CN107523624B

CN107523624B - Multi-cross isothermal amplification method combining AUDG and self-avoiding molecule recognition system

Info

Publication number: CN107523624B
Application number: CN201710848631.2A
Authority: CN
Inventors: 叶长芸; 王毅; 王艳
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2017-09-19
Filing date: 2017-09-19
Publication date: 2020-06-05
Anticipated expiration: 2037-09-19
Also published as: CN107523624A

Abstract

The invention discloses a multi-cross isothermal amplification method combining Antarctic thermosensitive uracil deoxyribonuclease (AUDG) and a self-avoiding molecule recognition system (SAMRS), which is characterized in that SAMRS modification is carried out on 4 bases from a penultimate base to a penultimate base at the 3 'end of a primer in multi-cross displacement amplification, hapten is marked at the 5' end of an amplification primer C1 or C2, AUDG enzyme, deoxyuracil and biotinylated deoxycytidine are introduced into the amplification system, and an amplification product is detected by combining macromolecular nano biosensing based on a multi-cross displacement amplification technology. The method aims at that the amplification product of IS6110 specific sequence of the mycobacterium tuberculosis complex can be visually detected by a macromolecular nano biosensor. The method is convenient, rapid, sensitive and specific, and is suitable for detecting various nucleotide fragments.

Description

Multi-cross isothermal amplification method combining AUDG and self-avoiding molecule recognition system

Technical Field

The invention discloses a method for detecting a microorganism target gene by multi-cross displacement isothermal amplification, belonging to the technical field of microorganisms and molecular biology.

Background

In the fields of modern biology and medicine, nucleic acid amplification is an indispensable technology, and has been widely used in the fields of basic research, clinical diagnosis, archaeological research, epidemic disease research, transgenic research, and the like. Among the developed nucleic acid amplification techniques, Polymerase Chain Reaction (PCR) is the first established in vitro nucleic acid amplification technique, and has epoch-making significance, and the technique is now widely used in the bio-related field. However, PCR technology is limited by laboratory conditions and relies on complex and expensive thermal cycling equipment for nucleic acid amplification. In addition, the detection of PCR products is complicated, and a set of complicated procedures and equipment are required. These disadvantages limit the widespread use of this technology, particularly in economically lagging regions and in the field of rapid diagnostics. Therefore, there is a great need for the development of simple, rapid and sensitive nucleic acid amplification methods for biological and medical related research fields. In order to overcome the disadvantages of PCR amplification techniques, a number of isothermal amplification techniques have been developed. Compared with PCR technology, the isothermal amplification technology does not depend on thermal cycle amplification equipment, and has high reaction speed and good sensitivity. Therefore, the isothermal amplification technology is beneficial to realizing rapid amplification, on-site diagnosis and convenient detection. There are over 10 kinds of isothermal amplification techniques developed so far, and the techniques are widely used for loop-mediated isothermal amplification (LAMP), cross amplification (CPA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), helicase-dependent isothermal amplification (HDA), and the like. However, these isothermal techniques require multiple enzymes to work simultaneously to achieve nucleic acid amplification, relying on expensive reagents and complex procedures. Therefore, the practicability, convenience and operability of these isothermal amplification methods are to be improved, especially in the field of rapid diagnosis and in less developed areas.

In order to overcome the disadvantages of the PCR technology and the existing isothermal Amplification technology and achieve sensitive, convenient, fast and specific Amplification of nucleic acid sequences, the inventors have recently established a new nucleic acid Amplification technology, named Multiple Cross Displacement Amplification (MCDA), the related content of which is disclosed in CN104946744A, which is a part of the specification of the present application as a prior art document. The MCDA realizes nucleic acid amplification under the condition of constant temperature, only uses a constant temperature displacer, and has the advantages of high amplification speed, sensitive reaction and high specificity.

Similar to loop-mediated isothermal amplification (LAMP) and cross amplification (CPA), the technical bottleneck of MCDA is in the interpretation of the results, i.e. the detection of the amplification products. By far the most common means of product detection mainly include colour indicators, electrophoresis, real-time turbidity. However, these three detection techniques are only applicable to singleplex detection (i.e., detection of a single target). In addition, when the amplification product is interpreted by the color indicator, a result simulating two functions appears, the time consumption of the electrophoresis interpretation result is long, cross contamination is easy to appear, the method is not suitable for field detection, and special instruments and equipment are needed when turbidity interpretation is carried out. In order to overcome the disadvantages of the three detection technologies, the MCDA technology is more widely and economically applied in the fields of biology, medicine and health. Recently, the inventor has developed a nano biosensing technology which relies on the MCDA technology and realizes rapid and sensitive detection by combining the MCDA technology with the nano biosensing technology on the basis of the MCDA, and the technology is named as a multi-Cross isothermal Amplification label-based gold nano specific magnetic Flow Biosensor (MCDA-LFB), and related contents are disclosed in CN 201610872509.4; CN 201610942289.8; CN 201610982015.1; CN201710566164.4, which is incorporated by reference herein as prior art document.

In order to apply the MCDA amplification product to the nano-sensing technology, the traditional strategy is to add two labeled primers in an MCDA amplification system at the same time, wherein one primer is labeled with biotin at the 5 'end, and the other primer is labeled with hapten at the 5' end. When the MCDA amplification is complete, a dual-label amplification product is constructed (the dual-label product is derived from two labeled primers), one end labeled with biotin and the other end labeled with hapten. However, traditional strategies for constructing double-labeled MCDA products can lead to false positive results, even without amplification. The false positive results are derived from hybridization between labeled primers. Therefore, in order to overcome the disadvantages of the traditional labeling strategy, the invention designs a new detection strategy, and the technology only needs one labeled primer to construct a dual-labeled amplification product, so that the amplification product is suitable for the biosensing technology.

