CN102643924B - Primer group and kit for rapidly detecting Listeria monocytogenes by utilizing LAMP (Loop-mediated Isothermal Amplification) technology - Google Patents

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

Primer sets and test kit for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection

The present patent application is that the patent No. is: 200810121029.X, and patent name is: monotonic increasing Listeria hymenial veil mediated isothermality amplification technique is primer, test kit and detection method for rapid detection, 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 primer, test kit and detection method for rapid detection.

Background technology

Listeria monocytogenes ( listeria monocytogenes, Lm) (hereinafter to be referred as " Listeria monocytogenes ") is a kind of important zoonosis pathogenic bacterium, is extensively present in soil, animal and fishery products etc., mainly by food transmissions such as Milk and milk products, vegetables, fishery products, meat products.This bacterium has been listed in one of large food pathogenic nineties 4, becomes the essential items for inspection of many state food health.

Traditional Listeria monocytogenes biochemistry detection method whole process at least needs 7～10 d, and detection limit is lower, wastes time and energy.Immunization is than very fast, but monoclonal antibody preparation is more difficult, easily produces cross reaction, poor specificity.And PCR or fluorescence quantifying PCR method are quick, special, sensitivity is very high, but need expensive PCR instrument.Once had a small amount of bibliographical information to utilize mRNA template with RT-PCR, to detect live body list for iap gene abroad and increase listeria spp, but need to hybridize to show detected result with film, time length, technical sophistication, expense are high, are difficult for applying.Fluorescence dye can detect viable bacteria, but technical sophistication needs high end instrument.Therefore set up a kind of easy, sensitive, quick, special method necessary.

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, the a set of two pairs of special primers of 6 zone design for target-gene sequence to be measured, utilize the strand displacement archaeal dna polymerase (Bst DNA polymerase) can specificity under 65 ℃ of left and right isothermal conditions, efficiently, carry out fast nucleic acid amplification, amplification can directly judge or detect its turbidity by naked eyes to amplification by product magnesium pyrophosphate precipitation, also the preferred SYBR Green of the fluorescence dye of available combination two strands I dyeing, can judge by naked eyes.Two pairs of primers that increase due to LAMP technology are 6 sections for target gene, thereby there is the specificity higher than PCR, under isothermal condition, not need the specific apparatus such as PCR instrument simultaneously, and the pre-treatment of sample is very simple, amplification efficiency advantages of higher more in the unit time, has caused people's concern.

(application number is Chinese invention patent: 200710030435.0,200710030437.X, 200710132320.2,200710026389.7,200810052321.0,200810015001.8,200810093986.6) disclose respectively the method that adopts ring mediated isothermal amplification gene engineering tested for pathogens.But, also do not use at present ring mediated isothermal amplification gene engineering to detect the report of the test kit of Listeria monocytogenes.

Summary of the invention

For solve the detection method of existing Listeria monocytogenes not easy, sensitive low, the time long, 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 object of the present invention is to provide uses the test kit of primer for above-mentioned detection; The 3rd object of the present invention is to provide uses the kit test method of primer for above-mentioned detection.Test kit of the present invention and detection method have easy, sensitive, quick, special good feature, can be extensively with being widely used in the fields such as food, fishery products, makeup and health care.

In order to realize first above-mentioned object, the present invention adopts following technical scheme:

Monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection primer, described primer is a set of Listeria monocytogenes characteristic primer sets, a set of primer sets is comprised of two pairs of primers, pair of primers is 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 object, the present invention adopts following technical scheme:

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 technique scheme.

As preferably, the 20-30 μ M outer primer 1 that described primer sets is 1:1:4:4 by volume ratio, outer primer 2, inner primer 1 form with inner primer 2.

As preferably, 10 * Thermopol reaction buffer that described reaction solution is is 5:2:1:2 by volume ratio, 7.5～12.5mM dNTP, 100～200mM MgSO ₄form with 25～37.5M trimethyl-glycine.As preferred again, described 10 * Thermopol reaction buffer, containing 200 mM pH8.8 trihydroxy methyl aminomethane hydrochlorides, 100 mM Repone K, 100 mM ammonium sulfate, 20 mM magnesium sulfate and concentration of volume percent is 1% triton x-100.

As preferably, the described every microlitre of Bst archaeal dna polymerase is containing 8～16Ge activity unit.

As preferably, described sample pretreatment liquid is by 20mM pH 8.0 Tris-HCl, 2mM EDTA, and concentration of volume percent is 1.2% triton x-100.

As preferably, this described test kit also comprises developer, and developer is fluorescence dye.Preferably fluorescence dye is SYBR Green I;

As preferably, described positive control solution is Listeria monocytogenes reference culture genomic dna.

In order to realize the 3rd above-mentioned object, the present invention adopts following technical scheme:

Monotonic increasing Listeria hymenial veil mediated isothermality amplification technique method for quick, adopts the test kit described in any one above-mentioned technical scheme, and the method comprises the following steps:

1) extraction of Listeria monocytogenes DNA: A, get 50ul incubated overnight enrichment liquid in eppendorf pipe, the centrifugal 2min of 1000rpm, abandons supernatant; B, add 80ul sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline; In C, boiling water, boil after 10min cooling 10min immediately; The centrifugal 2min of D, 10000rpm, supernatant 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 pipe preparing is reacted to 1～1.5h, 80 ℃ of termination reactions in 60～65 ℃;

4) analyze judgement reaction product result: in reaction product, add 2.5ul fluorescence dye, mix, standing 5min, if shows green is positive, orange negative; Or, by visual inspection, identify, relatively show with negative control pipe, detector tube occurs obviously muddy positive, has no muddy negative.

