CN110819717A - Graphene oxide-containing amplification system and application thereof in colorectal cancer marker detection - Google Patents
Graphene oxide-containing amplification system and application thereof in colorectal cancer marker detection Download PDFInfo
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Abstract
The invention discloses an amplification system containing graphene oxide and application thereof in colorectal cancer marker detection. The amplification system comprises: isothermal amplification buffer, MgSO4The kit comprises a dNTP mixture, a target specific primer group, DNA polymerase, an amplification template and 5-30 ng of graphene oxide. The invention verifies the mechanism of graphene oxide for inhibiting non-specific amplification in LAMP, optimizes the optimal addition amount of graphene oxide in an LAMP amplification system, and applies the amplification system to the detection of rectal cancer markers.
Description
Technical Field
The invention belongs to the technical field of nucleic acid amplification, and particularly relates to an amplification system containing graphene oxide and application of the amplification system in colorectal cancer marker detection.
Background
Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification technology, and is a powerful alternative to PCR due to its low cost, high stability, high sensitivity, high specificity and rapidity. Since the invention of 2000, LAMP has been widely used in many fields such as disease diagnosis, food safety, and environmental monitoring ("Loop-mediated isothermal amplification of DNA", 2000; 28(12): e63-e 63).
The LAMP technology uses 4-6 primers, respectively recognizes 6-8 regions of a target sequence DNA, combines Bst DNA polymerase with strand displacement activity, and has higher specificity compared with PCR. The simultaneous initiation of DNA synthesis by multiple primers makes this technique highly specific. LAMP is carried out under isothermal conditions (60-65 ℃) and produces a large amount of DNA, enabling amplification of approximately 10 hours in 1 hour9And (4) copying.
However, the use of LAMP for amplifying trace amounts of nucleic acids is hampered by false positive results, and the non-specific mechanism of generation leading to false positive results is not yet clear. Therefore, various methods have been developed to remove background amplification, suppress false positives and improve LAMP specificity, such as designing strand displacement fluorescent probes (anal. chem., 2016, 88(7), 3562-; the Janus probe system broadens the detection range by inhibiting false positives (Chem Sci., 2018,9(2), 392-; the use of uracil-DNA-glycosylase eliminates DNA-carrying contamination during amplification (Chem Commun, 2014,50(28), 3747-3749); single-stranded binding proteins are added to the amplification system to block the interaction of template and thus largely suppress false positives (anal. chem.2018,90(18), 11033-11039.).
Although these reports above suggest that non-specific amplification of LAMP can be effectively inhibited using different means. However, these techniques are not suitable for further practical applications. For example, uracil-DNA-glycosylase may also chemotranscribe mRNA in LAMP, which is not suitable for reverse transcription LAMP. Strand displacement probes require the involvement of fluorescently labeled nucleic acids, increase cost, and require longer signal readout times. In addition, the signal intensity of dye-labeled probes is limited and unstable, greatly reducing the sensitivity of LAMP. On the other hand, DNA binding dyes (such as SYBR Green I or SYTO) are commonly used to monitor DNA amplification in real time because their fluorescence signal is significantly enhanced upon binding to double stranded DNA (dsdna) products. However, these fluorescent dyes also intercalate into double strands due to primer dimers and other mismatches, generating strong false fluorescent signals, which are more likely to cause false positive results particularly in LAMP reaction systems with high primer concentrations.
Cyclooxygenase-2 (COX-2) mRNA is an important target for diagnosing, prognosing and monitoring colorectal cancer, the detection method for detecting the target is single at present, mainly adopts PCR detection, but the PCR detection usually needs reverse transcription and temperature-variable amplification equipment, consumes longer time and has limited sensitivity. Therefore, it is necessary to develop a rapid, sensitive and simple method for detecting cyclooxygenase-2 mRNA, especially for the clinical early diagnosis of cancer.
Graphene Oxide (GO) is an important two-dimensional (2D) nanomaterial, has a large specific surface area, has hydrophilic groups such as hydroxyl and carboxyl on the surface, and has excellent properties and wide application. GO has the ability to specifically bind single-stranded dna (ssdna), via pi-pi stacking interactions and hydrogen bonding, whereas double-stranded dna (dsdna) has a weaker affinity for GO surfaces because single nucleobases have formed complementary pairings. In addition, GO has been shown to efficiently quench the fluorescence of various dyes by energy resonance transfer, thereby reducing background and increasing signal-to-noise ratio. In recent years, there have been a number of reports on the use of GO in the biotechnology field. For example, the addition of different polymer-modified GO such as polyethylene glycol to a PCR system can significantly improve its specificity (patent application No.: CN 105647909A). GO can also be used as a carrier for immunodetection (patent application No. CN108660187A), and nucleic acid detection such as microRNA (2012 in anal. chem., 84(10), 4587-. There are no reports of GO for improving LAMP specificity.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problem of false positive in LAMP amplification in the prior art, the application provides an amplification system containing graphene oxide and simultaneously provides a new method for detecting colorectal cancer marker cyclooxygenase-2 mRNA.
Means for solving the problems
In view of the problems in the prior art, the present inventors have conducted intensive studies and repeated experiments, inspired by the unique affinity of Graphene Oxide (GO) for nucleotide primers and its ability to quench background fluorescence, and provided a loop-mediated isothermal amplification system for inhibiting false positive by graphene oxide, and applied the amplification system to detect a rectal cancer marker cyclooxygenase-2 mRNA, thereby completing the present invention. Namely, the present invention is as follows:
the first purpose of the invention is to provide a graphene oxide-based loop-mediated isothermal amplification system capable of avoiding false positive, wherein the isothermal amplification system comprises: the kit comprises a primer, a DNA dye, Bst DNA polymerase and sheet graphene oxide, wherein the graphene oxide specifically adsorbs the single-chain primer through pi bonds and hydrogen bond action, and the surface of the graphene oxide can also adsorb the DNA dye.
