CN110819728A - Detection technology combining multi-cross displacement amplification and gold nano detection and application - Google Patents
Detection technology combining multi-cross displacement amplification and gold nano detection and application Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a detection technology combining multi-cross displacement amplification and gold nano detection and application thereof in gene detection, wherein a marker is marked at the 5' end of an amplification primer C1 in the multi-cross displacement amplification, and the method can be used for visually detecting amplification products of specific genes pgaD (labeled fluorescein isothiocyanate FITC) of acinetobacter baumannii and carbapenem drug-resistant genes blaOXA-23-like (labeled digoxigenin Dig) of the acinetobacter baumannii by a gold nano biosensor.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a detection technology combining multi-cross displacement amplification and gold nano detection and application thereof.
Background
Nucleic acid detection, i.e., nucleic acid amplification detection technology, generally refers to detection technology that screens specific genes by means of amplifying DNA or RNA, and nucleic acid amplification technology includes PCR amplification technology, isothermal amplification technology, etc., and compared with PCR and its derivative technology, isothermal amplification technology has the characteristics of no dependence on thermocycling amplification equipment, constant fixed temperature in the whole amplification process, fast reaction speed, strong sensitivity, high specificity, etc., and is beneficial to realizing fast amplification, convenient detection and on-site diagnosis. At present, the isothermal amplification technologies widely applied include Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), helicase dependent isothermal amplification (HDA), loop-mediated isothermal amplification (LAMP), cross amplification (CPA) and the like, and the detection is not convenient, fast, sensitive and specific.
Disclosure of Invention
In view of the above problems, the present invention has been made to provide a detection technique combining multiple cross-over displacement amplification and gold nano-detection and its application in gene detection that overcomes or at least partially solves the above problems.
The embodiment of the invention provides a detection technology combining multi-cross displacement amplification and gold nano detection, which comprises the following steps:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
Further, the marker includes one of: fluorescein isothiocyanate FITC, digoxigenin Dig.
Further, in the MCDA reaction, the reaction temperature is constant and ranges from 61 ℃ to 65 ℃.
Further, the temperature was 63 ℃.
Further, in the detection, the detection object and the detection buffer solution are sequentially dripped on the surface of the detection device.
Further, the components of the detection apparatus include: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
Based on the same invention concept, the embodiment of the invention also provides application of a detection technology combining multi-cross displacement amplification and gold nano detection, which is used for detecting the acinetobacter baumannii specific gene pgaD and the acinetobacter baumannii carbapenem drug-resistant gene blaOXA-23-like.
Further, in the above-mentioned case,
for detecting said pgaD, the label is said FITC and the substance in the gold-labeled pad comprises one of: said SA-G, said B-BSA, said anti-FITC;
when the blaOXA-23-like is used for detecting the blaOXA-23-like, the marker is the Dig, and substances in a gold-labeled pad comprise one of the following substances: said SA-G, said B-BSA, said anti-Dig.
Further, in the above-mentioned case,
for the detection of said pgaD, the primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, and an amplification primer C1 shown in SEQ ID NO: 4, and the amplification primer D1 is shown as SEQ ID NO: 5, and the amplification primer R1 is shown as SEQ ID NO: 6, and the amplification primer R2 is shown as SEQ ID NO: 7, and the amplification primer D2 is shown as SEQ ID NO: 8, and the amplification primer C2 is shown as SEQ ID NO: 9, and the cross primer CP2 is shown as SEQ ID NO: 10, and a replacement primer F2;
when used to detect the blaOXA-23-like, primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 11, and a replacement primer F1 shown as SEQ ID NO: 12, as shown in seq id NO: 13, and an amplification primer C1 shown in SEQ ID NO: 14, and the amplification primer D1 shown in SEQ ID NO: 15, and the amplification primer R1 is shown as SEQ ID NO: 16, and the amplification primer R2 is shown as SEQ ID NO: 17, and an amplification primer D2 shown in SEQ ID NO: 18, and the amplification primer C2 is shown as SEQ ID NO: 19, and a cross primer CP2 shown as SEQ ID NO: 20, substitution primer F2.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
according to the detection method provided by the embodiment of the invention, markers are marked at the 5' end of an amplification primer C1 in multi-cross displacement amplification, and the method can be used for visually detecting amplification products of specific genes pgaD (labeled fluorescein isothiocyanate FITC) of acinetobacter baumannii and carbapenem drug-resistant genes blaOXA-23-like (labeled digoxigenin Dig) of the acinetobacter baumannii by a gold nano biosensor.
