CN106544444A - The dry powdered LAMP quick detection kits of streptococcus fecalis and its using method - Google Patents
The dry powdered LAMP quick detection kits of streptococcus fecalis and its using method Download PDFInfo
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Abstract
The invention discloses the dry powdered LAMP quick detection kits of streptococcus fecalis and the method for quick based on which.Kit includes lyophilized isothermal duplication agent, redissolves liquid, positive control dry powder, negative control, nitrite ion, lysate and confining liquid, freeze in isothermal duplication agent containing dNTPs,BstArchaeal dna polymerase, amplimer and freeze drying protectant.Primer pair in this kit answers eight sections of streptococcus fecalis specific gene sequences, nitrite ion is added after reaction, yin and yang attribute colour development difference is obvious, have the advantages that high specificity, sensitivity be high, easily operated, result judges simple, be suitable for food security, beverage, water and other raw and auxiliary material quality controls and safely and fast detect.Kit of the present invention is easy to normal temperature to transport, and using simply, fast, without the need for specialized equipment, reduces cost simultaneously widens products application scope, is more suitable for field quick detection and basic unit's remote districts coherent detection, is with a wide range of applications.
Description
Technical field
The present invention relates to a kind of dry powdered LAMP quick detection reagents and the method for quick based on which, more particularly to
A kind of dry powdered LAMP quick detection kits of streptococcus fecalis and the method for quick based on which.
Background technology
Streptococcus fecalis is commonly distributed in the enteron aisle of humans and animals, is excreted with excrement, is a kind of heat-resisting Pseudomonas, in temperature
Still well-grown in 44.5 ± 0.2 DEG C of degree.It is highly prone to excrement and directly or indirectly pollutes as the water source of drinking water, this
If having enteric infectious disease in a little pollution sources, then may contain virulence factor living in water source, contaminated drinking water can make one
Group produces disease, indicator bacteria of the streptococcus fecalis as health bacteria in drinking water, particularly significant, the excrement in water hygiene detection
Streptococcus can reflect whether water body is polluted by excrement or other pollution sources, whether there is so as to understand in water body indirectly
The danger of pathogen must not be detected in natural mineral water according to the rules to take measures in time to be administered and to be protected
Streptococcus fecalis.The conventional biochemical method that now can be used for streptococcus fecalis inspection in bottled water has rubbing method, many test tube MPN methods and filter membrane
Method, is to improve detection efficiency, it is also possible to Enzyme-multiplied immune technique, Nucleic Acid Probe Technique, the inspection of nucleic acid amplification technologies equimolecular biology
Survey method, is used for quickly detecting and result primary dcreening operation.These technologies attempt to break through traditional mould such as traditional morphology, biochemical reaction
Formula, it is not necessary to separating-purifying is carried out to object bacteria, and is directly detected with the enrichment liquid of sample or sample so that detected to standard
Really, quick and inexpensive direction is developed.
LAMP technology is a kind of new external isothermal duplication spy by exploitations such as Japanese researchers Notomi in 2000
The technology of heteronuclear acid fragment.The technology utilizes two pairs of special primers and the Bst archaeal dna polymerases with strand-displacement activity.Make reaction
In template two ends primer junction circulation there is cyclic single strand structure, primer is smoothly combined simultaneously with template under isothermal conditions
Carry out strand displacement amplification reaction.LAMP is used for quick detection without the need for complex instrument, with result interpretation it is simple, sensitivity is high, special
The opposite sex advantage that good, detection range is extensive and application cost is low.In actual applications, it is intended to prepare corresponding solution before detection
Reaction system, and wherein composition is more, easily causes reagent contamination, detection interval pollution and result that frequent operation causes to be missed
Difference etc., in the reaction system, portion of reagent needs stored frozen, Bst archaeal dna polymerases as required to need strictly at -20 DEG C in addition
Under the conditions of preserve, temperature is too high to affect its enzymatic activity, cause detection reagent to fail.Therefore the high temperature of the reagent is transported for long-distance
And scene application etc. bring inconvenience, also improve corresponding cost.Meanwhile, the current detection method is not yet in food security, doctor
The fields such as medicine are promoted and are come, especially in the quick detection of water body, as nonstandard method, current existing LAMP products only pin
To testing process, the mode nothing for being related to sample pre-treatments is referred to.And based on the fast of loop-mediated isothermal amplification technique on market
Speed detection streptococcus fecalis kit it is less, and can be used for normal temperature transport dry-type like product not yet occur.
