CN110218775A - PCR fluorescence visual colorimetric determination detection method and kit based on ruthenium complex - Google Patents
PCR fluorescence visual colorimetric determination detection method and kit based on ruthenium complex Download PDFInfo
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- CN110218775A CN110218775A CN201910512615.5A CN201910512615A CN110218775A CN 110218775 A CN110218775 A CN 110218775A CN 201910512615 A CN201910512615 A CN 201910512615A CN 110218775 A CN110218775 A CN 110218775A
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- 238000003752 polymerase chain reaction Methods 0.000 description 53
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- YVXNAGNLAPDXJU-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;quinoxalino[2,3-f][1,10]phenanthroline;ruthenium(2+) Chemical compound [Ru+2].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.C1=CC=C2C3=NC4=CC=CC=C4N=C3C3=CC=CN=C3C2=N1 YVXNAGNLAPDXJU-UHFFFAOYSA-N 0.000 description 16
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- 238000003753 real-time PCR Methods 0.000 description 4
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- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 1
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- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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Abstract
The invention discloses a kind of PCR fluorescence visual colorimetric determination detection method and kit based on ruthenium complex.The stopped pipe visual colorimetric determination detection of PCR reaction is carried out using ruthenium complex as fluorescent dye, as the result is shown, amplification system is displayed in red in the presence of the target dna of PCR reaction amplification, there is no then colourless, and compared with SYBR Green I and EvaGreen, ruthenium complex can produce more obvious color contrast as fluorescent dye, have the advantages that the detection of PCR intuitive, time saving, convenient, inexpensive.
Description
Technical field
The present invention relates to the detections of polymerase chain reaction (PCR), and in particular to using with core in PCR reaction system
The ruthenium complex of acid molecule " photoswitch " behavior carries out the method that visualization fluorescence quickly detects as fluorescence combination dye.
Background technique
Fluorescent dye only needs simple mild physical method excitation (illumination) and detects, special in research molecular biology
Instruction tracking is carried out when being nucleic acid.Here fluorescent dye refers mainly to that nucleic acid can be specifically bound and changes the chemical combination of the characteristics of luminescence
Object, when only needing to qualitatively judge, fluorescent dye shows the advantage of easy low cost.Fluorescent dye is in molecular biology
The most common application is quantitative PCR and gel electrophoresis dyeing.EB (ethidium bromide) itself does not shine in the UV lamp, but with list
Chain, double-strand even then issue bright fluorescent orange after triple strand dna combines, and cheap, high sensitivity, but have high cause
Abnormal property, threatens the safety of experiment operator.SYBR Green I is the fluorescent dye of alternative EB, and safety coefficient is high, spirit
Sensitivity is high, is widely applied in quantitative PCR fluorescent dye determination, but has experiment discovery SYBR Green I to swash in high temperature and light source
There are the phenomenons that stability is poor when hair, also can generate biggish inhibition to reaction during amplification, and as import
Commercial reagents, higher cost.Thus, it is necessary to the fluorescent dye application that exploiting economy is environmentally friendly, stability is good, practical
In detection of nucleic acids and portable kit.
Barton seminar has found ruthenium (II) complex [Ru (bpy) in early 1990s2(dppz)]2+To double-strand
The molecular light switch effect of DNA, i.e. ruthenium (II) complex itself do not shine, in conjunction with showing strong luminescence phenomenon after duplex DNA.Ruthenium
(II) complex has that small toxicity, environmentally friendly and high sensitivity, fluorescence background be low, water-soluble strong, Stokes (stoke '
S) it is displaced the features such as big, and ruthenium complex can use excited by visible light, and emit feux rouges, opposite ultraviolet source excitation pair
Operator's vision impairment is small.Ruthenium complex can be used in the detection of exploitation nucleic acid fluorescent as DNA binding dye and nucleic acid is examined
In disconnected reagent, for example, Chinese patent " LAMP fluorescence visual colorimetric determination detection method and kit based on ruthenium complex " is (public
The number of opening: CN109022545A).But higher dye strength limits its application in the detection of PCR fluorescence visual colorimetric determination.
