CN101368204B - Fast detection primer and reagent kit for enterobacter sakazakii hymenial veil mediated isothermality amplification technique - Google Patents
Fast detection primer and reagent kit for enterobacter sakazakii hymenial veil mediated isothermality amplification technique Download PDFInfo
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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 an enterobacter sakazaii. The primer used for the fast detection of the loop-mediated isothermal amplification technology of the enterobacter sakazaii is a set of character primer group of the enterobacter sakazaii; 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 the strain, 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 enterobacter sakazaii 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
Technical field
The present invention relates to biological detection reagent, be specifically related to a kind of enterobacter sakazakii hymenial veil mediated isothermality amplification technique rapid detection primer, test kit and detection method.
Background technology
Enterobacter sakazakii (Enterobacter sakazakii), have another name called yellow enterobacter cloacae, a kind of Gram-negative bacteria for enterobacter, be renamed as Enterobacter sakazakii after 1980, be the newfound in recent years a kind of pathogenic bacterium that cause extensive concern in milk and the milk preparation course of processing and the finished product, can cause that infant and adult diseases comprise baby's meningitis, pyemia, septicemia and enterocolitis etc., and may cause nervous dysfunction, cause serious sequela and even dead.The well-known milk powder Ceng Zifa of manufacturing enterprise of the 2002-2003 U.S. has recalled the infant formula powder that contains Enterobacter sakazakii, and after this Enterobacter sakazakii becomes the focus of attracting attention in the world in the milk powder.1961 to 2003 research reports have shown that Enterobacter sakazakii causes that infant and premature infant's overall mortality rate are up to 80%.FAO/WHO (Food and Argriculture OrganizationFAO/World Health Organization) lists Enterobacter sakazakii in cause infant infection, disease and main causes of death " A " quasi-microorganism category in the expert consulting meeting of holding in Geneva in February, 2004, the serious threat mankind's (especially infant) health, and bring heavy economic losses for food (especially milk and milk preparation) industry.
Therefore, strict requirement is all made to the Enterobacter sakazakii in the food (especially milk and milk preparation) by national governments.U.S. FDAs in 2002 worldwide take the lead in having set up the examination criteria of Enterobacter sakazakii; China's successively issue " Enterobacter sakazakii method of inspection part 1 in the milk powder: separate and method of counting " (SN/T1632.1-2005), " Enterobacter sakazakii method of inspection part 2 in the milk powder: PCR method " (SN/T1632.2-2005) and " the Enterobacter sakazakii method of inspection the 3rd part in the milk powder: fluorescence PCR method " industry standard such as (SN/T1632.3-2005), milk and milk preparation to most of import all require to carry out the check of Enterobacter sakazakii, and requirement must not detect.Therefore, Enterobacter sakazakii has become the essential items for inspection of many state food health, and its detection method causes people's attention just day by day.
Classical conventional bacterium Physiology and biochemistry method of inspection whole process needs 6～10d 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, technical sophistication, expense height, is 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, does not still have at present the test kit that utilizes ring mediated isothermal amplification gene engineering detection Enterobacter sakazakii and the report of detection method.
Summary of the invention
The detection method that has Enterobacter sakazakii (Enterobacter sakazakii) 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 enterobacter sakazakii 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 widely used in fields such as food (especially milk and milk preparation), fishery products, makeup and health care.
