CN107557440A - A kind of combination AUDG and the method for loop-mediated isothermal amplification for keeping away molecular recognition system certainly - Google Patents

Abstract

The invention discloses a kind of method of loop-mediated isothermal amplification for combining South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG) and keeping away molecular recognition system (SAMRS) certainly, methods described holds 4 bases of bit base second from the bottom to the 5th bit base reciprocal to carry out SAMRS modifications to the primer 3 ' in loop-mediated isothermal amplification, and in primer LF 5 ' end mark haptens, AUDG enzymes are introduced in amplification system, Brdurd and biotinylated dideoxycytosine, based on loop-mediated isothermal amplification technology, 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

A kind of combination AUDG and the method for loop-mediated isothermal amplification for keeping away molecular recognition system certainly

Technical field

The invention discloses a kind of method of loop-mediated isothermal amplification detection microorganism target gene, belongs to microorganism and divides Sub- biology techniques field.

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..In these isothermal amplification technologies, most popular technology For loop-mediated isothermal amplification.LAMP realizes nucleic acid amplification under constant temperature, using only a kind of constant temperature substituted enzyme, amplification rate It hurry up, be quick on the draw, specificity height.

The technical bottleneck of loop-mediated isothermal amplification (LAMP) is the detection of the interpretation, i.e. amplified production of result.To current Untill, most common product detection means mainly include color indicator, electrophoresis, real-time turbidity.However, these three detection techniques It is suitable only for single detection (detection of i.e. single target).In addition, during color indicator interpretation amplified production, it may appear that simulation All right one way or the other result, it is time-consuming longer during electrophoresis sentence read result, easily there is cross pollution, be not suitable for Site Detection, implement turbidity Special instrument and equipment is needed during interpretation.In order to overcome the inferior position of these three detection techniques, make LAMP technology biology, medicine and Health field application is more extensive, more economical.In the recent period, inventor is examined LAMP technology and nano biological based on LAMP technology Survey technology is combined, the nanobiosensor technology develop and rely on LAMP technology, realized quick, sensitive detection, the technology quilt It is named as nucleic acid diagnostic techniques (the Loop-mediated Isothermal of loop-mediated isothermal amplification combination gold nano bio-sensing Amplification Label-based Gold Nanoparticles Lateral Flow Biosensor,LAMP- LFB), related content is disclosed in CN201610576270.6, and the patent document forms the application explanation as prior art document One part of book.

In order to which LAMP amplified productions are applied into nanosensor technology, traditional strategy be in LAMP 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 LAMP 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 LAMP products of conventional measures structure double labelling easily cause false positive results, even It need not expand and lead to false positive results.The false positive results derive from the hybridization between labeled primer.Therefore, in order to gram The inferior position of conventional tag strategy is taken, the present invention devises a kind of new inspection policies, and the technology only needs the primer of a mark just Double target amplified productions can be built, so as to which amplified production is applied into biosensor technique.

In order to which LAMP 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, LAMP, due to Technology design, the technology easily produces false positive results, the false positive results are mainly by drawing Hybridization between thing, in primer caused by autogamy or hybridization of missing the target.In order to overcome conventional tag strategy, cross pollution and technology are set False positive results caused by meter, in the present invention, inventor utilize the primer singly marked, South Pole temperature-sensitive uracil deoxyribose Base enzyme and molecular recognition system is kept away certainly, based on LAMP technology, develop LAMP combinations nanosensor, keep away molecular recognition certainly System and South Pole temperature-sensitive uracil picodna glycosyl enzymatic nucleic acid diagnostic techniques (Loop-mediated isothermal amplification combined with biosensor,self-avoiding molecular recognition systems,antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid and prevention of carryover contamination；AUDG-SAMRS-LAMP-LFB). In order to verify the feasibility of AUDG-SAMRS-LAMP-LFB technologies, mycobacterium tuberculosis complex (Mycobacterium Tuberculosis complex, MTC) AUDG-SAMRS-LAMP-LFB technologies are applied to, establish detection MTC AUDG- SAMRS-LAMP-LFB diagnostic methods.

