CN108034700A - A kind of isothermal amplification detection method of molecular beacon mediation and its application - Google Patents
A kind of isothermal amplification detection method of molecular beacon mediation and its application Download PDFInfo
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- CN108034700A CN108034700A CN201810069855.8A CN201810069855A CN108034700A CN 108034700 A CN108034700 A CN 108034700A CN 201810069855 A CN201810069855 A CN 201810069855A CN 108034700 A CN108034700 A CN 108034700A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of isothermal amplification detection method and its application by molecular beacon mediation and syncaryon acid layer analysis detection technique.This method can be used to detect the pathogen genome in a variety of biofluid media, such as a variety of unknown contaminated bacterias in blood platelet or the small babesia through Transfusion Transmission.The isothermal duplication product of molecular beacon mediation is detected by the present invention with nucleic acid chromatography method, isothermal amplification technique is enhanced to real-time test (fast bed side diagnostic techniques, Point Of Care Technology, POCT) the possibility of direction conversion exploitation, and kit can further be made, by in the field acquisition extensive use such as emergent/war wound emergency aid and treatment, inspection and quarantine, the detection for the sexually transmitted disease that happens suddenly and monitoring, field quick detection (POCT), have a extensive future.
Description
Technical field
The invention belongs to field of molecular detection, is analysed more particularly to one kind by molecular beacon mediation and syncaryon acid layer
The isothermal amplification detection method of detection technique and its application in real-time test.
Background technology
Comply with the trend that clinical examination develops to high accuracy analysis direction, molecule diagnosis is as having highest technology content
In-vitro diagnosis method, development is swift and violent in the world, only 6,000,000,000 yuan nearby of world's molecule diagnosis market scale valuation in 2015.
Molecule diagnosis refers to that applied molecular biology method detects internal inhereditary material structure or the diagnostic techniques of expression change, its
Core is gene diagnosis.The routine techniques of molecule diagnosis includes PCR (Polymerase chain
Reaction, PCR), DNA sequencing, single nucleotide polymorphism (SNP), biochip technology etc..Serologic test depends on human body
The generation of immune response, in the clinical examination and diagnosis of infectious disease, considers depositing for window phase and the abnormal crowd of immune function
In a sense, molecule diagnosis is upgrading and supplement to immunodiagnosis.However, molecule diagnosis clinical implementation with
It is high using the limitation for receiving all many conditions, such as to operating personnel and environmental requirement, the height of instrument and equipment is relied on, is being produced
There is also difficulty etc. in the technological layer of product and specifications of quality management.
In recent years, in clinical examination, real-time test (point-of-care testing, POCT) theory rapid rising,
All field of medical examination are almost covered from simple dry chemical technology to sensing and biochip, its detection project.The author
Think, Protocols in Molecular Biology POCT fields application, particularly molecule diagnosis correlation technique and product POCTization, not only
Great impetus is played in development that can be to following POCT, can also drive clinical expansion and the application of molecular diagnostic techniques.
Standard PCR technology has been unable to meet quick, accurate base because of the problems such as dependence to instrument is high and proliferation time is long
Because of the demand of diagnosis.The isothermal amplification technique to grow up on the basis of round pcr, its main feature is that whole amplified reaction (removal of impurities
Hand over step outside) carry out at the same temperature, it is not necessary to carry out in PCR reactions multi-cycle denaturation, annealing, extension and etc..
Some isothermal amplification techniques mainly include strand displacement isothermal duplication (strand displacement amplification,
SDA), rolling ring isothermal duplication (rolling circle amplification, RCA), amplification of nucleic acid sequences technology (nucleic
Acid sequence based amplification, NASBA), ring mediated isothermal amplification (loop-mediated
Isothermal amplification, LAMP), cross primer mediation isothermal duplication (Cross-Priming
Amplification, CPA) etc..Above-mentioned isothermal amplification technique greatly reduces dependence of the nucleic acid amplification to instrument, Er Qie
After removing the time interval of alternating temperature repeatedly, also greatly shortened the time required to amplification.Wherein, loop-mediated isothermal amplification technique (LAMP)
Because high sensitivity, high specificity, rapidly and efficiently, technology relative maturity, be most widely used, existing relevant technical products.But
It is that LAMP amplified productions are made of series gradient DNA not of uniform size, the result interpretation of its commercial reagent box is dependent on generation
Magnesium pyrophosphate precipitates, and need to be equipped with transmissometer and be detected.If isothermal amplification technique can be with simple and rapid result interpretation mode phase
With reference to can more meet to examine by bed or the needs of real-time test.
The content of the invention
To solve the problems, such as that the nucleic acid isothermal amplification technical result interpretation of routine is difficult, implements limited, of the invention first
A purpose is to provide a kind of isothermal that detection technique is analysed by molecular beacon (molecular beacon) mediation and syncaryon acid layer
Amplification detection method.
Isothermal amplification detection method provided by the present invention, is in the DNA with loop-stem structure i.e. molecular beacon
Under the mediation of (molecular beacon), gene (DNA) fragment that dissociated state is in primer pair carries out isothermal duplication, point
Sub- beacon is marked with chromogenic substrate A, and primer is marked with chromogenic substrate B, so as to make one end of amplified production with colour developing
Substrate A label, the other end carry chromogenic substrate B labels, and amplified production can be anti-with mark chromogenic reaction thing A respectively and colour developing
Answer and chromogenic reaction occurs on the nucleic acid chromatograph test strip of two coloured particle bands of thing B, realize and isothermal amplification is carried out
The method of nucleic acid chromatography detection.
