CN116042915B - Kit and method for detecting novel coronaviruses by graphene oxide-multiplex qPCR - Google Patents
Kit and method for detecting novel coronaviruses by graphene oxide-multiplex qPCR Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C01B32/00—Carbon; Compounds thereof
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- C01B32/198—Graphene oxide
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a kit and a method for detecting novel coronaviruses by graphene oxide-multiplex qPCR, belonging to the field of microbial molecular diagnosis. The present invention provides a light-emitting diode having a specific sp 2 And sp (sp) 3 Hybridized proportion graphene oxide, and a complex of the graphene oxide and a target gene detection forward primer. The complex is used for multiplex qPCR detection, so that the detection sensitivity can be remarkably improved while the complex has excellent specificity, the novel coronavirus can be rapidly, sensitively and accurately detected, and the complex has popularization and application values.
Description
Technical Field
The invention belongs to the field of microbial molecular diagnosis, and particularly relates to a kit and a method for detecting novel coronaviruses by graphene oxide-multiplex qPCR.
Background
The novel coronavirus is mainly transmitted through the respiratory tract, is easy to transmit in the crowd and is easy to cause aggregated infection, so that the detection of the novel coronavirus is particularly important. Currently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered as a gold standard for novel coronavirus detection. However, conventional RT-qPCR does not adequately meet the clinical needs of screening for new coronavirus infections and increases the potential risk of secondary transmission of new coronaviruses. Multiplex qPCR allows simultaneous detection of different target genes, but is less useful in clinical diagnostics due to the complexity of the system and the interference that may exist between different amplification reactions. Thus, there is a need for a new or improved, high sensitivity multiplex qPCR system to improve the accuracy of new coronavirus detection.
GO is an oxidized derivative of graphene, sp with hexagonal honeycomb structure 2 Regions and sp linked to oxygen-containing functional groups 3 A carbon base region. sp (sp) 2 The domains give GO an affinity for aromatics and have fluorescence quenching ability. In addition, is connected to sp 3 The hydrophilic groups of the domains stabilize GO in aqueous solution. GO adsorbs ssDNA by pi-pi stacking and hydrogen bonding. Based on the characteristic, research shows that GO-qPCR has good detection performance in disease diagnosis; in addition, there are reports of improving sensitivity and specificity of multiplex PCR reaction by using GO, for example, chinese patent application publication No. CN105603055a, in which GO is directly added into multiplex PCR reaction system, so as to improve specificity and yield of target product, and help to inhibit non-specific amplification, but the performance characteristics of GO used by GO are unknown.
However, for multiplex qPCR detection, the system composition is more complex than that of multiplex PCR, and the influence factors are numerous, and particularly, what influence is hardly expected on the multiplex qPCR detection effect can be caused by the performance of GO itself, the preparation process, the dosage of GO in the reaction system and other parameters, so that further exploration is still needed to improve the sensitivity and specificity of multiplex qPCR detection more significantly and the accuracy of novel coronavirus detection.
Disclosure of Invention
The invention aims to provide a kit and a method for detecting novel coronaviruses by graphene oxide-multiplex qPCR with high sensitivity.
The invention provides graphene oxide, wherein sp in the graphene oxide 2 Hybridized carbon atoms and sp 3 The number ratio of the hybridized carbon atoms is 1 (0.5-1.5).
Further, sp in the graphene oxide 2 Hybridized carbon atoms and sp 3 The number ratio of hybridized carbon atoms was 1:1.
Further, the graphene oxide is prepared by reacting a manganese-embedded graphite solution with hydrogen peroxide, wherein the volume ratio of the manganese-embedded graphite solution to the hydrogen peroxide is 5 (1-5), and is preferably 5:2.5;
the manganese-embedded graphite solution is prepared by the following method:
(1) Adding concentrated sulfuric acid into graphite powder and potassium nitrate, and uniformly mixing to obtain a system A; uniformly mixing potassium permanganate, potassium nitrate and concentrated sulfuric acid to obtain a system B;
(2) And adding the system B into the system A in multiple times, uniformly mixing, sealing, naturally settling for 20-40 days, and uniformly stirring.