In order to further optimize the above technical solution, the technical measures taken by the present invention further include:
further, the addition amount of the graphene oxide is 20 ng.
Furthermore, cDNA of colorectal cancer marker cyclooxygenase-2 mRNA is selected as template DNA, namely the cDNA shown in SEQ ID NO: 5 as a target sequence; for the target sequence, the sequence shown as SEQ ID NO: 1 to 4 as primers.
Further, the fluorescent dye is any one of SYTO-9, SYBR Green, Cyto9, LC Green and Evagreen; preferably SYTO-9.
Further, the isothermal amplification system was 25 μ L (ultra pure water added to final volume), whereinComprises the following components in percentage by weight: 20mM Tris-HCl, 10mM (NH)4)2SO4、150mM KCl，8mM MgSO40.1% Tween 20, 1.4mM dNTP mixture, target specific primer mixture (1.6 mu M FIP, 1.6 mu M BIP, 0.2 mu M F3 and 0.2 mu M B3), 1 xSYTO-9, 8U Bst2.0WarmStart DNA polymerase and 20ng graphene oxide to obtain an isothermal amplification system, and performing loop-mediated isothermal amplification by using sample plasmid DNA as a template to obtain a loop-mediated isothermal amplification product.
Further, the temperature of the isothermal amplification reaction is 65 ℃ and the reaction time is 60 min.
Further, F3 primer is labeled by fluorescent FAM, graphene oxide and F3 in the mixed solution, and the quenching of FAM fluorescence by graphene oxide indicates the interaction and adsorption between the graphene oxide and the primer.
Further, the reduction of SYTO-9 fluorescence in the loop-mediated isothermal amplification translation system containing the graphene oxide shows that the graphene oxide can reduce the background fluorescence signal in the isothermal amplification system.
The second purpose of the invention is to provide the application of the reverse transcription loop-mediated isothermal amplification system based on the graphene oxide in colorectal cancer detection.
Further, the isothermal amplification system was 25 μ L (ultra pure water added to the final volume) containing the following components and their concentrations: 20mM Tris-HCl, 10mM (NH)4)2SO4、150mM KCl、8mM MgSO40.1% Tween 20, 1.4mM dNTP mixture, target specific primer mixture (1.6 mu M FIP, 1.6 mu M BIP, 0.2 mu M F3 and 0.2 mu M B3), 1 xSYTO-9, 8U Bst2.0WarmStart DNA polymerase, 5U ANV reverse transcriptase and 20ng graphene oxide to obtain an isothermal amplification system, and performing reverse transcription loop-mediated isothermal amplification by using total RNA extracted from colorectal cancer cells as a template to obtain an amplification product.
Further, total RNA extracted from colorectal cancer cells (HT-29), pancreatic cancer cells (PANC-1), lung cancer cells (HepG2), cervical cancer cells (HeLa) and normal colon cells (CCD-18Co) was selected for reverse transcription loop-mediated isothermal amplification to evaluate the specificity of the method.
In a specific embodiment, the present invention provides an amplification system comprising graphene oxide, the amplification system comprising: isothermal amplification buffer, MgSO4The kit comprises a dNTP mixture, a target specific primer group, DNA polymerase, an amplification template and 5-30 ng of graphene oxide. Preferably, the graphene oxide has a mass of 20ng, the DNA polymerase is Bst2.0WarmStart DNA polymerase, and the isothermal amplification buffer contains 20mM Tris-HCl and 10mM (NH)4)2SO4、150mM KCl，2mM MgSO4And 0.1% Tween 20; more preferably, the amplification system further comprises a fluorescent dye.
Further, the amplification template comprises a coding sequence of cyclooxygenase-2 mRNA, and the target-specific primer set comprises F3, B3, FIP and BIP; preferably, the nucleotide sequence of the coding sequence is as shown in SEQ ID NO: 5, the sequences of F3, B3, FIP and BIP in the target-specific primer group are respectively shown as SEQ ID NO: 1 to 4.
The invention also provides a method for inhibiting loop-mediated isothermal amplification false positive by using graphene oxide, which is characterized by comprising the following steps:
1) preparing an aqueous solution containing 5-30 ng of graphene oxide;
2) mixing the aqueous solution of graphene oxide obtained in the step 1) with a loop-mediated isothermal amplification reaction system, wherein the loop-mediated isothermal amplification reaction system comprises: isothermal amplification buffer, MgSO4A dNTP mix, a target specific primer set, a DNA polymerase and an amplification template;
3) isothermal amplification was performed on a PCR instrument.
In the method, the mass of the graphene oxide in the step 1) is 20 ng; the DNA polymerase in the step 2) is Bst2.0WarmStart DNA polymerase, and the isothermal amplification buffer in the step 2) contains 20mM Tris-HCl and 10mM (NH)4)2SO4、150mM KCl，2mM MgSO4And 0.1% Tween 20; preferably, the loop-mediated isothermal amplification reaction system in step 2) further comprises a fluorescent dye. The amplification template in the step 2) comprises a ringA coding sequence for oxidase-2 mRNA, the target-specific primer set comprising F3, B3, FIP, and BIP; preferably, the nucleotide sequence of the coding sequence is as shown in SEQ ID NO: 5, the sequences of F3, B3, FIP and BIP in the target-specific primer group are respectively shown as SEQ ID NO: 1 to 4.