The chromogenic substrate A is preferably biotin, and chromogenic reaction thing A is preferably Avidin, and the chromogenic substrate B is preferably
Fitc, chromogenic reaction thing B are preferably Fitc antibody.
In addition to amplified production is marked and is detected with biotin/avidin and Fitc/Fitc antibody, also it can use other
Material (the chromogenic substrate/aobvious of chromogenic reaction can occur for such as digoxin/DigiTAb or fluorescent dye/fluorescent dye antibody
Colour response thing) amplified production is marked and detected, the fluorescent dye include Fitc, Cy3, Cy5, AlexaFluor,
Rhodamine, FAM etc..
Isothermal amplification detection method provided by the present invention, it may include following steps：
The first step, aim sequence (Target) is found in the conservative region in determinand genome, according to aim sequence
Complementary series structure with loop-stem structure molecular beacon (molecular beacon), the corresponding ring sequence of its ring structures
It is the complementary series of aim sequence；The stem termination of molecular beacon is subjected to chromogenic substrate A marks, further according to the stem in molecular beacon
The corresponding stem sequence design of bar is used for the primer sequence (Primer) of constant-temperature amplification, and primer is carried out chromogenic substrate B marks；
Second step, using the genomic DNA of sample to be tested as template, under the mediation of molecular beacon, isothermal is carried out with primer
Amplification, heat denatured can untie the stem ring double-strand of complementary pairing in molecular beacon, and molecular beacon becomes chain, ring from hairpin
Sequence and template complementary pairing, can replace template sequence under the action of polymerase, shape with the primer of stem sequence complementation
Into target double-stranded sequence amplified production to be detected, one end of amplified production carries chromogenic substrate A labels, and the other end is with colour developing bottom
Thing B labels；
3rd step, is chromatographed with the nucleic acid with the coloured particle band for marking chromogenic reaction thing A and chromogenic reaction thing B respectively
Test strips carry out amplified production nucleic acid chromatography detection, and the coloured particle band of mark chromogenic reaction thing A is believed for detection molecules
The chromogenic substrate A labels of mark or amplified production, the coloured particle band of mark chromogenic reaction thing B are used to detect the aobvious of amplified production
Color substrate B label, result interpretation is carried out according to chromogenic reaction result, if there are two developed band (chromogenic substrate A/ chromogenic reactions
Thing A bands and chromogenic substrate B/ chromogenic reaction thing B bands) illustrate containing target gene (result is positive) in sample to be tested, if only
There is a developed band (chromogenic substrate A/ chromogenic reaction thing A bands) and illustrate that (result is cloudy without target gene in sample to be tested
Property).
In above-mentioned isothermal amplification detection method, the first step, for detectable substance (such as certain pathogen) genome
In the sequence design of the molecular beacon of the loop-stem structure of DNA, aim sequence is generally according to the 16S rDNA of detectable substance genomic DNA
The part different from other several sequences makes choice in the consensus of conservative region or different testing sample genomic DNA, point
The length of sub- beacon ring structures is 15-30 nucleotide, and ring sequence can be with aim sequence complementary pairing, and the length of cane is 8-
12 nucleotide, the G/C content of cane is 40%-60% or 70%-80%, is the 3 ' ends and 5 ' terminations unrelated with aim sequence
Tail complementary pairing sequence, molecular beacon is in hair clip type on space structure.
The molecular beacon falls within present invention.
The chromogenic substrate A is preferably biotin, and chromogenic reaction thing A is preferably Avidin, and the chromogenic substrate B is preferably
Fitc, chromogenic reaction thing B are preferably Fitc antibody.
It also can use other such as digoxin/DigiTAbs or fluorescent dye/fluorescent dye antibody that chromogenic reaction can occur
Material (chromogenic substrate/chromogenic reaction thing) amplified production is marked and detected, the fluorescent dye include Fitc, Cy3,
Cy5, AlexaFluor, Rhodamine, FAM etc..
The second step, can to the reaction system (including Mg ion concentrations etc.) of the isothermal duplication mediated by molecular beacon and
Reaction condition (including reaction temperature, reaction time etc.) is verified and optimized, and finds the high optimal reaction system of amplification efficiency
And reaction condition, with the sensitivity and specificity of lifting detection.
In the reaction system of the isothermal duplication of molecular beacon mediation, first configure amplification buffer and (include DTT, MgCl2、
DNTPs etc.), template, primer and polymerase etc. are then added, forms complete reaction system, preferable 20 μ L reaction system bags
Include：1 μ L of template, 1 μ L of molecular beacon, primer (20nM) 1 μ L, TrisHCl (250mM) 4 μ L, DTT (20mM) 1 μ L, MgCl2
1 μ L, NE Buffer2 of (100mM) 1 μ L, dNTPs (1mM), 2 μ L, 4.2 2 μ L, DMSO (1.2 μ of μ L, BSA (10mg/mL) of distilled water
L), 0.6 μ L of Klenow polymerases (5000U/mL).