The invention also provides a graphene oxide-primer complex, which is formed by compounding the graphene oxide and a gene primer, wherein the gene primer is a primer of a novel coronavirus specific target gene.
Further, the gene primer is a forward primer of a novel coronavirus RdRP gene and/or E gene; preferably, the forward primer sequence of the RdRP gene is shown in SEQ ID NO. 1; the forward primer sequence of the E gene is shown as SEQ ID NO. 4.
Further, the complex is prepared by mixing graphene oxide solution with a gene primer, incubating for 25-35 minutes at 50 ℃, and then performing ultrasonic treatment for 25-35 minutes; the concentration of the graphene oxide solution is 5.21-23.46. Mu.g/mL, preferably 10.11-13.44. Mu.g/mL, and more preferably 13.44. Mu.g/mL.
The invention also provides a kit for detecting the novel coronavirus, which comprises the graphene oxide-primer complex.
Further, the graphene oxide-primer complex is a complex formed by compositing graphene oxide with forward primers of novel coronavirus RdRP genes and E genes respectively; the kit also comprises reverse primers of RdRP gene and E gene of the novel coronavirus; the RdRP gene reverse primer sequence is shown as SEQ ID NO. 2; the reverse primer sequence of the E gene is shown as SEQ ID NO. 5;
preferably, the kit further comprises detection probes of a novel coronavirus RdRP gene and an E gene, wherein the sequence of the detection probe of the RdRP gene is shown as SEQ ID NO.3, and the sequence of the detection probe of the E gene is shown as SEQ ID NO. 6; the detection probe is modified with a fluorescence reporting group and a fluorescence quenching group;
more preferably, the kit further comprises a forward primer, a reverse primer and a detection probe of the reference gene; the detection probe is modified with a fluorescent reporter group and a fluorescent quenching group. The internal reference gene is preferably an RNase P gene, the forward primer sequence of the RNase P gene is shown as SEQ ID NO.7, the reverse primer sequence is shown as SEQ ID NO.8, and the probe sequence is shown as SEQ ID NO. 9.
Further, the kit further comprises a probe premix and enzyme-free water.
The invention also provides a method for detecting novel coronaviruses, comprising the following steps:
(1) The components of the kit are uniformly mixed with a sample to be tested, and incubated;
(2) Detecting the amplified product; the detection is agarose gel electrophoresis, fluorescence detection or colloidal gold test strip detection;
preferably, the kit comprises detection probes for the RdRP gene, the E gene and the RNase P gene of the coronavirus; the detection amplification product is a fluorescent detection, more preferably a real-time fluorescent PCR detection.
The invention has the beneficial effects that: the invention adopts the improved Hummers method to adjust the oxidation degree of GO to prepare the catalyst with proper sp 2 And sp (sp) 3 GO with a proportion, and a GO-forward primer complex constructed by using forward primers for adsorbing target genes, and is applied to a reaction system for detecting novel coronaviruses by multiple qPCR and other sp 2 And sp (sp) 3 Compared with a reaction system for detecting novel coronaviruses by multiple qPCR established by the GO-forward primer complex in proportion, the detection sensitivity is remarkably improved; meanwhile, the kit has excellent specificity, can realize rapid, sensitive and accurate detection of novel coronaviruses, and has popularization and application values.
The term "concentrated sulfuric acid" in the present invention refers to sulfuric acid solutions having a concentration of greater than or equal to 70%, with a concentration of 98% being most commonly used.
"novel coronavirus" refers to SARS-Cov-2.
The "RdRP gene" of the present invention is: the RNA-dependent RNA polymerase (RdRp) gene; gene ID 30897989.
"E gene" is an encodings gene; gene ID 43740570.
The "RNase P gene" is: the human ribonuclease P gene; gene ID 10556.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a graph showing the results of characterization of graphene oxide of the present invention.
Fig. 2 shows the screening results of graphene oxide preparation process and graphene oxide-forward primer complex preparation process (Control is a conventional multiple qPCR assay, i.e., a qPCR assay with no graphene modification of the forward primer).
FIG. 3 shows the results of multiplex qPCR assays for different viruses using the methods of the present invention.