In order to adapt the MCDA amplification product for the detection of biosensing technology, opening the reaction tube is a necessary step, which causes a large amount of the amplification product to volatilize in the form of aerosol, thereby causing cross-contamination and producing a false positive result. Furthermore, similar to LAMP and CPA techniques, MCDA can produce false positive results due to technical design, mainly caused by self-assembly between primers, self-assembly within primers, or off-target hybridization. In order to overcome the false positive results caused by the traditional labeling strategy, cross contamination and technical design, in the invention, the inventor utilizes a single-labeled primer, an Antarctic thermosensitive uracil deoxyribosylase and a self-avoiding molecule recognition system, and develops an MCDA combined nano-sensing, self-avoiding molecule recognition system and an Antarctic thermosensitive uracil deoxyribosylase nucleic acid detection technology [ Multiple cross discovery amplification with nanoparticles-based molecular fluorescence biosensor (LFB), and a self-imaging molecular recognition system (SAMRS) and a digital synthesis amplification with specific nucleic acid-DNA-glycosylation enzyme (DG) for a single detection and identification of a nucleic acid and interaction; AUDG-SAMRS-MCDA-LFB ]. In order to verify the feasibility of the AUDG-SAMRS-MCDA-LFB technology, Mycobacterium Tuberculosis Complex (MTC) is applied to the AUDG-SAMRS-MCDA-LFB technology, and an AUDG-SAMRS-MCDA-LFB diagnosis method for detecting MTC is established.

Disclosure of Invention

Based on the above purpose, the present invention firstly provides a method for detecting a target gene by combining multi-cross substitution isothermal amplification with macromolecule nano biosensing, which comprises the following steps:

(1) extracting a genome of a sample to be detected;

(2) providing a primer modified with SAMRS at 4 bases from the penultimate base to the penultimate base at the 3' end, wherein the modification can increase the specificity of the primer so that the primer is specifically combined with the target template; but also reduces the formation of primer dimers and auto-hybridization within primers comprising: and replacement primers F1 and F2, wherein the sequence of the replacement primer F1 is shown in SEQ ID NO: 1, and the sequence of the replacement primer F2 is shown as SEQ ID NO: 2 is shown in the specification; and cross primers CP1 and CP2, wherein the sequence of the cross primer CP1 is shown as SEQ ID NO: 3, the sequence of the cross primer CP2 is shown as SEQ ID NO: 4 is shown in the specification; providing amplification primers C1 and C2, D1 and D2, R1 and R2, wherein the sequence of the primer C1 is shown as SEQ ID NO: 5, the sequence of the primer C2 is shown as SEQ ID NO: 6 is shown in the specification; the sequence of primer D1 is shown in SEQ ID NO: 7, the sequence of the primer D2 is shown as SEQ ID NO: 8, the sequence of the primer R1 is shown as SEQ ID NO: 9, the sequence of the primer R2 is shown as SEQ ID NO: 10, and providing a modified primer labeled with a hapten at the 5' end of any one of the primers; the primer IS designed aiming at the IS6110 specific sequence of the target gene of the mycobacterium tuberculosis complex.

(3) Under the existence of Antarctic thermosensitive uracil deoxyribonuclease, chain-shifted polymerase, a melting temperature regulator, a primer, dNTP and biotinylated deoxyuracil, using genome nucleic acid of a sample to be detected as a template to amplify DNA at constant temperature;

(4) and (4) detecting the amplification product in the step (3) by using a macromolecular nano biosensor.

In a preferred embodiment, the modified primer labeled with hapten at the 5' end is the amplification primer C1.

In a more preferred embodiment, the hapten labeled at the 5' end is fluorescein.

More preferably, the polymeric nano biosensor comprises a back plate 1, wherein a sample plate 2, a binding plate 3, a nitrocellulose membrane 4 and a water absorption plate 5 are sequentially arranged on the back plate 1, a detection line 41 and a control line 42 are sequentially arranged on the nitrocellulose membrane 4, and the binding plate 3, the detection line 41 and the control line 42 are sequentially coated with a colored group modified avidin polymeric nanoparticle 6, an anti-fluorescein antibody 7 and a biotin-coupled bovine serum albumin 8.

In a preferred embodiment, the isothermal amplification is performed in an environment of 60-62 ℃.

In a more preferred embodiment, the isothermal amplification is performed at 61 ℃.

Secondly, the invention also provides a group of primer sequences for isothermal amplification of specific sequences of Mycobacterium tuberculosis complex IS6110, which IS characterized in that the sequences comprise: as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 3, and the cross primer CP1 is shown as SEQ ID NO: 4, as shown in SEQ ID NO: 5, and the amplification primer C1 shown in SEQ ID NO: 6, and the amplification primer C2 is shown as SEQ ID NO: 7, and the amplification primer D1 is shown as SEQ ID NO: 8, and the amplification primer D2 is shown as SEQ ID NO: 9, and the amplification primer R1 is shown as SEQ ID NO: 10, and a modified primer labeled with a hapten at the 5' end of any of the above primers.