The detection principle of MCDA is based on the fact that DNA is in a dynamic equilibrium state at about 65 ℃, when any primer extends towards the complementary part of double-stranded DNA in a base pairing mode, the other strand is dissociated and becomes a single strand, under the premise, different specific primers are utilized to identify a specific region of a target gene, under the action of strand displacement type DNA polymerase, the 3' tail end of an outer primer section is used as a starting point to pair with a template DNA complementary sequence, and strand displacement DNA synthesis is started.
The LFB detection principle is based on the binding of an antibody (coated on the LFB) and a hapten (labeled at the 5' end of the primer). When positive amplification products exist, the hapten marked amplification sequences are combined with the antibodies coated on the detection line area and are red, so that the amplification products are visually detected. Compared to other methods of detection of amplification products, LFB is relatively simple, rapid and objective, and can display the amplification result in a few minutes.
In conclusion, the MCDA amplification technology has exponential amplification effect on the original signal, and the LFB detection technology is simple, rapid and objective. The combination of MCDA amplification technology and LFB detection technology can amplify the original nucleic acid sequence exponentially, greatly improve the detection precision, shorten the detection time and realize the accurate detection of target molecules. Compared with the existing detection method, the MCDA-LFB can more quickly and accurately identify the drug resistance of the acinetobacter baumannii and the carbapenem thereof.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 is a schematic diagram showing the position and orientation of the MCDA primer design in the example of the present invention, wherein A represents pgaD and B represents blaOXA-23-like;
FIG. 2 is a schematic diagram illustrating the principle of MCDA amplification in an embodiment of the present invention;
FIG. 3 is a schematic diagram of detection of a gold nano biosensor in an embodiment of the invention;
FIG. 4 is a map of the results of the validation of MCDA primers (pgaD and blaOXA-23-like) in the examples of the present invention;
FIG. 5 is a graph showing the results of an optimum reaction temperature test of MCDA (pgaD and blaOXA-23-like) in the example of the present invention;
FIG. 6 is a graph showing the results of detecting the sensitivity of Acinetobacter baumannii and carbapenem resistance genes thereof by MCDA-LFB in the example of the present invention.
FIG. 7 is a diagram showing a lower limit result of double MCDA detection in the example of the present invention;
FIG. 8 is a diagram showing the results of the specificity of the double MCDA-LFB detection of Acinetobacter baumannii and carbapenem drug-resistant genes thereof in the example of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the application provides a detection technology combining multi-cross displacement amplification and gold nano detection, which comprises the following steps:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
In the present application, the marker includes one of:
fluorescein isothiocyanate FITC, digoxigenin Dig.
In the MCDA reaction, the reaction temperature is constant, and the temperature range is 61-65 ℃.
In the present application, the temperature is 63 ℃.
In the application, the detection is carried out, the detection object and the detection buffer solution are sequentially dripped on the surface of the detection device.
In this application, the component parts of the detection instrument include: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
Based on the same invention concept, the application also provides an application of a detection technology combining multi-cross displacement amplification and gold nano detection, which is used for detecting acinetobacter baumannii specific gene pgaD and acinetobacter baumannii carbapenem drug-resistant gene blaOXA-23-like.
In the present application, it is preferred that,
for detecting said pgaD, the label is said FITC and the substance in the gold-labeled pad comprises one of: said SA-G, said B-BSA, said anti-FITC;
when the blaOXA-23-like is used for detecting the blaOXA-23-like, the marker is the Dig, and substances in a gold-labeled pad comprise one of the following substances: said SA-G, said B-BSA, said anti-Dig.
In the present application, it is preferred that,
for the detection of said pgaD, the primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, and an amplification primer C1 shown in SEQ ID NO: 4, and the amplification primer D1 is shown as SEQ ID NO: 5, and the amplification primer R1 is shown as SEQ ID NO: 6, and the amplification primer R2 is shown as SEQ ID NO: 7, and the amplification primer D2 is shown as SEQ ID NO: 8, and the amplification primer C2 is shown as SEQ ID NO: 9, and the cross primer CP2 is shown as SEQ ID NO: 10, and a replacement primer F2;
when used to detect the blaOXA-23-like, primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 11, and a replacement primer F1 shown as SEQ ID NO: 12, as shown in seq id NO: 13, and an amplification primer C1 shown in SEQ ID NO: 14, and the amplification primer D1 shown in SEQ ID NO: 15, and the amplification primer R1 is shown as SEQ ID NO: 16, and the amplification primer R2 is shown as SEQ ID NO: 17, and an amplification primer D2 shown in SEQ ID NO: 18, and the amplification primer C2 is shown as SEQ ID NO: 19, and a cross primer CP2 shown as SEQ ID NO: 20, substitution primer F2.