FIG. 4 shows the comparison of the detection results of the method of the present invention with the conventional multiplex qPCR method (Control is the conventional multiplex qPCR detection, i.e., the qPCR detection with forward primer without graphene modification).
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
The primer and probe sequences related to the invention are as follows (please provide the sequences of the primer and probe):
RdRP gene forward primer sequence (SEQ ID NO. 1): GTGARATGGTCATGTGTGGCGG
RdRP gene reverse primer sequence (SEQ ID NO. 2): CARATGTTAAASACACTATTAGCATA
RdRP gene detection probe sequence (SEQ ID NO. 3): CAGGTGGAACCTCATCAGGAGATGC
E Gene forward primer sequence (SEQ ID NO. 4): ACAGGTACGTTAATAGTTAATAGCGT
E gene reverse primer sequence (SEQ ID NO. 5): ATATTGCAGCAGTACGCACACA E Gene detection probe sequence (SEQ ID NO. 6): ACACTAGCCATCCTTACTGCGCTTCG
The forward primer sequence of RNase P gene (SEQ ID NO. 7): AGATTTGGACCTGCGAGCG
RNase P gene reverse primer sequence (SEQ ID NO. 8): GAGCGGCTGTCTCCACAAGT
RNase P gene detection probe sequence (SEQ ID NO. 9): TTCTGACCTGAAGGCTCTGCGCG
Wherein the gene detection probe sequence is modified as follows:
RdRP gene detection probe sequence label: a fluorescence report group JOE is modified at the 5 'end, and a fluorescence quenching group BHQ-1 is modified at the 3' end;
e, sequence marking of a gene detection probe: a fluorescent reporter group ROX is modified at the 5 'end, and a fluorescent quenching group BHQ-2 is modified at the 3' end;
RNase P gene detection probe sequence labeling: and a fluorescent reporter group HEX is modified at the 5 'end, and a fluorescent quenching group BHQ-1 is modified at the 3' end.
Example 1 preparation of graphene oxide of the present invention
(1) At room temperature, 0.5g of graphite powder and 0.5g of KNO 3 And 23ml of concentrated H 2 SO 4 Added to beaker 1.
(2) 6g KMnO was added at room temperature 4 、1g KNO 3 And 46ml of concentrated H 2 SO 4 Added to beaker 2.
(3) Beaker 1 and beaker 2 were placed on a magnetic stirrer, respectively, while stirring thoroughly at the same rotational speed for 1.5 hours.
(4) 4ml of the mixed solution in beaker 2 was added to beaker 1 every 20 minutes until the mixed solution in beaker 2 was completely added to beaker 1.
(5) Stirring the mixed solution in the beaker 1 overnight, filling the mixed solution into a blue-mouth bottle for sealing and preserving, and naturally settling for 1 month to obtain the manganese-embedded graphite.
(6) After sedimentation, the manganese-embedded graphite is evenly mixed, 5ml of the mixture is added into a beaker, and 15ml of deionized water is added and stirred for 20 minutes.
(7) 2.5ml H was added to the beaker 2 O 2 And immediately stop stirring.
(8) Placing the solution into 3500Da dialysis bags, and dialyzing the dialysis bags in deionized water; and (3) until the pH value of the solution in the dialysis bag is 7.0, and collecting the product in the dialysis bag to obtain the graphene oxide solution.
EXAMPLE 2 construction of graphene oxide-forward primer complexes of the invention
(1) The graphene oxide solution obtained in example 1 was diluted to 13.44. Mu.g/mL and dispersed uniformly by sonication for 20 minutes.
(2) Graphene oxide solution and forward primers of RdRP gene, E gene and RNase P gene) were prepared according to 1:1.
(3) The mixture in (2) was incubated in a constant temperature heater at 50℃for 30 minutes and then sonicated for an additional 20 minutes.
(4) The mixture in (3) was centrifuged in a centrifuge for 1 hour at 15000rpm.
(5) And (3) after centrifugation, removing the upper layer solution of the mixed solution in the step (4) to respectively obtain a graphene oxide-RdRP gene forward primer complex, a graphene oxide-E gene forward primer complex and a graphene oxide-RNase P gene forward primer complex.