In the reaction condition of the isothermal duplication of molecular beacon mediation, denaturation temperature generally handles at least 5 at >=95 DEG C
Minute, template is added after high temperature or cooled to room temperature, amplification temperature is 37 DEG C -45 DEG C, and proliferation time is generally 30 points
Clock, can also be appropriately extended (30-90 minutes), and preferable reaction condition is：First molecular beacon and template are mixed, are placed in water
95 DEG C or directly boil 5min in bath and be denatured, cooled to room temperature after denaturation, by molecular beacon and and template
Mixture be added in the EP pipes containing reaction solution, then add primer, be eventually adding Klenow polymerases or Bst polymerization
Enzyme, is blown and beaten with liquid-transfering gun and mixed, and EP pipes are put into thermostat in the range of temperature is 37 DEG C -45 DEG C and are expanded, are expanded
The increasing time is generally 30 minutes, can also be appropriately extended to 40-90 minutes.
3rd step, carries out isothermal amplification the nucleic acid chromatography detecting test paper that the coloured particle of result interpretation marks
Bar, its coloured particle can be that colloid gold particle, rare-earth fluorescent nano particle, latex particle, golden magnetic nano particle etc. can be used in
The particle for directly developing the color or developing the color under certain condition.
Specifically, isothermal amplification detection method provided by the present invention, it may include following steps：
1) according to the common sequence of the 16S rDNA conservative regions of detectable substance genomic DNA or different testing sample genomic DNA
The part different from other several sequences selection aim sequence (Target) in row, has loop-stem structure according to aim sequence design
Molecular beacon (beacon) nucleotide sequence, molecular beacon with chromogenic substrate A mark, further according to molecular beacon stem structure
Nucleotide sequence designs primer (Primer) sequence, and primer is marked with chromogenic substrate B；
2) isothermal duplication of molecular beacon mediation：Using the genomic DNA of sample to be tested as template, in step 1) molecular beacon
Mediation under, carry out isothermal amplification with step 1) primer；
20 μ L reaction systems include：1 μ L of template, 1 μ L of molecular beacon, primer (20nM) 1 μ L, TrisHCl (250mM) 4 μ
L, DTT (20mM) 1 μ L, MgCl2(100mM) 1 μ L, dNTPs (1mM) 1 μ L, NE Buffer22 μ L, distilled water 4.2 μ L, BSA
(10mg/mL) 2 μ L, DMSO (1.2 μ L), 0.6 μ L of Klenow polymerases (5000U/mL)；
Operation is：First molecular beacon and template are mixed, 95 DEG C is placed in water-bath or directly boils 5min and carry out
Denaturation, cooled to room temperature after denaturation, the EP containing reaction solution is added to by molecular beacon and with the mixture of template
Guan Zhong, then adds primer, is eventually adding Klenow polymerases, is blown and beaten and mixed with liquid-transfering gun, EP pipes are put into thermostat
In in temperature to be expanded in the range of 37 DEG C -45 DEG C, proliferation time is generally 30 minutes, can be also appropriately extended to 40-90 points
Clock；After reaction, one end of amplified production carries chromogenic substrate A labels, and the other end carries chromogenic substrate B labels；
3) nucleic acid chromatography detection：With with the coloured particle band for marking chromogenic reaction thing A and chromogenic reaction thing B respectively
Nucleic acid chromatograph test strip step 2) amplified production is carried out the detection of nucleic acid chromatography (detection is with a nucleic acid chromatograph test strip every time,
But have two coloured bands on each, one is detection (test) band, and one is control (control) band.),
Result interpretation is carried out according to chromogenic reaction result, if there are two developed band (chromogenic substrate A/ chromogenic reactions thing A bands and colour developings
Substrate B/chromogenic reaction thing B bands) illustrate containing target gene (result is positive) in sample to be tested, if only there is a developed band
(chromogenic substrate A/ chromogenic reaction thing A bands) illustrates in sample to be tested without target gene (result is negative).
Second object of the present invention be to provide it is a kind of by molecular beacon mediation and syncaryon acid layer analysis detection technique etc.
Warm amplification detection kit.
It is aobvious that the kit of the present invention includes the molecular beacon of mark chromogenic substrate A, the primer of mark chromogenic substrate B, mark
The nucleic acid chromatograph test strip of colour response thing A and chromogenic reaction thing B.
The chromogenic substrate A is preferably biotin, and chromogenic reaction thing A is preferably Avidin, and the chromogenic substrate B is preferably
Fitc, chromogenic reaction thing B are preferably Fitc antibody.
In addition to amplified production is marked and is detected with biotin/avidin and Fitc/Fitc antibody, also it can use other
Material (the chromogenic substrate/aobvious of chromogenic reaction can occur for such as digoxin/DigiTAb or fluorescent dye/fluorescent dye antibody
Colour response thing) amplified production is marked and detected, the fluorescent dye include Fitc, Cy3, Cy5, AlexaFluor,
Rhodamine, FAM etc..
The sequence for determining or designing in above method falls within the present invention.Specifically include：
It is with the aim sequence of contaminated bacteria detection often for blood platelet：5’-gca taa tgg cgc cga cga ccg
tg-3’；Molecular beacon sequences are：5’-tct tgg aca caC GTA TTA CCG CGG CTG CTG GCA Ctg tgt
Cca aga-3 ', wherein Italic capitals thickened portion are ring structure；Primer sequence is：5'-tct tgg aca cag t-3'；
For small babesia detection aim sequence be：
5 '-ggtgattcataataaa-3 ' (are matched) with sequence B；
5 '-agtgacaagaaataacaatacaggg-3 ' (are matched) with sequence C；
5 '-gattggaggtcgtca-3 ' (are matched) with sequence D；
5 '-ggacggtagggtattgg-3 ' (are matched) with sequence E；
5 '-ggaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence F；
5 '-gaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence G.