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 Bst archaeal dna polymerase and design according to target-gene sequence, six isolated areas on specific recognition target sequence, start endless chain replacement(metathesis)reaction, in target DNA district, start complementary strand synthetic, go round and begin again stem--the circular DNA mixture of the Cauliflower structure that is formed with a lot of rings of result complementary sequence on same chain.Carry out in loop-mediated isothermal amplification (being called for short LAMP reaction) process the pyrophosphate ion of separating out from dNTP and the Mg reaction soln ²⁺in conjunction with, produce by product-----magnesium pyrophosphate milky white precipitate, can be by visual inspection result of determination.LAMP reaction is in 45 to 90 minutes, to complete under constant temperature (65 ℃ of left and right) condition.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is simplified, overcome normal PCR intrinsic detection time long, easily pollute and the shortcoming such as testing cost height.In addition, this detection method requires lower to testing staff's technical quality, and actually operating is very easy, does not need special reagent and plant and instrument, is conducive to set up rapid screening system with low cost.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 fluorescence real-time quantitative PCR technology) are compared, can find that this technology is equivalent to or is better than round pcr in the 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 testing cost is far below fluorescent quantitative PCR technique.Domestic not yet have the test kit of this respect to sell at present.In national standard, take microorganism separation and Culture and Morphological Identification at present as master, in conjunction with the passing method of biochemical analysis and serological typing evaluation, and preliminary evaluation needs 2-3 days, completes probation report and needs 10 to 15 days; Adopt the gene diagnosis kit detection in the present invention only to need 2 hours.And, in reaction solution of the present invention, having added fluorescence dye, 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: apply six sections, whether four primers, just can judge the existence of target substance according to whether increasing, and positive rate can reach and be greater than 99.9 ﹪, and false positive rate is less than 0.1 ﹪; (3) efficiently 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: by visual inspection, identify, without other any analytical procedures such as electrophoresis, the pyrophosphate ion of separating out from dNTP and the Mg reaction soln ²⁺in conjunction with, producing by product-----magnesium pyrophosphate milky white precipitate, can identify by visual inspection; Add after fluorescence dye, positive findings colour developing is for green, and negative findings is orange, 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 set of primer sets, and a set of primer sets comprises two pairs of primers, Bst archaeal dna polymerase, reaction solution, sample pretreatment liquid, the 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 comprised 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 form; Wherein, to contain 200 mM pH8.8 trihydroxy methyl aminomethane hydrochlorides, 100 mM Repone K, 100 mM ammonium sulfate, 20 mM magnesium sulfate and concentration of volume percent be 1% triton x-100 to 10 * Thermopol reaction buffer;

(3) Bst archaeal dna polymerase is Bst DNA polymerase, and every microlitre is containing 8 activity units;

(4) sample pretreatment liquid is 20mM pH 8.0 Tris-HCl, 2mM EDTA, and concentration of volume percent is that 1.2% triton x-100 forms;

(5) developer is fluorescence dye, preferably SYBR Green I;

(6) positive control solution is Listeria monocytogenes genomic dna.

embodiment 2 detection methods

Test sample: the test samples such as a little food or body fluid are placed in to 37 ℃ of cultivations of enrichment liquid.

(1), the pre-treatment to test sample: extract according to a conventional method DNA gene:

A, get 50ul incubated overnight enrichment liquid in eppendorf pipe, centrifugal 2 minutes of 1000rpm, abandons supernatant;

B, add 80ul sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline;

In C, boiling water, boil after 10 minutes cooling 10 minutes immediately;

Centrifugal 2 minutes of D, 10000rpm, supernatant 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 5.0 ul, Bst polymerase Large Fragment 1ul (8U), ready template DNA 2ul, adds 14.5ul;

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

(3), analyze judgement reaction product result: in reaction product, add 2.5ul fluorescence dye (SYBRGREENI), mix, standing 5min, if shows green is positive, orange negative.

Sequence table

The <110> China Measures Institute

<120> primer sets and test kit for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique rapid detection

<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 (1)

1. monotonic increasing Listeria hymenial veil mediated isothermality amplification technique quick detection kit, is characterized in that: this test kit comprises primer sets, reaction solution, Bst archaeal dna polymerase, sample pretreatment liquid, developer and positive control solution;

Described primer sets is comprised of two pairs of primers, and pair of primers is outer primer, and 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;

The 20-30 μ M outer primer 1 that primer sets is 1:1:4:4 by volume ratio, outer primer 2, inner primer 1 form with inner primer 2;

10 * Thermopol reaction buffer that described reaction solution is is 5:2:1:2 by volume ratio, 7.5～12.5mM dNTP, 100-200mM MgSO ₄form with 25～37.5M trimethyl-glycine;

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 magnesium sulfate and concentration of volume percent;

The every microlitre of Bst archaeal dna polymerase is containing 8～16Ge activity unit;

Sample pretreatment liquid is by 20mM pH 8.0 Tris-HCl, 2mM EDTA, and concentration of volume percent is that 1.2% triton x-100 forms;

Developer is fluorescence dye;

Positive control solution is Listeria monocytogenes reference culture genomic dna.