The present application will be described in detail with reference to the drawings and examples.
Material method
1. Reagents and equipment involved in the invention:
anti-fluorescein isothiocyanate antibody (anti-FITC), anti-digoxigenin antibody (anti-Dig), gold nanoparticle-coupled streptavidin (SA-G) and biotin-coupled bovine serum albumin (B-BSA) were purchased from Resenbio. The backing sheet, sample pad, gold pad, fibrous membrane and absorbent pad were purchased from Jie-Yi company. A deoxyribonucleic acid Isothermal Amplification Kit (Isotermal Amplification Kit) was purchased from North Kyoto Haita Zhengyuan technology, Inc. DNA extraction kits (QIAamp DNA minikites) were purchased from Qiagen, Germany. DL1000 DNA Marker was purchased from Takara Bio engineering (Dalian) Ltd. The other reagents are all commercial parting 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 Rongy and research Co. The PCR instrument is a Sensoquest Labcycler, a product of Sensoquest in Germany; 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.
The strains used in the experiments of the invention are from the preserved strains after clinical isolated culture and identification of the department of laboratory of the first Steel Hospital of Beijing university (see Table 1).
TABLE 1 sources of the strains
2. Primer design
The invention designs two sets of MCDA amplification primers aiming at Acinetobacter baumannii specific gene pgaD and carbapenem drug-resistant gene blaOXA-23-like thereof, and a primer design schematic diagram is shown in figure 1. Primer sequences and modifications are shown in table 2.
TABLE 2 primer sequences and modifications
nt, nucleotide; mer, monomeric unit, monomer unit.
MCDA amplification
Standard MCDA reaction system: the concentrations of the cross primers CP1 and CP2 were 1.6. mu.M, the concentrations of the displacement primers F1 and F2 were 0.4. mu.M, the concentrations of the amplification primers C1, C2, D1, D2, R1 and R2 were 0.8. mu.M, 12.5. mu.L of 2 × reaction buffer, 0.4mM biotin-11-dUTP, 1.25. mu.L of Bst DNA polymerase (10U), 1. mu.L of DNA template, and supplemented with deionized water to 25. mu.L. The whole reaction is kept at the constant temperature of 63 ℃ for 60min and at 85 ℃ for 5min to terminate the reaction.
Dual MCDA (pgaD prefixed with p-and blaOXA-23-like prefixed with b-) -reaction system: the concentrations of the crossover primers p-CP1 and p-CP2 were 0.8. mu.M, the concentrations of the displacement primers p-F1 and p-F2 were 0.2. mu.M, the concentrations of the amplification primers p-C1, p-C2, p-D1, p-D2, p-R1 and p-R2 were 0.4. mu.M, the concentrations of the crossover primers b-CP1 and b-CP2 were 1.6. mu.M, the concentrations of the displacement primers b-F1 and b-F2 were 0.2. mu.M, the concentrations of the amplification primers b-C1, b-C2, b-D1, b-D2, b-R1 and b-R2 were 0.8. mu.M, 12.5. mu.L of 2 × reaction buffer, 0.4mM of DNA-dBIP, 1.25. mu.25. mu.L of BsU, and 1. mu.25. mu.L of DNA template DNA supplemented with deionized water. The whole reaction is kept at the constant temperature of 63 ℃ for 60min and at 85 ℃ for 5min to terminate the reaction.
4. Design and principle of biodetector (LFB)
Design of LFB: as shown in fig. 3, the LFB comprises five parts, a sample pad, a gold label pad, a fibrous membrane, a bibulous pad and a back sheet. Firstly, a sample pad, a gold mark pad, a fiber membrane and a water absorption pad are sequentially assembled on a back plate. Then, SA-G (streptavidin coupled with gold nanoparticles), anti-FITC (fluorescein isothiocyanate antibody) or anti-Dig (digoxigenin antibody), and B-BSA (bovine serum albumin coupled with biotin) are respectively coated on the gold label pad, the detection line and the Control Line (CL), and are dried for later use.
