CN108315390A - Visualization mixed dye for LAMP detections - Google Patents
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Abstract
The invention discloses a kind of mixed dyes for LAMP visual detection, and for the mixed dye of LAMP visual detection, it includes, and the volume ratio of reagent A and reagent B are 1.8~2.2:1, the reagent A is that calcein powder and manganese chloride powder spend nuclease water dissolution, calcein a concentration of 600~650µM, a concentration of 10~15mM of manganese chloride；The reagent B is that hydroxynaphthol blue powder spends nuclease water dissolution, a concentration of 13~17mM；The invention has the advantages that：Compared with current common dyes, which makes LAMP reaction results be easier interpretation, and color distortion is more obvious between positive and negative result, and high degree avoids the erroneous judgement of result；（2）The novel mixed dye is made an addition in advance in LAMP amplification reaction solutions, after so that LAMP is reacted, it is not necessary to open pipe lid again, avoid and cause pollution of nucleic acid to laboratory environment；（3）The novel mixed dye is that non-DNA is embedded in type dye, small to LAMP reaction interference, does not influence the sensitivity of LAMP detections.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of visualization mixed dye for LAMP detections.
Background technology
Loop-mediated isothermal amplification technique（Loop-mediated isothermal amplification, LAMP）It is a kind of
Develop faster nucleic acid amplification technologies in recent years.It at a constant temperature can quickly expand target nucleic acid sequence,
And then realize the detection of object.Currently, the reading of LAMP results be broadly divided into Turbidity measurement, agarose gel electrophoresis detection and
Fluorescence/visualization dyestuff detects 3 class methods.But since Turbidity measurement and electrophoresis detection all rely on instrument and need more complex behaviour
Make, thus fluorescence/visualization dye detecting method is increasingly favored by scientist.This method need to only be reacted in LAMP to be started
Preceding that dyestuff is added in reaction system, after reaction, negative reaction reagent and positive reaction reagent will present out color distortion, from
And realize the quick interpretation of reaction result.
Currently, having the following two kinds using more extensive dyestuff：1. hydroxynaphthol blue（Hydroxynaphthol blue,
HNB）.HNB reacts visualization indicator as LAMP and was reported for the first time in 2009.Its principle is：In LAMP amplified reactions
Nucleic acid synthesis in, the upper a large amount of pyrophosphate ions of dNTP are dissociated out, and pyrophosphate ion can with react
Mg in system2+Specific binding, makes Mg in reaction system2+Concentration declines, and then leads to HNB-Mg2+Compound is reduced.To
Make positive reaction that blue be presented under natural light, purple is presented in negative reaction.2. calcein：Calcein is reacted as LAMP
Visualization indicator was reported for the first time in 2008.It is mainly by differentiating result under the excitation of 365nm blue lights.Its
There is also differents between positive and negative result under natural light, but are not obvious.Its principle is：First in calcein
Mn is added2+, Mn2+The green fluorescence of calcein is set to be quenched.After LAMP amplified reactions occur, the by-product pyrophosphoric acid of generation
Ion and Mn2+In conjunction with and discharge calcein, cancellation state releases.The result finally presented is the sun under the excitation of 365nm blue lights
Property reaction send out strong green fluorescence, negative reaction is sent out compared with weak green fluorescence.Under natural light, positive reaction is in light green color, cloudy
Property reaction be in shallow orange.
Above two dyestuff is usually used alone.But there is a common defects for they, are exactly negative and positive
As a result color distortion is not apparent enough between.Especially under natural light, when object concentration to be checked is too low, weakly positive result is tied with negative
Fruit is difficult to differentiate between, so as to cause the inaccuracy of result interpretation.
Invention content
The present invention be solve to be used alone when LAMP react hydroxynaphthol blue and the colour developing of calcein indicator it is negative and
Not apparent enough the problem of color distortion between positive findings, and a kind of mixed dye for LAMP visual detection is provided.
For the mixed dye of LAMP visual detection, it includes, and the volume ratio of reagent A and reagent B are 1.8~2.2:1,
The reagent A is that calcein powder and manganese chloride powder spend nuclease water dissolution, calcein a concentration of 600~650µ
M, a concentration of 10~15mM of manganese chloride；The reagent B is that hydroxynaphthol blue powder spends nuclease water dissolution, a concentration of 13~
17 mM；
A concentration of the 625 of calcein in the reagent AµM, a concentration of 12.5 mM of manganese chloride；Hydroxyl in the reagent B
A concentration of 15 mM of base naphthol blue.