The invention further provides application of the amplification system comprising the graphene oxide in preparation of a kit for detecting rectal cancer markers. Wherein the rectal cancer marker is cyclooxygenase-2; preferably, the coding sequence of the cyclooxygenase-2 is shown in SEQ ID NO: 5, respectively.
The invention further provides an application of graphene oxide in inhibiting non-specific amplification in loop-mediated isothermal amplification, which is characterized by comprising the following steps:
1) preparing an aqueous solution containing 5-30 ng of graphene oxide;
2) mixing the aqueous solution of graphene oxide obtained in the step 1) with a loop-mediated isothermal amplification reaction system, wherein the loop-mediated isothermal amplification reaction system comprises: isothermal amplification buffer, MgSO4A dNTP mix, a target specific primer set, a DNA polymerase and an amplification template;
3) isothermal amplification was performed on a PCR instrument.
Preferably, the mass of the graphene oxide in the step 1) is 20 ng.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the technical scheme, the graphene oxide-containing amplification system and the application thereof in colorectal cancer detection have the following beneficial effects:
1) the mechanism of Graphene Oxide (GO) for inhibiting non-specific amplification in LAMP is verified, namely GO absorbs LAMP primers, so that mismatching of nucleic acid chains is reduced, an amplification system is effectively stabilized, and the amplification specificity is greatly improved. While GO reduces background fluorescence due to DNA staining dyes (e.g., SYTO-9)
2) The inhibition effect of GO with different addition amounts on false positive in an LAMP reaction system is explored, and the final optimization result is as follows: when the amount of GO added into the LAMP reaction system is 20ng, false positive is completely inhibited, positive amplification is normal, and the effect is optimal.
3) The LAMP method containing the optimized addition amount of GO is used for detecting cyclooxygenase-2 mRNA in colorectal cancer cells, and has excellent specificity and sensitivity.
4) The GO-based LAMP detection method is low in cost, reliable and simple to operate, and has good application prospects in various fields of nucleic acid detection.
5) The invention applies GO to LAMP for the first time and verifies that GO is relative to MoS2The inhibition effect of AuNPs on the LAMP amplification efficiency of the target is smaller, and the performance of the AuNPs is superior to that of MoS2And AuNPs.
6) The invention verifies that the GO-based LAMP has higher sensitivity (detection limit is 10) compared with the traditional PCR reaction2copies/. mu.L), the reaction is faster.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail as follows:
drawings
FIG. 1 shows a schematic diagram of graphene oxide for suppressing non-specific amplification in loop-mediated isothermal amplification.
Fig. 2 shows a characterization of graphene oxide taken under a transmission electron microscope.
Fig. 3 shows the effect of different addition amounts of graphene oxide on the inhibition of false positives generated by the LAMP reaction system. Wherein, A to F in FIG. 3 correspond to the addition amounts of 5ng, 10ng, 15ng, 20ng, 25ng and 30ng of graphene oxide, respectively, and the positive sample shown in the figure is a plasmid containing a COX-2 target sequence, and the negative sample is ultrapure water.
A and B in FIG. 4 show real-time fluorescence LAMP graphs with no graphene oxide added and 20ng graphene oxide added, respectively, and C in FIG. 4 shows an agarose gel electrophoresis image of the LAMP amplification product. GO (-) in C of fig. 4 indicates no addition of graphene oxide, GO (+) indicates addition of 20ng of graphene oxide, M represents DNA molecular weight standard Marker, "+" represents positive sample, "-" represents negative sample.
Figure 5 shows the effect of different nanomaterials on the inhibition of LAMP false positive amplification and on LMAP amplification efficiency. In FIG. 5, A is a negative control group containing no target sequence, and B is a target group in FIG. 5.
Fig. 6 shows the quenched fluorescence intensity of the graphene oxide-adsorbed fluorescently labeled F3 primer at different addition amounts.
FIG. 7 shows that 20ng of graphene oxide was able to reduce the background fluorescence generated by the DNA dye SYTO-9 in the LAMP reaction system.
Fig. 8 shows the effect comparison of graphene oxide applied in LAMP and conventional PCR. Wherein a in fig. 8 shows a fluorescence amplification curve of the LAMP detection sensitivity based on graphene oxide; b in fig. 8 shows a standard curve for graphene oxide-based LAMP detection; c in FIG. 8 shows a fluorescence amplification curve of conventional PCR detection sensitivity; d in fig. 8 shows a standard curve for graphene oxide based PCR detection.
Figure 9 shows the sensitivity and specificity of fluorescent LAMP containing graphene oxide. Wherein A in FIG. 9 shows real-time fluorescent amplification curves for samples of different levels of COX-2mRNA target sequences; b in fig. 9 shows a standard curve of the LAMP detection system containing graphene oxide; c in fig. 9 shows an agarose gel electrophoresis of LAMP reaction products containing graphene oxide with varying amounts of COX-2mRNA target sequences; d in fig. 9 shows a real-time fluorescent LAMP amplification curve from total RNA extracted from different cancer cells as a sample.