The difficult point of dry-type LAMP quick detection kits exploitation mainly has following：1) keep Bst in freeze-drying process
The vigor of archaeal dna polymerase is not lost substantially；2) design suitable primer；3) develop the color not obvious enough, it is difficult to distinguish result.
In order to keep the vigor of Bst archaeal dna polymerases, addition freeze drying protectant is that, than more conventional selection, conventional is lyophilized
Protective agent is trehalose, but its protected effect is relatively difficult to ensure product quality than relatively limited.
The content of the invention
It is an object of the invention to provide the dry powdered LAMP quick detection kits of streptococcus fecalis and the quick inspection based on which
Survey method.
The technical solution used in the present invention is：
A kind of quick detection kit for being applied to streptococcus fecalis in water, including lyophilized isothermal duplication agent, redissolve liquid, the positive
Control dry powder, negative control, nitrite ion, lysate and confining liquid, are polymerized containing dNTPs, Bst DNA in freezing isothermal duplication agent
Enzyme, outer primer, inner primer, ring primer and freeze drying protectant, freeze drying protectant is by trehalose, glycine and bovine serum albumin(BSA) group
Into.
Lyophilized isothermal duplication agent consisting of before lyophilized：0.9～1.6mmol/L dNTPs, 0.3～0.4U/ μ L Bst
Archaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/
L ring primer LF/LB and freeze-drying composite protectant containing 3%～6% (w/v) trehalose, 0.01%-0.1% (w/v) glycine,
0.1%-1% bovine serum albumin(BSA)s, balance of aseptic ultra-pure water.
Lyophilized isothermal duplication agent consisting of before lyophilized：0.9～1.6mmol/L dNTPs, 0.3～0.4U/ μ LBst
Archaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/
L ring primer LF/LB and freeze-drying composite protectant containing 4%～5% (w/v) trehalose, 0.04%-0.07% (w/v) glycine,
0.4%-0.7% bovine serum albumin(BSA)s, balance of aseptic ultra-pure water.
Redissolve liquid and contain 2～9mmol/L MgSO4, 8～12mmol/L KCl, 15～20mmol/L Tris-HCl, 8～
12mmol/L(NH4)2SO4, 0.1～0.3%Triton X-100 and 0.5～1.5mol/L glycine betaines.
The sequence of outer primer F3/B3, inner primer FIP/BIP and ring primer LF/LB is as follows：
Streptococcus fecalis-F3：TGGGCATTTTTCTTTCGG；
Streptococcus fecalis-B3：CATTTAAAAACCAACGTACTTGA；
Streptococcus fecalis-FIP：CAAACGTTGAGTCGCCGTTGCCAATAGTTCGCAACCGA；
Streptococcus fecalis-BIP：GAAGGAGTGACCAACACAGAGACGACTCTCCAGCCAAATCG；
Streptococcus fecalis-LF：TGTTCCGAAATCCGTTTCTATCA；
Streptococcus fecalis-LB：CAAATTGAGCGAGACTATCCGT.
Nitrite ion by 0.3～1.5mmol/L calcein and 2.4～7.2mmol/L MnCl2It is mixed to get, calcium is yellow
Final concentration of 15～75 μm of ol/L, MnCl of green element2Final concentration of 120～360 μm of ol/L.
Lysate contains：10～20mmol/L Tris-HCl (pH 8.3), 1mmol/L EDTA, 0.3～1% (w/v)
SDS。
Confining liquid is atoleine.
In a kind of water, the method for quick of streptococcus fecalis, comprises the steps：
1) water sampling suction filtration, filter membrane are placed in Zengjing Granule in streptococcus fecalis enriched medium；
2) separate microorganism after the completion of Zengjing Granule, extracts nucleic acid after cracking；
3) will redissolve in the lyophilized isothermal duplication agent of liquid addition, and be completely dissolved lyophilized isothermal duplication agent, obtain reactant liquor；
4) operate addition extraction is obtained in the reactant liquor in different pipes respectively sample nucleic, the positive right by isothermal duplication
According to and negative control, reaction terminate after add nitrite ion, result is judged.
Used as the further improvement of above-mentioned method for quick, enriched medium is sodium azide liquid of glucose culture
Base.
The invention has the beneficial effects as follows：
1) the dry powdered preparation technology of the reagent adds compound freeze drying protectant, greatly increases reagent stability, reagent
Shelf-life durations can be extended to 2 years, be easy to normal temperature to transport, and save cost of transportation, be exposed to room temperature base after 72 hours before use
This does not affect using effect.