LAMP amplification reaction solutions, 10µIn L reaction systems, the mixing dye for LAMP visual detection is added
Material 0.4~0.7µL；
The mixed dye 0.6µL；
Described 10µL reaction systems are：
5 M glycine betaines 1.6µL
10 mM dNTP mixed liquors 1.4µL
10 × isothermal amplification buffer solution 1.0µL
100 mM Adlerikas 0.6µL
Mixed dye 0.6µL
Primer 2 .4µL
8000 U/ mL Bst DNA polymerases 0.4µL
DNA profiling 2.0µL。
The present invention provides a kind of mixed dye for LAMP visual detection, the mixing for LAMP visual detection
Dyestuff, it includes, and the volume ratio of reagent A and reagent B are 1.8~2.2:1, the reagent A is calcein powder and chlorination
Manganese powder end spends nuclease water dissolution, calcein a concentration of 600~650µM, a concentration of 10~15mM of manganese chloride；The examination
Agent B is that hydroxynaphthol blue powder spends nuclease water dissolution, a concentration of 13~17mM；The invention has the advantages that：With at present it is common
Dyestuff is compared, which makes LAMP reaction results be easier interpretation, and color distortion is brighter between positive and negative result
Aobvious, high degree avoids the erroneous judgement of result；（2）The novel mixed dye is made an addition in advance in LAMP amplification reaction solutions, is made
After LAMP reactions, it is not necessary to open pipe lid again, avoid and cause pollution of nucleic acid to laboratory environment；（3）The novel mixed dye is
Non- DNA is embedded in type dye, small to LAMP reaction interference, does not influence the sensitivity of LAMP detections.
Description of the drawings
Reaction result photo and ultraviolet-visible absorption spectra under natural light in Fig. 1 embodiments 2：A）Contain novel mixing
The LAMP reaction results of dyestuff；B）LAMP reaction results containing single HNB dyestuffs；C）Contain single calcein dyestuff
LAMP reaction results；
The LAMP detection sensitivities that Fig. 2 contains different dyes compare；No. 1-7 pipe：The SA- being added into LAMP amplification reaction solutions
DNA plasmid template concentrations are followed successively by 2.15 × 106 copies/ µL, 2.15 × 105 copies/ µL, 2.15 × 104
copies/ µL, 2.15 × 103 copies/ µL, 2.15 × 102 copies/ µL, 2.15 × 101 copies/ µL, 2.15
×100 copies/ µL；NTC is managed：It is added into LAMP amplification reaction solutions and removes nuclease water.
Specific implementation mode
Staphylococcus aureus（Staphylococcus aureus, SA）It is encountered pathogenic bacteria, therefore be to detect SA
Example is further described the specific implementation mode of the present invention, but the invention is not limited in these embodiments.
The selection of 1 SA nucleic acids characteristic sequences of embodiment and the design and synthesis of corresponding LAMP primer
According to species specificity, SA'snuc300 bp conservative nucleic acid segments are selected as feature target sequence in gene.For this section
Sequence, with 6 corresponding LAMP primers of computer software pair（F3-nuc、B3-nuc、LF-nuc、LB-nuc、FIP-nuc、
BIP-nuc）It is designed；All primers and the DNA plasmid template containing SA nucleic acids characteristic sequences are by raw work bioengineering
（Shanghai）Limited liability company is on behalf of synthesis.Characteristic sequence and primer sequence are as shown in table 1.
2 novel mixed dye of embodiment, single calcein dyestuff and single HNB dyestuffs are visual in LAMP detections
Change effect to compare
1, novel mixed dye, single calcein dyestuff and the configuration of single HNB dyestuffs storing liquid
It weighs certain mass calcein powder to be placed in same container with manganese chloride powder, spends nuclease water dissolution, make molten
A concentration of the 625 of calcein in liquidµM, a concentration of 12.5 mM of manganese chloride；Solution pH value is adjusted to alkalescent, is fully mixed
Even, the solution of acquisition is reagent A, i.e., single calcein dyestuff storing liquid, 4 DEG C or -20 DEG C preserve for use；
Certain mass HNB powder is weighed, nuclease water dissolution is spent, makes a concentration of 15 mM of HNB in solution, mixes well, obtain
The solution obtained is reagent B, i.e., single HNB dyestuffs storing liquid, 4 DEG C or -20 DEG C preserve for use；
Reagent A and reagent B are pressed 2:1 volume ratio mixes well, and the solution of acquisition is novel mixed dye, 4 DEG C or -20 DEG C guarantors
It deposits for use.