Detection principle of LFB: mu.L of MCDA product was added directly to the LFB pad area by dripping, and then 120. mu.L of detection buffer was added to the sample pad area, and the MCDA product was wicked from bottom to top (from the sample pad to the absorbent pad). When the MCDA product reached the gold-labeled pad, one end of the double-labeled product (i.e., the biotin-labeled end) reacted with SA-G (streptavidin coupled to gold nanoparticles). When the product continues to move, the other end of the dual-standard product (i.e. the fluorescein isothiocyanate labeled end or the digoxigenin labeled end) is combined with the antibody in the detection line region, and the dual-standard product is fixed in the detection line region. With the accumulation of the product in the detection line area, the color reaction is carried out through SA-G (gold nanoparticle coupled streptomycin avidin) at the other end, so that the MCDA product is visually detected. In addition, excess SA-G (streptavidin coupled to gold nanoparticles) can be directly reacted with B-BSA (bovine serum albumin coupled to biotin) in the CL (quality control line) region to determine whether the LFB function is normal.
Result verification
1. Construction of detectable products by MCDA amplification
The MCDA reaction system comprises 10 primers, 10 regions for recognizing target sequences, 2 crossed inner primers, namely CP1 and CP2(Cross Primer, CP), 2 replacement primers, namely F1 and F2, and 6 amplification primers, namely D1, C1, R1, D2, C2 and R2. To construct a detectable product, a hapten such as Fluorescein Isothiocyanate (FITC) or digoxigenin (Dig) was labeled at the 5' end of primer C1 and the newly labeled primer was named C1. CP1 comprises C1s (the complement of the C1 region) and P1, i.e., 5' -C1 s-P1; CP2 comprises C2s (the complement of the C2 region) and P2, i.e., 5' -C2 s-P2. The two crossed primers CP1 and CP2 are the main primers mediating MCDA amplification; the replacement primers F1 and F2 play a replacement role in the MCDA reaction, and replace the cross primers CP1 and CP 2; the six amplification primers D1, C1, R1, D2, C2 and R2 were able to accelerate the MCDA reaction and increase the amount of MCDA product, as shown in FIG. 2.
Under a predetermined constant temperature condition, when a double-stranded DNA is in a dynamic equilibrium state of half dissociation and half binding, and any one primer is subjected to base pairing extension to a complementary site of the double-stranded DNA, the other strand is dissociated and becomes a single strand. First, under the action of Bst DNA polymerase, the 3' -end of the P1 segment of CP1 primer was used as the starting point, and the primer was paired with the corresponding DNA complementary sequence, thereby initiating the strand displacement DNA synthesis. The F1 primer is complementary to the F1s sequence at the front end of C1s, and synthesizes self DNA while first displacing a DNA strand synthesized with the CP1 primer by the action of a DNA polymerase having a strand displacement activity with the 3' end as the origin. Finally, the DNA strand synthesized from the F1 primer forms a double strand with the template DNA. However, the DNA strand synthesized first by the cross primer CP1 is strand-displaced by the F1 primer to generate a single strand, and the single strand D1s, C1s, R1s, P2s and F2s regions can be combined with the amplification primers D1, C1, R1, the cross primer CP2 and the displacement primer F2 in sequence to perform the strand displacement amplification (steps 1 and 2). The C1 primer amplifies and displaces the amplified strand of D1, generating a short fragment C1s-D1 product that is capable of binding to the C1 and CP1 primers, initiating strand displacement amplification, and going to cycle amplification 1 (step 3 and cycle 1). The R1 primer amplifies and displaces the C1 x amplified strand, generating a short fragment C1s-C1 product that is capable of binding to the C1 and CP1 primers, initiating strand displacement amplification, and going to cycle amplification 1 (step 4 and cycle 2). In cycle amplification 2, as MCDA amplification proceeds, a large amount of ditag is formed, labeled with Fluorescein Isothiocyanate (FITC) or digoxigenin (Dig), and biotin, respectively (fig. 2). The dual-labeled product can be detected by a gold nano-biosensor, thereby performing visual detection (fig. 3). Thus, in the detection of target sequences using the MCDA-LFB technique, a detectable product can be constructed using the C1 primer.
2. Validation of the feasibility of MCDA primers (pgaD and blaOXA-23-like)
After MCDA amplification, two detection methods were used to discriminate the MCDA amplification result (fig. 4). Firstly, adding visible dye (such as Malachite green reagent, Malachite green, MG) into the reaction mixture, the positive reaction tube changes from colorless or light blue to blue, and the negative reaction tube remains colorless or light blue. In addition, a more straightforward and simple method is to detect the product by LFB.
