CN112501261A - Fluorescence detection method for nucleic acid amplified isothermally by using nano plasma resonance chip - Google Patents
Fluorescence detection method for nucleic acid amplified isothermally by using nano plasma resonance chip Download PDFInfo
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Abstract
The invention relates to a fluorescence detection method for nucleic acid amplified isothermally by using a nano plasma resonance chip. Specifically, the invention relates to a method for isothermal amplification nucleic acid fluorescence detection of target nucleic acid by using a nano plasma resonance chip. The invention also relates to an isothermal amplification nucleic acid fluorescence detection kit for detecting the target nucleic acid. The invention can effectively improve the nucleic acid amplification speed and the detection sensitivity, the lower limit of nucleic acid detection reaches or exceeds the conventional PCR nucleic acid detection level, and the rapid high-flux high-sensitivity nucleic acid detection can be realized on a portable instrument.
Description
Technical Field
The present invention relates to the field of nucleic acid amplification and detection. Specifically, the invention relates to a method for isothermal amplification nucleic acid fluorescence detection of target nucleic acid by using a nano plasma resonance chip. The invention also relates to an isothermal amplification nucleic acid fluorescence detection kit for detecting the target nucleic acid.
Background
The existing Isothermal Amplification (Isothermal Amplification) nucleic acid detection technology such as LAMP, RPA and ERA is realized by putting all reaction components including a DNA sample, a forward primer, a reverse primer, polymerase, recombinase, a fluorescent probe and the like into a PCR Amplification tube to perform DNA Amplification reaction in a constant temperature environment, and performing real-time acquisition and recording of a fluorescent signal in the Amplification reaction through a fluorescent reader.
The main disadvantages of the prior art are two: 1. because the reaction system is very complicated, the DNA template, the primer and various proteases are highly mixed, and a plurality of reactions are simultaneously carried out and mutually influence in space, so that the inhibition of the sample reaction with ultralow concentration (< 10^4 copies/mL) is caused, the lower detection limit of the conventional isothermal amplification nucleic acid detection technology used in several isothermal amplification nucleic acid detection products such as IDNo of Yapei company and the like is limited to more than 10^4 copies/mL, and the high-sensitivity detection close to single molecule level can not be carried out like the conventional thermal cycle PCR method. The speed of conventional isothermal amplification for higher concentration sample detection is likewise limited. 2, because the isothermal amplification reaction is highly sensitive to temperature, the temperature stability and uniformity control in the reaction system have high requirements on the overall constant temperature of the detection system, otherwise, the problems of poor reaction speed and poor detection time repeatability occur, the reaction efficiency is particularly influenced, and the reaction time is prolonged.
Disclosure of Invention
Compared with the conventional PCR nucleic acid amplification detection based on temperature rise and fall cycle control, the nucleic acid isothermal amplification technology only needs to carry out nucleic acid detection at constant temperature, not only has higher amplification speed, but also has small requirements on equipment and instruments, thereby being particularly suitable for the application scene of mobile rapid nucleic acid detection. The Nano plasma Resonance (Nano SPR) chip enhanced isothermal amplification nucleic acid fluorescence detection technology further improves the nucleic acid amplification speed and the detection sensitivity, the lower limit of nucleic acid detection reaches or exceeds the conventional PCR nucleic acid detection level, and the rapid high-flux high-sensitivity nucleic acid detection can be realized on a portable instrument.
The invention fixes the nucleic acid isothermal amplification reaction on the nano plasma resonance chip, and utilizes the optical resonance enhanced fluorescence effect, the thermal stability effect and the primer surface fixing effect of the chip to greatly improve the sensitivity and speed of the conventional isothermal amplification nucleic acid fluorescence detection, thereby achieving the purpose of rapid and high-sensitivity nucleic acid detection.