Molecular beacon sequences are：
Sequence B：5’-cttggacacaTTTATTATGAATCACCtgtgtccaag-3’；
Sequence C：5’-cttggacacaCCCTGTATTGTTATTTCTTGTCACTtgtgtccaag-3’；
Sequence D：5’-cttggacacaTGACGACCTCCAATCtgtgtccaag-3’；
Sequence E：5’-cttggacacaCCAATACCCTACCGTCCtgtgtccaag-3’；
Sequence F：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCCtgtgtccaag-3’；
Sequence G：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCtgtgtccaag-3’；
Italic capitals thickened portion is ring structure sequence in sequence B, C, D, E, F, G.
Primer sequence is：cttggacaca.
The present invention provides a kind of isothermal amplification detection method that detection technique is analysed by molecular beacon mediation and syncaryon acid layer.
This method can be used to detect the pathogen genome in a variety of biofluid media, such as a variety of unknown contaminated bacterias in blood platelet or
Small babesia through Transfusion Transmission.The present invention carries out the isothermal duplication product of molecular beacon mediation with nucleic acid chromatography method
Detection, enhance isothermal amplification technique to real-time test (diagnostic techniques by fast bed, Point-Of-Care Technology,
POCT) the possibility of direction conversion exploitation, and kit can be further developed into, will be in emergent/war wound emergency aid and treatment, inspection inspection
The field acquisition extensive use such as epidemic disease, detection and monitoring, the field quick detection (POCT) of the sexually transmitted disease that happens suddenly, application prospect are wide
It is wealthy.
The isothermal amplification detection method of the present invention has the following advantages：
1. precision instrument is not required, can complete to detect using any thermostat (such as water-bath, constant-temperature metal bath)；
2. operating process is easy, low, the direct interpretation of testing result is required experimenter, can be complete through self-study or simple training
Into detection；
3. kit can be prepared into, it is quick, portable, it is detected whenever and wherever possible.
The present invention is described in further details with reference to specific embodiment.
Brief description of the drawings
Fig. 1 is the isothermal amplification method schematic diagram that molecular beacon mediation and syncaryon acid layer analyse detection technique；
Fig. 2 is the secondary structure for molecular beacon (beacon) ring of various bacteria 16S rDNA conservative regions design；
Fig. 3 is that the isothermal amplification method that detection technique is analysed by molecular beacon mediation and syncaryon acid layer leads to a variety of contaminated bacterias
With the testing result of sequence；
Fig. 4 is to analyse the isothermal amplification method of detection technique to bacillus cereus by molecular beacon mediation and syncaryon acid layer
Plasmids detection result；
Fig. 5 is the two of molecular beacon (beacon) ring designed for the genomic DNA conserved sequence of small babesia
Level structure；
Fig. 6 is to analyse the isothermal amplification method of detection technique to synthesize small bar by molecular beacon mediation and syncaryon acid layer
The western worm target sequence testing result of shellfish.
Embodiment
The present invention is intended to provide a kind of detected by molecular beacon (molecular beacon) mediation and the analysis of syncaryon acid layer
The isothermal amplification detection method of technology, to solve, conventional nucleic acid isothermal amplification technical result interpretation is difficult, implements limited ask
Topic.
Principle such as Fig. 1 institutes of the isothermal amplification method of molecular beacon mediation of the present invention and syncaryon acid layer analysis detection technique
Show, be under the mediation of the DNA with loop-stem structure i.e. " molecular beacon " (molecular beacon), with " primer " in
The genetic fragment (" target sequence " in Fig. 1) of dissociated state carries out isothermal duplication；Since molecular beacon is with chromogenic substrate A marks
(" Bio " in Fig. 1), primer has chromogenic substrate B marks (" Fitc " in Fig. 1), so as to make the one end of " amplified production "
With chromogenic substrate A labels, the other end carries chromogenic substrate B labels；Amplified production can be with mark chromogenic reaction thing A respectively
With occur chromogenic reaction on the nucleic acid chromatograph test strip of two coloured particle bands of chromogenic reaction thing B, according to colour developing result into
Row is judged so as to fulfill nucleic acid chromatography detection is carried out to isothermal amplification.
Principle in accordance with the above, it is proposed by the present invention by molecular beacon mediation and syncaryon acid layer analysis detection technique etc.
Warm amplification detection method, it may include following steps：
The first step, aim sequence (target sequence) is found in the conservative region in determinand genome, according to aim sequence
Complementary series structure with loop-stem structure molecular beacon (the ring sequence in loop-stem structure is the complementary series of aim sequence),
And the stem termination of molecular beacon is subjected to chromogenic substrate A marks；
Stem sequence design in molecular beacon is used for the primer sequence (Primer) of constant-temperature amplification, and primer is carried out
Chromogenic substrate B is marked.