Example 3 graphene oxide-multiplex qPCR detection method of the present invention
(1) Graphene oxide-multiplex qPCR system: the total volume of each reaction was 25 μl, including: 12.5 mu L of probe premix which is a commercially available productII Probe qPCR SuperMix, beijing full-scale gold biotechnology Co., ltd.), rdRP gene, E gene and RNase P geneThe reverse primer (10. Mu.M) of each of the 0.5. Mu. L, rdRP gene, E gene and RNase P gene probes (10. Mu.M) of each of the 0.5. Mu. L, rdRP gene, E gene and RNase P gene graphene oxide-forward primer complexes were 1. Mu.L (the corresponding primer concentration in the complex was 5. Mu.M), 3.5. Mu.L of enzyme-free water and 1. Mu.L of each of the DNA standard control templates. The sample to be measured was added in an amount of 1. Mu.L. The RNase P gene is a reference gene.
(2) The above systems were mixed and then subjected to real-time fluorescence detection on a qTOWER 2.2 fluorescent quantitative gradient thermocycler (analytical Jena AG, jena, germany). The reaction procedure is divided into two steps: the first step: 94 ℃ for 30 seconds; and a second step of: 94 ℃ for 5 seconds; 60℃for 30 seconds (fluorescence signal acquisition) (. Times.40 cycles).
Comparative example 1 preparation of graphene oxide
According to the procedure of example 1, H 2 O 2 The dosage of the graphene oxide is adjusted to 10mL, and the rest conditions are unchanged, so that the graphene oxide is prepared.
Comparative example 2 construction of graphene oxide-forward primer Complex
Using the graphene oxide prepared in comparative example 1, graphene oxide-forward primer complexes of RdRP gene, E gene and RNase P gene were prepared according to the protocol of example 2.
Comparative example 3 graphene oxide-multiplex qPCR assay
Multiplex qPCR assays were performed using the graphene oxide-forward primer complex prepared in comparative example 2, with reference to the protocol of example 3.
The following experiments prove the beneficial effects of the invention.
Experimental example 1 characterization of graphene oxide
Detecting an object: graphene oxide prepared in example 1 and comparative example 1.
Direct observation of sp surrounded by disordered regions with TEM 2 Lattice region, revealing GO at 2.5mL and 10mL H 2 O 2 In the structure (fig. 1a and 1 b). At 2.5mL H 2 O 2 The GO prepared in the method contains rich and uniform sp 2 Lattice. However, at 10mL H 2 O 2 GO prepared in (C)Many disordered regions, only a few typical sp 2 Lattice.
AFM imaging determines the size of the GO sheets and compares the specific surface areas of the two types of GO. Fig. 1c and 1d show the morphology of GO samples visualized by AFM imaging. At 2.5mL of H 2 O 2 The average lateral dimension of GO is about 2 μm. Will H 2 O 2 After increasing the volume of 10mL, the transverse dimension of GO decreases.
Thus, TEM and AFM results confirm that 2.5mL of H 2 O 2 It is possible to synthesize GO with an optimal sp2-sp3 ratio and a large specific surface area.
Two prominent bands, 1374cm, are shown in the raman spectrum of GO -1 And 1598cm -1 Respectively corresponding to D (sp 3 Hybridization of C) and G (sp) 2 Hybridization C) band (fig. 1 e). The intensity ratio of D-band and G-band (ID/IG) is used to describe the degree of disorder of graphene. At 2.5mL and 10mL H 2 O 2 The calculated ID/IG ratio of GO was 2.63 and 8.28, respectively. To further explore the groups on the GO surface, we performed FT-IR studies, the results are shown in figure 1 f. At 2.5mL H 2 O 2 The GO spectra of (a) show characteristic absorption bands of 3419, 1734, 1626, 1279 and 1052cm-1, respectively, caused by O-H stretching vibration, c=o stretching vibration, c=c stretching vibration, epoxy C-O-C bending vibration and C-O stretching vibration. Notably, H is 2 O 2 An increase in volume of 10ml would result in a peak disappearance of 1734 cm-1 (c=o) due to the epoxy functionality being surmounted by H 2 O 2 And (5) breaking. Thus, we analyzed H using Raman and FT-IR 2 O 2 Effect of volume on GO synthesis. Excess H 2 O 2 The epoxy functional groups are destroyed, thus reducing the specific surface area and increasing the proportion of disordered regions.