Visual color change method: MCDA synthesizes DNA and simultaneously generates a large amount of pyrophosphate ions which can capture manganese ions combined with calcein, so that the calcein returns to a free state to fluoresce. The luminescent mixture is capable of binding with magnesium ions generated during the reaction, resulting in enhanced fluorescence. The result can be interpreted by visually detecting a color change by fluorescence, the positive control tube changing from colorless or bluish to blue, and the negative control tube remaining colorless or bluish unchanged, see fig. 4A and 4C. A1 and C1 indicated positive amplification (Acinetobacter baumannii blaOXA-23-like gene carrying strain (SG-AB001) template was added to the reaction tube as a positive control), A2 and C2 indicated negative amplification (Klebsiella pneumoniae template was added to the reaction tube as a negative control), A3 and C3 indicated negative amplification (Staphylococcus aureus template was added to the reaction tube as a negative control), and A4 and C4 indicated blank control reactions (1. mu.l of double distilled water instead of template as a blank control). Only positive control shows positive amplification, which indicates that MCDA primers designed aiming at specific genes and used for detecting the drug resistance of acinetobacter baumannii and carbapenem thereof can be used.
LFB detection: the products of FIGS. 4A and 4C were subjected to LFB detection. Since the hapten marked by the MCDA primer aiming at the acinetobacter baumannii and the carbapenem drug-resistant detection thereof is FITC (fluorescein isothiocyanate) and Dig (digoxigenin), the acinetobacter baumannii detection is positive when red bands appear in TL1 and CL, and the carbapenem drug-resistant detection is positive when red bands appear in TL2 and CL. The result of MCDA amplification is judged by an LFB detection method, the positive control shows the expected result, and the negative control and the blank control only show CL red bands, so that the MCDA-LFB technology and the MCDA primer designed by the research are feasible and can be used for detecting the target sequence (fig. 4B and 4D).
3. Determination of the optimum reaction temperature for the MCDA technique
Under the condition of a standard reaction system, adding an MCDA primer corresponding to a template of acinetobacter baumannii and carbapenem drug-resistant strains (SG-AB001), wherein the template concentration is 10 pg/ul. The reaction is carried out under different constant temperature conditions (61-65 ℃), and the result is detected by a real-time turbidimeter, so that an amplification dynamic curve chart of the MCDA primer can be obtained (figure 5). As shown in FIG. 5, robust amplification curves were seen at different temperatures. According to the earliest time of the peak appearance of the amplification curve, 63 ℃ is recommended as the optimal reaction temperature suitable for the MCDA primer amplification involved in the patent.
Sensitivity of MCDA-LFB for detecting acinetobacter baumannii and carbapenem drug resistance thereof
After the genomic DNA of the Acinetobacter baumannii and the carbapenem drug-resistant strain (SG-AB001) thereof which are subjected to the serial dilution is subjected to the standard MCDA amplification reaction, 4 detection methods are used for judging the MCDA amplification result.
First, a real-time turbidimeter was used to analyze MCDA amplification (fig. 6a1 and fig. 6a 2). And (3) carrying out standard MCDA amplification reaction on the SG-AB001 genome DNA subjected to continuous dilution, and simultaneously monitoring the amplification condition in real time by using a real-time turbidimeter, wherein the result shows that: SG-AB001 MCDA was detected as low as 100fg and a positive amplification turbidity curve was observed. And when the content of the SG-AB001 genome in the reaction system is 10fg or less, or no SG-AB001 genome template exists, a positive amplification turbidity curve does not appear, and a negative result is shown. Fig. 6a1 and 6a2 read MCDA amplification results visually using a real-time turbidimeter.
Secondly, the results of MCDA amplification were detected by visual dye method (fig. 6B1 and fig. 6B 2). Adding visible dye (such as Malachite Green (MG) reagent) into the reaction mixture, wherein the reaction is positive when the reaction solution changes from colorless to blue, and negative when the reaction solution still keeps colorless. And (3) detection and display: the detection range of SG-AB001 MCDA can be as low as 100fg, and positive amplification tubes turn blue (FIG. 6B 1: 1-4, FIG. 6B 2: 1-4). When the content of SG-AB001 genome in the reaction system was 10fg or less, or no SG-AB001 genome template was present, the reaction mixture was colorless and showed a negative result (FIG. 6B 1: 5-8, FIG. 6B 2: 5-8). Fig. 6B1 and 6B2 read MCDA amplification results visually using dye method: 1-8 in FIGS. 6B1 and 6B2 indicate that the template amounts of SG-AB001 were 10ng, 10pg, 1pg, 100fg, 10fg, 1fg, negative control (Klebsiella pneumoniae), blank control (double distilled water).