Detailed Description
The embodiments of the present invention are described as examples of the present invention, and the present invention is not limited to the embodiments described below. Any equivalent modifications and substitutions to the embodiments described below are within the scope of the present invention for those skilled in the art. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Example 1 principle of graphene oxide for inhibiting non-specific amplification in LAMP amplification
One major limitation of real-time fluorescent LAMP is that it is susceptible to non-specific amplification, the mechanism of which is complex and varies with different reaction systems. Compared with PCR, LAMP has high primer concentration, and nonspecific strand hybridization easily occurs between primers and a target sequence, so LAMP is easily subjected to false positive. In addition, fluorescence-based LAMP methods cannot distinguish between double-stranded dna (dsdna) structures resulting from hybridization of specific amplicons with non-specific strands, resulting in false positive signals.
The mechanism of Graphene Oxide (GO) for inhibiting LAMP non-specificity is mainly based on the adsorption effect of GO and a primer and the capability of GO for quenching background fluorescence.
As shown in fig. 1: GO preferentially binds to single-stranded primers through pi-pi stacking interactions and hydrogen bonding, while it has a weak affinity for negatively charged dsDNA with functional groups relatively trapped within the double helix structure. Based on the unique property, the combination of GO and the primer can weaken the interaction between the primer and the primer, and between the target sequence and the target sequence, and the primer can be complementarily paired with the target sequence for amplification only when the target sequence exists, so that the formation of primer dimer or other mismatched strand hybridization is avoided, and the inhibition of false positive is realized.
In addition, GO also has some affinity with DNA staining dyes (e.g., SYTO-9, SYBR Green, Cyto9, LC Green, Evagreen, etc.) to generate Fluorescence Resonance Energy Transfer (FRET), thereby reducing background fluorescence and improving signal-to-noise ratio. When a target sequence is not added into the LAMP reaction system, the primers stay on the surface of GO due to the adsorption effect of GO.
Example 2 comparison of the effect of graphene oxide at different concentrations on inhibiting LAMP false positive
2.1 pretreatment of graphene oxide
Graphene oxide (product number G139803-250mg, Shanghai Aladdin Biotechnology Co., Ltd.) with an average diameter of 1-4 μm is selected and dispersed in an aqueous solution, and the graphene oxide is subjected to ultrasonic treatment for 5min by using an ultrasonic cleaning machine with a power of 50W before use, so that the graphene oxide is uniformly dispersed in the solution.
2.2 test methods and test materials
In the process of optimizing the dosage of GO, the addition amounts are respectively set to be 5ng, 10ng, 15ng, 20ng, 25ng and 30 ng. A small amount of GO is dispersed in 50% ethanol, and is dripped onto a copper mesh by a pipette gun to be naturally dried for characterization by a transmission electron microscope. A characterization graph of GO under a transmission electron microscope is shown in FIG. 2, wherein GO is in a lamellar shape, and the specific surface area of GO is large, so that the GO is beneficial to adsorbing primers. In the embodiment, a series of F3 primers with different addition amounts of GO to adsorb fluorescent labels are utilized, and the condition that the fluorescence of the primers is quenched is utilized to illustrate that GO specifically interacts with the primers, so that the mismatch between primer chains is reduced. Similarly, the effect of GO in reducing background fluorescence is shown by comparing the fluorescence change before and after GO is added into a LAMP reaction system containing SYTO-9 dye.
Colorectal cancer cyclooxygenase-2 (COX-2) mRNA is selected as a target sequence, an amplification template sequence is obtained from a GenBank database, the sequence number of the cyclooxygenase-2 (COX-2) mRNA is NC-000963.3, the length of the selected amplification template is 297bp (336bp-632bp), and the specificity of the selected sequence is 100% through NCBI website sequence comparison. Primers for amplification were designed by the Primer Explorer V5(http:// Primer Explorer. jp/e/index. html. Eiken Chemicals Corporation, Tokyo, Japan) website. The specific amplification template may be a plasmid containing the coding sequence of COX-2 complementary DNA (cDNA) or COX-2 mRNA. Plasmids and primers were synthesized by Shanghai. The fluorescent dye is any one of SYBR green, SYTO-9 and the like, and SYTO-9 is preferable.
A plasmid containing a COX-2 target sequence was selected as a positive sample, and ultrapure water was selected as a negative sample.
2.3 LAMP reaction System containing GO
GO-based LAMP reaction system 25 μ L (ultrapure water added to final volume) containing:
2.5. mu.L of isothermal amplification buffer (20mM Tris-HCl, 10mM (NH)4)2SO4，150mM KCl，2mM MgSO4，0.1％Tween 20)；
1.5μL 6mM MgSO4；
3.5 μ L of 1.4mM dNTP mix (Shanghai Producer, cat # B500056);
0.9. mu.L of a target-specific primer mixture (primers synthesized by Shanghai Biotech, Inc., containing 1.6. mu.M FIP, 1.6. mu.M BIP, 0.2. mu. M F3, and 0.2. mu. M B3);
1.0. mu.L of 8000units/ml Bst2.0WarmStart DNA polymerase;
0.5. mu.L of DNA dye;
5.0 μ L of sample.
5.0 μ L of sonicated 5ng, 10ng, 15ng, 20ng, 25ng and 30ng GO were added to each reaction.
The reaction was finally carried out in a PCR tube using a PCR instrument with LAMP reaction temperature set at 65 ℃ for 60 min. 0.5 μ L of 10,000units/ml AMV reverse transcriptase was also added to the GO-based reverse transcription LAMP reaction system. All reagents above are provided by NEB corporation unless specifically stated otherwise.