In a preferred embodiment, the 4 bases at the 3' end of the primer are modified with SAMRS, where A is modified to 2-aminopurine, T is modified to 2-thiothymine, C is modified to N4-acetylcytosine, and G is modified to hypoxanthine.

In a more preferred embodiment, the modified primer labeled with hapten at the 5' end is the amplification primer C1.

Preferably, the hapten labeled is fluorescein.

The invention uses SAMRS component to modify the primer, and then SAMRS component [ A, 2-aminopurine (2-aminopurine); t, 2-thiothymine (2-thiothymine); c, N4-ethylcytosine (N4-acetylcytosine); g, hypoxanthine (hypoxanthine) ] can only pair with the natural base, i.e. a: t, T: a, C: g, G: C) therefore, after the end of the primer is modified by the SAMRS component, the SAMRS primer can be ensured to be only combined with a corresponding target but not combined with a non-target, so that the specificity of the primer is increased, the specificity of the method is ensured, and false positive results caused by off-target hybridization are eliminated. In addition, the SAMRS component can only pair with natural bases (i.e., A: T, T: A, C: G, G: C), and the SAMRS component cannot pair with each other (i.e., there is no A: T and C: G pair), so that primers containing the SAMRS component cannot hybridize to each other or cannot hybridize to each other within the primers, and hybridization between the primers (i.e., no dimer can be formed between the primers) or self-hybridization within the primers (i.e., no secondary structure can be formed within the primers) is eliminated, thereby ensuring high efficiency and specificity of the primers, and eliminating false positive results due to primer dimer and secondary structure.

The method provided by the invention aims at that the amplification product of IS6110 specific sequence of the mycobacterium tuberculosis complex can be visually detected by a macromolecular nano biosensor. The method is convenient, rapid, sensitive and specific, and is suitable for detecting various nucleotide fragments. The whole reaction amplification time of the invention is only 45 minutes, and the detection range is 2 multiplied by 10⁶～2×10¹Copy/microliter, with extremely high sensitivity. In the present invention, the AUDG degradation ability is 1 × 10^-14Grams per microliter. Therefore, the AUDG-SAMRS-MCDA method provided by the invention can effectively eliminate the pollutants.

The AUDG-SAMRS-MCDA-LFB technology is specifically evaluated by taking common mycobacteria types and nonmycobacteria genome nucleic acid as templates, and the result shows that only the members belonging to MTC generate a positive result through AUDG-SAMRS-MCDA-LFB detection, and no false positive or false negative result is generated, so that the AUDG-SAMRS-MCDA-LFB method established by the invention has excellent specificity.

Drawings

FIG. 1A schematic representation of the principle of MCDA amplification;

FIG. 1B is a schematic structural diagram of a polymer nano biosensor;

FIG. 1C is a diagram showing the detection result of the polymer nano biosensor;

FIG. 2A is a map of the results of MCDA primer validation;

FIG. 2B is a graph of the results of SAMRS-MCDA primer validation;

FIG. 3. evaluation of MCDA using different strain templates and a blank control;

FIG. 4.SAMRS-MCDA evaluation using different strain templates and a blank;

FIG. 5 is a graph of results of standard SAMRS-MCDA optimum reaction temperature tests;

FIG. 6A is a graph of sensitivity results for the SMARS-MCDA method;

FIG. 6B is a graph of sensitivity results for the MCDA method;

FIG. 7 is a schematic diagram of the principle of the AUDG-SAMRS-MCDA decontamination molecule;

FIG. 8A. AUDG-SAMRS-MCDA decontaminate daughter results map;

FIG. 8B SAMRS-MCDA decontaminate daughter results map in the absence of AUDG;

FIG. 9 is a graph of the results of the optimal response time test of the AUDG-SAMRS-MCDA-LFB technique

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

1. Reagents involved in the invention:

the backing sheet, sample pad, gold pad, fibrous membrane and absorbent pad were purchased from Jie-Yi company. Anti-digoxin antibody (anti-Dig), biotinylated calf serum (B-BSA) was purchased from Abcam. Chromogen (purple red) modified, avidinated polymeric nanoparticles (Dye streptavidin coated polymer nanoparticles, SA-DNPs, 129nm) were purchased from Bangs Laboratories. DNA extraction kits (QIAamp DNA minikites; Qiagen, Hilden, Germany) were purchased from Qiagen, Germany. Isothermal amplification reagents and color-developing agents (HNB) were purchased from Zhengyuan Biotech, Inc. of Heitai, Beijing. Biotinylated deoxycytidine (Biotin-14-dCTP), deoxyuracil (dUTP) was purchased from Thermo Scientific. Antarctic thermosensitive uracil deoxyribosylase (AUDG), deoxycytidine (dCTP), deoxythymine (dTTP), deoxyadenine (dATP) and deoxyguanine (dGTP) were purchased from NewEngland Biolabs. DL50DNA Marker was purchased from Takara Bio Inc. The other reagents were all commercial analytical pure products.

The main instruments used in the experiment of the invention: constant temperature real time turbidimeter LA-320C (Eiken Chemical Co., Ltd., Japan) was purchased from Japan Rongyan Co. The electrophoresis equipment is a product of Beijing Junyi Oriental electrophoresis equipment Co.Ltd; the Gel imaging system was Bio-Rad Gel Dox XR, product Bio-Rad, USA.