Subsequently, 2.5mL H was used 2 O 2 The synthesized GO was further analyzed with XPS and UV-Vis. The chemical composition of the GO surface and the atomic ratio of the elements are described by XPS. Thus, the unwind C1s peak for each functional group is c=c (284.6 eV), C-C (285.7 eV), C-O (287.0 eV), c=o (288.0 eV) and O-c=o (289.0 eV) (fig. 1g) A. The invention relates to a method for producing a fibre-reinforced plastic composite The carbon and oxygen contents were 63.36 and 33.57% and the carbon to oxygen ratio was 1.89. The uv-visible spectrum of GO shows that GO exhibits c=c pi-pi at 230nm * N-pi transition and 295nm c=o * Characteristic absorption peaks of the transition (fig. 1 h).
Further determining sp according to peak areas of different functional groups corresponding to XPS test results 2 -sp 3 Is about 1:1, as shown in table 1:
table 1: sp (sp) 2 -sp 3 Peak area and ratio of (2)
The above results indicate that the present invention successfully synthesizes a light sp with a large specific surface area and a suitable (about 1:1) 2 -sp 3 A proportion of water-soluble GO.
Experimental example 2, optimization of the degree of oxidation, solution concentration and Compound preparation temperature screening of graphene oxide of the present invention
(1) Oxidation degree screening of graphene oxide
The results of the real-time fluorescence detection of the systems of example 3 and comparative example 3 are shown in FIG. 2 a. It can be seen that the Ct value of the multiplex qPCR detection of each gene forward primer modified by GO prepared by the process of example 1 of the present invention is significantly lower than that of the GO modified product of comparative example 1, and the detection sensitivity is significantly higher.
(2) Graphene oxide solution concentration screening
The dilution factors of the GO solution in step (1) of example 2 were adjusted to be diluted to a concentration of 5.21. Mu.g/mL, 7.45. Mu.g/mL, 10.11. Mu.g/mL, 13.44. Mu.g/mL (example 2), 15.58. Mu.g/mL, 19.12. Mu.g/mL, 21.75. Mu.g/mL and 23.46. Mu.g/mL, respectively. A graphene oxide-forward primer complex was constructed in the same manner as in example 2. And multiplex qPCR assays were performed as in example 3.
As shown in FIG. 2b, it can be seen that the Ct value of the complex prepared from graphene solution with concentration of 10.11-13.44. Mu.g/mL for multiplex qPCR detection is significantly lower and the sensitivity is significantly higher.
(3) Preparation temperature screening of composites
Adjusting the incubation temperatures of graphene oxide and forward primer in step (3) of example 2, to be: 4 sets of graphene oxide-forward primer complexes were constructed at 35℃at 50 ℃ (example 2), 65℃and 80℃respectively, and multiplex qPCR assays were performed as in example 3.
As shown in fig. 2c, it can be seen that the Ct value can be significantly reduced and the sensitivity can be improved when the temperature is set to 50 ℃.
Experimental example 3 evaluation of Performance of graphene oxide-multiplex qPCR detection of novel coronaviruses of the present invention
(1) Specificity assessment
The specificity of GO-multiplex qPCR was assessed using nine common respiratory viruses, including two RNA viruses and seven DNA viruses. Two RNA viruses (coronavirus HKU1 and parainfluenza virus) and seven DNA viruses (enterovirus, adenovirus, rhinovirus, human cytomegalovirus, varicella-zoster virus, mumps virus and measles virus). The novel coronaviruses and these 9 viruses were detected simultaneously using GO-multiplex qPCR and traditional multiplex qPCR. The results are shown in FIG. 3, and the other viruses except the specific genes RdRP and E of the novel coronavirus have no amplification curve, so that the method has good specificity.
