CN107523624A - A kind of combination AUDG and the more intersection constant-temperature amplification methods for keeping away molecular recognition system certainly - Google Patents
A kind of combination AUDG and the more intersection constant-temperature amplification methods for keeping away molecular recognition system certainly Download PDFInfo
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Abstract
The invention discloses a kind of more intersection constant-temperature amplification methods for combining South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG) and keeping away molecular recognition system (SAMRS) certainly, primer 3 ' during methods described expands to more cross substitutions holds 4 bases of bit base second from the bottom to the 5th bit base reciprocal to carry out SAMRS modifications, and in amplimer C1 or C2 5 ' end mark haptens, AUDG enzymes are introduced in amplification system, Brdurd and biotinylated dideoxycytosine, based on more cross substitution amplification techniques, amplified production is detected with reference to high molecular nanometer bio-sensing.Methods described can be by high molecular nanometer biology sensor Visual retrieval for the amplified production of the IS6110 distinguished sequences of mycobacterium tuberculosis complex.Methods described is convenient, quick, sensitive, special, is suitable for various nucleotide fragments detections.
Description
Technical field
The invention discloses a kind of method of more cross substitution constant-temperature amplification detection microorganism target gene, belong to microorganism
And technical field of molecular biology.
Background technology
In modern biology and medical domain, nucleic acid amplification is a kind of indispensable technology, has been widely used in base
The fields such as plinth research, clinical diagnosis, archaeological research, epidemic research, transgenic research.In the nucleic acid amplification technologies developed
In, PCR (Polymerase Chain Reaction, PCR) is first beyond body nucleic acid being established amplification
Technology, have an epoch-marking significance, the technology has been widely used in biological association area.However, round pcr carries out nucleic acid expansion
During increasing, limited by laboratory condition, the heat circulating equipment dependent on complex and expensive.In addition, the detection ratio of PCR primer
It is more complicated, it is necessary to a set of complicated flow and equipment.These inferior positions limit the extensive use of the technology, especially backward in economy
Area and quick diagnosis field.Therefore, for biology and medical science Related Research Domain, be highly desirable development it is simple,
Quickly, sensitive nucleic acid amplification method.In order to overcome the inferior position of PCR amplification techniques, arisen at the historic moment many isothermal amplification technologies.
Compared with round pcr, isothermal amplification technology is independent of thermal cycling amplification equipment, and reaction speed is fast, and sensitiveness is good.Therefore, it is permanent
Warm amplification technique is advantageously implemented rapid amplifying, field diagnostic and easy detection.Up to the present, the constant-temperature amplification skill developed
Art has more than 10 to plant, it is widely used have loop-mediated isothermal amplification (LAMP), intersect amplification (CPA), rolling circle amplification (RCA),
Constant-temperature amplification (HDA) that strand displacement amplification (SDA), unwindase rely on etc..However, these constant temperature technologies realize nucleic acid amplification needs
A variety of enzymes work simultaneously, the reagent dependent on costliness, complicated operating procedure.Therefore the practicality of these constant-temperature amplification methods,
Convenience, operability are up for improving, especially in quick diagnosis field and low developed area.
In order to overcome the inferior position of round pcr and existing isothermal amplification technology, sensitive, convenient, quick and special expansion is realized
Increase nucleotide sequence, inventor has been set up a kind of new nucleic acid amplification technologies in the recent period, is named as more cross substitution amplifications
(Multiple Cross Displacement Amplification, MCDA), related content is disclosed in CN104946744A,
The patent document forms a part of present specification as prior art document.MCDA realizes nucleic acid under constant temperature
Amplification, using only a kind of constant temperature substituted enzyme, amplification rate is fast, is quick on the draw, specificity height.
Similar to loop-mediated isothermal amplification (LAMP) and intersect amplification (CPA), MCDA technical bottleneck result interpretation,
That is the detection of amplified production.Up to the present, most common product detection means mainly include color indicator, electrophoresis, in real time
Turbidity.However, these three detection techniques are suitable only for substance detection (detection of i.e. single target).In addition, color indicator
During interpretation amplified production, it may appear that simulate all right one way or the other result, it is time-consuming longer during electrophoresis sentence read result, easily there is cross pollution,
It is not suitable for Site Detection, implements to need special instrument and equipment during turbidity interpretation.In order to overcome the bad of these three detection techniques
Gesture, make MCDA technologies more extensive, more economical in biology, medicine and health field application.In the recent period, inventor is based on MCDA,
MCDA technologies are combined with nano biological detection technique, the MCDA technologies that rely on is developed, realizes the nanometer of quick, sensitive detection
Biosensor technique, the technology are named as the nucleic acid diagnostic techniques for intersecting constant-temperature amplification combination gold nano bio-sensing more
(Multiple Cross Displacement Amplification label-based gold Nanoparticles
Lateral Flow Biosensor, MCDA-LFB), related content is disclosed in CN201610872509.4；
CN201610942289.8；CN201610982015.1；CN201710566164.4, these patent documents are as prior art text
Part forms a part of present specification.
In order to which MCDA amplified productions are applied into nanosensor technology, traditional strategy be in MCDA amplification systems simultaneously
The primer of two marks of addition, a primer hold mark haptens in 5 ' end mark biotins, another primer 5 '.Work as MCDA
Amplification is finished, and double target amplified productions are fabricated (primer that this pair of target product originates from two marks), one end mark biology
Element, other end mark haptens.However, the MCDA products of conventional measures structure double labelling can cause false positive results, not even
Amplification is needed to lead to false positive results.The false positive results derive from the hybridization between labeled primer.Therefore, in order to overcome
The inferior position of conventional tag strategy, the present invention devise a kind of new inspection policies, and the technology only needs the primer of a mark with regard to energy
The double target amplified productions of structure, so as to which amplified production is applied into biosensor technique.