2. Designing a primer:

in order to verify and evaluate the AUDG-SAMRS-MCDA-LFB technology and establish a rapid, sensitive and specific AUDG-SAMRS-MCDA-LFB detection system aiming at MTC pathogens. The invention designs an amplification primer aiming at the specific insertion sequence 6110(IS 6110; Genbank No. X17348) of the MTC, and aims to verify the feasibility, sensitivity, specificity and reliability of the AUDG-SAMRS-MCDA-LFB technology.

The primer design result is as follows:

IS6110 specific sequence exists in all MTC members, the specificity IS good, and the MTC can be distinguished from other closely similar mycobacteria. MCDA primers are designed by using Primer design software PrimeExplorer V4(Eiken Chemical) (http:// PrimeExplorer. jp/e /) and Primer design software Primer Premier 5.0, and sequence alignment analysis is carried out on the obtained specific primers in NCBI database (http:// blast. NCBI. nlm. nih. gov/blast. cgi) to exclude possible non-specific matching of the primers and other species sequences, and finally optimized MCDA amplification primers are obtained and are used for detecting all members of MTC. On the basis of the standard MCDA primer, the SAMRS component is used for modifying the standard MCDA primer to construct a SAMRS-MCDA primer, and the SAMRS-MCDA primer is used for establishing SAMRS-MCDA amplification. Standard MCDA and SAMRS-MCDA primer sequences and modifications are shown in Table 1.

TABLE 1 Standard MCDA primer sequences

C1, FITC at the 5' end (this primer was used in MCDA-LFB detection system); FITC, fluorescein isothiocyanate (fluorescein).

nt, nucleic (nucleotide); mer, monomeric (monomeric units).

SAMRS-MCDA primer sequence modifications are shown in Table 2.

TABLE 2 SAMRS-MCDA primer sequence modifications

SAMRS, self-association molecular recognition system (Sedan molecule recognition System). SAMRS-C1, 5' labeled FITC (this primer was used in AUDG-SAMRS-MCDA-LFB detection system). A, 2-aminopurine (2-aminopurine); t, 2-thiothymine (2-thiothymine); c, N4-ethylcytosine (N4-acetylcytosine); g, hypoxanthine.

3. Design of lateral flow nanosensors (LFBs)

The design of the lateral flow nanosensor (LFB) is shown in FIG. 1B. The LFB comprises a back plate 1, a sample plate 2, a binding plate 3, a nitrocellulose membrane 4 and a water absorption plate 5 are sequentially arranged on the back plate 1, and a detection line 41 and a control line 42 are sequentially arranged on the nitrocellulose membrane 4. In FIG. 1B, Biotin is Biotin, BSA is bovine serum albumin, Biotin-BSA is biotinylated bovine serum albumin, FITC is fluorescein, anti-FITC is an anti-fluorescein antibody, FITC/Biotin-labelledtarget amplicon is fluorescein-and Biotin-labeled amplicon, Polymer nanoparticles are Polymer nanoparticles, SA is avidin, and SA-DNP is avidin-labeled Polymer nanoparticles. Firstly, a sample plate, a combination plate, a fiber membrane and a water absorption plate are sequentially assembled on a back plate. Then, SA-DNPs, anti-FITC and B-BSA are respectively coated on a gold label pad, a detection line 41(TL1) and a control line 42(CL), and the gold label pad, the detection line and the control line are dried for later use

Detection principle of LFB: the MCDA product was added dropwise to the sample pad area of the LFB, followed by the addition of a buffer drop to the sample pad area of the LFB. The MCDA product moves from bottom to top (from the sample pad to the absorbent pad) under the action of the siphon. When the MCDA product reaches the conjugate pad, biotin labeled onto the product reacts with the SA-DNPs. When the product continues to move, one end (hapten labeled end, namely FITC labeled end) of the double-labeled product is combined with the antibody (namely anti-FITC antibody) in the TL (detection line) area, and the double-labeled product is fixed in the detection line area. As the products accumulate in the detection line region, a color reaction proceeds by the bound SA-DNPs, thereby performing visual detection of the MCDA products. In addition, excess SA-DNPs can react with B-BSA in the CL (quality control line) region to perform direct color reaction, and the function of LFB can be judged to be normal.

Interpretation of LFB results (fig. 1C): a red band appeared in the CL region only, indicating a negative control, with no positive product (II); the CL and TL regions show simultaneous red bands, indicating a positive result (I) for detection of the target; when the LFB does not have the red line strip, indicating that the LFB is failed; when red stripes appear on TL, CL has no red stripes, representing that the results are not feasible and need to be re-detected.

Interpretation of the results of MCDA and SAMRS-MCDA amplification:

after amplification of MCDA and SAMRS-MCDA, three detection methods were used for the amplification discrimination. First, a visible dye (e.g., HNB reagent) is added to the reaction mixture, the color of the positive reaction tube changes from purple to sky blue, and the original purple color of the negative reaction tube is maintained. Secondly, the MCDA product can be subjected to agarose electrophoresis and then the amplicon is detected, and because the product contains amplified fragments with different sizes, the electrophoresis pattern of the positive amplified product is in a specific ladder shape, and no band appears in the negative reaction. A more straightforward and simple method is to detect the product by LFB.

Visual color change method: MCDA and SAMRS-MCDA synthesize DNA and generate a large amount of pyrophosphate ions which can be combined with magnesium ions in a reaction system to form insoluble substances, so that the pH value of the solution is changed, and the color of a reaction mixed solution is changed. The results can be interpreted by visually detecting a color change, with the positive reaction tube changing from purple to sky blue and the negative reaction remaining unchanged purple, as shown in FIGS. 2A (left panel) and 2B (left panel).