Third, LFB was used to detect MCDA products (fig. 6C1 and 6C 2). The results show that: the detection range of SG-AB001 MCDA-LFB can be as low as 100fg, and LFB has red lines in TL1 and CL, TL2 and CL regions (FIG. 6C 1: 1-4, FIG. 6C 2: 1-4). And when the SG-AB001 genome content in the reaction system is 10fg and below, or no SG-AB001 genome template exists, LFB shows a red line only in the CL region, indicating a negative result (FIG. 6C 1: 5-8, FIG. 6C 2: 5-8). Fig. 6C1 and 6C2 read MCDA amplification results using LFB visualization: 1-8 in FIGS. 6C1 and 6C2 indicate that the template amounts of SG-AB001 were 10ng, 10pg, 1pg, 100fg, 10fg, 1fg, negative control (Klebsiella pneumoniae), blank control (double distilled water).
Fourthly, MCDA products were detected by agarose gel electrophoresis (fig. 6D1 and fig. 6D 2). Because the products contain amplified fragments with different sizes, the electrophoretogram of the positive amplified product is in a step shape, and the negative reaction does not have any band. Electrophoresis detection and display: the detection range of SG-AB001 MCDA can be as low as 100fg, and positive reactions have a step-shaped band (FIG. 6D 1: 1-4, FIG. 6D 2: 1-4). When the content of SG-AB001 genome in the reaction system is 10fg or less, or no SG-AB001 genome template is present, no specific step-like band appears, and a negative result is obtained (FIG. 6D 1: 5-8, FIG. 6D 2: 5-8). FIGS. 6D1 and 6D2 illustrate the results of MCDA amplification by electrophoresis; 1-8 in FIGS. 6D1 and 6D2 indicate that the template amounts of SG-AB001 were 10ng, 10pg, 1pg, 100fg, 10fg, 1fg, negative control (Klebsiella pneumoniae), blank control (double distilled water).
5. Sensitivity of double MCDA (modified methyl cellulose) for detecting acinetobacter baumannii and carbapenem drug resistance thereof
After double MCDA amplification reaction using serially diluted genomic DNA of Acinetobacter baumannii and carbapenem-resistant strain thereof (SG-AB001), the MCDA product was detected using LFB (FIG. 7).
The results show that: the detection range of SG-AB001 double MCDA-LFB can be as low as 100fg, and LFB has red lines in TL1, TL2 and CL regions (FIG. 7: 1-4). When the SG-AB001 genome content in the reaction system was 10fg or less, or no SG-AB001 genome template was present, LFB showed a red line only in the CL region, indicating a negative result (FIG. 7: 5-8). Figure 7 reads the dual MCDA amplification results using LFB visualization: FIGS. 7, 1-8, show the amounts of the template SG-AB001 as 10ng, 10pg, 1pg, 100fg, 10fg, 1fg, negative control (Klebsiella pneumoniae), and blank control (double distilled water).
6. Determination of specificity of Dual MCDA-LFB technology
And (3) evaluating the specificity of the double MCDA-LFB technology by taking common pathogenic bacteria and conditional pathogenic bacteria DNA as templates. (the strain information is shown in Table 1, and the specificity results are shown in FIG. 8). In the figure, No. 1-9 show the detection results of carbapenem resistant (carrying blaOXA-23-like) acinetobacter baumannii, and LFB has red lines in TL1, TL2 and CL regions; in the figure, No. 10-12 shows the detection result of non-carbapenem resistant (carrying blaOXA-23-like) acinetobacter baumannii, and LFB shows red lines in TL1 and CL areas; in the figure, the numbers 13-27 are as follows: and the LFB only has a red line in a CL region. From the figure, it can be seen that the double MCDA-LFB technology can accurately identify the acinetobacter baumannii and the carbapenem drug resistance thereof, and the specificity of the double MCDA-LFB method is good.
Example 1
The present embodiment provides a detection technique combining multi-cross displacement amplification and gold nano-detection, including:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
The marker includes at least one of: fluorescein isothiocyanate FITC, digoxigenin Dig.
The temperature was 63 ℃.
And during detection, the detection object and the detection buffer solution are sequentially dripped on the surface of the detection device.
The volume ratio of the detection object to the detection buffer solution is 1: 600.
The component parts of the detection apparatus comprise: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
Example 2
The present embodiment provides a detection technique combining multi-cross displacement amplification and gold nano-detection, including:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
The marker includes at least one of: fluorescein isothiocyanate FITC, digoxigenin Dig.
The temperature was 65 ℃.
And during detection, the detection object and the detection buffer solution are sequentially dripped on the surface of the detection device.
The volume ratio of the detection object to the detection buffer solution is 1: 600.