Second step, with the genomic DNA (including target sequence) of sample to be tested for template, under the mediation of molecular beacon, is used
Primer carries out isothermal duplication.In amplified reaction, heat denatured can untie the stem ring double-strand of complementary pairing in molecular beacon, point
Sub- beacon becomes chain from hairpin, its middle ring sequence and template complementary pairing, and can be with the primer sequence of stem sequence complementation
Template sequence (target sequence) is replaced under the action of polymerase, so as to obtain expanding available for the double-stranded sequence of color developing detection
Increase production thing, one end of the amplified production carries chromogenic substrate A labels, and the other end carries chromogenic substrate B labels；
3rd step, is chromatographed with the nucleic acid with the coloured particle band for marking chromogenic reaction thing A and chromogenic reaction thing B respectively
Test strips carry out amplified production nucleic acid chromatography detection, mark the coloured particle band of chromogenic reaction thing A to be used in the test strips
The chromogenic substrate A labels of detection molecules beacon or amplified production, the coloured particle band of mark chromogenic reaction thing B, which is used to detect, to be expanded
Increase production the chromogenic substrate B labels of thing；
Result interpretation is carried out according to chromogenic reaction result, if there are two developed band (chromogenic substrate A/ chromogenic reaction thing A bars
Band and chromogenic substrate B/ chromogenic reaction thing B bands) illustrate to contain target gene i.e. aim sequence (result is positive) in sample to be tested,
If only there is a developed band (chromogenic substrate A/ chromogenic reaction thing A bands) to illustrate to be free of target gene (result in sample to be tested
It is negative).
The present invention is described in detail with specific embodiment below.Embodiment is implemented under premised on technical solution of the present invention,
There is shown detailed embodiment and specific operating process, it will help understands the present invention, but should not become to this
The limitation of invention.
Method therefor is conventional method unless otherwise instructed in embodiment, and specific steps can be found in：《Molecular Cloning: A Laboratory
Guide》(《Molecular Cloning:A Laboratory Manual》Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
The percent concentration is mass/mass (W/W, unit g/ that this area usually defines unless otherwise instructed
100g) percent concentration, mass/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/
100mL) percent concentration.
The acquirement approach of various biomaterials described in embodiment be only to provide it is a kind of test obtain approach with up to
To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to carrying in embodiment
Show and be replaced.
Molecular beacon and primer used are synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 1, the isothermal amplification detection method detection blood by molecular beacon mediation and syncaryon acid layer analysis detection technique
A variety of contaminated bacterias in platelet
By a variety of in the isothermal amplification method detection blood platelet of molecular beacon mediation and syncaryon acid layer analysis detection technique
Contaminated bacteria (is carried out at the same time detection), comprises the following steps：
1) with DNAMAN softwares to a variety of functions on common pollutant bacteria in blood platelet (such as pseudomonas aeruginosa, staphylococcus aureus,
The genomic DNA of staphylococcus epidermis, serratia marcescens, bacillus cereus etc. is compared, according to detectable substance genome
The 16S rDNA conservative regions selection aim sequence (Target) of DNA, finally definite aim sequence is 5 '-gca taa tgg
cgc cga cga ccg tg-3’。
According to the nucleotide sequence of molecular beacon of the aim sequence design with loop-stem structure：
5 '-tct tgg aca caC GTA TTA CCG CGG CTG CTG GCA Ctg tgt cca aga-3 ', sequence
Italic capitals thickened portion is ring structure in row, which is the molecule for detecting a variety of contaminated bacteria conserved sequences in blood platelet
Beacon.
The molecular beacon sequences of design are screened with UNA Fold softwares, obtain preferable loop-stem structure.For more
(A, B, C are stem rings to the secondary structure of the molecular beacon (beacon) of kind bacterial 16 S rDNA conservative region designs as shown in Figure 2
Secondary structure).
Further the stem primers sequence in molecular beacon, primer sequence are：5'-tct tgg aca
cag t-3'
2) sequence conventional method synthetic molecules beacon and the primer according to step 1) design, wherein molecular beacon biology
Element mark, primer are marked with Fitc.
3) isothermal duplication of molecular beacon mediation：Using the genomic DNA of sample to be tested (various bacteria pollution blood platelet) as
Template, under the mediation of molecular beacon, isothermal amplification is carried out with primer.
20 μ L reaction systems include：1 μ L of template, 1 μ L of molecular beacon, primer (20nM) 1 μ L, TrisHCl (250mM) 4 μ
L, DTT (20mM) 1 μ L, MgCl2(100mM) 1 μ L, dNTPs (1mM) 1 μ L, NE Buffer22 μ L, distilled water 4.2 μ L, BSA
(10mg/mL) 2 μ L, DMSO (1.2 μ L), 0.6 μ L of Klenow polymerases (5000U/mL)；
Reaction process is：First molecular beacon and template are mixed, 95 DEG C is placed in water-bath or directly boils 5min and carry out
Denaturation, cooled to room temperature after denaturation, the EP containing reaction solution is added to by molecular beacon and with the mixture of template
Guan Zhong, then adds primer, is eventually adding Klenow polymerases, is blown and beaten and mixed with liquid-transfering gun, EP pipes are put into thermostat
In in temperature to be expanded in the range of 37 DEG C -45 DEG C, proliferation time is generally 30 minutes, and (amplified reaction can also be appropriately extended
Time is 30-90 minutes).After reaction, one end of amplified production carries biotin label, and the other end is marked with Fitc
Label.
4) nucleic acid chromatography detection：Chromatographed with the nucleic acid with the coloured particle band for marking Avidin and Fitc antibody respectively
Test strips (being purchased from Yousida Biological Technology Co., Ltd., Hangzhou) carry out amplified production nucleic acid chromatography detection, first in 96 orifice plates
Supporting sodium citrate buffer solution (SSC buffer solutions, pH=7.0) is added, then takes 9 μ L samples (i.e. the amplified production of step 3)) to drip
Among the sample pad of test strips, the test strips for having added sample are inserted into the hole containing buffer solution, in 15-30 minutes
Observation is as a result, photograph to record chromogenic reaction result.