2) constituent part reagent in reaction system is pressed corresponding proportion combination by the dry powdered reagent, and Jing redissolution liquid is instant
Use, reduce solution allocation step, using more convenient, accurate, be more suitable for basic unit's Site Detection；
3) course of reaction can be carried out under isothermal conditions, without the need for special matched reagent and instrument, greatly reduce
Testing cost；
4) needed for reacting, template amount is few, and sensitivity is high, and minimum inspection limit can as little as 9CFU/Test；
5) three pairs of primers are designed and are screened using target-gene sequence, specifically recognize eight independent zones on target sequence
Domain, with high degree of specificity, realizes that reaction is completed in 35～50min, efficient and quick；
6) by adding the nitrite ion for being mixed by calcein and manganese chloride according to a certain percentage before reaction, can be by naked eyes
The change of color change or fluorescence monitoring instrument to fluorescence pattern carries out interpretation to result, and the color change difference of yin and yang attribute is bright
It is aobvious, interpretation intuitive and convenient, and the Aerosol Pollution for causing of uncapping after avoiding reaction.
Specific embodiment
A kind of quick detection kit for being applied to streptococcus fecalis in water, including lyophilized isothermal duplication agent, redissolve liquid, the positive
Control dry powder, negative control, nitrite ion, lysate and confining liquid, are polymerized containing dNTPs, Bst DNA in freezing isothermal duplication agent
Enzyme, outer primer, inner primer, ring primer and freeze drying protectant, freeze drying protectant is by trehalose, glycine and bovine serum albumin(BSA) group
Into.
Lyophilized isothermal duplication agent consisting of before lyophilized：0.9～1.6mmol/L dNTPs, 0.3～0.4U/ μ L Bst
Archaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/
L ring primer LF/LB and freeze-drying composite protectant containing 3%～6% (w/v) trehalose, 0.01%-0.1% (w/v) glycine,
0.1%-1% bovine serum albumin(BSA)s, balance of aseptic ultra-pure water.
Lyophilized isothermal duplication agent consisting of before lyophilized：0.9～1.6mmol/L dNTPs, 0.3～0.4U/ μ LBst
Archaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/
L ring primer LF/LB and freeze-drying composite protectant containing 4%～5% (w/v) trehalose, 0.04%-0.07% (w/v) glycine,
0.4%-0.7% bovine serum albumin(BSA)s, balance of aseptic ultra-pure water.
Redissolve liquid and contain 2～9mmol/L MgSO4, 8～12mmol/L KCl, 15～20mmol/L Tris-HCl, 8～
12mmol/L(NH4)2SO4, 0.1～0.3%Triton X-100 and 0.5～1.5mol/L glycine betaines.
The sequence of outer primer F3/B3, inner primer FIP/BIP and ring primer LF/LB is as follows：
Streptococcus fecalis-F3：TGGGCATTTTTCTTTCGG；
Streptococcus fecalis-B3：CATTTAAAAACCAACGTACTTGA；
Streptococcus fecalis-FIP：CAAACGTTGAGTCGCCGTTGCCAATAGTTCGCAACCGA；
Streptococcus fecalis-BIP：GAAGGAGTGACCAACACAGAGACGACTCTCCAGCCAAATCG；
Streptococcus fecalis-LF：TGTTCCGAAATCCGTTTCTATCA；
Streptococcus fecalis-LB：CAAATTGAGCGAGACTATCCGT.
Nitrite ion by 0.3～1.5mmol/L calcein and 2.4～7.2mmol/L MnCl2It is mixed to get, calcium is yellow
Final concentration of 15～75 μm of ol/L, MnCl of green element2Final concentration of 120～360 μm of ol/L.
Lysate contains：10～20mmol/L Tris-HCl (pH 8.3), 1mmol/L EDTA, 0.3～1% (w/v)
SDS.Other conventional lysates can also be used.
Confining liquid is atoleine.Can be using other confining liquids.
In a kind of water, the method for quick of streptococcus fecalis, comprises the steps：
5) water sampling suction filtration, filter membrane are placed in Zengjing Granule in streptococcus fecalis enriched medium；
6) separate microorganism after the completion of Zengjing Granule, extracts nucleic acid after cracking；
7) will redissolve in the lyophilized isothermal duplication agent of liquid addition, and be completely dissolved lyophilized isothermal duplication agent, obtain reactant liquor；
8) operate addition extraction is obtained in the reactant liquor in different pipes respectively sample nucleic, the positive right by isothermal duplication
According to and negative control, reaction terminate after add nitrite ion, result is judged.