The content of the invention

Based on foregoing invention purpose, present invention firstly provides a kind of loop-mediated isothermal amplification combination high molecular nanometer biology The method of sensing detection target gene, the described method comprises the following steps：

(1) genome of detected sample is extracted；

(2) 4 bases provided in bit base second from the bottom to the 5th bit base reciprocal at 3 ' ends are drawn by what SAMRS was modified Thing, the primer include：SEQ ID NO：Primers F 3 shown in 1, SEQ ID NO：Primer B3, SEQ ID NO shown in 2：3 institutes Primers F IP, the SEQ ID NO shown：Primer BIP, SEQ ID NO shown in 4：Primer LF, SEQ ID NO shown in 5：Shown in 6 Primer LB, wherein, select any of the above primer 5 ' end mark haptens；

(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 primer LF.

In the technical scheme that one is more highly preferred to, the haptens in 5 ' end marks is digoxin.

Particularly preferably, the high molecular nanometer biology sensor includes a backboard 1, set gradually on the backboard 1 Equipped with sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 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 pad 3, detection line 41 and the region of control line 42 High molecular nanometer particles 6, anti digoxin 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 59-61 DEG C of environment.

In the technical scheme that one is more highly preferred to, the constant-temperature amplification is carried out in 60 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, the sequence include：Such as SEQ ID NO：Primers F 3 shown in 1, SEQ ID NO：Primer B3, SEQ shown in 2 ID NO：Primers F IP, SEQ ID NO shown in 3：Primer BIP, SEQ ID NO shown in 4：Primer LF, SEQ ID shown in 5 NO：Primer LB shown in 6, wherein, select 5 ' ends of any of the above primer to mark haptens.

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 primer LF.

Preferably, the haptens marked is digoxin.

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, and 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 caused false positive results that miss the target are eliminated.In addition, SAMRS into Dividing can only be with natural base pairing (i.e. A*：T, T*：A, C*：G, and G*：C), can not be matched (i.e. not between SAMRS compositions A* be present：T* and C*：G* match), therefore, between the primer containing SAMRS compositions can not hybridize or primer in can not hybridize, eliminate Self hybridization in hybridization (dimer can not be formed between primer) or primer between primer (can not be formed two in primer Level structure), the high efficiency and specificity of primer are ensure that, it is thus eliminated that the false sun as caused by primer dimer and secondary structure Property result.

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 90 minutes, and detection range is 2 × 10⁷~2 × 10¹Copy/microlitre, With high sensitivity.In the present invention, AUDG degraded can be up to 1 × 10^-14Gram/microlitre.Therefore the AUDG- in the present invention SAMRS-LAMP 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 LAMP-LFB technologies, as a result shows, only belongs to MTC member, is detected through AUDG-SAMRS-LAMP-LFB Positive findings is produced, non-false positive and false negative result produce, and illustrate the AUDG-SAMRS-LAMP-LFB methods that the present invention establishes With excellent specificity.

Brief description of the drawings

Figure 1A .LAMP 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 .LAMP primer the result collection of illustrative plates；

Fig. 2 B.SAMRS-LAMP primer the result collection of illustrative plates；

Fig. 3 are with different bacterial strain templates and blank control evaluation LAMP method；

Fig. 4 are with different bacterial strain templates and blank control evaluation SAMRS-LAMP methods；

Fig. 5 standard SAMRS-LAMP optimal reaction temperature test result collection of illustrative plates；

The sensitivity results collection of illustrative plates of Fig. 6 A.SMARS-LAMP methods；

The sensitivity results collection of illustrative plates of Fig. 6 B.LAMP methods；

Fig. 7 .AUDG-SAMRS-LAMP, which are eliminated, pollutes sub- principle schematic；

Fig. 8 A.AUDG-SAMRS-LAMP, which are eliminated, pollutes sub- result collection of illustrative plates；

SAMRS-LAMP, which is eliminated, when Fig. 8 B.AUDG are not present pollutes sub- result collection of illustrative plates.

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 hyclone albumen (B-BSA) is purchased from Abcam companies.Coloured groups (aubergine) modification, Avidin Change high molecular nanometer particles (Dye streptavidin coated polymer nanoparticles, SA-DNPs, 129nm) buy in Bangs Laboratories.DNA extraction kit (QIAamp DNA minikits；Qiagen, Hilden, Germany) it is purchased from German Qiagen companies.Constant-temperature amplification reagent and developer (Hydroxynaphthol, HNB) purchase Buy from Beijing Haitai positive element bio tech ltd.Biotinylated dideoxycytosine (Biotin-14-dCTP), deoxidation urine Pyrimidine (dUTP) is bought from Thermo Scientific companies.South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG), take off Oxygen cytimidine (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-LAMP-LFB technologies and establish for MTC pathogen quick, sensitive and Special AUDG-SAMRS-LAMP-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-LAMP-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 LAMP 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 LAMP amplimers, the primer are directed to the detection of all members of MTC.On the basis of standard LAMP primer, with SAMRS components The LAMP primer of standard is modified, builds SAMRS-LAMP primers, the primer is used to establish SAMRS-LAMP amplifications.Standard LAMP and SAMRS-LAMP primer sequences and modification see Tables 1 and 2 respectively.