The component parts of the detection apparatus comprise: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
Example 3
The present embodiment provides a detection technique combining multi-cross displacement amplification and gold nano-detection, including:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
The marker includes at least one of: fluorescein isothiocyanate FITC, digoxigenin Dig.
The temperature was 61 ℃.
And during detection, the detection object and the detection buffer solution are sequentially dripped on the surface of the detection device.
The volume ratio of the detection object to the detection buffer solution is 1: 600.
The component parts of the detection apparatus comprise: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
Example 4
The embodiment provides an application of a detection technology combining multi-cross displacement amplification and gold nano detection, which is used for detecting acinetobacter baumannii specific genes pgaD.
During detection, the used marker is the FITC, and substances in the gold-labeled pad comprise one of the following substances: said SA-G, said B-BSA, said anti-FITC;
the primers are selected from the following sequence combinations:
as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, and an amplification primer C1 shown in SEQ ID NO: 4, and the amplification primer D1 is shown as SEQ ID NO: 5, and the amplification primer R1 is shown as SEQ ID NO: 6, and the amplification primer R2 is shown as SEQ ID NO: 7, and the amplification primer D2 is shown as SEQ ID NO: 8, and the amplification primer C2 is shown as SEQ ID NO: 9, and the cross primer CP2 is shown as SEQ ID NO: 10, and a replacement primer F2;
example 5
The present example provides an application of a detection technique combining multi-cross displacement amplification and gold nano-detection for detecting carbapenem-resistant gene blaOXA-23-like of Acinetobacter baumannii.
During detection, the marker is the Dig, and substances in the gold-labeled pad comprise one of the following substances: said SA-G, said B-BSA, said anti-Dig;
the primers are selected from the following sequence combinations:
as shown in SEQ ID NO: 11, and a replacement primer F1 shown as SEQ ID NO: 12, as shown in seq id NO: 13, and an amplification primer C1 shown in SEQ ID NO: 14, and the amplification primer D1 shown in SEQ ID NO: 15, and the amplification primer R1 is shown as SEQ ID NO: 16, and the amplification primer R2 is shown as SEQ ID NO: 17, and an amplification primer D2 shown in SEQ ID NO: 18, and the amplification primer C2 is shown as SEQ ID NO: 19, and a cross primer CP2 shown as SEQ ID NO: 20, substitution primer F2.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A detection technique that combines multiple cross-exchange amplification with gold nano-detection, comprising:
setting a first arbitrary sequence F1s and a second arbitrary sequence P1s from the 3 'end of the target gene fragment, setting a third arbitrary sequence F2 and a fourth arbitrary sequence P2 from the 5' end of the target gene fragment, setting a fifth arbitrary sequence C1 at the 5 'end of the second arbitrary sequence P1s, and/or setting a sixth arbitrary sequence C2s at the 3' end of the fourth arbitrary sequence P2;
providing a replacement primer F1, wherein the primer F1 comprises a sequence complementary to the sequence F1s, providing a cross primer CP1, wherein the primer CP1 comprises a sequence C1s which is complementary to the sequence C1 and a sequence P1 in sequence from the 5 'end, providing a replacement primer F2, wherein the primer comprises a sequence complementary to the sequence F2s, providing a cross primer CP2, and wherein the primer CP2 comprises a sequence C2s which is complementary to the sequence C2 and a sequence P2 in sequence from the 5' end;
providing amplification primers comprising an amplification primer C1 comprising the sequence C1, and/or an amplification primer C2 comprising the sequence C2s complementary, and labeling a label at the 5' end of the amplification primer C1 to obtain an amplification primer C1;
under the existence of Bst DNA polymerase, strand displacement active DNA polymerase and primers, using the target gene segment as a template to amplify DNA at constant temperature to obtain a marked MCDA target gene segment; the primer comprises: displacement primers F1 and F2, crossover primers CP1 and CP2, amplification primers D1, C1, R1, D2, C2 and R2;
and (3) taking the double-label MCDA gene fragment as a detection object, and detecting the detection object by adopting a gold nano detection method.
2. The detection technique combining multiple cross-displacement amplification and gold nano-detection according to claim 1, wherein the label comprises one of: fluorescein isothiocyanate FITC, digoxigenin Dig.
3. The detection technology combining multi-cross displacement amplification and gold nano detection as claimed in claim 1, wherein in the MCDA reaction, the reaction temperature is constant and the temperature range is 61-65 ℃.
4. The detection technique combining multiple cross-displacement amplification and gold nano-detection as claimed in claim 3, wherein the temperature is 63 ℃.
5. The detection technology combining the multi-cross displacement amplification and the gold nano detection as claimed in claim 1, wherein the detection is performed by sequentially dripping the detection object and the detection buffer solution on the surface of the detection device.