In order to which MCDA amplified productions to be applied to the detection of biosensor technique, it is a necessary step to open reaction tube
Suddenly, the step causes substantial amounts of amplified production to volatilize in the form of an aerosol, so as to cause cross pollution, produces false positive knot
Fruit.In addition, being similar to LAMP and CPA technologies, due to Technology design, MCDA can produce false positive results, the false positive
As a result mainly as caused by autogamy in the autogamy between primer, primer or hybridization of missing the target.In order to overcome conventional tag strategy, intersect
False positive results caused by pollution and Technology design, in the present invention, inventor are phonetic using the primer, South Pole temperature-sensitive urine singly marked
Pyridine picodna glycosyl enzyme and keep away molecular recognition system certainly, based on MCDA technologies, develop MCDA combinations nanosensor,
From keeping away molecular recognition system and South Pole temperature-sensitive uracil picodna glycosyl enzymatic nucleic acid detection technique [Multiple cross
displacement amplification coupled with nanoparticles-based lateral flow
Biosensor (LFB), self-avoiding molecular recognition systems (SAMRS) and
antarctic thermal sensitive uracil-DNA-glycosylase(AUDG)for simultaneous
detection of nucleic acid sequence and elimination of carryover
contamination；AUDG-SAMRS-MCDA-LFB].In order to verify the feasibility of AUDG-SAMRS-MCDA-LFB technologies, knot
The compound group of core mycobacteria (Mycobacterium tuberculosis complex, MTC) is applied to AUDG-SAMRS-
MCDA-LFB technologies, establish detection MTC AUDG-SAMRS-MCDA-LFB diagnostic methods.
The content of the invention
Based on foregoing invention purpose, present invention firstly provides a kind of more cross substitution constant-temperature amplification combination high molecular nanometers
The method of bio-sensing testing goal gene, the described method comprises the following steps：
(1) genome of detected sample is extracted；
(2) 4 bases provided in 3 ' end bit base second from the bottom to the 5th bit bases reciprocal are drawn by what SAMRS was modified
Thing, the modification can increase the specificity of primer, be combined with To Template with making primer specificity；But also primer can be reduced
Autologous hybridization in the formation of dimer and primer, the primer include：Replace primers F 1 and F2, the sequence of the displacement primers F 1
Row such as SEQ ID NO：Shown in 1, the sequence such as SEQ ID NO of the displacement primers F 2：Shown in 2；Cross primer CP1 and CP2, institute
State cross primer CP1 sequence such as SEQ ID NO：Shown in 3, the sequence such as SEQ ID NO of the cross primer CP2：Shown in 4；
Amplimer C1 and C2, D1 and D2, R1 and R2, primer C1 sequence such as SEQ ID NO are provided：Shown in 5, primer C2 sequence is such as
SEQ ID NO：Shown in 6；Primer D1 sequence such as SEQ ID NO：Shown in 7, primer D2 sequence such as SEQ ID NO：Shown in 8,
Primer R1 sequence such as SEQ ID NO：Shown in 9, primer R2 sequence such as SEQ ID NO：Shown in 10, above-mentioned is simultaneously provided in
5 ' ends of one primer are marked with the Mdification primer of haptens；It for target gene is mycobacterium tuberculosis complex that above-mentioned primer, which is,
IS6110 distinguished sequences and design.
(3) in South Pole temperature-sensitive uracil picodna glycosyl enzyme, chain shift-type polymerase, melting temperature conditioning agent, draw
In the presence of thing, dNTP, and biotinylated Brdurd, template constant temperature is used as using the genomic nucleic acids of detected sample
DNA amplification；
(4) amplified production of high molecular nanometer biology sensor detecting step (3) is used.
In a preferable technical scheme, the Mdification primer that haptens is marked with 5 ' ends is amplimer C1.
In the technical scheme that one is more highly preferred to, the haptens in 5 ' end marks is fluorescein.
Particularly preferably, the high molecular nanometer biology sensor includes a backboard 1, set gradually on the backboard 1
Equipped with sample panel 2, board 3, nitrocellulose filter 4 and water sucting plate 5, detection is set gradually on the nitrocellulose filter 4
Line 41 and control line 42, it is coated with the Avidin of coloured groups modification successively in board 3, detection line 41 and the region of control line 42
High molecular nanometer particles 6, anti-fluorescein antibody 7 and the bovine serum albumin 8 of biotin coupling of change.
In a preferable technical scheme, the constant-temperature amplification is carried out in 60-62 DEG C of environment.
In the technical scheme that one is more highly preferred to, the constant-temperature amplification is carried out in 61 DEG C of environment.
Secondly, present invention also offers one group to be used for constant-temperature amplification mycobacterium tuberculosis complex IS6110 distinguished sequences
Primer sequence, it is characterised in that the sequence includes：Such as SEQ ID NO：Displacement primers F 1 shown in 1, such as SEQ ID NO：2
Shown displacement primers F 2, such as SEQ ID NO：Cross primer CP1 shown in 3, such as SEQ ID NO：Cross primer shown in 4
CP2, such as SEQ ID NO：Amplimer C1 shown in 5, such as SEQ ID NO：Amplimer C2 shown in 6, such as SEQ ID NO：7
Shown amplimer D1, such as SEQ ID NO：Amplimer D2 shown in 8, such as SEQ ID NO：Amplimer R1 shown in 9,
Such as SEQ ID NO：Amplimer R2 shown in 10, the 5 ' ends for being simultaneously provided in any of the above-described primer are marked with the modification of haptens
Primer.
In a preferable technical scheme, 4 bases at 3 ' ends of the primer are modified by SAMRS, and wherein A is repaiied
Adorn and be modified to 2- thio-thymines for 2-aminopurine, T, C is modified to N4- acetylcytosines, and it is fast that G is modified to time Huang
Purine.
In the technical scheme that one is more highly preferred to, the Mdification primer that haptens is marked with 5 ' ends is amplimer C1.
Preferably, the haptens marked is fluorescein.
After the present invention uses SAMRS compositions to Modify to primer, [(2- amino is fast by A*, 2-aminopurine for SAMRS compositions
Purine)；T*, 2-thiothymine (2- thio-thymines)；C*, N4-ethylcytosine (N4- acetylcytosines)；G*,
Hypoxanthine (hypoxanthine)] can only be with natural base pairing, i.e. A*：T, T*：A, C*：G, G*：C), therefore, primer
In end after the modification of SAMRS compositions, it ensure that SAMRS primers can only combine with corresponding target, it is impossible to and non-target combination,
So as to increase the specificity of primer, the specificity of ensuring method, the false positive results caused by hybridization that miss the target are eliminated.In addition,
SAMRS compositions can only be with natural base pairing (i.e. A*：T, T*：A, C*：G, G*：C), can not be matched between SAMRS compositions
(i.e. in the absence of A*：T* and C*：G* match), therefore, between the primer containing SAMRS compositions can not hybridize or primer in can not be miscellaneous
Hand over, eliminating the hybridization (dimer can not be formed between primer) between primer or self hybridization in primer (can not form
Secondary structure in primer), the high efficiency and specificity of primer are ensure that, it is thus eliminated that being led by primer dimer and secondary structure
The false positive results of cause.