In order to realize first above-mentioned purpose, the present invention adopts following technical scheme:
Enterobacter sakazakii hymenial veil mediated isothermality amplification technique rapid detection primer, described primer are a cover Enterobacter sakazakii characteristic primer sets, and a cover primer sets is become by two pairs of primers, and a pair of primer is an outer primer, and a pair of is inner primer, has 6 cover primers, and sequence is respectively:
Primer sets one:
Outer primer 1:GCAATATTCCCCACTGCTGC
Outer primer 2:GGAGGGGGATAACTACTGGA
Inner primer 1:CGACGATCCCTAGCTGGTCTGATTTTTCTGGACCGTGTCTCAGTT
Inner primer 2:TCCCATCTGGGCACATCTGATGTTTTAACGTCTACGGACCAAAGTG or primer sets two:
Outer primer 1:AGTGTGGCTGGTCATCCT
Outer primer 2:CGGACGGGTGAGTAATGTCT
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTCTCAGACCAGCTAGGGATCG
Inner primer 2:AGGTCCCCCACTTTGGTCCGTATTTTGGAAACTGCCTGATGGAGG or primer sets three:
Outer primer 1:TGTGGCTGGTCATCCTCTC
Outer primer 2:TGCTGCTCTGCTGACGA
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTAGACCAGCTAGGGATCGTC
Inner primer 2:TTGGTCCGTAGACGTTATGCGGTTTTATGTCTGGGAAACTGCCTG or primer sets four:
Outer primer 1:TCCTCCCCGCTGAAAGT
Outer primer 2:TGCCCAGATGGGATTAGCT
Inner primer 1:GGCAGCAGTGGGGAATATTGCATTTTGGCCTTCTTCATACACGCG
Inner primer 2:CCGTGTCTCAGTTCCAGTGTGGTTTTGGTGGGGTAACGGCTCA or primer sets five:
Outer primer 1:ACTTTACAACCCGAAGGCC
Outer primer 2:AGCTAGTAGGTGGGGTAACG
Inner primer 1:GGCAGCAGTGGGGAATATTGCATTTTTTCATACACGCGGCATGG
Inner primer 2:CGTAGGAGTCTGGACCGTGTCTTTTTTCACCTAGGCGACGATCC or primer sets six:
Outer primer 1:TGTGGCTGGTCATCCTCTC
Outer primer 2:TGCTGCTCTGCTGACGA
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTAGACCAGCTAGGGATCGTC
Inner primer 2:TGGTCCGTAGACGTTATGCGGTTTTTATGTCTGGGAAACTGCCTG.
In order to realize second above-mentioned purpose, the present invention adopts following technical scheme:
Enterobacter sakazakii hymenial veil mediated isothermality amplification technique quick detection kit, this test kit comprise 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 pH8.0Tris-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 an Enterobacter sakazakii reference culture genomic dna.
In order to realize the 3rd above-mentioned purpose, the present invention adopts following technical scheme:
The enterobacter sakazakii 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 Enterobacter sakazakii 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 LddH2O;
3) ring mediated isothermal amplification of Enterobacter sakazakii: 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 isothermal amplication, abbreviation LAMP) method of rapid detection sample Enterobacter sakazakii 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, serological typing and PCR identify etc., 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, also can add fluorescence dye in the reaction solution of the present invention, qualification result is more visual and clear.
Advantage of the present invention is (1), do 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 M 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 detection safely and fast that can be widely used in Enterobacter sakazakii in the food (especially milk and milk preparation).
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 sets has 6 covers, is respectively:
Primer sets one:
Outer primer 1:GCAATATTCCCCACTGCTGC
Outer primer 2:GGAGGGGGATAACTACTGGA
Inner primer 1:CGACGATCCCTAGCTGGTCTGATTTTTCTGGACCGTGTCTCAGTT
Inner primer 2:TCCCATCTGGGCACATCTGATGTTTTAACGTCTACGGACCAAAGTG or primer sets two:
Outer primer 1:AGTGTGGCTGGTCATCCT
Outer primer 2:CGGACGGGTGAGTAATGTCT
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTCTCAGACCAGCTAGGGATCG
Inner primer 2:AGGTCCCCCACTTTGGTCCGTATTTTGGAAACTGCCTGATGGAGG or primer sets three:
Outer primer 1:TGTGGCTGGTCATCCTCTC
Outer primer 2:TGCTGCTCTGCTGACGA
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTAGACCAGCTAGGGATCGTC
Inner primer 2:TTGGTCCGTAGACGTTATGCGGTTTTATGTCTGGGAAACTGCCTG or primer sets four:
Outer primer 1:TCCTCCCCGCTGAAAGT
Outer primer 2:TGCCCAGATGGGATTAGCT
Inner primer 1:GGCAGCAGTGGGGAATATTGCATTTTGGCCTTCTTCATACACGCG
Inner primer 2:CCGTGTCTCAGTTCCAGTGTGGTTTTGGTGGGGTAACGGCTCA or primer sets five:
Outer primer 1:ACTTTACAACCCGAAGGCC
Outer primer 2:AGCTAGTAGGTGGGGTAACG
Inner primer 1:GGCAGCAGTGGGGAATATTGCATTTTTTCATACACGCGGCATGG
Inner primer 2:CGTAGGAGTCTGGACCGTGTCTTTTTTCACCTAGGCGACGATCC or primer sets six:
Outer primer 1:TGTGGCTGGTCATCCTCTC
Outer primer 2:TGCTGCTCTGCTGACGA
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTAGACCAGCTAGGGATCGTC
Inner primer 2:TGGTCCGTAGACGTTATGCGGTTTTTATGTCTGGGAAACTGCCTG
Primer sets 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 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 8-16 activity unit;