The main difference of the technical implementation scheme of the invention is that a forward Primer (5 'SH F Primer) for modifying SH by 5' is fixed on the surface of the nano plasma resonance chip in advance, and the site which is not combined on the surface of the chip is sealed so as to avoid quenching fluorescent group by the chip in the subsequent reaction process. Then, the pre-mixed and preheated isothermal amplification reaction systems such as LAMP, RPA and ERA are quickly added into a 96-well plate provided with a chip, and the 96-well plate is placed in a 37 ℃ constant-temperature microplate reader for real-time acquisition and recording of fluorescence signals.
Without being bound by theory, because the forward primer is fixed on the surface of the chip in advance, and only the reverse primer and the rest amplification components are contained in the solution, the modification of the surface of the chip reduces the steric hindrance caused by the combination of the recombinase and the corresponding template with the two primers to a certain extent, so that the FRET probe and the reverse (R) primer are more efficiently positioned to a complementary chain, and the release of the fluorescent group and the amplification efficiency of the reaction system are indirectly enhanced. Along with the reaction, part of the amplified molecules are always fixed on the surface of the chip; this portion of the amplicon will also generate a signal based on the characteristics of the chip itself. Meanwhile, the gold-plated chip with the nano structure can well keep the constant temperature around a reaction system on the surface of the chip, so that the enzyme is always in the best reaction activity.
In the whole reaction process, the reaction is always divided into two phases to participate in the reaction, the amplification reaction of a forward primer and a template is mainly carried out on the surface of the chip, meanwhile, in the next round of reaction amplification, one template strand is released into a solution and is used as a target for recognition by a reverse primer and a FRET probe, a large number of amplicons are continuously generated in the direction of an R primer, simultaneously, an Exo III cutting FRET probe is activated to release a fluorescent group, the other strand in an amplification product reaches the surface of the chip and is used as a F primer to complete further amplification, the whole reaction is continuously circulated until a substrate is exhausted, the reaction is basically saturated, fluorescent substances are also continuously accumulated along with the reaction, and the amplification degree is judged according to the intensity of the collected fluorescent signals.
In one aspect, the invention relates to a method for isothermal amplification nucleic acid fluorescence detection of target nucleic acid using nanoplasmon resonance chip. The method of the invention may comprise the following steps in order: (1) fixing a forward primer of a 5' modified SH for amplifying a target nucleic acid on the surface of the nano plasma resonance chip; (2) optionally blocking the sites of the forward primer on the surface of the nanoplasmon resonance chip, to which the 5' modified SH is not bound, with a blocking reagent; (3) contacting an isothermal amplification reaction system with the nano plasma resonance chip and carrying out isothermal amplification nucleic acid reaction; and (4) collecting and recording fluorescence signals generated by the reaction of step (3) in real time using a fluorescence detection instrument. In the method, the surface of the chip may be washed after step (1) to remove the non-immobilized primer.
In one aspect, the nanoplasmon resonance chip may be a nanostructured gold-plated chip.
In one aspect, in step (2), unbound sites on the chip surface can be blocked to avoid quenching of the fluorescent group by the chip during subsequent reactions. The blocking reagent used in step (2) may be a thiol compound, for example a hydrocarbon containing one or more thiol groups and optionally one or more hydroxyl groups, having up to 10 (e.g. 1 to 10 or 2 to 6) carbon atoms, for example an alkane, for example a straight or branched alkane. Preferably the blocking reagent may be 6-mercapto-1-hexanol.
In one aspect, in step (3), the contacting and reacting can be performed in a well plate, such as a 96 well plate. Pre-mixed pre-heated isothermal amplification reaction systems such as LAMP, RPA and ERA can be added to the well plate with the chip.
In one aspect, the fluorescence detector device may be a microplate reader. In step (4), real-time acquisition and recording of the fluorescent signal can be performed in a 37 ℃ thermostated microplate reader.
