3

<210>4

<211>48

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>4

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>5

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>6

<210>7

<211>47

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>7

<210>8

<211>45

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>8

<210>9

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>9

<210>10

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>10

<210>11

<211>51

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>11

<210>12

<211>48

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>12

<210>13

<211>20

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>13

<210>14

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>14

<210>15

<211>46

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>15

<210>16

<211>47

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>16

<210>17

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>17

<210>18

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>18

<210>19

<211>51

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>19

<210>20

<211>48

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>20

<210>21

<211>18

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>21

<210>22

<211>21

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>22

<210>23

<211>44

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>23

<210>24

<211>47

<212>DNA

＜213〉artificial sequence

＜223〉primer

<400>24

Claims (10)

1. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection primer, it is characterized in that: described primer is a cover Listeria monocytogenes characteristic primer sets, one 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, have six cover primers, 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.

2. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit, it is characterized in that: this test kit comprises any one group of 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 200mM pH8.8 trihydroxy methyl aminomethane hydrochloride, 100mM Repone K, 100mM ammonium sulfate, 20mM 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 is characterized in that: sample pretreatment liquid is by 20mM pH 8.0 Tris-HCl, and 2mM EDTA, concentration of volume percent are 1.2% triton x-100.

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 a fluorescence 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.

10. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique method for quick is characterized in that: adopt as any described test kit of claim of claim 2～9, 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 precipitation gained thalline in centrifuge tube; Cool off 10min immediately after boiling 10min in C, the boiling water; The centrifugal 2min of D, 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 LBst archaeal dna polymerase, 2 μ L template DNA to be checked or positive control solution and 14.5 μ L ddH ₂O;

3) ring mediated isothermal amplification of Listeria monocytogenes: the PCR pipe for preparing is reacted 1～1.5h, 80 ℃ of termination reactions in 60～65 ℃;

4) analysis and judgement reaction product result: add the 2.5ul fluorescence dye in reaction product, 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 by visual inspection.