(2) Sensitivity assessment
Three plasmids containing RdRP, E and RNase P target sequences were used to determine the sensitivity of the GO-multiplex qPCR method. Each plasmid template was from 10 7 To 10 1 Copy/. Mu.L was serially diluted. Thus, each plasmid concentration was tested simultaneously using two methods to compare the sensitivity of the graphene oxide-multiplex qPCR of the present invention to detect novel coronaviruses and conventional multiplex qPCR to detect novel coronaviruses. The results are shown in FIG. 4, the detection limit of the graphene oxide-multiplex qPCR on the novel coronavirus is 10 copies/reaction, and compared with the conventional multiplex qPCR, the detection limit is improved by 10 times.
Therefore, the method has high detection sensitivity and specificity to the novel coronavirus.
In summary, the present invention employs a modified Hummers method to regulate GO oxidationPreparation of the appropriate sp 2 And sp (sp) 3 The GO-forward primer complex is constructed by using the forward primer of the target gene, and is applied to a reaction system for detecting novel coronaviruses by multiple qPCR, so that the sensitivity of the multiple qPCR is obviously improved, and the detection limit is reduced by 10 times compared with the traditional multiple qPCR; meanwhile, the kit has excellent specificity, can realize rapid, sensitive and accurate detection of novel coronaviruses, and has popularization and application values.
Claims (3)
1. A kit for detecting novel coronaviruses is characterized in that,
the method comprises a graphene oxide-primer complex, wherein the complex is formed by compounding graphene oxide and a gene primer, and the gene primer is a primer of a novel coronavirus specific target gene;
sp in the graphene oxide 2 Hybridized carbon atoms and sp 3 The number ratio of hybridized carbon atoms is 1:1; the quantitative ratio is determined according to peak areas of different functional groups corresponding to XPS test results;
the gene primer is a forward primer of a novel coronavirus RdRP gene and an E gene; the sequence of the RdRP gene forward primer is shown as SEQ ID NO. 1; the forward primer sequence of the E gene is shown as SEQ ID NO. 4;
the complex is prepared by mixing graphene oxide solution with a gene primer, incubating for 25-35 minutes at 50 ℃, and performing ultrasonic treatment for 25-35 minutes; the concentration of the graphene oxide solution is 13.44 mug/mL;
the graphene oxide is prepared by reacting a manganese-embedded graphite solution with hydrogen peroxide, and the volume ratio of the manganese-embedded graphite solution to the hydrogen peroxide is 5:2.5;
the manganese-embedded graphite solution is prepared by the following method:
(1) Adding concentrated sulfuric acid into graphite powder and potassium nitrate, and uniformly mixing to obtain a system A; uniformly mixing potassium permanganate, potassium nitrate and concentrated sulfuric acid to obtain a system B;
(2) Adding the system B into the system A for multiple times, uniformly mixing, sealing, naturally settling for 20-40 days, and uniformly stirring;
the kit also comprises reverse primers of RdRP gene and E gene of the novel coronavirus; the RdRP gene reverse primer sequence is shown as SEQ ID NO. 2; the reverse primer sequence of the E gene is shown as SEQ ID NO. 5;
the kit also comprises detection probes of a novel coronavirus RdRP gene and an E gene, wherein the sequence of the detection probe of the RdRP gene is shown as SEQ ID NO.3, and the sequence of the detection probe of the E gene is shown as SEQ ID NO. 6; the detection probe is modified with a fluorescent reporter group and a fluorescent quenching group.
2. The kit of claim 1, further comprising a forward primer, a reverse primer and a detection probe for a reference gene; the detection probe is modified with a fluorescent reporter group and a fluorescent quenching group.