Used as the further improvement of above-mentioned method for quick, enriched medium is sodium azide liquid of glucose culture
Base.
This kit is based on loop-mediated isothermal amplification technique quick detection streptococcus fecalis, is using three pairs of special primers and tool
There are the Bst archaeal dna polymerases of strand-displacement activity, eight on specific recognition target sequence isolated area, and realize in isothermy
Under (60～65 DEG C) carry out strand displacement amplification reaction, overcoming normal PCR reaction needs to obtain single-stranded mould by thermal denaturation process
The shortcomings of plate, detection time length, easy pollution and high cost, while the method is carried out in mild temperature condition, it is easy to operate, it is right
The technical quality requirement of testing staff is also low, is easy to the quick screening cheap to large sample cost of implementation.It is at present domestic mainly with
Microorganism be separately cultured with Morphological Identification based on, identify etc. that passing method carries out food with reference to biochemical identification and serological typing
Microbiology related check, its Preliminary Identification are generally required 2～3 days, and being finally completed probation report then needs 10～15 days, but are adopted
Detected from collection sample with this kit and complete only to need 12～14 hours to detection, and this kit is developed the color using calcein
Liquid so that sentence read result by by naked eyes, it is more visual and clear.
If no special instructions, the present invention in w/v be mass volume ratio, the percentage in composition if no special instructions,
Refer to mass percent.
With reference to experiment, technical scheme is further illustrated.
The screening of streptococcus fecalis LAMP primer
Inventor devises 6 groups of primers, and difference is as follows：
Table 1, different streptococcus fecalis LAMP primer sequences
Note：In table, in group 1,6 primers ACE-F3, ACE-B3, ACE-FIP, ACE-BIP, ACE-LF, ACE-LB are compiled successively
Number be 1～6, group 2 in 6 primer ACE-F3, ACE-B3, ACE-FIP, ACE-BIP, ACE-LF, ACE-LB number consecutivelies be 7
～12, the numbering of other groups is similar to.
Using 6 groups of optimum primer sequence combinations, containing ring primer, test through the reaction system of unified optimization,
By agarose gel electrophoresis observing response product, primer 1 only needs 35min to have obvious amplified band, primer 2 and primer 3
In 45min or so, primer 4, primer 5 and primer 6 are in 50min or so.
The preparation of kit：
Primer：Primer sets 1 in table 1.
Lyophilized isothermal duplication agent：Composition such as table 2 before lyophilized.
The composition table of table 2, different lyophilized detection agents
The preparation method of lyophilized detection agent：Mixing under gnotobasis needs the related component mixed liquor of frozen dried, encapsulation
In container, it is dried according to normal freeze-drying program, you can obtain lyophilized isothermal duplication agent.
Redissolve liquid：Composition is as follows,
| Component | Final concentration |
| KCl | 8mmol/L |
| Tris-HCl, pH 8.8 | 16mmol/L |
| (NH4)2SO4 | 9mmol/L |
| MgSO4 | 8mmol/L |
| Triton X-100 | 0.2% |
| Glycine betaine | 1.2mol/L |
| Water | Surplus |
Mix multiple solution component under gnotobasis, be placed in container.
Positive control dry powder：The trehalose of 3% (w/v) is separately added into the streptococcus fecalis Genomic DNA solution of purification, point
In dress and container, the above-mentioned lyophilized program of same Jing prepares positive control dry powder.
Nitrite ion：By the MnCl of the calcein and 7.2mmol/L of 0.3mmol/L2Equal-volume is mixed to get, and calcium is yellowish green
Final concentration of 15 μm of ol/L, MnCl of element2Final concentration of 360 μm of ol/L, be placed in container.
Confining liquid：Atoleine, it is aseptic subpackaged, it is placed in container.
Lysate：Containing 10～20mmol/L Tris-HCl (pH 8.3), 1mmol/L EDTA, 0.3～1% (w/v)
SDS, is placed in container.