In one aspect, the target nucleic acid may be DNA or RNA. The target nucleic acid may be single-stranded or double-stranded. For example, the target nucleic acid may be derived from the genome of a pathogen. Virosomes may be bacteria, viruses, fungi and parasites. In one aspect, the target nucleic acid is derived from the genome of a novel coronavirus. For example, the target nucleic acid may be the N gene of the novel coronavirus or the orf1ab gene. The target nucleic acid may also be referred to as an amplification template or template, in the case of double strands, each strand may be bound by a forward primer and a reverse primer, respectively. The target nucleic acid may be comprised in the sample to be detected. The sample can be from a patient, subject, environment, product, and the like. The sample from the patient or subject may be saliva, sputum, a deep throat or nasal sample, aspirates, urine, blood, serum, stool, and the like. The environmental sample may be a hospital, factory, market, food processing plant, wholesale market derived sample. The sample from the product may be a sample from a food product, a seafood product, a medical product.
In one aspect, an isothermal amplification reaction system can comprise a target nucleic acid, a reverse primer for amplifying the target nucleic acid, an enzyme and/or protein required to perform an isothermal amplification nucleic acid fluorescence detection reaction, and a fluorescent probe for detecting an amplicon. In one aspect, the isothermal amplification reaction system may comprise a polymerase, a recombinase, and optionally a single-stranded DNA binding protein and an exonuclease. These reagents or reactants are not immobilized on the chip surface. Therefore, they are usually present in a free form in the reaction system. In the isothermal amplification reaction system, various reagents or reactants are present in the form of a solution. After the reaction system is contacted with the chip, the forward primer of the 5' modified SH remains fixed on the surface of the chip, and other reaction reagents or reactants exist in a free form in the reaction solution. In one aspect, the fluorescent probe used to detect the amplicon can be a FRET probe. It is understood by those skilled in the art that the forward and reverse primers mentioned herein are used in pairs for amplifying the same target nucleic acid, e.g. a certain double stranded DNA or a certain viral gene. The amplicon generated by the immobilized forward primer may remain immobilized on the chip surface while the amplicon generated by the free reverse primer remains free. The exonuclease may be exonuclease III.
The chip of the invention can be fixed with a plurality of forward primers of 5' modified SH and used for amplifying a plurality of different target nucleic acids. Accordingly, the reaction system may comprise a plurality of corresponding reverse primers for amplifying said plurality of different target nucleic acids.
In one aspect, the isothermal amplification reaction system is an isothermal amplification reaction system selected from LAMP, RPA and ERA. Isothermal amplification nucleic acid fluorescence detection is performed using loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), or enzymatic recombinant isothermal amplification (ERA) techniques. Isothermal amplification nucleic acid fluorescence detection can also be performed using nucleic acid sequence dependent amplification (NASBA), Rolling Circle Amplification (RCA), Single Primer Isothermal Amplification (SPIA), helicase dependent DNA isothermal amplification (HDA), Strand Displacement Amplification (SDA). The isothermal amplification reaction system of the present invention may be any one of those amplification reaction systems. These amplification techniques are well known in the art, and the principles and reagents used are described in the relevant technical literature and can be determined by the skilled person.
In one aspect, the invention relates to an isothermal amplification nucleic acid fluorescence detection kit for detecting a target nucleic acid. The kit of the present invention may comprise (a) a nanoplasmon resonance chip, (b) a forward primer for amplifying a 5' modified SH of a target nucleic acid, and (c) isothermal amplification reaction reagents and/or reactants. In this aspect, a forward primer for amplifying a 5' modified SH of a target nucleic acid may be immobilized on the surface of the nanoplasmon resonance chip. Isothermal amplification reaction reagents and/or reactions comprise a reverse primer for amplifying a target nucleic acid. The reaction reagents and/or reactants may further comprise enzymes and/or proteins required to perform the isothermal amplified nucleic acid fluorescence detection reaction, and a fluorescent probe for detecting amplicons. In one aspect, the reaction reagents and/or reactants may comprise a polymerase, a recombinase, and optionally a single-stranded DNA binding protein and an exonuclease. These reagents or reactants are not immobilized on the chip surface. Therefore, they are usually present in a free form in the reaction system. In one aspect, the fluorescent probe used to detect the amplicon can be a FRET probe. It is understood by those skilled in the art that the forward and reverse primers mentioned herein are used in pairs for amplifying the same target nucleic acid, e.g. a certain double stranded DNA or a certain viral gene. The exonuclease may be exonuclease III.