3. The kit of claim 1 or 2, further comprising a probe premix, and enzyme-free water.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102502612A (en) * | 2011-11-21 | 2012-06-20 | 南京工业大学 | Method for preparing grapheme through oxidation reduction |
| CN102978201A (en) * | 2012-12-24 | 2013-03-20 | 厦门大学 | Application of graphene in polymerase chain reaction as reinforcing agent |
| CN102998447A (en) * | 2012-12-03 | 2013-03-27 | 广西壮族自治区兽医研究所 | Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor |
| CN105460922A (en) * | 2015-11-29 | 2016-04-06 | 福建医科大学 | Partially reduced GO (graphene oxide) fluorescence resonance energy transfer nano-probe and preparation method thereof |
| MX2016007399A (en) * | 2016-06-07 | 2017-12-06 | Centro De Investigacion En Mat Avanzados S C | Method for the partial reduction of graphene oxide by means of mild reducers at room temperature. |
| CN112626209A (en) * | 2020-12-22 | 2021-04-09 | 绵竹市人民医院 | miRNA marker for ovarian cancer diagnosis, application thereof and diagnosis kit |
| CN113025476A (en) * | 2020-06-17 | 2021-06-25 | 山东大学 | Double-layer micro-fluidic chip, kit and method for detecting novel coronavirus |
| CN113265448A (en) * | 2020-02-14 | 2021-08-17 | 成都中医药大学 | Method for improving sensitivity and specificity of real-time fluorescence PCR (polymerase chain reaction) based on graphene oxide |
| WO2021215556A1 (en) * | 2020-04-22 | 2021-10-28 | (주)오상헬스케어 | Method for preparing coronavirus diagnosis kit, coronavirus diagnosis kit prepared thereby, and method for diagnosing coronavirus by using same |
| KR20220009233A (en) * | 2020-07-15 | 2022-01-24 | 울산과학기술원 | Reduced graphene oxide and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11731875B2 (en) * | 2020-01-15 | 2023-08-22 | University Of Wyoming | Methods for production of graphene oxide |
-
2022
- 2022-09-30 CN CN202211213523.5A patent/CN116042915B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102502612A (en) * | 2011-11-21 | 2012-06-20 | 南京工业大学 | Method for preparing grapheme through oxidation reduction |
| CN102998447A (en) * | 2012-12-03 | 2013-03-27 | 广西壮族自治区兽医研究所 | Electrochemical immunosensor for detecting H5N1 subtype avian influenza viruses and manufacturing method of electrochemical immunosensor |
| CN102978201A (en) * | 2012-12-24 | 2013-03-20 | 厦门大学 | Application of graphene in polymerase chain reaction as reinforcing agent |
| CN105460922A (en) * | 2015-11-29 | 2016-04-06 | 福建医科大学 | Partially reduced GO (graphene oxide) fluorescence resonance energy transfer nano-probe and preparation method thereof |
| MX2016007399A (en) * | 2016-06-07 | 2017-12-06 | Centro De Investigacion En Mat Avanzados S C | Method for the partial reduction of graphene oxide by means of mild reducers at room temperature. |
| CN113265448A (en) * | 2020-02-14 | 2021-08-17 | 成都中医药大学 | Method for improving sensitivity and specificity of real-time fluorescence PCR (polymerase chain reaction) based on graphene oxide |
| WO2021215556A1 (en) * | 2020-04-22 | 2021-10-28 | (주)오상헬스케어 | Method for preparing coronavirus diagnosis kit, coronavirus diagnosis kit prepared thereby, and method for diagnosing coronavirus by using same |
| CN113025476A (en) * | 2020-06-17 | 2021-06-25 | 山东大学 | Double-layer micro-fluidic chip, kit and method for detecting novel coronavirus |
| KR20220009233A (en) * | 2020-07-15 | 2022-01-24 | 울산과학기술원 | Reduced graphene oxide and preparation method thereof |
| CN112626209A (en) * | 2020-12-22 | 2021-04-09 | 绵竹市人民医院 | miRNA marker for ovarian cancer diagnosis, application thereof and diagnosis kit |
Non-Patent Citations (3)
| Title |
|---|
| Effect of hydrogen peroxide on properties of graphene oxide in Hummers method;Myung Jin Yoo;Carbon;第141卷;摘要，第2节实验及第3节结果讨论部分 * |
| 氧化石墨烯对聚合酶链式反应扩增效率、灵敏度和特异性的影响;蒋城等;环境化学;第36卷(第4期);第724-729页 * |
| 陈润峰著.有机化学与光电材料实验教程.南京：东南大学出版社,2019,第233-234页. * |
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