Stability experiment
The lyophilized isothermal duplication agent for taking example 1～6 respectively carries out stability experiment, 4 DEG C of preservations, at quarterly intervals using reagent
Box carries out the reaction effect checking of product, the results are shown in Table 3：
Table 3, the stability of different lyophilized isothermal duplication agent
Freeze drying protectant is combined using example 1 carries out the lyophilized of reaction reagent, after liquid nitrogen pre-freeze and freeze-drying, reagent
Freeze-dried powder can keep higher stability, and most long shelf-life durations were 21-24 month, far above 12 months of similar-type products
Shelf-life, during 24 months shelf-lifves, its inspection limit is declined slightly, but sensitivity still can reach 65CFU/TEST or so, it is sufficient to meet
The needs of detection.In addition to only the single protectant freeze-dried powder containing 5% trehalose (w/v) has moisture absorption phenomenon, remaining complex protection
Agent keeps good form after being lyophilized into powderous preparations in the range of the respective shelf-life, does not subside and hollow out phenomenon.
Minimum inspection limit and the comparison of sensitivity
1) the pure bacterium bacterium colony of picking streptococcus fecalis (ATCC29212) is distinguished with aseptic inoculation ring, is entered with sterile saline
(extension rate is respectively 10 to row gradient dilution0, 101, 102, 103, 104, 105, 106, 107, 108), and each dilution bacterium is taken respectively
100 μ L spirals of liquid are inoculated into TSA flat boards and are cultivated and counted
2) above dilution 100 μ L of bacterium solution, 6000r/min centrifugation 5min are taken respectively, are abandoned supernatant, are added 10 μ L lysates abundant
Suspension thalline is precipitated, and 99 DEG C of heating cracking thalline 10min, thermal cracking terminate rear 12000r/min centrifugations 15min, take supernatant and be
The thalline templet gene for slightly carrying；
3) DNA is slightly carried more than and enters performing PCR and LAMP reactions for template, streptococcus fecalis PCR primer is：
PF-5’-GGGCATTTTTCTTTCGGGATTA-3’(SEQ ID NO：37)
PR-5’-CCGCTTGCCTCGCCTTCTA-3’(SEQ ID NO：38)
4) PCR amplification conditions are：94 DEG C of denaturations 5min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, 72
DEG C extend 10min, 35 circulation
5) DNA is slightly carried more than carries out LAMP reactions for template, takes out freeze-dried type detection reaction tube, and often pipe is separately added into above-mentioned
20 μ L of liquid are redissolved, dry powder is completely dissolved in treating pipe, being separately added into the above each dilution, bacterium solution is corresponding slightly carries 2.5 μ L of DNA profiling,
2.5 μ L of aseptic ultra-pure water, add 20 μ L/ duct occlusion liquid after fully mixing, tight to cover, and arrange 64 DEG C of isothermal reactions 50min；
6) with LAMP amplified productions agarose gel electrophoresis come interpretation, plate count determines minimum inspection to above PCR
Test limit.
As a result：Agarose gel electrophoresis result shows that the minimum inspection of streptococcus fecalis PCR is limited 105At multiple dilutions,
LAMP is minimum, and inspection is limited in 106At multiple dilutions.Corresponding colony counting adopts GB 4789.2-2010 Food Microbiology
Inspection total plate count bioassay standard, streptococcus fecalis is 106It is 9CFU at multiple dilutions, the sensitivity of LAMP detections is far above PCR.
The detection of pure bacterium
1) sample treatment：
In Screening and Identification for pure bacterium, with oese picking single bacterium colony semi-ring, the nothing containing 30 μ L lysates is transferred to
In bacterium PCR pipe, 99 DEG C of thermal crackings 10min of water-bath or metal bath after fully mixing；Cracking is gone in centrifuge after terminating
12000r/min is centrifuged 10min, takes supernatant as sample and slightly carries genomic DNA, is shown in Table 4 from bacterial strain：
Table 4
2) isothermal amplification
Freeze-dried type detection reaction tube is taken out, often pipe is separately added into 20 μ L of above-mentioned redissolution liquid, and dry powder is completely dissolved in treating pipe, point
Not Jia Ru more than the 2.5 μ L of DNA profiling that slightly carry of all kinds of bacterium, wherein, take 1 pipe as negative control, 1 pipe as positive control, point
Do not add 2.5 μ L of corresponding contrast agents, after add 2.5 μ L of nitrite ion in all pipes respectively, add 20 μ after fully mixing
L confining liquids, tight to cover, Jing Biometra professional PCR instruments carry out isothermal duplication, 64 DEG C of isothermal reactions 50min；
3) interpretation after reacting
After reaction terminates, color change before and after contrast reaction is the positive if fluorescence green is presented, and greenish orange color is then feminine gender,
If yin and yang attribute control reaction pipe result is not inconsistent with above-mentioned situation, this testing result is invalid, should detect again, entirely observe
Journey must not open reaction lid.Testing result is shown in Table 5
Table 5
As a result, Jing present invention detection, in the quick detection of the streptococcus fecalis and non-streptococcus fecalis category selected by the above, excrement hammer
The reference culture and separation strains that bacterium is chosen occurs obvious fluorescence green in detection architecture, and selected non-streptococcus fecalis to belong to each bacterium anti-
Without color change before and after answering, it is still greenish orange color, it is seen that the present invention has fabulous specificity, can effectively detect streptococcus fecalis.