Example 1 MCDA amplification

Principle of MCDA reaction

The MCDA reaction system comprises 10 primers, 10 regions for recognizing target sequences, and 2 crossed internal primers, namely CP1 and CP2(Cross Primer, CP), 2 Displacement primers (Displacement Amplification), namely F1 and F2, and 6 Amplification primers, namely D1, C1, R1, D2, C2 and R2. To construct detectable products, any of the 10 primers were selected, labeled at the 5' end with hapten (FITC, fluorescein), and the newly labeled primers were designated F1, F2, CP1, CP2, C1, C2, D1, D2, R1 and R2. In the present invention, the principle of the present invention is illustrated by an example of C1.

Under a predetermined constant temperature condition, the double-stranded DNA in the reaction system is in a dynamic equilibrium state of half dissociation and half binding, and when any one primer performs base pairing extension to the complementary part of the double-stranded DNA, the other strand dissociates to become a single strand. First, under the action of Bst 2.0DNA polymerase, the CP1 primer P1 segment 3' end was used as the starting point, and the primer was paired with the corresponding DNA complementary sequence, thereby initiating strand displacement DNA synthesis (FIG. 1A, step 1). The upstream F1 primer displaced the product formed by amplification of CP1 primer, which was able to bind five primers simultaneously (C1 ×, D1, R1, CP2 and F2, step 2). When the C1 primer anneals to the target sequence, biotinylated deoxycytidine (Biotin-14-dCTP) was integrated into the amplicon by Bst 2.0 dnase. As MCDA amplification proceeded, a large amount of ditag (hapten labeled at one end, Biotin labeled in the middle) was formed, which originated from labeled C1 primer and biotinylated deoxycytidine (Biotin-14-dCTP) that had infiltrated the amplicon. The double-labeled product can be detected by a polymer nano biosensor, so that visual detection is carried out.

In FIG. 1A, FITC is fluorescein, C1 is C1 primer, C1 is fluorescein-labeled C1 primer, Biotin is Biotin, and Biotin-14-dCTP is Biotin-labeled dCTP.

Detailed MCDA amplification is described in the inventor's prior patent CN 104946744A.

2. Standard MCDA reaction system:

the concentration of the cross primer CP1 was 40pmol, the concentration of the cross primer CP2 was 40pmol, the concentrations of the displacement primers F1 and F2 were 10pmol, the concentrations of the amplification primers C1, C2, R1, R2, D1 and D2 were 20pmol, 2M Betain, 8mM MgSO₄2.5. mu.L of 10 XBst DNA polymerase buffer, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM dCTP, 0.02mM biotin-14-dCTP, 1.4mM dGTP, 10U of strand-displacing DNA polymerase, 1U of Antarctic thermosensitive uracil deoxyribonuclease, 1. mu.L of template, supplemented with deionized water to 25. mu.L. The whole reaction was kept at a constant temperature of 61 ℃ for 1 hour and 80 ℃ for 5min to terminate the reaction.

3. Verification of feasibility of MCDA primers

FIG. 2A shows the validation of MCDA primers for MTC, 2A1 shows positive amplification [ 2X 10 added to reaction tube³Copy (10pg) of MTC template as a positive control]2A2 shows negative amplification (Staphylococcus aureus template was added to the reaction tube and cross reaction was detected as a negative control), 2A3 shows negative amplification (Klebsiella pneumoniae template was added to the reaction tube and a negative control was used), and 2A4 shows negative amplification (1. mu.l of double distilled water was used instead of the template and used as a blank control). Only the positive control showed positive amplification, indicating that MCDA primers designed for MTC-specific IS6110 sequences to detect MTC were available.

Electrophoresis detection method: the product of 2A (left panel) is subjected to electrophoresis detection, and as the amplification product of MCDA comprises a DNA fragment mixture of a stem-loop structure and a multi-loop cauliflower-like structure formed by a plurality of short fragments with different sizes and a series of target sequences with inverted repeats, a stepwise pattern formed by zones with different sizes is displayed on a gel after electrophoresis, which is shown in figure 2A (right panel). The amplification result of MCDA is judged by an electrophoresis detection method, the expected result appears in positive reaction, and no amplification band appears in negative reaction and blank control, so that the feasibility of MCDA and SAMRS-MCDA primers designed in the research is further verified, and the primers can be used for target sequence amplification detection.

LFB detection: the product of 2A (left panel) was subjected to LFB detection, and since the MCDA primer (C1) labeled hapten for MTC detection was FITC, when TL and CL appeared as red bands, it was indicated as MTC detection positive. The MCDA amplification result is judged by an LFB detection method, the expected result appears in a positive reaction, but only CL red bands appear in a negative reaction and a blank control, and the MCDA-LFB technology and MCDA primers designed by the research are feasible and can be used for detecting the target sequence (figure 2A, middle graph).

4. The MCDA evaluation method by using different strain templates and blank controls:

a total of 64 reactions were used to evaluate MCDA, including a positive control reaction [ signal 1, 2X 10 added to the reaction tube³Copy (10pg) of MTC template as a positive control]31 negative control reactions (signal 2-32, non-MTC template added to the reaction tube as negative control) and 32 blank control reactions (signal 33-64, 1 microliter of double distilled water instead of template as blank control reaction). MCDA incubated at 61 ℃ for 1 hour yielded a total of four false positive results (FIG. 3). Two of these false positive results are referred to as non-specific amplifications resulting from off-target hybridization between the MCDA primers and the non-specific template (signals 18 and 25). The remaining two false positive results are called self-amplifications, which result from self-matching between or within primers, without any template in the primer reaction (signals 37 and 57). Thus, standard MCDA can lead to false positive results.