As a result interpretation：If there are two developed band (biotin/avidin band and Fitc/Fitc antibody bands) to illustrate to treat
Containing target gene (result positive) in sample, if only occur a developed band (biotin/avidin band) illustrate it is to be measured
Without target gene in sample (result is negative).
By the isothermal amplification method of molecular beacon mediation and syncaryon acid layer analysis detection technique to a variety of common in blood platelet
The testing result of contaminated bacteria universal genetic group such as Fig. 3 (blank is distilled water, and negative control is the blood platelet not being contaminated by bacterial)
It is shown, it can be seen that to use this method to the Monitoring lower-cut of the bacterium universal sequence of synthesis for 10pM, show this method to synthesis
Template there is higher amplification efficiency.Using polyacrylamide gel electrophoresis (polyacrylamide gel
Electrophoresis PAGE) or nucleic acid chromatography method can to molecular beacon mediate isothermal duplication result be detected.
Contrast and experiment shows that nucleic acid chromatography detection method sensitivity is better than PAGE.
Specificity experiments：Functions on common pollutant bacteria genome in blood platelet is expanded using constructed molecular beacon, is sent out
Existing six kinds of contaminated bacterias can be expanded smoothly, and HBV, CMV etc. are viral without obvious amplified band, this shows the isothermal amplification method
With good specificity.
By the isothermal amplification method of molecular beacon mediation and syncaryon acid layer analysis detection technique to bacillus cereus plasmid
Sensitivity technique result such as Fig. 4 (T1 add template be 1011Copies/mL, T2 1010Copies/mL, T3 are
109Copies/mL, T4 108Copies/mL shown in), it can be seen that when the template number for adding amplification pipe reaches 1010copies/
Contain 10 in mL, i.e. 1 μ L7Can smoothly it be expanded during copies templates.
Embodiment 2, by molecular beacon mediation and syncaryon acid layer analysis detection technique isothermal amplification method detect small bar
The western worm of shellfish
Small babe west is detected by the isothermal amplification detection method of molecular beacon mediation and syncaryon acid layer analysis detection technique
Worm, comprises the following steps：
1) genome sequence of the small babesia to be caused a disease by investigating literature search to people, HQ285838,
AY027815 is Duncan babesia system, and BDU16370 is difference babesia system, and AF097993 is Theileria,
Sequence alignment is carried out to the genomic DNA of 4 kinds of small babesias with DNAMAN softwares, selects small babesia system consensus
In sequence, definite aim sequence are as a purpose the part different from other several sequences：
5 '-ggtgattcataataaa-3 ' (are matched) with sequence B；
5 '-agtgacaagaaataacaatacaggg-3 ' (are matched) with sequence C；
5 '-gattggaggtcgtca-3 ' (are matched) with sequence D；
5 '-ggacggtagggtattgg-3 ' (are matched) with sequence E；
5 '-ggaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence F；
5 '-gaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence G.
It is as follows according to the nucleotide sequence of molecular beacon of the aim sequence design with loop-stem structure：
B：5’-cttggacacaTTTATTATGAATCACCtgtgtccaag-3’；
C：5’-cttggacacaCCCTGTATTGTTATTTCTTGTCACTtgtgtccaag-3’；
D：5’-cttggacacaTGACGACCTCCAATCtgtgtccaag-3’；
E：5’-cttggacacaCCAATACCCTACCGTCCtgtgtccaag-3’；
F：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCCtgtgtccaag-3’；
G：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCtgtgtccaag-3’.
Italic capitals thickened portion is ring structure sequence in sequence B, C, D, E, F, G.
The molecular beacon sequences of design are screened with UNA Fold softwares, obtain preferable loop-stem structure.For micro-
The secondary structure of molecular beacon B, C, D of small babesia design are as shown in Figure 5.
Primer sequence is further designed according to the nucleotide sequence of molecular beacon stem structure, above-mentioned molecular beacon uses phase
Same stem structure, accordingly the nucleotides sequence designed for the primer of amplifier molecule beacon be classified as：
Primer sequence：cttggacaca.
2) synthetic molecules beacon and primer, molecular beacon biotin labeling, primer digoxigenin labeled.
3) isothermal duplication of molecular beacon mediation：Using the genomic DNA of sample to be tested (small babesia) as template,
Under the mediation of molecular beacon, isothermal duplication is carried out with primer, reaction system and reaction condition are same as Example 1.Reaction terminates
Afterwards, one end of amplified production carries biotin label, and the other end carries digoxin label.
4) nucleic acid chromatography detection：With the nucleic acid layer with the coloured particle band for marking Avidin and DigiTAb respectively
Analyse test strips (be purchased from Yousida Biological Technology Co., Ltd., Hangzhou) to amplified production carry out nucleic acid chromatography detection, detection method and
Embodiment 1 is identical, and result interpretation is carried out according to chromogenic reaction result, if occur two developed band (biotin/avidin band and
Digoxin/DigiTAb band) illustrate containing target gene (result is positive) in sample to be tested, if only there is a developed band
(biotin/avidin band) illustrates in sample to be tested without target gene (result is negative).
Isothermal amplification method by molecular beacon mediation and syncaryon acid layer analysis detection technique is western to the small babe synthesized
Shown in the testing result of worm target sequence such as Fig. 6 (blank is distilled water, and negative control is unrelated sequences), it can be seen that to small bar
The Monitoring lower-cut of the western worm target sequence of shellfish shows that the isothermal amplification method has higher amplification efficiency in 10pM.