In one aspect, the reaction reagents or reactants used in the kits of the invention are those used in the following isothermal amplification techniques: loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), enzymatic recombinant isothermal amplification (ERA), nucleic acid sequence-dependent amplification (NASBA), Rolling Circle Amplification (RCA), Single Primer Isothermal Amplification (SPIA), helicase-dependent DNA isothermal amplification (HDA), and Strand Displacement Amplification (SDA), etc., with loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), and enzymatic recombinant isothermal amplification (ERA) being preferred.
In one aspect, the kit further comprises a blocking reagent for blocking sites of the nanoplasmon resonance chip to which the forward primer of the 5' modified SH is not bound on the surface. The blocking reagent may be a thiol compound, for example a hydrocarbon containing one or more thiol groups and optionally one or more hydroxyl groups, having up to 10 (e.g. 1 to 10 or 2 to 6) carbon atoms, for example an alkane, for example a straight or branched alkane. Preferably the blocking reagent may be 6-mercapto-1-hexanol.
In one aspect, the amplification reaction systems, reagents and reactants used in the methods and kits of the invention may comprise materials necessary to carry out isothermal amplification reactions and detection reactions, such as the reaction reagents and reactants mentioned above. Besides, dNTPs, metal ions required for enzyme activity such as magnesium ions, buffers, fluorescent labels, solvents, and the like may be contained. They may also include proteins required for the reaction, such as single-stranded nucleic acid binding proteins, e.g., single-stranded DNA binding proteins. The enzymes required for carrying out the isothermal amplification nucleic acid fluorescence detection reaction include enzymes required for the amplification reaction, for example, polymerases including DNA or RNA polymerases, recombinases, reverse transcriptases, rnases, helicases, and the like; and enzymes required for the fluorescent detection reaction, for example exonucleases including DNA exonucleases. The enzymes or proteins required for different types of isothermal amplification reactions may be different and the skilled person can determine the corresponding enzymes or proteins.
In one aspect, the target nucleic acid detected by the methods and kits of the invention is derived from the genome of a novel coronavirus, for example selected from the group consisting of the N gene and ORF1ab gene of a novel coronavirus.
The term "SH 5' -modified forward primer" means a primer in which-OH at the 5' -terminal nucleotide residue is substituted with-SH, and includes a forward primer in which-OH at the phosphate group of the 5' -terminal nucleotide residue is substituted with-SH. The forward primer for 5' modified SH may be prepared using conventional DNA synthesis methods, and may be synthesized, for example, by a supplier of a specialized DNA synthesis service. Primer synthesis methods for modifying SH functionality are known and are also used by numerous DNA synthesis facilitators.
The nanoplasmon resonance chip may be coated with a gold atomic layer or have a gold atomic surface. The thiol (-SH) group of the forward primer and/or blocking reagent that modifies SH at 5' binds to a gold atom on the surface of the chip, thereby allowing the primer/blocking reagent to attach to the surface of the chip. The bonding of the thiol group to the gold atom is stable and, without being bound by theory, may be a physical attachment and/or the formation of various chemical bonds, including covalent bonds, ionic bonds, hydrogen bonds, van der waals forces, and the like, such as Au-S covalent bonds. Methods for immobilization are well known to those skilled in the art.
In one aspect, the fluorescence detection reaction of the isothermal amplified nucleic acids in the methods of the invention can be performed at about 30 ℃ to 45 ℃, e.g., 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, or 44 ℃, preferably 37 ℃.
In one aspect, primers and probes for amplifying the N gene and ORF1ab gene of the novel coronavirus are shown in the following table. These primers and/or probes may be used in the methods of the invention or comprised in a kit.
TABLE 1
Drawings
FIG. 1 shows the method for isothermal amplification nucleic acid fluorescence detection of target nucleic acid using nanoplasmon resonance chip and the reagents used in the method. FIG. 2 details the principle of amplification and the reagents used in the method of the invention.