2, the LAMP amplification reaction solutions configuration containing novel mixed dye
3, the LAMP amplification reaction solutions configuration containing single calcein dyestuff
4, the LAMP amplification reaction solutions configuration containing single HNB dyestuffs
5, LAMP reacts
The above-mentioned LAMP amplification reaction solutions containing novel mixed dye, single calcein dyestuff and single HNB dyestuffs are placed in
It under the conditions of 63 DEG C, reacts 1 hour, terminates reaction within 10 minutes under the conditions of being subsequently placed at 80 DEG C.
6, result is read
Under natural light, reaction solution color change is visually observed.The results are shown in Figure 1, and the positive containing novel mixed dye is anti-
Answer liquid in dark purple blue, negative reaction liquid is in light gray；Positive reaction liquid containing single calcein dyestuff is in light green color, negative
Reaction solution is in shallow orange；For positive reaction liquid containing single HNB dyestuffs in blue, negative reaction liquid is in purple.Sentence according to naked eyes
It is disconnected, it can be seen that color distortion is the most apparent between the reagent positive and negative result containing novel mixed dye, it is easiest to visualize
As a result interpretation.
7, ultraviolet-visible absorption spectra scanning
Absorption spectrum scanning is carried out to the reagent after above-mentioned reaction using ultraviolet visible light spectrophotometer.As a result such as Fig. 1 institutes
Show, the positive and negative reaction solution spectrum morphological differences containing novel mixed dye is maximum, and positive findings are bimodal pattern, negative findings
For single peak type；Positive and negative reaction solution spectrum morphological differences containing single calcein dyestuff and single HNB dyestuffs is smaller,
For single peak type, and positive and negative spectrum wave crest is at a distance of relatively close.This further proves the reagent containing novel mixed dye relatively containing single
The reagent of one dyestuff is obviously easier to distinguish positive and negative as a result, effect of visualization is obviously improved.
LAMP detection of the embodiment 3 containing novel mixed dye, single calcein dyestuff and single HNB dyestuffs is sensitive
Degree compares
Novel mixed dye, single calcein dyestuff and single HNB dyestuffs storing liquid configuration method and LAMP amplification reaction solutions
Configuration method（It is added without DNA profiling）With embodiment 2；Will after 10 times of doubling dilutions of SA plasmid templates be added LAMP reaction solutions in into
Row reaction, reaction condition are：It is placed under the conditions of 63 DEG C, reacts 1 hour, terminate reaction within 10 minutes under the conditions of being subsequently placed at 80 DEG C.
The results are shown in Figure 2, the LAMP inspections containing novel mixed dye, single calcein dyestuff and single HNB dyestuffs
Survey method is 21.5 copies/ to the concentration limit of SA plasmid templatesµL illustrates that novel mixed dye is not right
The sensitivity of LAMP method has an impact；Also, the reagent containing novel mixed dye more easily discriminates weakly positive（Purple）With
It is negative（It is light grey）Between color distortion.Although under the excitation of 365nm blue lights, reagent the moon containing single calcein dyestuff,
Positive difference is equally more apparent, but it to excitation light source, there are dependences；The application of novel mixed dye makes LAMP reagents put
This dependence to instrument has been taken off, result interpretation can be completed by naked eyes under natural light, it is quick at the scene fully to show it
The application potential of context of detection.