In packaging drinking water of the present invention, the detection method of streptococcus fecalis is as follows：
1) sample treatment：Taking 250mL water samples to be checked carries out suction filtration, after filter membrane is placed in into 100mL streptococcus fecalis Zengjing Granules
In base (sodium azide glucose liquid culture medium), under the conditions of 37 ± 1 DEG C, increase bacterium 8-12h.Inhale enrichment liquid 1mL to 1.5mL specifications
Sterile centrifugation tube in, 6000r/min centrifugation 5min abandon supernatant, add 30 μ L lysates, abundant suspension thalline to flick tube wall
Eliminate bubble, 99 DEG C of heating 10min, 12000r/min centrifugation 15min, the DNA that supernatant is as slightly carried；
2) isothermal amplification：Freeze-dried type detection reaction tube is taken out, often pipe is separately added into 20 μ L of above-mentioned redissolution liquid, in treating pipe
Dry powder is completely dissolved, and is separately added into the 2.5 μ L of DNA profiling that above each sample is slightly carried, wherein taking 1 pipe as negative control, 1 pipe is made
For positive control, be separately added into 2.5 μ L of corresponding contrast agents, after respectively in all pipes add 2.5 μ L of nitrite ion, fully
20 μ L confining liquids are added after mixing, tight to cover, Jing Biometra professional PCR instruments carry out isothermal duplication, 64 DEG C of isothermals
Reaction 45min；
3) interpretation after reacting：After reaction terminates, in observing response pipe, solution is fluorescent green by the greenish orange discoloration before detecting,
It is identical with positive control pipe change, indicate that streptococcus fecalis detects, conversely, without color change, indicating without detection.
SEQUENCE LISTING
<110>Guangdong Huan Kai bio tech ltd
Huankai Microbes Tech Co., Ltd., Guangdong
<120>The dry powdered LAMP quick detection kits of streptococcus fecalis and its using method
<130> ACE
<160> 38
<170> PatentIn version 3.5
<210> 1
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<212> DNA
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tgggcatttt tctttcgg 18
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<211> 38
<212> DNA
<213>Artificial primer
<400> 10
caacggcgac tcaacgtttg acggatagtc tcgctcaa 38
<210> 11
<211> 20
<212> DNA
<213>Artificial primer
<400> 11
ggttgcgaac taattggtga 20
<210> 12
<211> 22
<212> DNA
<213>Artificial primer
<400> 12
acgattgaag gagtgaccaa ca 22
<210> 13
<211> 19
<212> DNA
<213>Artificial primer
<400> 13
ggcatttttc tttcgggat 19
<210> 14
<211> 18
<212> DNA
<213>Artificial primer
<400> 14
gactctccag ccaaatcg 18
<210> 15
<211> 41
<212> DNA
<213>Artificial primer
<400> 15
cgttgctgtt ccgaaatccg acgcttatca ccaatagttc g 41
<210> 16
<211> 43
<212> DNA
<213>Artificial primer
<400> 16
gcgactcaac gtttgacgat ataaaaaaac ggatagtctc gct 43
<210> 17
<211> 19
<212> DNA
<213>Artificial primer
<400> 17
tttctatcac attcggttg 19
<210> 18
<211> 21
<212> DNA
<213>Artificial primer
<400> 18
tgaaggagtg accaacacag a 21
<210> 19
<211> 18
<212> DNA
<213>Artificial primer
<400> 19
tgggcatttt tctttcgg 18
<210> 20
<211> 23
<212> DNA
<213>Artificial primer
<400> 20
catttaaaaa ccaacgtact tga 23
<210> 21
<211> 38
<212> DNA
<213>Artificial primer
<400> 21
caaacgttga gtcgccgttg ccaatagttc gcaaccga 38
<210> 22
<211> 41
<212> DNA