Amplified production energy of the method provided by the present invention for the IS6110 distinguished sequences of mycobacterium tuberculosis complex
By high molecular nanometer biology sensor Visual retrieval.Methods described is convenient, quick, sensitive, special, is suitable for various nucleotides
Fragment detects.Whole reaction proliferation time of the invention is only 45 minutes, and detection range is 2 × 106~2 × 101Copy/micro-
Rise, there is high sensitivity.In the present invention, AUDG degradation capability is up to 1 × 10-14Gram/microlitre.Therefore in the present invention
AUDG-SAMRS-MCDA methods can effectively eliminate pollution.
AUDG-SAMRS- is carried out as template using common mycobacteria type and the genomic nucleic acids of non-branch bacillus
The Evaluation on specificity of MCDA-LFB technologies, as a result shows, only belongs to MTC member, is detected through AUDG-SAMRS-MCDA-LFB
Positive findings is produced, non-false positive and false negative result produce, and illustrate the AUDG-SAMRS-MCDA-LFB methods that the present invention establishes
With excellent specificity.
Brief description of the drawings
Figure 1A .MCDA expand principle schematic；
Figure 1B high molecular nanometer biosensor structure schematic diagrames；
Fig. 1 C. high molecular nanometer biology sensors testing result display figure；
Fig. 2A .MCDA primer the result collection of illustrative plates；
Fig. 2 B.SAMRS-MCDA primer the result collection of illustrative plates；
Fig. 3 are with different bacterial strain templates and blank control evaluation MCDA methods；
Fig. 4 are with different bacterial strain templates and blank control evaluation SAMRS-MCDA methods；
Fig. 5 standard SAMRS-MCDA optimal reaction temperature test result collection of illustrative plates；
The sensitivity results collection of illustrative plates of Fig. 6 A.SMARS-MCDA methods；
The sensitivity results collection of illustrative plates of Fig. 6 B.MCDA methods；
Fig. 7 .AUDG-SAMRS-MCDA, which are eliminated, pollutes sub- principle schematic；
Fig. 8 A.AUDG-SAMRS-MCDA, which are eliminated, pollutes sub- result collection of illustrative plates；
SAMRS-MCDA, which is eliminated, when Fig. 8 B.AUDG are not present pollutes sub- result collection of illustrative plates；
The optimum reacting time test result collection of illustrative plates of Fig. 9 .AUDG-SAMRS-MCDA-LFB technologies
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to protection scope of the present invention.
1. involved reagent in the present invention：
Backboard, sample pad, gold standard pad, tunica fibrosa and adsorptive pads are purchased from Jie-Yi companies.Anti digoxin antibody (anti-
Dig), biotinylated calf serum (B-BSA) is purchased from Abcam companies.Coloured groups (aubergine) modification, Avidin
High molecular nanometer particles (Dye streptavidin coated polymer nanoparticles, SA-DNPs, 129nm) are purchased
Buy in Bangs Laboratories.DNA extraction kit (QIAamp DNA minikits；Qiagen, Hilden,
Germany German Qiagen companies) are purchased from.Constant-temperature amplification reagent and developer (Hydroxynaphthol, HNB) purchase are certainly northern
Capital Haitai positive element bio tech ltd.Biotinylated dideoxycytosine (Biotin-14-dCTP), Brdurd
(dUTP) buy from Thermo Scientific companies.South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG), deoxidation born of the same parents
Pyrimidine (dCTP), deoxythymidine (dTTP), deoxyadenine (dATP) and deoxy-guanine (dGTP) are bought from New
England Biolabs companies.DL50DNA Marker are purchased from precious bioengineering (Dalian) Co., Ltd.Remaining reagent is city
Sell analysis net product.
The key instrument used in present invention experiment：The real-time transmissometer LA-320C of constant temperature (Eiken Chemical Co.,
Ltd, Japan) it is purchased from Japanese Rong Yan companies.Electrophoresis equipment is Beijing Jun Yi east electrophoresis equipment Co., Ltd product；Gel into
As system is Bio-Rad Gel Dox XR, U.S.'s Bio-Rad products.
2. design of primers：
In order to verify, evaluate AUDG-SAMRS-MCDA-LFB technologies and establish for MTC pathogen quick, sensitive and
Special AUDG-SAMRS-MCDA-LFB detection architectures.The present invention is directed to the MTC (IS6110 of special insetion sequence 6110；
Genbank no.X17348) design amplimer, it is intended to verify feasibility, the sensitivity of AUDG-SAMRS-MCDA-LFB technologies
Property, specificity and reliability.
Design of primers result：
IS6110 distinguished sequences are present in all MTC members, and its specificity is good, can be by MTC and other close phases
Near Mycobacterium, which differentiates, to be separated.Utilize primer-design software PrimerExplorer V4 (Eiken Chemical)
(http：//primerexplorer.jp/e/) and the design MCDA primers of primer-design software Primer Premier 5.0, and
By the specific primer of acquisition in ncbi database (http：//blast.ncbi.nlm.nih.gov/Blast.cgi) in carry out
Sequence alignment analysis, matched with excluding primer and other species sequences non-specificity that may be present, after finally being optimized
MCDA amplimers, the primer are directed to the detection of all members of MTC.On the basis of standard MCDA primers, with SAMRS components
The MCDA primers of standard are modified, build SAMRS-MCDA primers, the primer is used to establish SAMRS-MCDA amplifications.Standard
MCDA and SAMRS-MCDA primer sequences and modification be shown in Table 1.
The standard MCDA primer sequences of table 1
C1*, 5' end flag F ITC (primer is used for MCDA-LFB detection architectures)；FITC, fluorescein
Isothiocyanate (fluorescein).
Nt, nucleitid (nucleotides)；Mer, monomeric (monomeric unit).
The modification of SAMRS-MCDA primer sequences is shown in Table 2.
Table 2.SAMRS-MCDA primer sequences are modified
SAMRS, self-avoiding molecular recognition system (keeping away molecular recognition system certainly).