FIGS. 3 and 4 show the effect of amplifying and detecting the N gene of the novel coronavirus by the method of the present invention. FIG. 3 shows the fluorescence intensity of a sample containing the copy number of each gene (N gene) at 1 hour of amplification. FIG. 4 is the fluorescence intensity of a sample containing the copy number of each gene (N gene) after 20 minutes of amplification.
FIGS. 5 and 6 show the effect of amplifying and detecting the orf1ab gene of the novel coronavirus by the method of the present invention. FIG. 5 is the fluorescence intensity of a sample containing the copy number of each gene (orf 1ab gene) at 1 hour of amplification. FIG. 6 is the fluorescence intensity of a sample containing the copy number of each gene (orf 1ab gene) after 20 minutes of amplification.
FIG. 7 shows the relationship between fluorescence intensity of isothermal amplification assay of the present invention and copy number of nucleic acid template in a sample.
Detailed Description
Examples
The experimental method comprises the following steps:
1. modifying the surface of the chip:
the F-SH primer is fixed on the surface of the chip, and the surface of the chip is washed to remove the unfixed primer. This step corresponds to "forward primer (SH) modified nanoplasmon chip" in FIG. 1.
2. Sealing the surface of the chip:
and adding a blocking group solution (blocking reagent) to the surface of the modified chip for incubation, and then washing the chip to remove all residual solution. This step corresponds to "6-mercapto-1-hexanol (MCH)" and "MCH-blocking modified nanoplasma chip unbound sites" in figure 1.
ERA nucleic acid isothermal amplification reaction:
and reagents such as polymerase, recombinase, single-stranded DNA binding protein, exonuclease and dNTP are stored in the form of mixed freeze-dried powder. Dissolving the mixed freeze-dried powder by using a dissolving agent before reaction. The forward-SH primer, reverse primer and fluorescent probe for detecting the N gene and orf1ab gene are shown in Table 1. The isothermal amplification detection reaction was performed at 37 ℃. Adding the reaction quantities according to the proportion, fully mixing uniformly, centrifuging for a short time, opening a cover of the 8-connection pipe, reversely buckling the cover on a desktop, adding an activator into the center of the cover, centrifuging for a short time, fully mixing uniformly, placing the mixture in a constant-temperature water bath kettle at 37 ℃ for preheating for 5min, taking out the mixture, immediately adding the mixture into a micropore plate chip hole of the integrated forward primer modified nano plasma resonance chip, and avoiding bubbles; quickly putting the fluorescent powder into a pre-preheated fluorescent microplate reader, and collecting corresponding fluorescent signals by adopting a fluorescent mode. These steps correspond to the "Enzymatic Recombinant Amplification (ERA) reaction system", "ExoIII cleaved FRET probe releases fluorophore", "ERA indicates that the amplification reaction reaches equilibrium" and the display shows the results in fig. 1.
Figure 1 depicts the experimental procedure described above in a schematic way.
FIG. 2 shows the interaction and reaction of various molecules with each other during amplification in the above experimental procedure. Because the 5' SH forward primer is fixed on the surface of the chip, the solution only contains the reverse primer and the rest amplification components, the modification of the surface of the chip reduces the steric hindrance caused by the combination of the recombinase and the corresponding template with the two primers to a certain extent, so that the FRET probe and the reverse (R) primer are more efficiently positioned to a complementary strand, and the release and amplification efficiency of fluorescent groups (such as FAM and BHQ1) of a reaction system are indirectly enhanced. Along with the reaction, part of the amplified molecules are always fixed on the surface of the chip; this portion of the amplicon will also generate a signal based on the characteristics of the chip itself. Meanwhile, the gold-plated chip with the nano structure can well keep the constant temperature around a reaction system on the surface of the chip, so that the enzyme is always in the best reaction activity.
In this example, the DNA sample to be tested contained the N gene and orf1ab gene at different copy numbers.
Results and comments on the experiments
The results of the experiment are shown in FIGS. 3 to 7. Copies indicates copy number, NTC indicates negative control, min indicates minutes.