The standard LAMP primer sequence of table 1 and modification

^aLF* is marked with digoxigenin (digoxin) at 5' ends, and the primer is used for LAMP-LFB detection architectures.

^bNt, nucleitid (nucleotides)；Mer, monomeric (monomeric unit).

The SAMRS-LAMP primer sequences of table 2 and modification

^aSAMRS, self-avoiding molecular recognition system (keeping away molecular recognition system certainly).

^bDig, digoxigenin (digoxin)；SAMRS-LF*, digoxin is marked with primer 5' ends, the primer is used for AUDG-SAMRS-LAMP-LFB detection architectures.

A*, 2-aminopurine (2-aminopurine)；T*, 2-thiothymine (2- thio-thymines)；C*, N4- Ethylcytosine (N4- acetylcytosines)；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 five parts, backboard 1, sample pad 2, knot Close pad 3, nitrocellulose filter 4 and adsorptive pads 5.First by sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5 successively It is assembled on backboard 1.Then by the high molecular nanometer particles 6 (SA-DNPs) of the Avidin, (anti-of anti digoxin antibody 7 Dig) and biotinylated hyclone albumen 8 (B-BSA) be coated on respectively pad 3 (gold standard pad), detection line 41 (TL) and Control line 42 (CL), it is standby after to be dried.

LFB Cleaning Principle：LAMP products are added drop-wise to LFB sample pad area, buffer solution is then added drop-wise to LFB's Sample pad area.LAMP products move (being moved from sample pad to adsorptive pads direction) from the bottom up under siphonage.Work as LAMP 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-Dig antibody) of one end (hapten-marked end, i.e. Dig mark end) and TL (detection line) region combines, by double marks 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 LAMP 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.

Embodiment 1.LAMP reaction amplifications

1.LAMP reaction principles

LAMP reaction systems include 6 primers, identify 8 regions of target sequence, including 2 inner primers, i.e. inner primer FIP And BIP；2 displacement primers, i.e. F3 and B3；2 ring primers, i.e. LF and LB.In order to build detectable product, it may be selected 6 and draw Any one primer in thing, in 5 ' end mark haptens (Dig, digoxin), the primer newly marked is named as F3*, B3*, FIP*, BIP*, LF* and LB*.In the present invention, the principle of the present invention is illustrated using LF* 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 the section of FIP primers Fs 2 as starting point, with corresponding DNA Complementary series matches, and starts strand displacement DNA synthesis (Figure 1A, step 1).It is in the F3 primers displacement FIP primer amplification shapes of upstream Into product, the product can be in combination with 3 primers (LF*, BIP and B3, steps 2).When LF* primer annealings to target sequence When, biotinylated dideoxycytosine (Biotin-14-dCTP) is incorporated into amplicon by Bst 2.0DNA enzymes.As LAMP expands The progress of increasing, substantial amounts of double mark products (one end marks haptens, central marker biotin) are formed, double mark products are originating from mark LF* primers and penetrate into biotinylated dideoxycytosine (Biotin-11-dCTP) in amplicon.This pair of target product energy Detected by high molecular nanometer biology sensor, so as to carry out visual detection.Applicant's earlier application is shown in detailed LAMP amplifications Chinese invention patent application CN201510162302.3 and CN201610576270.6.

2. the LAMP reaction systems of standard：

Preceding inner primer FIP concentration is 40pmol, and rear inner primer BIP concentration is 40pmol, and preceding displacement primers F 3 is with after Displacement primer B3 concentration is that 10pmol, front ring primer LF* and rear ring primer LB 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 biotin-14-dCTP, 1.4mM dGTP, 10U strand displacement archaeal dna polymerase, 1 μ L mould Plate, deionized water is added to 25 μ l.Whole reaction constant temperature 60 DEG C 1.5 hours, 80 DEG C of 5min terminating reactions.