SAMRS-C1*, 5' flag F ITC (primer is used for AUDG-SAMRS-MCDA-LFB detection architectures).A*, 2-aminopurine
(2-aminopurine)；T*, 2-thiothymine (2- thio-thymines)；(N4- acetyl born of the same parents are phonetic by C*, N4-ethylcytosine
Pyridine)；G*, hypoxanthine (hypoxanthine).
3. the design of lateral flow nano-sensor (LFB)
Figure 1B is shown in the design of lateral flow nano-sensor (LFB).LFB includes a backboard 1, on the backboard 1 successively
Setting is equipped with sample panel 2, board 3, nitrocellulose filter 4 and water sucting plate 5, is set gradually on the nitrocellulose filter 4
Detection line 41 and control line 42.In Figure 1B, Biotin is biotin, and BSA is bovine serum albumin(BSA), and Biotin-BSA is biotin
The bovine serum albumin(BSA) of change, FITC are fluorescein, and anti-FITC is anti-fluorescein antibody, FITC/Biotin-labelled
Target amplicon are the amplicon of fluorescein and biotin labeling, and Polymer nanoparticles are high molecular nanometer
Particle, SA are Avidin, and SA-DNP is the high molecular nanometer particles of Avidin.First by sample panel, board, tunica fibrosa and
Water sucting plate is assembled on backboard successively.Then SA-DNPs, anti-FITC and B-BSA are coated on gold standard pad, detection line respectively
41 (TL1) and control line 42 (CL), it is standby after to be dried
LFB Cleaning Principle：MCDA products are added drop-wise to LFB sample pad area, buffer solution is then added drop-wise to LFB's
Sample pad area.MCDA products move (being moved from sample pad to adsorptive pads direction) from the bottom up under siphonage.Work as MCDA
After product reaches pad, the biotin and the SA-DNPs that are tagged on product react.When product continues to move, double mark products
The antibody (i.e. anti-FITC antibody) of one end (hapten-marked end, i.e. FITC mark end) and TL (detection line) region combines, will be double
Mark product is fixed on detection line region.With accumulation of the product in detection line region, by the SA-DNPs of combination develop the color instead
Should, so as to carry out Visual retrieval to MCDA products.In addition, superfluous SA-DNPs can be anti-with the B-BSA in CL (nature controlling line) region
Should, direct chromogenic reaction is carried out, judges whether LFB function is normal.
The interpretation (Fig. 1 C) of LFB results：Only there are red stripes in CL regions, negative control are represented, without positive products
(II)；There are red stripes simultaneously in CL and TL regions, represent the test positive result (I) for target；When LFB occur without it is red
During lines band, LFB failures are represented；When red stripes occurs in TL, CL redfree bands, it is infeasible, it is necessary to again to represent result
Detection.
MCDA and SAMRS-MCDA amplification interpretations：
After MCDA and SAMRS-MCDA amplifications, three kinds of detection methods are used for amplification and differentiated.First, in reactant mixture
Middle addition visible dyes (such as HNB reagents), the color of positive reaction pipe are changed into sky blue from purple, and negative reaction pipe then keeps former
The purple come.Secondly, MCDA products can be by detecting amplicon, due to containing different size in product after agarose electrophoresis
Amplified fragments, therefore the electrophoretogram of positive amplification product in specificity it is stepped, negative reaction occurs without any band.More
Directly simple method is that product is detected by LFB.
Visible color method of changing：MCDA and SAMRS-MCDA produces substantial amounts of pyrophosphate ion while synthetic DNA,
The ion can be combined with the magnesium ion in reaction system, form insoluble material, so as to change the pH value of solution, make reaction
The color of mixed liquor changes.Color change sentence read result can be detected by visual observation, and positive reaction pipe is changed into day from purple
Blueness, negative reaction keep purple constant, see Fig. 2A (left figure) and 2B (left figure).
Embodiment 1.MCDA is expanded
1.MCDA reaction principles
MCDA reaction systems include 10 primers, identify 10 regions of target sequence, including 2 intersection inner primers, i.e. CP1
With CP2 (Cross Primer, CP), 2 displacement primers (Displacement amplification), i.e. F1 and F2,6 expansions
Increase primer (Amplification primer), i.e. D1, C1, R1, D2, C2 and R2.In order to build detectable product, 10 are selected
Any one primer in primer, in 5 ' end mark haptens (FITC, fluorescein), the primer newly marked is named as F1*, F2*,
CP1*, CP2*, C1*, C2*, D1*, D2*, R1* and R2*.In the present invention, the principle of the present invention is illustrated using C1* as example.
Under set constant temperature, the double-stranded DNA in reaction system is in the dynamic equilibrium shape of half dissociation and quasi integration
In state, complementary portions from any one primer to double-stranded DNA carry out base pairing extension when, another chain will dissociate, and become
It is single-stranded.First in the presence of Bst 2.0DNA polymerases, using 3 ' ends of CP1 primer P1 sections as starting point, with corresponding DNA
Complementary series matches, and starts strand displacement DNA synthesis (Figure 1A, step 1).It is in the F1 primers displacement CP1 primer amplification shapes of upstream
Into product, the product can be in combination with five primers (C1*, D1, R1, CP2 and F2, steps 2).When C1* primer annealings arrive
During target sequence, biotinylated dideoxycytosine (Biotin-14-dCTP) is incorporated into amplicon by Bst 2.0DNA enzymes.With
The progress of MCDA amplifications, forms substantial amounts of double mark products (one end marks haptens, central marker biotin), double mark products rise
Come from the C1* primers of mark and penetrate into biotinylated dideoxycytosine (Biotin-14-dCTP) in amplicon.This pair is marked
Product can be detected by high molecular nanometer biology sensor, so as to carry out visual detection.
In Figure 1A, FITC is fluorescein, and C1 is C1 primers, and C1* is fluorescein-labeled C1 primers, and Biotin is biology
Element, Biotin-14-dCTP are the dCTP of biotin labeling.
Patent CN104946744A early stage of inventor is shown in detailed MCDA amplifications.
2. the MCDA reaction systems of standard：
Cross primer CP1 concentration is 40pmol, and cross primer CP2 concentration is 40pmol, replaces primers F 1 and F2
Concentration is 10pmol, and amplimer C1*, C2, R1, R2, D1 and D2 concentration are 20pmol, 2M Betain, 8mM MgSO4,
2.5 μ L 10 × Bst DNA polymerase buffer liquid, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM's
DCTP, 0.02mM biotin-14-dCTP, 1.4mM dGTP, 10U strand displacement archaeal dna polymerase, 1U South Pole temperature-sensitive urine are phonetic
Pyridine picodna glycosyl enzyme, 1 μ L template, deionized water is added to 25 μ l.Whole reaction constant temperature 61 DEG C 1 hour, 80 DEG C
5min terminating reactions.