After the technical scheme of the invention is adopted, for the same detection reagent composition, the sensitivity of isothermal amplification nucleic acid detection can be improved by 2-3 orders of magnitude, the lower limit of detection can be reduced from 10^4 viruses/mL to 20 viruses/mL, and the detection time for a DNA sample with the same concentration is shortened to be within 1/3 of conventional detection. The NanoSPR chip enhanced isothermal amplification nucleic acid detection kit product, such as a new coronavirus nucleic acid detection kit, adopting the technical scheme of the invention can meet the requirement of detecting <100 copies/mL low-concentration virus samples within 15 minutes, which cannot be achieved by the new coronavirus nucleic acid detection kit based on the traditional isothermal amplification technology, including Yapeh ID No (4000 copies/mL) and Lucira (lower detection limit 900 copies/mL).
While the invention has been described in further detail with reference to specific preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (10)
1. A method for isothermal amplification nucleic acid fluorescence detection of a target nucleic acid using a nanoplasmon resonance chip, comprising the following steps in order:
(1) fixing a forward primer of a 5 'modified SH for amplifying a target nucleic acid on the surface of a nanoplasmon resonance chip, wherein the nanoplasmon resonance chip is coated with a gold atom layer or has a gold atom surface, and the gold atom on the chip surface can be combined with a thiol-SH of the forward primer of the 5' modified SH, so that the forward primer is attached to the chip surface;
(2) optionally blocking the sites of the forward primer on the surface of the nanoplasmon resonance chip, to which the 5' modified SH is not bound, with a blocking reagent;
(3) contacting an isothermal amplification reaction system with the nano plasma resonance chip and carrying out isothermal amplification nucleic acid fluorescence detection reaction; and
(4) and (3) collecting and recording fluorescence signals generated by the reaction in the step (3) in real time by using a fluorescence detection instrument.
2. The method according to claim 1, wherein the isothermal amplification reaction system in step (3) comprises a target nucleic acid, a reverse primer for amplifying the target nucleic acid, an enzyme and/or protein required for performing a fluorescent detection reaction of the isothermal amplification nucleic acid, and a fluorescent probe for detecting an amplicon.
3. The method of claim 2, wherein the isothermal amplification reaction system comprises a polymerase, a recombinase, and optionally a single-stranded DNA binding protein and an exonuclease.
4. The method of any one of claims 1-3, wherein the blocking reagent is a thiol-based compound.
5. The method of any one of claims 1-3, wherein the blocking reagent is 6-mercapto-1-hexanol.
6. The method of any one of claims 1-3, wherein the isothermal amplification reaction system is an isothermal amplification reaction system selected from LAMP, RPA, and ERA.
7. An isothermal amplification nucleic acid fluorescence detection kit for detecting a target nucleic acid, the kit comprising
(a) A nanoplasmon resonance chip, wherein the nanoplasmon resonance chip is coated with a gold atom layer or has a gold atom surface, the gold atom on the chip surface can be combined with a thiol-SH of a forward primer of a 5' modified SH, so that the forward primer is attached to the chip surface,
(b) a forward primer for amplifying a 5' modified SH of a target nucleic acid, and
(c) isothermal amplification reaction reagents or reactants.
8. The kit of claim 7, wherein a forward primer for amplifying 5' modified SH of a target nucleic acid may be immobilized on the surface of the nanoplasmon resonance chip.
9. The kit of claim 7 or 8, wherein the isothermal amplification reaction reagents or reactants comprise a reverse primer for amplifying a target nucleic acid, an enzyme and/or protein required to perform an isothermal amplification nucleic acid fluorescence detection reaction, and a fluorescent probe for detecting an amplicon.
10. The kit according to claim 7 or 8, wherein the kit further comprises a blocking reagent for blocking sites of the nanoplasmon resonance chip to which the forward primer of 5' modified SH is not bound on the surface.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115058493A (en) * | 2022-06-07 | 2022-09-16 | 浙江大学 | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybrid nucleic acid detection system and application |
| CN115058493B (en) * | 2022-06-07 | 2023-09-19 | 浙江大学 | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application |
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| CN112501261B (en) | 2021-12-07 |
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