3. verify the feasibility of LAMP primer

After LAMP amplifications, three kinds of detection methods are used for amplification and differentiated, first, add visual dye in the reactive mixture Expect (such as HNB reagents), positive reaction pipe color is changed into sky blue from purple, negative reaction pipe then keep original purple (Fig. 2A, Upper figure).Secondly, LAMP products can be different size of due to being contained in product by detecting amplicon after agarose electrophoresis Amplified fragments, thus the electrophoretogram of positive amplification product in specificity it is stepped, negative reaction occur without any band (Fig. 2A, Middle figure).More direct simple method is to be detected (Fig. 2A, figure below) to product by LFB.

Visible color method of changing：LAMP produces substantial amounts of pyrophosphate ion while synthetic DNA, and the ion can be with Magnesium ion in reaction system combines, and forms insoluble material, so as to change the pH value of solution, makes the color of reaction mixture Change.Color change sentence read result can be detected by visual observation, and positive reaction pipe is changed into sky blue, negative reaction from purple Keep purple constant, see Fig. 2A (above).

Fig. 2A represents the checking of the LAMP primer for MTC, and 2A1 represents that positive amplification [adds 2 × 10 in reaction tube³Copy The MTC templates of shellfish (10 pik), as positive control], 2A2 represents that negative amplification (adds staphylococcus aureus in reaction tube Template, determine whether cross reaction be present as negative control), 2A3 represents that negative amplification (adds kerekou pneumonia primary in reaction tube Bacterium template, determine whether cross reaction be present as negative control), 2A4 represents that (1 microlitre of distilled water replaces mould for negative amplification Plate, reacted as blank control).Only there is positive amplification in positive control, illustrates for IS6110 sequences Designs special MTC Detection MTC LAMP primer can use.

Electrophoresis assays：The product of 2A (above) is subjected to electrophoresis detection, because LAMP 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 (middle).Pass through electricity Swimming detection method interpretation LAMP amplification, there is expected result in positive reaction, and negative reaction and blank control do not go out What incumbent amplified band, the LAMP primer further demonstrated designed by this research is feasible, is detected available for target sequence amplification.

LFB is detected：The product of Fig. 2A (above) is subjected to LFB detections, due to the LAMP primer (LF*) detected for MTC The haptens of mark is Dig, therefore, it is positive that MTC detections is expressed as when red stripes occurs in TL and CL.Pass through LFB detection methods Interpretation LAMP amplifications, there is expected result in positive reaction, and CL red bars only occur in negative reaction and blank control Band (see Fig. 2A figure below), the designed LAMP-LFB technologies of this research are demonstrated, LAMP primer is feasible, can be used in purpose target sequence The detection of row.

4. with different bacterial strain templates and blank control evaluation LAMP method

64 are reacted for evaluating LAMP reactions altogether, including the reaction of positive control [signal 1, adds 2 in reaction tube ×10³The MTC templates of (10 pik) are copied, as positive control], the reaction of 31 negative controls (signal 2-32, in reaction tube plus Enter non-MTC templates, as negative control) and 32 blank controls reaction (signal 33-64,1 microlitre of distilled water replace template, Reacted as blank control).In 60 DEG C of constant temperature 1 hour, LAMP common properties gave birth to five false positive results (Fig. 3).The false sun of two of which Property result be referred to as non-specific amplification, amplification comes from the hybridization (signal that misses the target between LAMP primer and non-specific template 14 and 21).Remaining three false positive results is referred to as self amplification, amplification come between primer or in primer self Match somebody with somebody, there is no any template (signal 33,44 and 56) in primer reaction system.Therefore, standard LAMP easily causes false positive results.

Embodiment 2.SAMRS-LAMP reaction systems

1.SAMRS-LAMP reaction principles

SAMRS-LAMP reaction principle and common LAMP reactions, only common LAMP primer is 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.SAMRS-LAMP reaction systems：

SAMRS-FIP concentration is 40pmol, and SAMRS-BIP concentration is 40pmol, SAMRS-F3 and SAMRS-B3 Concentration is that 10pmol, SAMRS-LF* and SAMRS-LB concentration are 20pmol, 2M Betain, 8mM MgSO₄, 2.5 μ L's 10 × Bst DNA polymerase buffer liquid, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM dCTP, 0.02mM biotin-14-dCTP, 1.4mM dGTP, 10U strand displacement archaeal dna polymerase, 1 μ L template, add deionization Water is to 25 μ l.Whole reaction constant temperature 60 DEG C 1.5 hours, 80 DEG C of 5min terminating reactions.