The nucleotide sequences of the above target-specific primers are shown below:
F3：5’-ACCCACTCCAAACACAGT-3’(SEQ ID NO：1)；
B3：5’-TCCCAGCTTTTGTAGCCA-3’(SEQ ID NO：2)；
FIP:5’-TCGAAGGAAGGGAATGTTATTCACGCACTACATACTTACCCACTTC-3’(SEQ ID NO：3)；
BIP:5’-ATGAGTTATGTGTTGACATCCAGATTAGTCAGCATTGTAAGTTGGTG-3’(SEQ ID NO：4)。
the coding sequence of the COX-2cDNA contained in the amplification template is:
CTGAAACCCACTCCAAACACAGTGCACTACATACTTACCCACTTCAAGGGATTTTGGAACGTTGTGAATAACATTCCCTTCCTTCGAAATGCAATTATGAGTTATGTGTTGACATCCAGATCACATTTGATTGACAGTCCACCAACTTACAATGCTGACTATGGCTACAAAAGCTGGGAAGCCTTCTCTAACCTCTCCTATTATACTAGAGCCCTTCCTCCTGTGCCTGATGATTGCCCGACTCCCTTGGGTGTCAAAGGTAAAAAGCAGCTTCCTGATTCAAATGAGATTGTGGAA(SEQ ID NO：5)。
2.4 test results
As shown in FIG. 3, the fluorescent real-time LAMP experiment results show that the difference between the target sequence signal and the negative blank can be effectively enlarged by the GO. The effect of inhibiting false positives increased significantly as the amount of GO increased from 5ng to 30ng (see a-F in fig. 3, respectively). When 20ng of GO was added to the LAMP reaction system (see D in fig. 3), false positive amplification could be completely suppressed, while amplification of the target sequence was hardly affected. Amplification of the target sequence was also gradually inhibited when higher concentrations of GO were added (see E in figure 3). In the presence of 30ng GO (see F in FIG. 3), excess GO binds strongly to all primers, primers cannot bind to target sequences to extend amplification, and amplification signals for both target sequences and negative blanks disappear. This indicates that GO concentration is a key factor to improve LAMP specificity.
On the basis of the above test, the results of LAMP amplification were examined for the case where GO was not present and 20ng of GO was added, respectively, for the case where the plasmid containing the target sequence was positive and the ultrapure water was negative as controls (see A and B in FIG. 4, respectively). As can be seen from the figure, when no GO is present in the LAMP reaction system, a fluorescence signal (amplification signal) begins to appear when the GO is positive for about 3 minutes, but a false positive signal also begins to appear when the GO is negative for about 10 minutes (see a in fig. 4). When 20ng GO was added to both positive and negative amplification systems, we seen that the negative signal completely disappeared, while the positive signal remained normal, beginning to appear around 7 minutes (see B in FIG. 4). The results further verify that 20ng of GO completely inhibited LAMP false positives and did not affect the normal amplification of positives.
We performed agarose gel electrophoresis run-up validation on the positive and negative amplification products without and with GO, respectively, and dissolved agarose (Shanghai Biotech, cat # A500016) in 1 XTAE buffer (Shanghai Biotech, cat # B548101) with agarose concentration of 2% and electrophoresis apparatus parameters set to 110V for 45 minutes. After running the gel, the gel was photographed by a gel imager, and we can see that after adding GO, the positive amplification product presents a ladder-shaped band after classical LAMP amplification, and no band appears in the negative sample (see C in FIG. 4). The results further demonstrate that GO can effectively inhibit LAMP false positives.
The following tests further verify that: 20ng of graphene oxide can be used as the optimal using amount of the LAMP reaction system.
Example 3 effect of comparing different nanomaterials for inhibiting LAMP false positives
There are many nanomaterials in the prior art, including Graphene Oxide (GO), molybdenum disulfide (MoS)2) And gold nanoparticles (AuNPs) have been used to improve the specificity of PCR. Wherein, MoS2The specificity of PCR can be enhanced by combining primers and DNA staining dye with proper strength of adsorption. AuNPs interact with primers and DNA polymerase, which can enhance PCR specificity. However, there is no report in the prior art that the above nanomaterials are applied to LAMP.
We firstly explore how the three nano materials have the effect of improving the specificity of the three nano materials when applied to the LAMP system. Respectively mixing MoS2(Shanghai Aladdin Biotechnology Co., Ltd., a product number of M196564-100mg), 5nm AuNPs (Shanghai Soffy biomedicine Co., Ltd., a product number of Au010001), and graphene oxide are prepared into an aqueous solution, and the aqueous solution is subjected to ultrasonic treatment for 5min by using an ultrasonic cleaning machine with a power of 50W, so that the solution is uniformly dispersed.
The target group (i.e. B in fig. 5) further comprises in the standard LAMP reaction system: 5 μ L of 20ng ultrasonically uniform GO, 5 μ L of 55ng ultrasonically uniform MoS 25 μ L of 25ng of ultrasonically homogeneous AuNPs or no nanomaterial contained. Specifically, the standard LAMP reaction system comprises: 2.5. mu.L of isothermal amplification buffer (20mM Tris-HCl, 10mM (NH)4)2SO4，150mM KCl，2mM MgSO4，0.1％Tween 20)，1.5μL 6mM MgSO43.5 μ L of 1.4mM dNTP mix, 0.9 μ L of target specific primer mix (1.6 μ M FIP, 1.6 μ M BIP, 0.2 μ M F3 and 0.2 μ M B3), 1.0 μ L of 8000units/ml Bst2.0WarmStart DNA polymerase and 0.5 μ L of 1 XSSYTO-9 dye, 5.0 μ L of plasmid sample containing COX-2 target sequence.
The negative control group (i.e., a in fig. 5) is a standard LAMP system without target sequence.