Embodiment 3, prepare the isothermal amplification detection kit that detection technique is analysed by molecular beacon mediation and syncaryon acid layer
It is aobvious that the kit of the present invention includes the molecular beacon of mark chromogenic substrate A, the primer of mark chromogenic substrate B, mark
The nucleic acid chromatograph test strip of colour response thing A and chromogenic reaction thing B.
Molecular beacon is designed according to detectable substance, for the stem ring of detectable substance (such as certain pathogen) genomic DNA
In the sequence design of the molecular beacon of structure, aim sequence is generally according to the 16S rDNA conservative regions of detectable substance genomic DNA
Or the part different from other several sequences makes choice in the consensus of different testing sample genomic DNA, in molecular beacon
The length of ring structure is 15-30 nucleotide, and ring sequence can be with aim sequence complementary pairing, and the length of cane is 8-12 nucleosides
Acid, the G/C content of cane is 40%-60% or 70%-80%, is that 3 ' unrelated with aim sequence are held and 5 ' termination tails mutually recruit
To sequence, molecular beacon is in hair clip type on space structure.
Primer is designed according to the stem sequence of molecular beacon.
The chromogenic substrate A is preferably biotin, and chromogenic reaction thing A is preferably Avidin, and the chromogenic substrate B is preferably
Fitc, chromogenic reaction thing B are preferably Fitc antibody.
In addition to amplified production is marked and is detected with biotin/avidin and Fitc/Fitc antibody, also it can use other
Such as digoxin/DigiTAb or fluorescent dye/anti-fluorescent dye antibody can occur chromogenic reaction material (chromogenic substrate/
Chromogenic reaction thing) amplified production is marked and detected, the fluorescent dye include Fitc, Cy3, Cy5, AlexaFluor,
Rhodamine, FAM etc..
The application method of kit is referring to embodiment 1 and embodiment 2.
Claims (11)
1. a kind of isothermal amplification detection method that detection technique is analysed by molecular beacon mediation and syncaryon acid layer,
Under the mediation of DNA, that is, molecular beacon with loop-stem structure, isothermal expansion is carried out with primer pair target gene (DNA) fragment
Increase, the molecular beacon is marked with chromogenic substrate A, and the primer is marked with chromogenic substrate B；
With the nucleic acid chromatograph test strip with the coloured particle band for marking chromogenic reaction thing A and chromogenic reaction thing B respectively to expanding
Increase production thing and carry out nucleic acid chromatography detection, testing result is obtained according to the interpretation to the band that develops the color.
2. isothermal amplification detection method according to claim 1, it is characterised in that：The chromogenic substrate A is biotin, is shown
Colour response thing A is Avidin, and the chromogenic substrate B is Fitc, and chromogenic reaction thing B is Fitc antibody.
3. isothermal amplification detection method according to claim 2, it is characterised in that：Except with biotin/avidin and Fitc/
Fitc antibody is marked amplified production and detects outer, also can use other such as digoxin/DigiTAbs or fluorescent dye/glimmering
The material (chromogenic substrate/chromogenic reaction thing) that chromogenic reaction can occur for photoinitiator dye antibody etc. is marked and examines to amplified production
Survey, the fluorescent dye is including Fitc, Cy3, Cy5, AlexaFluor, Rhodamine, FAM etc..
4. according to any isothermal amplification detection methods of claim 1-3, it is characterised in that：The isothermal duplication detection side
Method, comprises the following steps：
The first step, determines the target sequence (aim sequence) of sample to be tested target gene, has stem ring knot according to aim sequence structure
The molecular beacon sequences of structure, make the complementary series that molecular beacon sequences middle ring sequence is aim sequence, and by the stem of molecular beacon
Termination is marked with chromogenic substrate A；
Stem sequence design in molecular beacon sequences is used for the primer sequence of constant-temperature amplification, and primer is carried out chromogenic substrate
B is marked；
Second step, using the genomic DNA of sample to be tested as template, under the mediation of molecular beacon, isothermal duplication is carried out with primer,
Obtain amplified production；
3rd step, carries out amplified production with the nucleic acid chromatograph test strip nucleic acid chromatography detection, according to the colour developing in test strips
Band carries out interpretation：If there are two developed band (chromogenic substrate A/ chromogenic reactions thing A bands and chromogenic substrate B/ chromogenic reaction things
B bands) it is determined as the positive, illustrate to contain target gene in sample to be tested, if only there is a developed band, (chromogenic substrate A/ develops the color
Reactant A band) it is determined as feminine gender, illustrate to be free of target gene in sample to be tested.
5. isothermal amplification detection method according to claim 4, it is characterised in that：The first step, in sample to be tested base
Because of (such as the common sequence of the 16S rDNA conservative regions of pathogen or different pathogens genomic DNA in the conservative region in group
The part different from other several sequences in row) find and determine aim sequence.
6. isothermal amplification detection method according to claim 4 or 5, it is characterised in that：The second step, 20 μ L reactants
System includes：1 μ L of template, 1 μ L of molecular beacon, primer (20nM) 1 μ L, TrisHCl (250mM) 4 μ L, DTT (20mM) 1 μ L,
MgCl21 μ L, NE Buffer2 of (100mM) 1 μ L, dNTPs (1mM), 2 μ L, distilled water 4.2 μ L, BSA (10mg/mL) 2 μ L, DMSO
(1.2 μ L), 0.6 μ L of Klenow polymerases (5000U/mL)；Reaction condition is：First molecular beacon and template are mixed, are placed in water
95 DEG C or directly boil 5min in bath and be denatured, cooled to room temperature after denaturation, by molecular beacon and and template
Mixture be added in the EP pipes containing reaction solution, then add primer, be eventually adding Klenow polymerases, blown with liquid-transfering gun
Beat and mix, EP pipes are put into thermostat in the range of temperature is 37 DEG C -45 DEG C and are expanded, proliferation time is generally 30
Minute, it can also be appropriately extended to 40-90 minutes.