3. verify the feasibility of MCDA primers
Fig. 2A represents the checking of the MCDA primers for MTC, and 2A1 represents that positive amplification [adds 2 × 10 in reaction tube3Copy
The MTC templates of shellfish (10pg), as positive control], 2A2 represents that negative amplification (adds staphylococcus aureus mould in reaction tube
Plate, determine whether cross reaction be present as negative control), 2A3 represents that negative amplification (adds Klebsiella Pneumoniae in reaction tube
Template, act on negative control), 2A4 represents negative amplification (1 microlitre of distilled water replaces template, as blank control).It is only positive
Property control there is positive amplification, the MCDA primers for illustrate to be directed to the detection MTC of the special IS6110 sequences Designs of MTC can use.
Electrophoresis assays：2A (left figure) product is subjected to electrophoresis detection, because MCDA amplified production contains many
The loop-stem structure and the DNA of polycyclic cauliflower spline structure that short-movie section not of uniform size and a series of target sequence of inverted repeats are formed
Fragment mixture, show the staged collection of illustrative plates of different size zone composition after electrophoresis on gel, see Fig. 2A (right figure).Pass through electricity
Swimming detection method interpretation MCDA amplification, there is expected result in positive reaction, and negative reaction and blank control do not go out
What incumbent amplified band, MCDA the and SAMRS-MCDA primers further demonstrated designed by this research are feasible, available for target sequence
Row augmentation detection.
LFB is detected：2A (left figure) product is subjected to LFB detections, due to MCDA primers (C1*) mark detected for MTC
The haptens of note is FITC, therefore, it is positive that MTC detections is expressed as when red stripes occurs in TL and CL.Sentenced by LFB detection methods
MCDA amplifications are read, there is expected result in positive reaction, and CL red stripes only occur in negative reaction and blank control,
Demonstrate that the designed MCDA-LFB technologies of this research, MCDA primers are feasible, can be used in purpose target sequence detection (Fig. 2A,
Middle figure).
4. with different bacterial strain templates and blank control evaluation MCDA methods：
64 are reacted for evaluating MCDA altogether, including positive control reaction [signal 1, add 2 in reaction tube ×
103The MTC templates of (10pg) are copied, as positive control], the reaction of 31 negative controls (signal 2-32, is added in reaction tube non-
MTC templates, as negative control) and 32 blank controls reaction (signal 33-64,1 microlitre of distilled water replace template, as
Blank control is reacted).In 61 DEG C of constant temperature 1 hour, MCDA common properties gave birth to four false positive results (Fig. 3).Two of which false positive knot
Fruit is referred to as non-specific amplification, amplification come between MCDA primers and non-specific template hybridization of missing the target (signal 18 and
25).Remaining two false positive results is referred to as self amplification, and amplification comes between primer or self matching in primer, draws
There is no any template (signal 37 and 57) in thing reaction system.Therefore, standard MCDA can cause false positive results.
Embodiment 2.SAMRS-MCDA reaction systems
1.SAMS-MCDA reaction principles
SAMS-MCDA reaction principle and common MCDA reactions, only common MCDA primers are entered with SAMRS compositions
Row modification, the specificity of primer is enhanced, so as to enhance the specificity of method, therefore eliminated by hybridization, the primer dimerization of missing the target
False positive results caused by body.
2.SAMS-MCDA reaction systems
Cross primer SAMRS-CP1 concentration is 40pmol, and cross primer SAMRS-CP2 concentration is 40pmol, displacement
Primer SAMRS-F1 and SAMRS-F2 concentration are 10pmol, amplimer SAMRS-C1*, SAMRS-C2, SAMRS-R1,
SAMRS-R2, SAMRS-D1 and SAMRS-D2 concentration are 20pmol, 2M Betain, 8mM MgSO4, 2.5 μ L 10 ×
Bst DNA polymerase buffer liquid, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM dCTP, 0.02mM's
Biotin-14-dCTP, 1.4mM dGTP, 10U strand displacement archaeal dna polymerase, 1U South Pole temperature-sensitive uracil deoxyribose
Base enzyme, 1 μ L template, deionized water is added to 25 μ l.Whole reaction constant temperature 61 DEG C 1 hour, 80 DEG C of 5min terminating reactions.
3. verify the feasibility of SAMRS-MCDA primers：
Fig. 2 B represent for MTC SAMRS-MCDA primers checking, 2B1 represent positive amplification (in reaction tube add 2 ×
103The MTC templates of copy, as positive control), 2B2 represents that negative amplification (adds staphylococcus aureus mould in reaction tube
Plate, determine whether cross reaction be present as negative control), 2B3 represents that negative amplification (adds Klebsiella Pneumoniae in reaction tube
Template, act on negative control), 2B4 represents negative amplification (1 microlitre of distilled water replaces template, as blank control).It is only positive
Property control there is positive amplification, the MCDA primers of description standard are remained able to be expanded, said after the modification of SAMRS compositions
The detection MTC of the bright IS6110 sequences Design special for MTC SAMRS-MCDA primers can use.
Electrophoresis assays：2B (left figure) product is subjected to electrophoresis detection, because SAMRS-MCDA amplified production includes
The loop-stem structure and polycyclic cauliflower sample knot that a series of target sequence of many short-movie sections not of uniform size and inverted repeats is formed
The DNA fragmentation mixture of structure, show the staged collection of illustrative plates of different size zone composition after electrophoresis on gel, see that Fig. 2 B are (right
Figure).By electrophoresis assays interpretation SAMRS-MCDA amplification, there is expected result in positive reaction, and negative anti-
Should not occur any amplified band with blank control, the SAMRS-MCDA primers further demonstrated designed by this research are feasible,
Detected available for target sequence amplification.
LFB is detected：Fig. 2 B (left figure) product is subjected to LFB detections.Due to the SAMRS-MCDA primers detected for MTC
(SAMRS-C1*) haptens of mark is FITC, therefore, it is positive that MTC detections is expressed as when red stripes occurs in TL and CL.It is logical
Cross LFB detection method interpretation SAMRS-MCDA amplifications, expected result occurs in positive reaction, and negative reaction and blank pair
According to only there are CL red stripes, the designed SAMRS-MCDA-LFB technologies of this research are demonstrated, SAMRS-MCDA primers are feasible,
It can be used in the detection (scheming in Fig. 2 B) of purpose target sequence.