6. The detection technique combining multi-cross displacement amplification and gold nano-detection as claimed in claim 5, wherein the volume ratio of the detection object to the detection buffer is 1: 600.
7. A detection technique combining multiple cross-over displacement amplification and gold nano-detection according to claim 5, wherein the components of the detection apparatus comprise: the sample pad, the gold label pad, the fiber membrane, the water absorption pad and the back plate are sequentially arranged from top to bottom; the gold-labeled pad comprises the following substances: gold nanoparticle-coupled streptomycin avidin SA-G, an anti-fluorescein isothiocyanate antibody anti-FITC, an anti-digoxigenin antibody anti-Dig and biotin-coupled bovine serum albumin B-BSA.
8. Use of a detection technique combining multiple cross-over displacement amplification and gold nano-detection according to any one of claims 1 to 7 for the detection of acinetobacter baumannii specific gene pgaD and acinetobacter baumannii carbapenem resistance gene blaOXA-23-like.
9. The use of a detection technique combining multiple cross-displacement amplification and gold nano-detection according to claim 8,
for detecting said pgaD, the label is said FITC and the substance in the gold-labeled pad comprises one of: said SA-G, said B-BSA, said anti-FITC;
for detecting said blaOXA-23-Iike, the marker is said Dig and the substance in the gold pad comprises one of the following: said SA-G, said B-BSA, said anti-Dig.
10. The use of a detection technique combining multiple cross-displacement amplification and gold nano-detection according to claim 8,
for the detection of said pgaD, the primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, and an amplification primer C1 shown in SEQ ID NO: 4, and the amplification primer D1 is shown as SEQ ID NO: 5, and the amplification primer R1 is shown as SEQ ID NO: 6, and the amplification primer R2 is shown as SEQ ID NO: 7, and the amplification primer D2 is shown as SEQ ID NO: 8, and the amplification primer C2 is shown as SEQ ID NO: 9, and the cross primer CP2 is shown as SEQ ID NO: 10, and a replacement primer F2;
when used to detect the blaOXA-23-like, primers are selected from the following combinations of sequences:
as shown in SEQ ID NO: 11, and a replacement primer F1 shown as SEQ ID NO: 12, as shown in SEQ id no: 13, and an amplification primer C1 shown in SEQ ID NO: 14, and the amplification primer D1 shown in SEQ ID NO: 15, and the amplification primer R1 is shown as SEQ ID NO: 16, and the amplification primer R2 is shown as SEQ ID NO: 17, and an amplification primer D2 as shown in seq id NO: 18, and the amplification primer C2 is shown as SEQ ID NO: 19, and a cross primer CP2 shown as SEQ ID NO: 20, substitution primer F2.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525909A (en) * | 2013-09-12 | 2014-01-22 | 周燕斌 | Real-time fluorescent loop-mediated isothermal amplification kit of acinetobacter baumannii |
| US20140356859A1 (en) * | 2011-04-08 | 2014-12-04 | American University Of Cairo (Auc) | Detection of nucleic acids using unmodified gold nanoparticles |
| CN106399517A (en) * | 2016-09-30 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology |
| CN109576349A (en) * | 2018-12-24 | 2019-04-05 | 北京大学首钢医院 | A kind of application intersects the method for constant-temperature amplification combination nanobiosensor technology detection Candida albicans more |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
| CN109811036A (en) * | 2019-03-15 | 2019-05-28 | 首都医科大学附属北京儿童医院 | The methods intersected amplification and combine bio-sensing detection mycobacterium tuberculosis complex more |
-
2019
- 2019-08-28 CN CN201910805561.1A patent/CN110819728A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140356859A1 (en) * | 2011-04-08 | 2014-12-04 | American University Of Cairo (Auc) | Detection of nucleic acids using unmodified gold nanoparticles |
| CN103525909A (en) * | 2013-09-12 | 2014-01-22 | 周燕斌 | Real-time fluorescent loop-mediated isothermal amplification kit of acinetobacter baumannii |
| CN106399517A (en) * | 2016-09-30 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology |
| CN109576349A (en) * | 2018-12-24 | 2019-04-05 | 北京大学首钢医院 | A kind of application intersects the method for constant-temperature amplification combination nanobiosensor technology detection Candida albicans more |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
| CN109811036A (en) * | 2019-03-15 | 2019-05-28 | 首都医科大学附属北京儿童医院 | The methods intersected amplification and combine bio-sensing detection mycobacterium tuberculosis complex more |
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