3. verify the feasibility of SAMRS-LAMP primers：

After SAMRS-LAMP amplifications, three kinds of detection methods are used for amplification and differentiated, first, add in the reactive mixture Visible dyes (such as HNB reagents), positive reaction pipe color are changed into sky blue from purple, and negative reaction pipe then keeps original purple (the upper figures of Fig. 2 B).Secondly, SAMRS-LAMP products can be by detecting amplicon, due to being contained in product after agarose electrophoresis Different size of amplified fragments, therefore the electrophoretogram of positive amplification product is stepped in specificity, negative reaction occurs without any Band (is schemed) in Fig. 2 B.More direct simple method is product to be detected by LFB (Fig. 2 B figure below).

Visible color method of changing：SAMRS-LAMP produces substantial amounts of pyrophosphate ion while synthetic DNA, the ion It can be combined with the magnesium ion in reaction system, form insoluble material, so as to change the pH value of solution, make reaction mixture Color change.Color change sentence read result can be detected by visual observation, and positive reaction pipe is changed into sky blue from purple, cloudy Property reaction keep purple it is constant, see Fig. 2 B (above).

Fig. 2 B represent for MTC SAMRS-LAMP 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, determine whether cross reaction be present as negative control), the negative amplification of 2B4 expressions (1 microlitre of distilled water replaces template, As blank control).There is positive amplification in only positive control, the LAMP primer of description standard after the modification of SAMRS compositions, Remain able to be expanded, illustrate that the SAMRS-LAMP primers for being directed to the detection MTC of the special IS6110 sequences Designs of MTC can With.

Electrophoresis assays：The product of Fig. 2 B (above) is subjected to electrophoresis detection, due to SAMRS-LAMP amplified production bag The loop-stem structure of many short-movie sections not of uniform size and a series of target sequence composition of inverted repeats and polycyclic cauliflower sample are contained The DNA fragmentation mixture of structure, show the staged collection of illustrative plates of different size zone composition after electrophoresis on gel, see Fig. 2 B (in Figure).By electrophoresis assays interpretation SAMRS-LAMP amplification, there is expected result in positive reaction, and negative anti- Should not occur any amplified band with blank control, the SAMRS-LAMP primers further demonstrated designed by this research are feasible, Detected available for target sequence amplification.

LFB is detected：The product of Fig. 2 B (above) is subjected to LFB detections, due to the SAMRS-LAMP primers detected for MTC (SAMRS-LF*) haptens of mark is Dig, 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-LAMP amplifications, expected result occurs in positive reaction, and negative reaction and blank pair According to only there are CL red stripes (see Fig. 2 B figure below), the designed SAMRS-LAMP-LFB technologies of this research, SAMRS- are demonstrated LAMP primer is feasible, can be used in the detection of purpose target sequence.

4. with different bacterial strain templates and blank control evaluation SAMRS-LAMP methods

64 are reacted for evaluating SAMRS-LAMP reactions, including positive control reaction [signal 1, a reaction tube altogether It is middle to add 2 × 10³The MTC templates of (10 pik) are copied, as positive control], and the reaction of 31 negative controls (signal 2-32, instead Ying Guanzhong adds non-MTC templates, as negative control) and 32 blank control reaction (signal 33-64,1 microlitre of distilled water generations For template, reacted as blank control).In 60 DEG C of constant temperature 1 hour, SAMRS-LAMP did not produced any false positive results (figure 4), or even proliferation time extends to 1.5 hours.Unique positive amplification comes from positive control (signal 1).Compared with LAMP method Speech, SAMRS-LAMP methods do not produce any false positive results.Therefore, SAMRS-LAMP methods are more suitable than the LAMP method of standard Close diagnostic field.

Embodiment 3.AUDG-SAMRS-LAMP reaction systems：

SAMRS-FIP concentration is 40pmol, and SAMRS-BIP concentration is 40pmol, SAMRS-F3 and SAMRS-B3 Concentration is that 10pmol, SAMRS-LF* and SAMRS-LB concentration are 20pmol, 2M Betain, 8mM MgSO₄, 2.5 μ L's 10 × Bst DNA polymerase buffer liquid, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM dCTP, 0.02mM biotin-14-dCTP, 1.4mM dGTP, 10U strand displacement archaeal dna polymerase, 1U South Pole temperature-sensitive uracil take off Oxygen glycosylase enzyme, 1 μ L template, deionized water is added to 25 μ l.Whole reaction constant temperature 60 DEG C 1.5 hours, 80 DEG C 5min terminating reactions.