7. according to the isothermal amplification detection method described in claim 4 or 5 or 6, it is characterised in that：3rd step, to isothermal
Amplification carries out the nucleic acid chromatography detecting test paper strip of the coloured particle mark of result interpretation, its coloured particle can be colloidal gold
Grain, rare-earth fluorescent nano particle, latex particle, golden magnetic nano particle etc. can be used in directly colour developing or develop the color under certain condition
Particle.
8. a kind of wanted by molecular beacon mediation and the isothermal amplification detection kit of syncaryon acid layer analysis detection technique, including right
Ask the molecular beacon of mark chromogenic substrate A mentioned in 1 to 7 any claim, the primer of mark chromogenic substrate B, mark aobvious
The nucleic acid chromatograph test strip of colour response thing A and chromogenic reaction thing B；
The chromogenic substrate A is preferably biotin, and chromogenic reaction thing A is preferably Avidin, and the chromogenic substrate B is preferably
Fitc, chromogenic reaction thing B are preferably Fitc antibody；Except being carried out with biotin/avidin and Fitc/Fitc antibody to amplified production
Mark and detection are outer, also can use other such as digoxin/DigiTAbs or fluorescent dye/fluorescent dye antibody to develop the color
The material (chromogenic substrate/chromogenic reaction thing) of reaction is marked and detects to amplified production, the fluorescent dye include Fitc,
Cy3, Cy5, AlexaFluor, Rhodamine, FAM etc..
9. the molecular beacon involved in any one of claim 1 to 7, it is characterised in that：The length of the molecular beacon ring structures
Spend for 15-30 nucleotide, ring sequence and aim sequence complementary pairing, the length of cane is 8-12 nucleotide, the GC of cane
Content is 40%-60% or 70%-80%, is unrelated with aim sequence 3 ' end and 5 ' termination tail complementary pairing sequences, molecule
Beacon is in hair clip type on space structure.
10. the sequence involved in any one of claim 1 to 7, it is characterised in that：
It is with the aim sequence of contaminated bacteria detection often for blood platelet：5’-gca taa tgg cgc cga cga ccg tg-
3’；Molecular beacon sequences are：5’-tct tgg aca caC GTA TTA CCG CGG CTG CTG GCA Ctg tgt cca
Aga-3 ', wherein Italic capitals thickened portion are ring structure；Primer sequence is：5'-tct tgg aca cag t-3'；
For small babesia detection aim sequence be：
5 '-ggtgattcataataaa-3 ' (are matched) with sequence B；
5 '-agtgacaagaaataacaatacaggg-3 ' (are matched) with sequence C；
5 '-gattggaggtcgtca-3 ' (are matched) with sequence D；
5 '-ggacggtagggtattgg-3 ' (are matched) with sequence E；
5 '-ggaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence F；
5 '-gaggtagtgacaagaaataacaatac-3 ' (are matched) with sequence G；
Molecular beacon sequences are：
Sequence B：5’-cttggacacaTTTATTATGAATCACCtgtgtccaag-3’；
Sequence C：5’-cttggacacaCCCTGTATTGTTATTTCTTGTCACTtgtgtccaag-3’；
Sequence D：5’-cttggacacaTGACGACCTCCAATCtgtgtccaag-3’；
Sequence E：5’-cttggacacaCCAATACCCTACCGTCCtgtgtccaag-3’；
Sequence F：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCCtgtgtccaag-3’；
Sequence G：5’-cttggacacaGTATTGTTATTTCTTGTCACTACCTCtgtgtccaag-3’；
Italic capitals thickened portion is ring structure sequence in sequence B, C, D, E, F, G；
Primer sequence is：cttggacaca.
11. the molecular beacon described in detection kit, claim 9 described in claim 8 or the sequence described in claim 10
It is listed in pathogen genome (a variety of unknown contaminated bacterias or through defeated in such as blood platelet for preparing and being used in a variety of biofluid media
Blood propagate small babesia) real-time test product in application.
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| CN108998509A (en) * | 2018-08-14 | 2018-12-14 | 陆欣华 | The constant-temperature amplification primer of nucleic acid and its application |
| CN109055500A (en) * | 2018-09-13 | 2018-12-21 | 中国人民解放军疾病预防控制所 | A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon |
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| CN108998509A (en) * | 2018-08-14 | 2018-12-14 | 陆欣华 | The constant-temperature amplification primer of nucleic acid and its application |
| US11926872B2 (en) | 2018-08-14 | 2024-03-12 | Xinhua LU | Method for isothermal amplification of nucleic acid using primers to generate a tandem repeat sequence of a target gene |
| CN109055500A (en) * | 2018-09-13 | 2018-12-21 | 中国人民解放军疾病预防控制所 | A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon |
| CN109517903A (en) * | 2018-11-28 | 2019-03-26 | 江南大学 | Strand displacement type archaeal dna polymerase induces sex taboo object in pig source in isothermal circulation amplified reaction detection halal food |
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