4. with different bacterial strain templates and blank control evaluation SAMRS-MCDA methods
64 are reacted for evaluating SAMRS-MCDA reactions, including positive control reaction [signal 1, a reaction tube altogether
It is middle to add 2 × 103The MTC templates of (10pg) are copied, as positive control], 31 negative control reactions (signal 2-32, reactions
Non- MTC templates are added in pipe, as negative control) and 32 blank control reactions (signal 33-64,1 microlitre of distilled water replacements
Template, answered as blank control).In 61 DEG C of constant temperature 1 hour, SAMRS-MCDA did not produced any false positive results (Fig. 4), very
Extended to 1.5 hours to proliferation time.Unique positive amplification comes from positive control (signal 1).For MCDA methods,
SAMRS-MCDA methods do not produce any false positive results.Therefore, SAMRS-MCDA methods are more suitable for than the MCDA methods of standard
Detection field.
The optimal reaction temperature measure of embodiment 3.SAMRS-MCDA technologies
Under the conditions of SAMRS-MCDA reaction systems, MTC templates and designed corresponding SAMRS-MCDA primers are added,
Its template concentrations is 2 × 103Copy/microlitre (10pg/ microlitres).Reaction carries out (59-66 DEG C) under constant temperature, application of results
Real-time transmissometer is detected, and is obtained different dynamic curve diagrams at different temperature, is seen Fig. 5.60-62 DEG C is proposed as
The optimal reaction temperature of SAMRS-MCDA primers.Subsequent authentication in the present invention selects 61 DEG C as constant temperature and carries out SAMRS-
MCDA is expanded.
The sensitivity evaluation of embodiment 4.SAMRS-MCDA-LFB detections
With the good template of serial dilution (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100aq/ microlitres~2 ×
106, 2 × 103, 2 × 102, 2 × 101, 2 × 100, 2 × 10-1With 2 × 10-2Copy/microlitre) carry out SAMRS-MCDA amplified reactions
Afterwards, detected with LFB and show result.Detected for MTC, SAMRS-MCDA-LFB detection range is 2 × 106~2 × 101Copy
Shellfish/microlitre, LFB occurs red line (Fig. 6 A, figure below, 6A1-6A4) in TL and CL regions.When genomic templates amount in reaction system
It is reduced to 2 × 100When copying and be following, only there is red line in CL regions in LFB, represents negative findings (Fig. 6 A, figure below, 6A5-
6A8).6A1 to 6A7 represents that MTC template amount is 10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100aq/ microlitre, 6A8 tables
Show blank control (1 microlitre of distilled water).Visualized with LFB read SAMRS-MCDA amplification and HNB (Fig. 6 A, in
Figure), real-time turbidity (Fig. 6 A, upper figure) read result it is consistent.In addition, compared with the MCDA methods of standard, SAMRS-MCDA side
The susceptibility of method is consistent with standard MCDA susceptibility.Fig. 6 B represent the susceptibility of MCDA methods, and upper figure is with real-time turbidity
MCDA susceptibility is detected, middle figure is with HNB detections MCDA susceptibility, and figure below is the susceptibility that MCDA is detected with LFB.
The South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG) of embodiment 5. removes cross pollution evaluation
In reaction system, due to adding Brdurd (dUTP), with the progress that SAMRS-MCDA is expanded, own
Amplified production be all infiltrated Brdurd (dUTP).When amplified production (amplicon for penetrating into Brdurd) enters amplification
During system, AUDG (such as room temperature) under normal temperature condition removes single-stranded or double-stranded middle Brdurd, so that single-stranded or double-stranded DNA
There are breach (Fig. 7, Nucleic acid, target nucleic acids；Carryover contamination, cross pollution；Hapten,
Haptens；Hapten-labeled primer, hapten-marked primer；Bst 2.0, Bst 2.0DNA polymerases；AUDG,
AUDG enzymes；Biotin, biotin；DCTP, dideoxycytosine；Biotin-14-dCTP, biotinylated dideoxycytosine；
DUTP, Brdurd).Because natural template does not include Brdurd, therefore AUDG makees to natural DNA without catalysis
With.When carrying out MCDA amplifications, temperature is higher (being more than 60 DEG C), declines with single-stranded or double-stranded DNA jaggy in thermodynamic activity
Solution, therefore cannot function as template and expanded, so as to eliminate the pollution products of reaction system, reach the mesh for removing cross pollution
's.In addition, AUDG can be inactivated immediately when temperature is more than 50 DEG C, so as to the amplified production newly synthesized that can not degrade, even if amplification
Brdurd is included in product.Therefore the AUDG enzymes selected in the present invention can be used to eliminate cross pollution, and not influence
MCDA normal amplification.
In order to confirm that AUDG can eliminate the amplified production that dUTP penetrates into as effective instrument, SAMRS-MCDA reactions
Amplified production is by serial dilution (1 × 10-12, 1 × 10-13, 1 × 10-14, 1 × 10-15, 1 × 10-16, 1 × 10-17, 1 × 10-18With 1
×10-19Gram/microlitre).Each dilution factor amplified production is used as template, is reacted for SAMRS-MCDA.The nothing in reaction system
In the presence of AUDG, the ability that SAMRS-MCDA can detect pollution is 1 × 10-18(Fig. 8 B, upper figure are fortune to gram/microlitre
With real-time turbidity interpretation SAMRS-MCDA result, middle figure is with HNB interpretations SAMRS-MCDA result, and figure below is utilization
LFB interpretations SAMRS-MCDA result)；In reaction system in the presence of AUDG, SAMRS-MCDA can detect pollution
The ability of son is 1 × 10-14(Fig. 8 A, upper figure are the result with real-time turbidity interpretation SAMRS-MCDA to gram/microlitre, and middle figure is fortune
With HNB interpretations SAMRS-MCDA result, figure below is the result with LFB interpretations SAMRS-MCDA).In normal conditions, expand
Caused by increasing production thing, the sub- concentration of pollution that can produce cross reaction is usually 1 × 10-18Gram/microlitre.In the present invention, AUDG's
Degraded can be up to 1 × 10-14Gram/microlitre.Therefore the AUDG-SAMRS-MCDA methods in the present invention can effectively eliminate pollution.