The novel mixed dye of the application of embodiment 4 detects food pollution analog sample
1, prepared by food pollution analog sample
It takes fresh meat gruel to be placed in ultraviolet disinfecting lower 2 hours, is interfered with removing the pathogen accumulated naturally in meat gruel；1g is taken to sterilize
Meat gruel afterwards is added 10 mL physiological saline and mixes well, food samples matrix is made.By a certain concentration staphylococcus aureus
（ATCC 23213）, vibrio parahemolyticus（ATCC 17802）, salmonella（ATCC 14028）, monocyte hyperplasia Liszt
Salmonella（ATCC 43251）, escherichia coli O157：H7（ATCC 35150）It is inoculated in the matrix, extracts above-mentioned analog sample
DNA is used for subsequent detection.
2, reagent configuration and LAMP reactions
Novel mixed dye configuration method and LAMP reaction solution configuration methods（It is added without DNA profiling）With embodiment 2；It is carried above-mentioned
The DNA profiling taken, which is added in LAMP reaction solutions, to be reacted, and reaction condition is：It is placed under the conditions of 63 DEG C, reaction 1 hour, then
Terminate reaction within 10 minutes under the conditions of being placed in 80 DEG C.
3, result is read
The results are shown in Table 2, and testing result is accurate, is easy to interpretation, and sensitivity, specificity are good.All experiments are repeated three times,
As a result consistent.
。
Claims (5)
1. for the mixed dye of LAMP visual detection, it includes, and the volume ratio of reagent A and reagent B are 1.8~2.2:1,
The reagent A is that calcein powder and manganese chloride powder spend nuclease water dissolution, calcein a concentration of 600~650µM, a concentration of 10~15mM of manganese chloride；The reagent B is that hydroxynaphthol blue powder spends nuclease water dissolution, a concentration of 13
~17 mM.
2. the mixed dye according to claim 1 for LAMP visual detection, it is characterised in that：The reagent A
A concentration of the 625 of middle calceinµM, a concentration of 12.5 mM of manganese chloride；Hydroxynaphthol blue is a concentration of in the reagent B
15 mM。
3. LAMP amplification reaction solutions, it is characterised in that：10µIn L reaction systems, it is added described in claim 1 for LAMP
The mixed dye 0.4~0.7 of Visual retrievalµL。
4. LAMP amplification reaction solutions according to claim 3, it is characterised in that：The mixed dye 0.6µL。
5. LAMP amplification reaction solutions according to claim 4, which is characterized in that 10µL reaction systems are：
5 M glycine betaines 1.6µL
10 mM dNTP mixed liquors 1.4µL
10 × isothermal amplification buffer solution 1.0µL
100 mM Adlerikas 0.6µL
Mixed dye 0.6µL
Primer 2 .4µL
8000 U/ mL Bst DNA polymerases 0.4µL
DNA profiling 2.0µL。
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| CN201810213791.4A CN108315390A (en) | 2018-03-15 | 2018-03-15 | Visualization mixed dye for LAMP detections |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
| CN111575359A (en) * | 2020-05-25 | 2020-08-25 | 尹秀山 | Detection of amplification products using a combination of pH sensitive dyes |
| CN114292903A (en) * | 2021-12-21 | 2022-04-08 | 翌圣生物科技(上海)股份有限公司 | LAMP (loop-mediated isothermal amplification) multidimensional visual detection color indicator and RNA/DNA detection premix solution |
| CN114317688A (en) * | 2022-01-27 | 2022-04-12 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Visual mixed dye and application thereof in LAMP detection |
| WO2023067510A1 (en) * | 2021-10-19 | 2023-04-27 | Life Technologies Corporation | Colorimetric detection of nucleic acids |
Citations (1)
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| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
Non-Patent Citations (1)
| Title |
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| MING HONG ET AL.: "A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex", 《WORLD JOURNAL OF MCIROBIOLOGY AND BIOTECHNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
| CN111575359A (en) * | 2020-05-25 | 2020-08-25 | 尹秀山 | Detection of amplification products using a combination of pH sensitive dyes |
| WO2023067510A1 (en) * | 2021-10-19 | 2023-04-27 | Life Technologies Corporation | Colorimetric detection of nucleic acids |
| CN114292903A (en) * | 2021-12-21 | 2022-04-08 | 翌圣生物科技(上海)股份有限公司 | LAMP (loop-mediated isothermal amplification) multidimensional visual detection color indicator and RNA/DNA detection premix solution |
| CN114317688A (en) * | 2022-01-27 | 2022-04-12 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Visual mixed dye and application thereof in LAMP detection |
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Application publication date: 20180724 |