It is PCR rapid reaction, accurate, but need to be easy to cause amplicon to pollute by electrophoresis confirmatory reaction product.It will be glimmering
Photoinitiator dye and the mixing of PCR reaction system, can achieve the purpose of stopped pipe detection, avoid electrophoresis verifying, reduce pollution, improve
Accuracy.For example, Chinese patent " a kind of real-time fluorescence quantitative PCR detection method and kit based on ruthenium complex "
The real-time fluorescence quantitative PCR detection method (publication number: CN108753933A) based on ruthenium complex is disclosed, but it is answered
With the real-time fluorescence PCR instrument for being limited to valuableness.
In the detection to nucleic acid amplification result, on the one hand, fluorescence visual colorimetric determination it is easy to operate, do not need expensive instrument
Device equipment, and quickly, intuitively.But to obtain apparent colorimetric effect, it usually needs higher dye strength.And another party
Face needs to reduce the concentration of dyestuff in reaction system, causes color developing effect poor in order to avoid dyestuff inhibits to react.
Summary of the invention
The purpose of the present invention is to provide a kind of PCR fluorescence visual colorimetric determination detection method and examination based on ruthenium complex
Agent box.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, comprising the following steps:
1) according to amplification target sequence design PCR primer and synthetic primer sequence；
2) DNA is extracted from sample to be tested, with the DNA of extraction for PCR reaction template, is contaminated by fluorescence of ruthenium complex
Material constructs substance Fluorescence PCR system in conjunction with the primer sequence of step 1)；In the PCR reaction system, PCR reaction template
Concentration be≤10ng/ μ L, the concentration of fluorescent dye is >=3 μM；
3) after step 2), the PCR reaction system is made to carry out PCR reaction under the conditions of stopped pipe according to setting program,
PCR reaction product (target sequence amplification for being combined with fluorescent dye) is obtained, utilizing under the conditions of stopped pipe to PCR reaction product can
Light-exposed (for example, blue light) is irradiated (excitation fluorescent dye), then according to PCR reaction product colour developing situation to amplification into
Row determines, if amplification is positive (i.e. from template amplification to target sequence), develops the color, if amplification be it is negative (i.e. from
Target sequence is arrived without amplification in template), then be still colourless (not developing the color).
Preferably, the step 1) is further comprising the steps of: verifying the amplification validity and specificity of primer sequence.
Preferably, the sample to be tested is selected from dairy products, lactogenesis or other biological sample.
Preferably, in the step 2), suitable DNA extraction method is selected according to the type of sample to be tested.
Preferably, the step 2) is further comprising the steps of: it is to investigate foundation with Ct value, fluorescence increment and plateau,
Determine the optium concentration of fluorescent dye in PCR reaction system.
Preferably, in the PCR reaction, the annealing temperature that single amplification cycles use is 53 DEG C -56 DEG C.
Preferably, the ruthenium complex is selected from [Ru (phen)2(dppz)]2+、[Ru(bpy)2(dppz)]2+、[Ru
(phen)2(dppx)]2+One of equal Ru-polypyridine complexes；In order to enhance fluorescence visual colorimetric analysis performance, or selected from logical
Other metal Rus for carrying out further structural modification to ligands such as phen, bpy, dppz of Ru-polypyridine complex and obtaining are crossed to match
Close object.
A kind of PCR fluorescence visual colorimetric determination detection kit based on ruthenium complex, including PCR reaction reagent (contain
Taq enzyme, dNTPs and buffer), above-mentioned ruthenium complex and anti-for realizing the stopped pipe of substance Fluorescence PCR system
It answers, the reaction tube of the excited by visible light of PCR reaction product and colour developing situation observation；In the PCR reaction system, PCR reacts mould
The concentration of plate is≤10ng/ μ L, and the concentration of ruthenium complex is >=3 μM.
The beneficial effects of the present invention are embodied in:
The present invention establishes a kind of substance fluorescent PCR by template and fluorescent dye concentration in control target sequence amplification
The detection architecture of amplification and visual colorimetric determination, after the completion of amplification, directly observation is produced by excited by visible light without open pipe
Raw color can judge amplification, can realize under the premise of pollution-free while target sequence obtains amplification
To quick, convenient, the intuitive qualitative detection of the amplification of target dna (target sequence), testing cost is reduced.