The optimum reacting time measure of embodiment 6.AUDG-SAMRS-MCDA-LFB technologies
Under the conditions of AUDG-SAMRS-MCDA reaction systems, at the same add for MTC amplification SAMRS-MCDA primers and
MTC templates (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100aq/ microlitres~2 × 10 after dilution6, 2 × 103, 2 ×
102, 2 × 101, 2 × 100, 2 × 10-1With 2 × 10-2Copy/microlitre).Reaction carries out (61 DEG C) under constant temperature, during constant temperature
Between respectively 25 minutes, 35 minutes, 45 minutes and 55 minutes.Detect and show with LFB：AUDG-SAMRS-MCDA-LFB technologies exist
During testing goal target, optimum reacting time is 45 minutes (Fig. 9).When AUDG-SAMRS-MCDA systems are in amplification step constant temperature
At 45 minutes, the horizontal template of minimum detection limit can be detected (Fig. 9 C).Fig. 9 C, LFB detection range are 10ng~100fg,
LFB occurs red line (LFB1-LFB4) in TL and CL regions.When genomic templates amount is reduced to 10fg and following in reaction system
When, only there is red line in CL regions in LFB, represents negative findings (LFB5-LFB7).Fig. 9 reads AUDG- with LFB visualizations
Amplification of the SAMRS-MCDA systems from 25 minutes to 55 minutes；LFB1 to LFB7 represents that MTC template amount is 10ng,
10pg, 1pg, 100fg, 10fg, 1fg, 100aq；LFB8 represents blank control (1 microlitre of distilled water).
The Evaluation on specificity of embodiment 7.AUDG-SAMRS-MCDA-LFB technologies
AUDG-SAMRS- is evaluated as template using common mycobacteria type and the genomic nucleic acids of non-branch bacillus
The specificity (table 3) of MCDA-LFB technologies.AUDG-SAMRS-MCDA-LFB technologies can detect MTC member, explanation exactly
The specificity of AUDG-SAMRS-MCDA-LFB methods is good, is shown in Table 2.
The bacterial strain of table 3 and specific detection result
aMycobacteria reference strain, from China national tuberculosis Reference Lab.
bATCC, American Type Culture Collection (American Type Culture Collection)；ZG-CDC,
Zigong Center for Disease Control and Prevention (Zi Gong Municipal Disease Control and Prevention Center)；
ICDC, National Institute for Communicable Disease Control and Prevention,
Chinese Center for Disease Control and Prevention (CDC, infectious disease
Prevention and control institute).
cP, positive (AUDG-SAMRS-MCDA-LFB detections are positive)；N, negative (AUDG-SAMRS-MCDA-
LFB detections are negative).Testing result illustrates in table 2, only belongs to MTC member, detects and produces through AUDG-SAMRS-MCDA-LFB
Raw positive findings, illustrate that established AUDG-SAMRS-MCDA-LFB methods being capable of precise Identification MTC, non-false positive and false the moon
Property result produce.
Sequence table
<110>Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an
<120>A kind of combination AUDG and the more intersection constant-temperature amplification methods for keeping away molecular recognition system certainly
<141> 2017-09-19
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 1
ccaacaagaa ggcgtactc 19
<210> 2
<211> 18
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 2
ttgaaccagt cgacccag 18
<210> 3
<211> 42
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 3
ctcgctgaac cggatcgatg tgcctgaaag acgttatcca cc 42
<210> 4
<211> 38
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 4
tagccgagac gatcaacggc ctcgacatcc tcgatgga 38
<210> 5
<211> 22
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 5
ctcgctgaac cggatcgatg tg 22
<210> 6
<211> 20
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 6
tagccgagac gatcaacggc 20
<210> 7
<211> 18
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 7
tactgagatc ccctatcc 18
<210> 8
<211> 19
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 8
ctatacaaga ccgagctga 19
<210> 9
<211> 17
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 9
gaccgacggt tggatgc 17
<210> 10
<211> 19
<212> DNA
<213> Mycobacterium tuberculosis complex
<400> 10
agctcctatg acaatgcac 19
Claims (10)
1. a kind of method for intersecting constant-temperature amplification combination high molecular nanometer bio-sensing testing goal gene, methods described include more
Following steps：
(1) genome of detected sample is extracted；
(2) primer that 4 bases provided in 3 ' end bit base second from the bottom to the 5th bit bases reciprocal are modified by SAMRS, institute
Stating primer includes：Replace primers F 1 and F2, the sequence such as SEQ ID NO of the displacement primers F 1：Shown in 1, the displacement primer
F2 sequence such as SEQ ID NO：Shown in 2；Cross primer CP1 and CP2, the sequence such as SEQ ID NO of the cross primer CP1：3
It is shown, the sequence such as SEQ ID NO of the cross primer CP2：Shown in 4；Amplimer C1 and C2, D1 and D2, R1 and R2 are provided,
Primer C1 sequence such as SEQ ID NO：Shown in 5, primer C2 sequence such as SEQ ID NO：Shown in 6；Primer D1 sequence such as SEQ
ID NO：Shown in 7, primer D2 sequence such as SEQ ID NO：Shown in 8, primer R1 sequence such as SEQ ID NO：Shown in 9, primer
R2 sequence such as SEQ ID NO：Shown in 10, the 5 ' ends for being simultaneously provided in any of the above-described primer are marked with the modification of haptens and drawn
Thing；
(3) South Pole temperature-sensitive uracil picodna glycosyl enzyme, chain shift-type polymerase, melting temperature conditioning agent, primer,
In the presence of dNTP, and biotinylated Brdurd, expanded using the genomic nucleic acids of detected sample as template constant temperature
Increase DNA；
(4) amplified production of high molecular nanometer biology sensor detecting step (3) is used.
2. according to the method for claim 1, it is characterised in that the Mdification primer that haptens is marked with 5 ' ends draws for amplification
Thing C1.
3. according to the method for claim 2, it is characterised in that the haptens in 5 ' end marks is fluorescein.
4. according to the method for claim 3, it is characterised in that the high molecular nanometer biology sensor includes a backboard
(1), set gradually on the backboard (1) equipped with sample panel (2), board (3), nitrocellulose filter (4) and water sucting plate
(5) detection line (41) and control line (42), are set gradually on the nitrocellulose filter (4), in board (3), detection line
(41) and control line (42) region is coated with high molecular nanometer particles (6), the anti-fluorescence of the Avidin of coloured groups modification successively
Plain antibody (7) and the bovine serum albumin (8) of biotin coupling.