The experimental results are shown in fig. 5, wherein a in fig. 5 shows the three nanomaterials (GO, MoS)2AuNPs) were able to inhibit LAMP false positive amplification. As shown in B of FIG. 5, MoS2And AuNPs also have inhibition on target sequence amplificationDuring the preparation, a target amplification signal appears only after about 20 min. The inhibition effect of GO on the LAMP amplification efficiency of the target is much smaller, and the performance of GO is superior to that of MoS2And AuNPs.
Example 4 Effect of different addition amounts of graphene oxide on LAMP primer adsorption
GO is specifically combined with the primer through pi bonds and hydrogen bonds by virtue of the unique property of the surface, so that the primer is adsorbed on the surface, and the hybridization and the mismatching between primer chains are prevented. Therefore, we further select a primer chain F3 in the LAMP amplification system, fluorescently label the primer chain F3 with FAM, and experimentally explore the fluorescence change of the F3 primer in the GO system to verify the interaction between the primer and GO.
Fluorescence spectra were measured by an Agilent Cary Eclpse fluorescence spectrometer, USA. 200 mu L of 10 mu M FAM labeled F3 primer is put into a four-side light-transmitting cuvette and put into a detection groove of a fluorescence spectrometer, the detection parameter is set to 491nm of excitation light, the scanning wavelength range is 500-650nm, the excitation and emission slit width is 5nm, and the PMT voltage is set to 600V. Under the excitation of 491nm excitation light, the FAM-labeled F3 primer generates strong fluorescence with a maximum emission wavelength of 523nm (as shown in FIG. 6). The fluorescence of the FAM-labeled F3 primer probe was affected to varying degrees when different amounts of GO (10 ng/. mu.L, 30 ng/. mu.L, 50 ng/. mu.L, 70 ng/. mu.L, and 90 ng/. mu.L, respectively) were added to the primer system.
As can be seen from FIG. 6, the fluorescence intensity of the FAM-labeled primer was gradually decreased with the increase of GO concentration, and when GO concentration was 90 ng/. mu.L, the fluorescence intensity decreased to about 14% of the original value, indicating that the FAM fluorescence labeled on the primer was quenched. The reason why the fluorescence primer of FAM labeled on the primer is quenched is that the primer is adsorbed on the GO sheet surface, so that the labeled fluorophore interacts with GO and generates Fluorescence Resonance Energy Transfer (FRET). This experimental result demonstrates that GO can adsorb the primer effectively.
Example 5 graphene oxide reduces background fluorescence generated by DNA dyes in LAMP reaction systems
DNA dyes are commonly used to monitor LAMP amplification in real time. During amplification, the target sequence is continuously amplified to produce new double-stranded DNA amplification products, and the DNA dye is inserted into the double strands to generate strong fluorescent signals. However, in the conventional LAMP amplification, because the concentration of primers is high, the mismatch between the primers is easy to generate a dimer, a hairpin and other structures, and a high concentration of DNA dye is also embedded in the primer, so that a fluorescence background signal is generated. We therefore further investigated the interaction between Graphene Oxide (GO) and DNA staining dye, preferably we investigated the effect of GO on the DNA staining dye SYTO-9.
The experimental group is that 5 mu L of 20ng of GO with uniform ultrasound is added into a standard LAMP system, and specifically the LAMP reaction system comprises 2.5 mu L of isothermal amplification buffer (20mM Tris-HCl, 10mM (NH)4)2SO4，150mM KCl，2mM MgSO4，0.1％Tween 20)，1.5μL 6mM MgSO43.5 μ L of 1.4mM dNTP mix, 0.9 μ L of target specific primer mix (1.6 μ M FIP, 1.6 μ M BIP, 0.2 μ M F3 and 0.2 μ M B3), 1.0 μ L of 8000units/ml Bst2.0WarmStart DNA polymerase and 0.5 μ L of 1 XSSYTO-9 dye. The control group was a standard LAMP system without GO.
No amplification template was added to both the test and control groups. The fluorescence intensities of the test group and the control group were measured by a PCR instrument. The results of fluorescence intensity measurement of SYTO-9 in the test group and the control group are shown in FIG. 7. According to the result of fig. 7, the fluorescence intensity of the LAMP reaction system is weakened after 20ng of GO is added, and the fact that GO can adsorb SYTO-9, help to quench the fluorescence generated by SYTO-9, reduce the background fluorescence at the beginning of the LAMP reaction, and improve the amplification performance is proved.
Example 6 comparison of the effects of graphene oxide in LAMP and conventional PCR
We evaluated the sensitivity of GO-based LAMP detection system and compared it to traditional PCR.
LAMP sensitivity experimental determination based on graphene oxide: in a standard LAMP reaction (i.e., containing 2.5. mu.L of isothermal amplification buffer (20mM Tris-HCl, 10mM (NH))4)2SO4，150mM KCl，2mM MgSO4，0.1％Tween 20)，1.5μL 6mM MgSO43.5 μ L of 1.4mM dNTP mix, 0.9 μ L of target specific primer mix (1.6 μ M FIP, 1.6 μ M BIP, 0.2 μ M F3 and 0.2 μ M B3), 1.0 μ L of 8000units/ml Bst2.0WarmStart DNA polymerase and 0.5 μ L of 1 XSSYTO-9 dye) with 20ng GO added and amplification with the addition of a plasmid containing COX-2 target sequence as amplification template, the final amplification template concentration being 10 in order8copies/μL、107copies/μL、106copies/μL、105copies/μL、104copies/μL、103copies/. mu.L and 102copies/. mu.L, with negative controls (no target sequence template, NC). The LAMP reaction was carried out in a PCR instrument at a constant temperature of 65 ℃ for one hour, and the fluorescence intensity was read in real time by the PCR instrument. And then, taking a fluorescence inflection point value (reaction threshold time, Tt) corresponding to the logarithm according to the amplification templates with different concentrations to obtain a standard curve for detecting the COX-2 target sequence based on the LAMP reaction of the graphene oxide. A in FIG. 8 is a GO-based LAMP real-time fluorescence curve for a range of concentrations of target sequence, ranging from 108copies/. mu.L to 102copies/. mu.L, results indicate that detection limit of GO-based LAMP is 102copies/. mu.L. Plotting the reaction threshold time (Tt) against the logarithm of the initial concentration of the target sequence (LogC) yields a GO-based LAMP reaction standard curve with a good linear relationship (R)20.994) (see B in fig. 8).