EXAMPLE 2 SAMRS-MCDA reaction System

Principle of SAMS-MCDA reaction

SAMS-MCDA is based on the same reaction principle as that of ordinary MCDA, and only the SAMRS component is used for modifying the ordinary MCDA primer, so that the specificity of the primer is enhanced, and the specificity of the method is enhanced, and the false positive result caused by off-target hybridization and primer dimer is eliminated.

SAMS-MCDA reaction System

The concentration of cross-primer SAMRS-CP1 was 40pmol, the concentration of cross-primer SAMRS-CP2 was 40pmol, the concentration of displacement primers SAMRS-F1 and SAMRS-F2 was 10pmol, the concentration of amplification primers SAMRS-C1, SAMRS-C2, SAMRS-R1, SAMRS-R2, SAMRS-D1 and SAMRS-D2 was 20pmol, 2M Betain, 8mM MgSO 3₄2.5. mu.L of 10 XBst DNA polymerase buffer, 1.4mM dATP, 0.7mM dTTP, 0.7mM dUTP, 1.38mM dCTP, 0.02mM biotin-14-dCTP, 1.4mM dGTP, 10U of strand-displacing DNA polymerase, 1U of Antarctic thermosensitive uracil deoxyribonuclease, 1. mu.L of template, supplemented with deionized water to 25. mu.L. The whole reaction was kept at a constant temperature of 61 ℃ for 1 hour and 80 ℃ for 5min to terminate the reaction.

3. Verifying the feasibility of the SAMRS-MCDA primer:

FIG. 2B shows validation of SAMRS-MCDA primers for MTC, and 2B1 shows positive amplification (2X 10 added to reaction tube)³Copied MTC template as a positive control), 2B2 for negative amplification (staphylococcus aureus template added to the reaction tube and a negative control to determine whether there is a cross reaction), 2B3 for negative amplification (klebsiella pneumoniae template added to the reaction tube and a negative control), and 2B4 for negative amplification (1 μ l of double distilled water instead of template as a blank control). Only the positive control shows positive amplification, which indicates that the standard MCDA primer can still be amplified after being modified by SAMRS component, and indicates that the SAMRS-MCDA primer for detecting MTC, which IS designed aiming at the MTC specific IS6110 sequence, can be used.

Electrophoresis detection method: the products of 2B (left panel) were electrophoretically detected, and since the SAMRS-MCDA amplification product contained a mixture of many short fragments of varying sizes and a series of DNA fragments with stem-loop structure and multi-loop cauliflower-like structure composed of the target sequence in inverted repeat, a stepwise pattern composed of bands of different sizes was visualized on the gel after electrophoresis, as shown in FIG. 2B (right panel). The amplification result of SAMRS-MCDA is judged by an electrophoresis detection method, the expected result appears in positive reaction, and no amplification band appears in negative reaction and blank control, so that the SAMRS-MCDA primer designed by the research is further verified to be feasible and can be used for target sequence amplification detection.

LFB detection: the product of FIG. 2B (left panel) was subjected to LFB detection. Since the hapten labeled with SAMRS-MCDA primer (SAMRS-C1) for MTC detection is FITC, when TL and CL appear as red bands, it is indicated as positive for MTC detection. The SAMRS-MCDA amplification results were read by LFB assay, positive reactions gave the expected results, while negative reactions and blank controls gave only CL red bands, confirming that the SAMRS-MCDA-LFB technique designed in this study, the SAMRS-MCDA primers were viable and could be used for the detection of the target sequence of interest (FIG. 2B panel).

4. Method for evaluating SAMRS-MCDA (S-specific amplified polymorphic DNA) by using different strain templates and blank control

A total of 64 reactions were used to evaluate the SAMRS-MCDA reaction, including a positive control reaction [ signal 1, addition of 2X 10 to the reaction tube³Copy (10pg) of MTC template as a positive control]31 negative control reactions (signal 2-32, non-MTC template added to the reaction tube as negative control) and 32 blank control reactions (signal 33-64, 1 microliter of double distilled water instead of template as blank control). SAMRS-MCDA did not produce any false positive results (FIG. 4) even when incubated at 61 ℃ for 1 hour, even with an extension of the amplification time to 1.5 hours. The only positive amplification was from the positive control (signal 1). The SAMRS-MCDA method does not produce any false positive results compared to the MCDA method. Therefore, the SAMRS-MCDA method is more suitable for the field of detection than the standard MCDA method.

Example 3 determination of optimal reaction temperature for SAMRS-MCDA technology

Adding MTC template and designed corresponding SAMRS-MCDA primer under the condition of SAMRS-MCDA reaction system, wherein the template concentration is 2 x 10³Copies/microliter (10 pg/microliter). The reaction was carried out at constant temperature (59-66 ℃), and the results were measured using a real-time turbidimeter, giving different dynamic profiles at different temperatures, see FIG. 5. 60-62 ℃ is recommended as the best SAMRS-MCDA primerThe reaction temperature. Subsequent validation in the present invention selects 61 ℃ as isothermal condition for SAMRS-MCDA amplification.