(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 an Enterobacter sakazakii reference culture 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 (or positive control solution) 2ul adds ddH
2O14.5ul;
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〉enterobacter sakazakii hymenial veil mediated isothermality amplification technique rapid detection primer, test kit and detection method
<160>24
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>1
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>2
<210>3
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>3
<210>4
<211>46
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>4
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>5
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>6
<210>7
<211>46
<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>19
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>9
<210>10
<211>17
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>10
<210>11
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>11
<210>12
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>12
<210>13
<211>17
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>13
<210>14
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>14
<210>15
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>15
<210>16
<211>43
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>16
<210>17
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>17
<210>18
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>18
<210>19
<211>44
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>19
<210>20
<211>44
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>20
<210>21
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>21
<210>22
<211>17
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>22
<210>23
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>23
<210>24
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉primer
<400>24
Claims (9)
1. enterobacter sakazakii hymenial veil mediated isothermality amplification technique rapid detection primer, it is characterized in that: described primer is a cover Enterobacter sakazakii 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:AGTGTGGCTGGTCATCCT
Outer primer 2:CGGACGGGTGAGTAATGTCT
Inner primer 1:GCCATCAGATGTGCCCAGATGGTTTTCTCAGACCAGCTAGGGATCG
Inner primer 2:AGGTCCCCCACTTTGGTCCGTATTTTGGAAACTGCCTGATGGAGG.
2. enterobacter sakazakii 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. enterobacter sakazakii hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: described primer sets is 1: 1: 4 by volume ratio: 4 20-30 μ M outer primer 1, outer primer 2, inner primer 1 are formed with inner primer 2.
4. enterobacter sakazakii hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2, it is characterized in that: reaction solution is to be 5: 2: 1 by volume ratio: 10 * Thermopol reaction buffer of 2,7.5～12.5mM dNTP, 100～200mM MgSO
4Form with 25～37.5M trimethyl-glycine..
5. enterobacter sakazakii hymenial veil mediated isothermality amplification technique quick detection kit according to claim 4 is characterized in that: it is 1% triton x-100 that 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. enterobacter sakazakii 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. enterobacter sakazakii 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. enterobacter sakazakii 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. enterobacter sakazakii hymenial veil mediated isothermality amplification technique quick detection kit according to claim 2 is characterized in that: positive control solution is an Enterobacter sakazakii reference culture genomic dna.
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| CN103757131A (en) * | 2013-12-17 | 2014-04-30 | 东北农业大学 | Lamp detection primers and Lamp detection method for discrimination of equine herpesvirus type 1/4 |
| CN106480020B (en) * | 2015-09-02 | 2019-09-24 | 上海产业技术研究院 | A kind of design method and its application of nucleic acid amplification reaction primer |
| CN111020009B (en) * | 2015-09-02 | 2022-07-22 | 上海产业技术研究院 | Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application |
| CN107012218B (en) * | 2017-04-06 | 2020-09-15 | 南方医科大学南方医院 | LAMP primer group, kit and rapid detection method for detecting enterobacter aerogenes |
| CN107576785A (en) * | 2017-08-25 | 2018-01-12 | 广州市雷德生物科技有限公司 | A kind of sample treatment solution and its application |
| CN111172325A (en) * | 2020-02-21 | 2020-05-19 | 北京天恩泽基因科技有限公司 | Multi-target double-dye isothermal amplification rapid detection method and kit |
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| WO2004104550A2 (en) * | 2003-05-16 | 2004-12-02 | Qualicon Incorporated | Rapid and specific detection of enterobacter sakazakii |
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