Further, present invention optimizes PCR reaction conditions, reduce the annealing temperature in PCR amplification circulation, thus
In low copy number template reaction system, it is ensured that the efficiency and specificity of primer amplification improve purpose product yield.
Further, Ru-polypyridine complex can compared with Eva Green and SYBR Green I as fluorescent dye
To generate more obvious color contrast.
Detailed description of the invention
Fig. 1 is that the excitation of ruthenium complex fluorescence, emission spectrum and Stokes (Stoke ' s) are displaced (A) and SYBR
The excitation of Green I fluorescence, emission spectrum and Stokes (Stoke ' s) are displaced (B) schematic diagram.
Fig. 2 is with various concentration gradient [Ru (bpy)2(dppz)]2+Visual colorimetric determination figure as dyestuff.
Fig. 3 is ruthenium complex [Ru (bpy)2(dppz)]2+(A), PCR common dyes Eva Green (B) and SYBR
The visual colorimetric determination comparison diagram of Green I (C).
Fig. 4 is sensitivity experiment visual colorimetric determination figure: number G-0, G-1, G-2, G-3, G-4, G-5 represent mountain containing various concentration
The reaction system of sheep genomic DNA.
Fig. 5 is the visual colorimetric determination figure of different actual samples: number 1,2,3,4,5,6 is represented to be mentioned using different dairy products samples
The template DNA taken, number G are represented using goat genomic DNA as the positive control of template.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples, the embodiment is only used for explaining this hair
It is bright, rather than limiting the scope of the invention.
The present invention for commercialization PCR dyestuff SYBR Green I and Eva Green that there are Stokes shifts is small,
Visual colorimetric determination comparison is unobvious to wait the problem of influencing practical application (referring to Fig. 1), passes through and investigates ruthenium complex property, optimization
The condition for selecting the ligand with molecular light switch behavior and fluorescence detection finally establishes a kind of based on polymerase chain reaction
(PCR) and by nucleic acid molecules light switch ruthenium (II) complex the nucleic acid amplification detection method of visual colorimetric determination is directly carried out.
(1) [Ru (bpy)2(dppz)]2+For visualizing the feasibility of PCR amplification detection
1, with paramagnetic particle method poba gene group DNA extraction kit, from commercially available fresh goat dairy (being pre-separated fat constituent)
Middle extraction goat genomic DNA designs species specificity according to the cytochrome b gene conserved sequence of goat and draws as template
Object synthesize by raw work biology (Shanghai) Co., Ltd., is purified through HPLC, and (the design of primers deadline is primer sequence as follows
In January, 2018):
F3:(5'-3') ACAATAGCCACAGCATTCAT
R3:(5'-3') ATCTGTGTCCGATGGAATTC
2, PCR reaction system, reaction condition optimization
[Ru (bpy) in reaction system is set2(dppz)]2+Concentration be respectively 0.5,1,1.5,2,3,4,6,8 μM, referring to
Fig. 2 can be seen in the figure from the visual colorimetric determination after the completion of PCR amplification, [Ru (bpy)2(dppz)]2+Concentration range of the concentration at < 3 μM
When interior, template group (+, contain template) and no template control (-, NTC) without apparent color difference, and in [Ru (bpy)2
(dppz)]2+When concentration >=3 μM, that is, it is red to observe that template group (+) is presented, no template control (-) is still colourless, illustrates metal Ru
Complex [Ru (bpy)2(dppz)]2+PCR could be reacted in certain concentration range and indicative function occurs.
In the above PCR reaction:
(1) 5 μ L of PCR reaction system (10 μ L): 2 × Taq PCR Master Mix, each 0.25 μ L (10 of primer (F3, R3)
μM), ruthenium complex [Ru (bpy)2(dppz)]2+1 μ L, 1 μ L of 7ng/ μ L template DNA solution, residue ultrapure water polishing.
(2) PCR response procedures: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35
A circulation；72 DEG C re-extend 5min.