Example 4 evaluation of sensitivity of SAMRS-MCDA-LFB detection

Serially diluted templates (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100 aq/microliter-2X 10. mu.l) were used⁶，2×10³，2×10²，2×10¹，2×10⁰，2×10^-1And 2X 10^-2Copy/microliter) was subjected to SAMRS-MCDA amplification reaction, and the results were shown by LFB detection. For MTC detection, the detection range of SAMRS-MCDA-LFB is 2 x 10⁶～2×10¹Copy/microliter, LFB appeared as red lines at the TL and CL regions (FIG. 6A, bottom panel, 6A1-6A 4). When the amount of the genomic template in the reaction system was reduced to 2X 10⁰At copy and below, LFB appeared red only in the CL region, indicating a negative result (FIG. 6A, bottom panel, 6A5-6A 8). 6A1 to 6A7 indicated template amounts of MTC of 10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100 aq/microliter, 6A8 indicated a blank control (1 microliter double distilled water). The amplification results of SAMRS-MCDA read using LFB visualization were consistent with HNB (FIG. 6A, middle panel) and real-time turbidity (FIG. 6A, top panel) readings. Furthermore, the sensitivity of the SAMRS-MCDA method is consistent with that of standard MCDA, as compared to the standard MCDA method. FIG. 6B shows the sensitivity of the MCDA method, the top graph shows the sensitivity of detecting MCDA using real-time turbidity, the middle graph shows the sensitivity of detecting MCDA using HNB, and the bottom graph shows the sensitivity of detecting MCDA using LFB.

Example 5 Antarctic thermosensitive uracil deoxyribonuclease (AUDG) removal Cross contamination evaluation

In the reaction system, as the SAMRS-MCDA amplification proceeds, all amplification products are infiltrated with deoxyuracil (dUTP) due to the addition of deoxyuracil (dUTP). When the amplification product (amplicon permeated with deoxyuracil) enters the amplification system, the AUDG removes deoxyuracil from single-stranded or double-stranded DNA under normal temperature conditions (e.g., room temperature), thereby nicking the single-stranded or double-stranded DNA (FIG. 7, Nucleic acid, target Nucleic acid; Carryover conjugation, cross-contamination; Hapten, Hapten; Hapten-labeled primer, Hapten-labeled primer; Bst 2.0, Bst 2.0DNA polymerase; AUDG, AUDG enzyme; Biotin, Biotin; dCTP, deoxycytidine; Biotin-14-dCTP, biotinylated deoxycytidine; dUTP, deoxyuracil). Since the native template does not contain deoxyuracil, AUDG does not catalyze native DNA. When MCDA amplification is carried out, the temperature is higher (more than 60 ℃), and single-stranded or double-stranded DNA with a gap is degraded under the action of heat, so that the single-stranded or double-stranded DNA cannot be used as a template for amplification, and therefore, a pollution product of a reaction system is eliminated, and the purpose of removing cross contamination is achieved. Furthermore, AUDG is immediately inactivated at temperatures greater than 50 ℃ and thus cannot degrade newly synthesized amplification products, even if deoxyuracil is included in the amplification products. Therefore, the AUDG enzyme selected in the invention can be used for eliminating cross contamination without influencing the normal amplification of MCDA.

To confirm that AUDG can be an effective tool to eliminate dUTP-infiltrated amplification products, the amplification products of the SAMRS-MCDA reaction were serially diluted (1X 10)^-12，1×10^-13，1×10^-14，1×10^-15，1×10^-16，1×10^-17，1×10^-18And 1X 10^-19Grams/microliter). The respective dilutions of the amplification product were used as templates for SAMRS-MCDA reactions. SAMRS-MCDA has a contaminant detection capability of 1X 10 in the absence of AUDG in the reaction system^-18Grams/microliter (FIG. 8B, top panel shows the results of interpreting SAMRS-MCDA using real-time turbidity, middle panel shows the results of interpreting SAMRS-MCDA using HNB, and bottom panel shows the results of interpreting SAMRS-MCDA using LFB); SAMRS-MCDA has a contaminant detection ability of 1X 10 in the presence of AUDG in the reaction system^-14Grams/microliter (FIG. 8A, top panel shows the results of interpreting SAMRS-MCDA using real-time turbidity, middle panel shows the results of interpreting SAMRS-MCDA using HNB, and bottom panel shows the results of interpreting SAMRS-MCDA using LFB). In the conventional case, the concentration of contaminants which are caused by amplification products and which are capable of generating cross-reactions is generally 1X 10^-18Grams per microliter. In the invention, the degradation energy of AUDG can reach 1 × 10^-14Grams per microliter. Therefore, the AUDG-SAMRS-MCDA method provided by the invention can effectively eliminate the pollutants.

Example 6 determination of optimal reaction time for the AUDG-SAMRS-MCDA-LFB technique

Under the condition of an AUDG-SAMRS-MCDA reaction system, SAMRS-MCDA primers aiming at MTC amplification and diluted MTC templates (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 100 aq/microliter-2X 10 are added simultaneously⁶，2×10³，2×10²，2×10¹，2×10⁰，2×10^-1And 2X 10^-2Copy/microliter). The reaction was carried out at constant temperature (61 ℃ C.) for 25 minutes, 35 minutes, 45 minutes and 55 minutes, respectively. LFB detection is used for displaying that: the optimal reaction time for the AUDG-SAMRS-MCDA-LFB technique to detect the target is 45 minutes (FIG. 9). When the AUDG-SAMRS-MCDA system was incubated for 45 minutes during the amplification step, the lowest detection-limiting level of template could be detected (FIG. 9C). FIG. 9C shows that LFB detection ranged from 10ng to 100fg, and the LFB appeared as red lines in the TL and CL areas (LFB1-LFB 4). When the amount of the genomic template in the reaction system was decreased to 10fg and below, LFB appeared as a red line only in the CL region, indicating a negative result (LFB5-LFB 7). FIG. 9 shows the LFB visualization reading of the amplification results of the AUDG-SAMRS-MCDA system from 25 minutes to 55 minutes; LFB1 to LFB7 indicate template amounts of 10ng, 10pg, 1pg, 100fg, 10fg, 1fg, 100aq for MTC; LFB8 represents a blank control (1 μ l double distilled water).