<213>Artificial primer
<400> 22
gaaggagtga ccaacacaga gacgactctc cagccaaatc g 41
<210> 23
<211> 22
<212> DNA
<213>Artificial primer
<400> 23
tgttccgaaa tccgtttcta tc 22
<210> 24
<211> 19
<212> DNA
<213>Artificial primer
<400> 24
tggccaaatt gagcgagac 19
<210> 25
<211> 18
<212> DNA
<213>Artificial primer
<400> 25
caccaatagt tcgcaacc 18
<210> 26
<211> 23
<212> DNA
<213>Artificial primer
<400> 26
tcacatttaa aaaccaacgt act 23
<210> 27
<211> 46
<212> DNA
<213>Artificial primer
<400> 27
actccttcaa tcgtcaaacg ttggaatgtg atagaaacgg atttcg 46
<210> 28
<211> 38
<212> DNA
<213>Artificial primer
<400> 28
gaccaacaca gagactggcc gatttgactc tccagcca 38
<210> 29
<211> 17
<212> DNA
<213>Artificial primer
<400> 29
agtcgccgtt gctgttc 17
<210> 30
<211> 21
<212> DNA
<213>Artificial primer
<400> 30
attgagcgag actatccgtt t 21
<210> 31
<211> 18
<212> DNA
<213>Artificial primer
<400> 31
ggatttcgga acagcaac 18
<210> 32
<211> 22
<212> DNA
<213>Artificial primer
<400> 32
tgaaatatct tctgtgacat cg 22
<210> 33
<211> 38
<212> DNA
<213>Artificial primer
<400> 33
ctcgctcaat ttggccagtc cgactcaacg tttgacga 38
<210> 34
<211> 45
<212> DNA
<213>Artificial primer
<400> 34
taaagtaggc gatttggctg gatgaggttc acatttaaaa accaa 45
<210> 35
<211> 21
<212> DNA
<213>Artificial primer
<400> 35
tctgtgttgg tcactccttc a 21
<210> 36
<211> 17
<212> DNA
<213>Artificial primer
<400> 36
gagtcaaatc aagtacg 17
<210> 37
<211> 22
<212> DNA
<213>Artificial sequence
<400> 37
gggcattttt ctttcgggat ta 22
<210> 38
<211> 19
<212> DNA
<213>Artificial sequence
<400> 38
ccgcttgcct cgccttcta 19
Claims (10)
1. a kind of quick detection kit for being applied to streptococcus fecalis in water, including lyophilized isothermal duplication agent, redissolves liquid, the positive right
According to dry powder, negative control, nitrite ion, lysate and confining liquid, freeze in isothermal duplication agent containing dNTPs,BstDNA is polymerized
Enzyme, outer primer, inner primer, ring primer and freeze drying protectant, it is characterised in that：Freeze drying protectant is by trehalose, glycine and ox
Seralbumin is constituted.
2. quick detection kit according to claim 1, it is characterised in that：Group of the lyophilized isothermal duplication agent before lyophilized
Become：0.9～1.6 mmol/L dNTPs, 0.3～0.4 U/ μ LBstArchaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer
F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/L ring primer LF/LB and lyophilized complex protections
Agent contains 3% ~ 6% (w/v) trehalose, 0.01%-0.1% (w/v) glycine, 0.1%-1% bovine serum albumin(BSA)s, balance of nothing
Bacterium ultra-pure water.
3. quick detection kit according to claim 2, it is characterised in that：Group of the lyophilized isothermal duplication agent before lyophilized
Become：0.9～1.6 mmol/L dNTPs, 0.3～0.4 U/ μ LBstArchaeal dna polymerase, 0.1～0.5 μm of ol/L outer primer
F3/B3,1.0～2.0 μm of ol/L inner primer FIP/BIP, 0.5～1.5 μm of ol/L ring primer LF/LB and lyophilized complex protections
Agent contains 4% ~ 5% (w/v) trehalose, 0.04%-0.07% (w/v) glycine, 0.4%-0.7% bovine serum albumin(BSA)s, balance of
Aseptic ultra-pure water.
4. the quick detection kit according to claims 1 to 3 any one, it is characterised in that：Redissolve liquid and contain 2～9
mmol/L MgSO4, 8～12 mmol/L KCl, 15～20 mmol/L Tris-HCl, 8～12 mmol/L (NH4)2SO4、
0.1～0.3% Triton X-100 and 0.5～1.5 mol/L glycine betaines.