The conventional PCR reaction is as follows: PCR reactions of COX-2 target sequences were performed using the SuperReal Premix Plus (SYBR Green) real-time PCR kit (Biotech, Inc., Kyoto, Beijing) according to the manufacturer's instructions. The reaction mixture (total volume 25. mu.L) contained 12.5. mu.L of 2 XSuperReal Premix Plus, 0.75. mu.L of 10. mu.M forward and reverse primers (wherein the nucleotide sequence of the forward primer is TTCAAATGAGATTGTGGGAAAATTGCT (SEQ ID NO: 6) and the nucleotide sequence of the reverse primer is AGATCATCTCTGCCTGAGTATCTT (SEQ ID NO: 7), both synthesized by Shanghai, 5. mu.L of the target (i.e., a plasmid containing the COX-2 target sequence) or ultrapure water. The reaction was carried out in a PCR instrument with the parameters set to: at 95 ℃ for 15min, 40 cycles of 95 ℃ for 10s and 60 ℃ for 32s, respectively. C in FIG. 8 is the PCR fluorescence curve for a range of concentrations of target sequence, ranging from 108copies/μL to 103copies/. mu.L, the result shows that the detection limit of PCR is 103copies/. mu.L. And a standard curve was obtained by plotting the cycle number (Ct) against the log of the initial concentration (LogC) of the target sequence (as shown in D in fig. 8).
From the above results, it can be obtained: compared with the traditional PCR reaction, the LAMP based on the graphene oxide has higher sensitivity and faster reaction.
Example 7 application of LAMP reaction System comprising graphene oxide in colorectal cancer detection
5.1 test methods
(1) Cell culture: colorectal cancer HT-29 cells and normal colon CCD-18Co cells were cultured in McCoy's 5A medium (Beijing Solebao technologies, Inc., cat # M9420) and RPMI 1640 medium (Beijing Solebao technologies, Inc., cat # M13800), respectively. SW-480 cells, pancreatic cancer PANC-1 cells, lung cancer HepG2 cells and cervical cancer HeLa cells were cultured in DMEM medium. All media contained 10% Fetal Bovine Serum (FBS) (v/v) (Sijiqing, cat # 11011-8611) and 1% mixed solution of penicillin streptomycin (100 ×) (Beijing Solebao technologies, cat # P1400). All cells were supplied by North Nay Bio Inc. Placing the cells and culture medium into sterilized 25cm2Placing the tissue culture bottle at 37 deg.C and 5% CO2And (5) culturing in a standard incubator.
(2) The extraction of total RNA of the cells is carried out by adopting a kit extraction method. The cells were centrifuged, lysate was added, the specific procedure was according to the supplier's instructions (Omega) and the final total RNA concentration was quantified using a micro uv-vis spectrophotometer and the obtained RNA was immediately used in the reverse transcription LAMP experiment.
(3) According to the sensitivity experimental determination of LAMP based on graphene oxide, the addition amount of GO in the LAMP reaction solution is 20ng, total RNA extracted from colorectal cancer cells (HT-29) is added as an amplification template for amplification, the final amplification concentration is 100 ng/mu L, 10 ng/mu L, 1 ng/mu L, 0.1 ng/mu L, 0.01 ng/mu L, 0.001/mu L, a positive control (plasmid containing COX-2 target sequence, PC) and a negative control (no target sequence template and NC) in sequence, the LAMP reaction is carried out in a PCR instrument at constant temperature of 65 ℃ for one hour, and the fluorescence intensity is read in real time by the PCR instrument. And then, taking a fluorescence inflection point value (reaction threshold time, Tt) corresponding to the logarithm according to the amplification templates with different concentrations to obtain a standard curve for detecting the COX-2 target sequence based on the LAMP reaction of the graphene oxide. And carrying out agarose gel electrophoresis detection on the LAMP reaction product.
Selecting different types of cancer cells as samples to be detected, extracting total RNA, performing fluorescent LAMP amplification based on 20ng GO, and verifying the specificity of the GO-based LAMP method for detecting colorectal cancer by detecting COX-2 targets.
The specificity of the LAMP system comprising graphene oxide was verified using total RNAs extracted from colorectal cancer cells (HT-29), pancreatic cancer cells (PANC-1), lung cancer cells (HepG2), cervical cancer cells (HeLa) and normal colon cells (CCD-18Co), respectively. Specifically, 25. mu.L of the LAMP reaction contains 2.5. mu.L of isothermal amplification buffer (20mM Tris-HCl, 10mM (NH))4)2SO4，150mM KCl，2mM MgSO4，0.1％Tween 20)，1.5μL 6mMMgSO43.5 μ L of 1.4mM dNTP mix, 0.9 μ L of target specific primer mix (1.6 μ M FIP, 1.6 μ M BIP, 0.2 μ M F3 and 0.2 μ M B3), 1.0 μ L of 8000units/ml Bst2.0WarmStart DNA polymerase, 0.5 μ LAMV reverse transcriptase and 0.5 μ L of SYTO-9 dye, 5 μ L of 20ng sonicated homogenous GO and total RNA extracted from different cancer cells were added separately. Amplification was carried out in a PCR apparatus at a constant temperature of 65 ℃ for 1 hour.