Example 7 evaluation of specificity of the AUDG-SAMRS-MCDA-LFB technique

The specificity of the AUDG-SAMRS-MCDA-LFB technique was evaluated using the genomic nucleic acids of common mycobacterial and nonmycobacterial species as templates (Table 3). The AUDG-SAMRS-MCDA-LFB technology can accurately detect MTC members, and the AUDG-SAMRS-MCDA-LFB method is good in specificity and is shown in Table 2.

TABLE 3 strains and results of specificity detection

^aReference strain of mycobacterium, derived from tuberculosis reference laboratory in China.

^bATCC, American Type Culture Collection (American Type Culture Collection); ZG-CDC, zing Center for Disease Control and preservation (from the Disease Prevention Control Center of Gong City); ICDC, National Institute for communicative Disease Control and preservation, Chinese Center for Disease Control and preservation (China Center for Disease Prevention and Control, Institute for infectious Disease Prevention and Control).

^cP, positive (AUDG-SAMRS-MCDA-LFB detection positive); n, negative (AUDG-SAMRS-MCDA-LFB detection negative). The detection results in the table 2 show that only the members belonging to the MTC generate positive results through the AUDG-SAMRS-MCDA-LFB detection, and the established AUDG-SAMRS-MCDA-LFB method can accurately identify the MTC without generating false positive and false negative results.

Sequence listing

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Claims

1. A method for detecting a target gene by combining multi-cross isothermal amplification and macromolecular nano biosensing of non-diagnostic purposes, which comprises the following steps:

(1) extracting a genome of a sample to be detected;

(2) providing a primer in which 4 bases from the penultimate base to the penultimate base at the 3' end are modified with SAMRS, A is modified with 2-aminopurine, T is modified with 2-thiothymine, C is modified with N4-acetylcytosine, and G is modified with hypoxanthine, the primer comprising: and replacement primers F1 and F2, wherein the sequence of the replacement primer F1 is shown in SEQ ID NO: 1, and the sequence of the replacement primer F2 is shown as SEQ ID NO: 2 is shown in the specification; and cross primers CP1 and CP2, wherein the sequence of the cross primer CP1 is shown as SEQ ID NO: 3, the sequence of the cross primer CP2 is shown as SEQ ID NO: 4 is shown in the specification; providing amplification primers C1 and C2, D1 and D2, R1 and R2, wherein the sequence of the primer C1 is shown as SEQ ID NO: 5, the sequence of the primer C2 is shown as SEQ ID NO: 6 is shown in the specification; the sequence of primer D1 is shown in SEQ ID NO: 7, the sequence of the primer D2 is shown as SEQ ID NO: 8, the sequence of the primer R1 is shown as SEQ ID NO: 9, the sequence of the primer R2 is shown as SEQ ID NO: 10, and providing a modified primer labeled with a hapten at the 5' end of any one of the primers;

2. The method according to claim 1, wherein the modified primer labeled with hapten at the 5' end is amplification primer C1.

3. The method of claim 2, wherein the hapten labeled at the 5' end is fluorescein.

4. The method according to claim 3, wherein the polymeric nanobiosensor comprises a back plate (1), a sample plate (2), a binding plate (3), a nitrocellulose membrane (4) and a water absorption plate (5) are sequentially disposed on the back plate (1), a detection line (41) and a control line (42) are sequentially disposed on the nitrocellulose membrane (4), and the binding plate (3), the detection line (41) and the control line (42) are sequentially coated with the colored group modified avidinated polymeric nanoparticles (6), the anti-fluorescein antibody (7) and the biotin-coupled bovine serum albumin (8).

5. The method of claim 1, wherein the isothermal amplification is performed in an environment of 60-62 ℃.

6. The method of claim 5, wherein the isothermal amplification is performed in an environment of 61 ℃.

7.A group of primer sequences for isothermal amplification of specific sequences of Mycobacterium tuberculosis complex IS6110 IS characterized by comprising the following sequences: as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 3, and the cross primer CP1 is shown as SEQ ID NO: 4, as shown in SEQ ID NO: 5, and the amplification primer C1 shown in SEQ ID NO: 6, and the amplification primer C2 is shown as SEQ ID NO: 7, and the amplification primer D1 is shown as SEQ ID NO: 8, and the amplification primer D2 is shown as SEQ ID NO: 9, and the amplification primer R1 is shown as SEQ ID NO: 10 and a modified primer labeled with a hapten at the 5' end of any of the above primers, wherein a is modified to 2-aminopurine, T is modified to 2-thiothymine, C is modified to N4-acetylcytosine, and G is modified to hypoxanthine.

8. The primer sequence of claim 7, wherein the modified primer labeled with hapten at the 5' end is amplification primer C1.

9. The primer sequence of claim 8, wherein the hapten is fluorescein.