Embodiment 4. determines the optimal reaction temperature of SAMRS-LAMP technologies

Under the conditions of SAMRS-LAMP reaction systems, MTC templates and designed corresponding SAMRS-LAMP primers are added, Its template concentrations is 2 × 10³Copy/microlitre (10 piks/microlitre).Reaction carries out (58-65 DEG C) under constant temperature, as a result transports Detected with real-time transmissometer, obtain different dynamic curve diagrams at different temperature, see Fig. 5.59-61 DEG C of recommended work For the optimal reaction temperature of SAMRS-LAMP primers.Subsequent authentication in the present invention selects 60 DEG C to be carried out as constant temperature SAMRS-LAMP is expanded.

Embodiment 5. evaluates the sensitivity of SAMRS-LAMP-LFB detections

With the good plasmid of serial dilution (100ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg microlitres~2 × 10⁷, 2 × 10⁴, 2 × 10³, 2 × 10², 2 × 10¹, 2 × 10⁰With 2 × 10^-1Copy/microlitre) carry out SAMRS-LAMP amplified reactions Afterwards, detected with LFB and show result.Detected for MTC, SAMRS-LAMP-LFB detection range is 2 × 10⁷~2 × 10¹Copy Shellfish/microlitre, LFB occurs red line (Fig. 6 A, figure below, 6A1-6A5) 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, 6A6- 6A8).6A1 to 6A7 represents that MTC template amount is 100ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg/ microlitres, 6A8 tables Show blank control (1 microlitre of distilled water).Visualized with LFB read SAMRS-LAMP amplification and real-time turbidity (Fig. 6 A, Upper figure) read result it is consistent.In addition, compared with the LAMP method of standard, the susceptibility and standard of SAMRS-LAMP methods LAMP susceptibility is consistent.Fig. 6 B represent the susceptibility of LAMP method, and upper figure is the susceptibility with real-time Turbidity measurement LAMP, Figure below is the susceptibility that LAMP is detected with LFB.

The South Pole temperature-sensitive uracil picodna glycosyl enzyme (AUDG) of embodiment 6. removes cross pollution

In reaction system, due to adding Brdurd (dUTP), with the progress that SAMRS-LAMP is expanded, own Amplified production be all infiltrated Brdurd (dUTP) (Fig. 7, first stage).When amplified production (penetrates into Brdurd Amplicon) when entering amplification system, AUDG (such as room temperature) under normal temperature condition removes single-stranded or double-stranded middle Brdurd, so as to Single-stranded or double-stranded DNA is set breach (Fig. 7) occur.Because natural template does not include Brdurd, therefore AUDG is to natural DNA is without catalytic action.When carrying out SAMRS-LAMP amplifications, temperature is higher (to be more than 50 DEG C, SAMRS-LAMP reaction temperature is 60 DEG C), degraded with single-stranded or double-stranded DNA jaggy under thermodynamic activity, therefore cannot function as template and expanded, so as to disappear Except the pollution products of reaction system, reach the purpose (Fig. 7, second stage) for removing cross pollution.Further, since AUDG is in temperature When degree is more than 50 DEG C, it can inactivate immediately, so as to the amplified production newly synthesized that can not degrade, even if being urinated in amplified production comprising deoxidation Pyrimidine.Therefore the AUDG enzymes selected in the present invention can be used to eliminate cross pollution, and not influence SAMRS-LAMP normal expansion Increase.