3, it is based on ruthenium complex [Ru (bpy)2(dppz)]2+Visualization PCR detection
(1) PCR reaction system: using 40 μM of ruthenium complexes [Ru (bpy)2(dppz)]2+, referring to the above system system
It is standby, ruthenium complex [Ru (bpy)2(dppz)]2+Optium concentration (4 μM) in the reaction system is increased according to Ct value, fluorescence
Amount and plateau, determine.
(2) PCR response procedures: referring to procedure above.
(3) visual colorimetric determination identification experimental result: is carried out directly under blue light transilluminator after the reaction was completed.Positive and negative knot
Fruit can be distinguished according to color, as shown in Fig. 3 (A), wherein template group (+) is red (positive findings), and no template control (-) is
Colourless (negative findings).
Using same reaction system, program, detected with dyestuff Eva Green, SYBR Green I, the results showed that,
Referring to shown in Fig. 3 (B), Fig. 3 (C), [Ru (bpy)2(dppz)]2+Color contrast is substantially better than using dyestuff Eva Green, SYBR
The Colorimetric results of Green I.
(2) ruthenium complex [Ru (bpy)2(dppz)]2+Visualize the sensitivity of PCR detection
(1) goat genomic DNA stoste is subjected to 10 times of gradient dilutions, makes its concentration in the reaction system be respectively
7ng/μL(G-0)、0.7ng/μL(G-1)、0.07ng/μL(G-2)、0.007ng/μL(G-3)、0.0007ng/μL(G-4)、
0.00007ng/μL(G-5)。
(2) experimental result: carrying out color observation under blue light transilluminator after amplification, as shown in figure 4, with different dilutions
The DNA (final concentration of 0.7 × 10 of degree-4~7ng/ μ L) be used as template, the color of inner reaction tube product be all it is red, i.e., it is minimum can
The template concentrations of detection reach 0.07pg/ μ L, show detection architecture sensitivity with higher.
(3) ruthenium complex [Ru (bpy)2(dppz)]2+Visualize the actual sample application of PCR detection
(1) choose in the market different dairy products (liquid milk, whole milk powder, evaporated milk powder and formula milk) sample on sale into
Row DNA is extracted and (is used paramagnetic particle method poba gene group DNA extraction kit, and be pre-separated fat constituent in sample), and mountain is arranged
Sheep genomic DNA is positive control.
(2) experimental result: reaction product is observed immediately under the irradiation of blue light transilluminator after amplification.Referring to Fig. 5,
There are the actual samples of target sequence DNA (through sequence verification) and positive control, and red is presented, and colourless, card is presented in no template control
It is bright to be based on ruthenium complex [Ru (bpy)2(dppz)]2+Fluorescent PCR visual colorimetric determination detection architecture applicability it is good.
(4) the fluorescent visual PCR detection kit based on ruthenium complex
By 2 × Taq PCR Master Mix (containing Taq enzyme, dNTPs and buffer), ruthenium complex [Ru
(bpy)2(dppz)]2+And ultrapure water is packed jointly, obtains fluorescent visual PCR detection kit.When use with lid,
Pipe shaft can be to be added packing composition according to reaction system in the reaction tube of light transmission, and primer and template DNA is added, and stopped pipe reaction is expanded
Direct (not open pipe) carries out visible light (blue light) excitation and observation after increasing, and testing result can be obtained.
(5) superiority possessed by the more typical dyestuff Eva Green and SYBR Green I of ruthenium complex
(1) ruthenium complex property under PCR high temperature and acid-base environment keeps stablizing.
(2) comparison is obvious: shining when free using ruthenium complex as fluorescent dye faint, with double-stranded DNA knot
" photoswitch " feature of fluorescence intensity substantial increase establishes detection system when conjunction.Due to the Stokes of ruthenium complex
(Stoke ' s) is displaced big (180nm), it can be observed that the positive is red, feminine gender is colourless, more traditional dye under blue light transilluminator
The positive observed by material SYBR Green I is in green, feminine gender in yellow green as a result, color more easily discriminates, as a result more
Obviously, reduce subjective identification error.