5. the quick detection kit according to claims 1 to 3 any one, it is characterised in that：It is outer primer F3/B3, interior
The sequence of primers F IP/BIP and ring primer LF/LB is as follows：
Streptococcus fecalis-F3：tgggcatttttctttcgg；
Streptococcus fecalis-B3：catttaaaaaccaacgtacttga；
Streptococcus fecalis-FIP：caaacgttgagtcgccgttgccaatagttcgcaaccga；
Streptococcus fecalis-BIP：gaaggagtgaccaacacagagacgactctccagccaaatcg；
Streptococcus fecalis-LF：tgttccgaaatccgtttctatca；
Streptococcus fecalis-LB：caaattgagcgagactatccgt.
6. the quick detection kit according to claims 1 to 3 any one, it is characterised in that：Nitrite ion by 0.3～
The MnCl of the calcein of 1.5 mmol/L and 2.4～7.2 mmol/L2It is mixed to get, final concentration of the 15～75 of calcein
μm ol/L, MnCl2Final concentration of 120～360 μm of ol/L.
7. the quick detection kit according to claims 1 to 3 any one, it is characterised in that：Lysate contains：10～
20 mmol/L Tris-HCl (pH 8.3), 1 mmol/L EDTA, 0.3～1%（w/v）SDS.
8. the quick detection kit according to claims 1 to 3 any one, it is characterised in that：Confining liquid is liquid stone
Wax.
9. in a kind of water streptococcus fecalis method for quick, comprise the steps：
1）Water sampling suction filtration, filter membrane are placed in Zengjing Granule in streptococcus fecalis enriched medium；
2）Separate microorganism after the completion of Zengjing Granule, extracts nucleic acid after cracking；
3）To redissolve in the lyophilized isothermal duplication agent of liquid addition, and be completely dissolved lyophilized isothermal duplication agent, obtain reactant liquor；
4）By isothermal duplication operate respectively in the reactant liquor in different pipes add extract obtain sample nucleic, positive control and
Negative control, reaction add nitrite ion after terminating, and result is judged.
10. method for quick according to claim 9, it is characterised in that：Enriched medium is sodium azide glucose
Fluid nutrient medium.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611263571.XA CN106544444B (en) | 2016-12-30 | 2016-12-30 | LAMP (loop-mediated isothermal amplification) rapid detection kit for streptococcus faecalis dry pulverization and use method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611263571.XA CN106544444B (en) | 2016-12-30 | 2016-12-30 | LAMP (loop-mediated isothermal amplification) rapid detection kit for streptococcus faecalis dry pulverization and use method thereof |
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| CN106544444B CN106544444B (en) | 2020-05-05 |
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| CN107988401A (en) * | 2017-12-29 | 2018-05-04 | 广东环凯微生物科技有限公司 | The dry powdered LAMP quick detection kits of salmonella |
| CN108277289A (en) * | 2017-12-29 | 2018-07-13 | 广东环凯生物科技有限公司 | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 |
| CN108285925A (en) * | 2017-12-29 | 2018-07-17 | 广东环凯微生物科技有限公司 | A kind of rugged Cronobacter sakazakii quick detection kit of slope |
| CN114525324A (en) * | 2022-03-31 | 2022-05-24 | 河南冠宇仪器有限公司 | LAMP (loop-mediated isothermal amplification) detection primer group and kit for Burkholderia gladioli and preparation method of freeze-dried microspheres |
| US11453907B2 (en) | 2020-03-23 | 2022-09-27 | The Broad Institute, Inc. | Crispr effector system based coronavirus diagnostics |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988401A (en) * | 2017-12-29 | 2018-05-04 | 广东环凯微生物科技有限公司 | The dry powdered LAMP quick detection kits of salmonella |
| CN108277289A (en) * | 2017-12-29 | 2018-07-13 | 广东环凯生物科技有限公司 | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 |
| CN108285925A (en) * | 2017-12-29 | 2018-07-17 | 广东环凯微生物科技有限公司 | A kind of rugged Cronobacter sakazakii quick detection kit of slope |
| US11453907B2 (en) | 2020-03-23 | 2022-09-27 | The Broad Institute, Inc. | Crispr effector system based coronavirus diagnostics |
| US11639523B2 (en) | 2020-03-23 | 2023-05-02 | The Broad Institute, Inc. | Type V CRISPR-Cas systems and use thereof |
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| CN114525324A (en) * | 2022-03-31 | 2022-05-24 | 河南冠宇仪器有限公司 | LAMP (loop-mediated isothermal amplification) detection primer group and kit for Burkholderia gladioli and preparation method of freeze-dried microspheres |
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