In order to confirm that AUDG can eliminate the amplified production that dUTP penetrates into as effective instrument, SAMRS-LAMP reactions Amplified production is by serial dilution (1 × 10^-13, 1 × 10^-14, 1 × 10^-15, 1 × 10^-16, 1 × 10^-17, 1 × 10^-18With 1 × 10^-19 Gram/microlitre).The amplified production of these dilution factors is used as template, is reacted for SAMRS-LAMP.The nothing in reaction system In the presence of AUDG, the ability that SAMRS-LAMP can detect pollution is 1 × 10^-18(Fig. 8 B, upper figure are fortune to gram/microlitre With real-time turbidity interpretation SAMRS-LAMP result, figure below is the result with LFB interpretations SAMRS-LAMP)；In reaction system In the presence of middle AUDG, the ability that SAMRS-LAMP can detect pollution is 1 × 10^-14(Fig. 8 A, upper figure are gram/microlitre With real-time turbidity interpretation SAMRS-LAMP result, figure below is the result with LFB interpretations SAMRS-LAMP).In conventional feelings Under condition, caused by amplified production, the sub- concentration of pollution that can produce cross reaction is usually 1 × 10^-18Gram/microlitre.The present invention In, AUDG degraded can be up to 1 × 10^-14Gram/microlitre.Therefore the AUDG-SAMRS-LAMP methods in the present invention can effectively disappear Depollution.

Embodiment 7.AUDG-SAMRS-LAMP-LFB Evaluation on specificity

AUDG-SAMRS- is evaluated as template using common mycobacteria type and the genomic nucleic acids of non-branch bacillus The specificity (table 2) of LAMP-LFB technologies.AUDG-LAMP-LFB technologies can accurately identification of M TC member, illustrate AUDG- The specificity of SAMRS-LAMP-LFB methods is good, is shown in Table 3.

The bacterial strain of table 3 and specific detection result

^aMycobacteria refers to bacterium, and these bacterium sources are in 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-LAMP-LFB detections are positive)；N, negative (AUDG-SAMRS-LAMP- LFB detections are negative).Testing result illustrates in table 2, only belongs to MTC member, detects and produces through AUDG-SAMRS-LAMP-LFB Raw positive findings, illustrate that established AUDG-SAMRS-LAMP-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 method for loop-mediated isothermal amplification for keeping away molecular recognition system certainly

<141> 2017-10-22

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Mycobacterium tuberculosis

<400> 1

atcgagcaag ccatctgg 18

<210> 2

<211> 18

<212> DNA

<213> Mycobacterium tuberculosis complex

<400> 2

tcgacatcct cgatggac 18

<210> 3

<211> 41

<212> DNA

<213> Mycobacterium tuberculosis complex

<400> 3

ggatcgatgt gtactgagat cccgcgtact cgacctgaaa g 41

<210> 4

<211> 38

<212> DNA

<213> Mycobacterium tuberculosis complex

<400> 4

agcggtcgga agctcctatg ttgatcagct cggtcttg 38

<210> 5

<211> 20

<212> DNA

<213> Mycobacterium tuberculosis complex

<400> 5

ctatccgtat ggtggataac 20

<210> 6

<211> 18

<212> DNA

<213> Mycobacterium tuberculosis complex

<400> 6

aatgcactag ccgagacg 18

Claims (10)

1. a kind of method of loop-mediated isothermal amplification combination high molecular nanometer bio-sensing testing goal gene, methods described include Following steps：

(1) genome of detected sample is extracted；

(2) primer that 4 bases provided in bit base second from the bottom to the 5th bit base reciprocal at 3 ' ends are modified by SAMRS, The primer includes：SEQ ID NO：Primers F 3 shown in 1, SEQ ID NO：Primer B3, SEQ ID NO shown in 2：Shown in 3 Primers F IP, SEQ ID NO：Primer BIP, SEQ ID NO shown in 4：Primer LF, SEQ ID NO shown in 5：Shown in 6 Primer LB, wherein, select 5 ' ends of any of the above primer to mark haptens；

(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 dideoxycytosine, 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 is primer LF。

3. according to the method for claim 2, it is characterised in that the haptens in 5 ' end marks is digoxin.

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 pad (2), pad (3), nitrocellulose filter (4) and adsorptive pads (5) detection line (41) and control line (42), are set gradually on the nitrocellulose filter (4), in pad (3), detection line (41) and control line (42) region is coated with the high molecular nanometer particles (6) of the Avidin of coloured groups modification, anti-ground height successively Pungent 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 59-61 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 60 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：Primers F 3 shown in 1, SEQ ID NO：Primer B3, SEQ ID NO shown in 2：3 institutes Primers F IP, the SEQ ID NO shown：Primer BIP, SEQ ID NO shown in 4：Primer LF, SEQ ID NO shown in 5：Shown in 6 Primer LB, wherein, select any of the above primer 5 ' end mark 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 primer LF.

10. primer sequence according to claim 9, it is characterised in that the haptens marked is digoxin.