5. according to the method for claim 1, it is characterised in that the constant-temperature amplification is carried out in 60-62 DEG C of environment
's.
6. according to the method for claim 5, it is characterised in that the constant-temperature amplification is carried out in 61 DEG C of environment.
7. one group of primer sequence for being used for constant-temperature amplification mycobacterium tuberculosis complex IS6110 distinguished sequences, it is characterised in that
The sequence includes：Such as SEQ ID NO：Displacement primers F 1 shown in 1, such as SEQ ID NO：Displacement primers F 2 shown in 2, such as
SEQ ID NO：Cross primer CP1 shown in 3, such as SEQ ID NO：Cross primer CP2 shown in 4, such as SEQ ID NO：Shown in 5
Amplimer C1, such as SEQ ID NO：Amplimer C2 shown in 6, such as SEQ ID NO：Amplimer D1 shown in 7, such as
SEQ ID NO：Amplimer D2 shown in 8, such as SEQ ID NO：Amplimer R1 shown in 9, such as SEQ ID NO：Shown in 10
Amplimer R2, the 5 ' ends for being simultaneously provided in any of the above-described primer are marked with the Mdification primer of haptens.
8. primer sequence according to claim 7, it is characterised in that the primer 3 ' end 4 bases by SAMRS
Modification, wherein A are modified to 2-aminopurine, and T is modified to 2- thio-thymines, and C is modified to N4- acetylcytosines, G
It is modified to hypoxanthine.
9. the primer sequence according to claim 7 or 8, it is characterised in that be marked with the Mdification primer of haptens at 5 ' ends
For amplimer C1.
10. primer sequence according to claim 9, it is characterised in that the haptens marked is fluorescein.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182366A (en) * | 2018-10-08 | 2019-01-11 | 广东菲鹏生物有限公司 | The preparation method of thermosensitive type uracil-DNA glycosylase |
| CN109811036A (en) * | 2019-03-15 | 2019-05-28 | 首都医科大学附属北京儿童医院 | The methods intersected amplification and combine bio-sensing detection mycobacterium tuberculosis complex more |
| CN110295241A (en) * | 2019-07-11 | 2019-10-01 | 深圳易致生物科技有限公司 | For detecting the primer sets of urinary tract infections and comprising the kit of the primer sets |
| CN110819724A (en) * | 2018-08-07 | 2020-02-21 | 中国疾病预防控制中心 | Nucleic acid sequence for detecting mycobacterium tuberculosis complex, kit, detection method and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736078A (en) * | 2008-11-05 | 2010-06-16 | 上海复星医药（集团）股份有限公司 | Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit |
| CN102373273A (en) * | 2010-08-26 | 2012-03-14 | 杭州优思达生物技术有限公司 | Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof |
| CN103725750A (en) * | 2012-10-10 | 2014-04-16 | 哈尔滨德歌生物科技有限公司 | Visual detection method of isothermal amplification products of nucleic acids |
| CN105483219A (en) * | 2015-12-11 | 2016-04-13 | 杭州优思达生物技术有限公司 | Mycobacterium tuberculosis complex nucleic acid detection method and kit |
| CN105658811A (en) * | 2013-08-19 | 2016-06-08 | 史蒂文·奔纳 | Helicase dependent amplification of DNA molecules using nucleotide analogs |
| CN105936941A (en) * | 2016-07-05 | 2016-09-14 | 吉林农业大学 | Mycobacterium tuberculosis PCR-LFB detection kit |
| CN107164541A (en) * | 2017-07-12 | 2017-09-15 | 中国疾病预防控制中心传染病预防控制所 | Many cross substitutions amplification of AUDG mediations combines the nucleic acid detection technique of bio-sensing |
-
2017
- 2017-09-19 CN CN201710848631.2A patent/CN107523624B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736078A (en) * | 2008-11-05 | 2010-06-16 | 上海复星医药（集团）股份有限公司 | Method for detecting viable bacteria of Mycobacterium tuberculosis through isothermal amplification of nucleic acid and kit |
| CN102373273A (en) * | 2010-08-26 | 2012-03-14 | 杭州优思达生物技术有限公司 | Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof |
| CN103725750A (en) * | 2012-10-10 | 2014-04-16 | 哈尔滨德歌生物科技有限公司 | Visual detection method of isothermal amplification products of nucleic acids |
| CN105658811A (en) * | 2013-08-19 | 2016-06-08 | 史蒂文·奔纳 | Helicase dependent amplification of DNA molecules using nucleotide analogs |
| CN105483219A (en) * | 2015-12-11 | 2016-04-13 | 杭州优思达生物技术有限公司 | Mycobacterium tuberculosis complex nucleic acid detection method and kit |
| CN105936941A (en) * | 2016-07-05 | 2016-09-14 | 吉林农业大学 | Mycobacterium tuberculosis PCR-LFB detection kit |
| CN107164541A (en) * | 2017-07-12 | 2017-09-15 | 中国疾病预防控制中心传染病预防控制所 | Many cross substitutions amplification of AUDG mediations combines the nucleic acid detection technique of bio-sensing |
Non-Patent Citations (1)
| Title |
|---|
| HONG M ET AL.: "A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex", 《WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819724A (en) * | 2018-08-07 | 2020-02-21 | 中国疾病预防控制中心 | Nucleic acid sequence for detecting mycobacterium tuberculosis complex, kit, detection method and application |
| CN110819724B (en) * | 2018-08-07 | 2022-02-11 | 中国疾病预防控制中心 | Nucleic acid sequence for detecting mycobacterium tuberculosis complex, kit, detection method and application |
| CN109182366A (en) * | 2018-10-08 | 2019-01-11 | 广东菲鹏生物有限公司 | The preparation method of thermosensitive type uracil-DNA glycosylase |
| CN109811036A (en) * | 2019-03-15 | 2019-05-28 | 首都医科大学附属北京儿童医院 | The methods intersected amplification and combine bio-sensing detection mycobacterium tuberculosis complex more |
| CN110295241A (en) * | 2019-07-11 | 2019-10-01 | 深圳易致生物科技有限公司 | For detecting the primer sets of urinary tract infections and comprising the kit of the primer sets |
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|---|---|
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