(3) PCR reaction is not pressed down as fluorescent dye in wider concentration range (0.5-10 μM) interior ruthenium complex
Production is used, and higher concentration ruthenium complex can be used, fluorescence is made to reach higher signal strength, facilitates observation, comparison colour developing
As a result.
(4) stopped pipe detects: detection reagent (dyestuff) is directly added into PCR reaction solution, is not needed and PCR reagent physics point
It cuts, directly carries out visual colorimetric determination in the case where not open pipe after reaction, it is pollution-free, improve accuracy.
(5) safety and environmental protection: exciting without using ultraviolet lamp, is excited with visible light (blue light), it is ensured that operator is pacified with eye
Entirely.
In short, detection method it is simple, it is at low cost, using excited by visible light, stopped pipe detection is pollution-free, by visual observation
Colorimetric intuitively obtains testing result, can be widely applied to the fields such as environment measuring, food safety.
<110>Shaanxi Tech Univ
<120>PCR fluorescence visual colorimetric determination detection method and kit based on ruthenium complex
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<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
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acaatagcca cagcattcat 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
atctgtgtcc gatggaattc 20
Claims (10)
1. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, it is characterised in that: the following steps are included:
1) according to amplification target sequence design PCR primer and synthetic primer sequence；
2) DNA is extracted from sample to be tested, with the DNA of extraction for PCR reaction template, using ruthenium complex as fluorescent dye,
In conjunction with the primer sequence of step 1), substance Fluorescence PCR system is constructed；In the PCR reaction system, PCR reaction template
Concentration is≤10ng/ μ L, and the concentration of fluorescent dye is >=3 μM；
3) after step 2), so that the PCR reaction system is carried out PCR reaction, PCR reaction product is obtained, to PCR reaction product
Excited by visible light is carried out, then amplification is determined according to PCR reaction product colour developing situation, if colour developing, expands knot
Fruit is the positive, if not developing the color, amplification is feminine gender.
2. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
In: the step 1) is further comprising the steps of: verifying the amplification validity and specificity of primer sequence.
3. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
In: the sample to be tested is selected from dairy products, lactogenesis or other biological sample.
4. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
In: in the step 2), the extracting method of DNA is selected according to the type of sample to be tested.
5. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
In: the step 2) is further comprising the steps of: being to investigate foundation with Ct value, fluorescence increment and plateau, determines PCR reactant
The optium concentration of fluorescent dye in system.
6. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
In: in the PCR reaction, the annealing temperature that amplification cycles use is 53 DEG C -56 DEG C.
7. a kind of PCR fluorescence visual colorimetric determination detection method based on ruthenium complex, feature exist according to claim 1
Ru-polypyridine complex is selected from: the ruthenium complex or selected from tying by the ligand to the Ru-polypyridine complex
Other ruthenium complexes of structure modification and acquisition.
8. a kind of PCR fluorescence visual colorimetric determination detection kit based on ruthenium complex, it is characterised in that: including as fluorescence
The ruthenium complex of dyestuff and the colour developing feelings of stopped pipe reaction and PCR reaction product for substance Fluorescence PCR system
The reaction tube of condition observation；In the PCR reaction system, the concentration of PCR reaction template is≤10ng/ μ L, the concentration of fluorescent dye
It is >=3 μM.
9. a kind of PCR fluorescence visual colorimetric determination detection kit based on ruthenium complex according to claim 8, feature
Be: the ruthenium complex is selected from Ru-polypyridine complex or selected from by the ligand progress to the Ru-polypyridine complex
Structural modification and other ruthenium complexes obtained.
10. a kind of PCR fluorescence visual colorimetric determination detection kit based on ruthenium complex according to claim 8, special
Sign is: the PCR reaction product is observed immediately after radiation of visible light, if colour developing, amplification is the positive, if not
Colour developing, then amplification is feminine gender.
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| CN111424073A (en) * | 2020-04-30 | 2020-07-17 | 陕西科技大学 | Closed-tube nucleic acid amplification detection method and device |
| 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 |
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| CN108753933A (en) * | 2018-06-22 | 2018-11-06 | 陕西科技大学 | A kind of real-time fluorescence quantitative PCR detection method and kit based on ruthenium complex |
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| 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 |
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