5.2 test results
To evaluate the sensitivity of the graphene oxide-based LAMP method, total RNA extracted from colorectal cancer cells (HT-29) at different concentrations was selected as an amplification template, and the real-time fluorescence amplification curve results are shown as a in fig. 9. This method showed very good amplification results for target concentrations in the range of 100 ng/. mu.L to 0.001 ng/. mu.L.
The time at which the amplification occurred in the fluorescent signal, i.e., the reaction threshold time (Tt), was plotted against the logarithm of the initial concentration (LogC) of the target sequence to obtain a standard curve of fluorescent LAMP containing graphene oxide (see B in fig. 9). From the standard curve shown in B of FIG. 9, it can be seen that there is a good linear relationship between Tt and LogCIs (R)20.993), the detection limit was 0.001 ng/. mu.l.
The amplified products were subjected to agarose gel electrophoresis (see C in fig. 9), which further demonstrated the accuracy of the experimental results.
As shown in D in FIG. 9, the Positive Control (PC) and colorectal cancer cells (HT-29) generated amplification reactions within 8 minutes and 20 minutes, respectively, while other cancer cells generated weak fluorescence signals at approximately 50 minutes.
These results indicate that the method provided by the present invention can identify colorectal cancer and distinguish other types of cancer by detecting COX-2mRNA, which indicates that the graphene oxide-based LAMP method shows very high specificity for the detection of COX-2mRNA in colorectal cancer.
Sequence listing
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Claims (10)
1. An amplification system comprising graphene oxide, the amplification system comprising: isothermal amplification buffer, MgSO4The kit comprises a dNTP mixture, a target specific primer group, DNA polymerase, an amplification template and 5-30 ng of graphene oxide.
2. The amplification system of claim 1, wherein the graphene oxide has a mass of 20ng, the DNA polymerase is Bst2.0WarmStart DNA polymerase, and the isothermal amplification buffer comprises 20mM Tris-HCl and 10mM (NH)4)2SO4、150mM KCl，2mM MgSO4And 0.1% Tween 20; preferably, the amplification system further comprises a fluorescent dye.
3. The amplification system of claim 1 or 2 wherein the amplification template comprises a coding sequence for cyclooxygenase-2 mRNA, the target-specific primer set comprising F3, B3, FIP, and BIP; preferably, the nucleotide sequence of the coding sequence is as shown in SEQ ID NO: 5, the sequences of F3, B3, FIP and BIP in the target-specific primer group are respectively shown as SEQ ID NO: 1 to 4.
4. A method for inhibiting loop-mediated isothermal amplification false positives by graphene oxide, comprising the steps of:
1) preparing an aqueous solution containing 5-30 ng of graphene oxide;
2) mixing the aqueous solution of graphene oxide obtained in the step 1) with a loop-mediated isothermal amplification reaction system, wherein the loop-mediated isothermal amplification reaction system comprises: isothermal amplification buffer, MgSO4A dNTP mix, a target specific primer set, a DNA polymerase and an amplification template;
3) isothermal amplification was performed on a PCR instrument.
5. The method for inhibiting the false positive of the loop-mediated isothermal amplification by using the graphene oxide according to claim 4, wherein the method comprises the following steps: oxygen in the step 1)The mass of the graphene is 20 ng; the DNA polymerase in the step 2) is Bst2.0WarmStart DNA polymerase, and the isothermal amplification buffer in the step 2) contains 20mM Tris-HCl and 10mM (NH)4)2SO4、150mM KCl，2mM MgSO4And 0.1% Tween 20; preferably, the loop-mediated isothermal amplification reaction system in step 2) further comprises a fluorescent dye.
6. The method according to claim 4 or 5, characterized in that: the amplification template in the step 2) comprises a coding sequence of cyclooxygenase-2 mRNA, and the target-specific primer group comprises F3, B3, FIP and BIP; preferably, the nucleotide sequence of the coding sequence is as shown in SEQ ID NO: 5, the sequences of F3, B3, FIP and BIP in the target-specific primer group are respectively shown as SEQ ID NO: 1 to 4.
7. Use of the amplification system comprising graphene oxide according to any one of claims 1 to 3 in the preparation of a kit for detecting a marker of rectal cancer.
8. The use of claim 8, the marker for rectal cancer is cyclooxygenase-2; preferably, the coding sequence of the cyclooxygenase-2 is shown in SEQ ID NO: 5, respectively.
9. The application of the graphene oxide in inhibiting the occurrence of nonspecific amplification in the loop-mediated isothermal amplification is characterized by comprising the following steps:
1) preparing an aqueous solution containing 5-30 ng of graphene oxide;
2) mixing the aqueous solution of graphene oxide obtained in the step 1) with a loop-mediated isothermal amplification reaction system, wherein the loop-mediated isothermal amplification reaction system comprises: isothermal amplification buffer, MgSO4A dNTP mix, a target specific primer set, a DNA polymerase and an amplification template;
3) isothermal amplification was performed on a PCR instrument.
10. The use according to claim 9, characterized in that the mass of graphene oxide in step 1) is 20 ng.
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