CN115807061A - Real-time digital LAMP nucleic acid detection method based on micro-droplets - Google Patents
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Abstract
The invention provides a real-time digital LAMP nucleic acid detection method based on micro-droplets, belonging to the technical field of molecular biology detection. The method comprises the steps of mixing an oil phase and an LAMP reaction solution in a microchannel, and cutting the mixture into a plurality of water-in-oil micro-droplets; the micro-droplets are tiled into a single layer; and carrying out LAMP reaction on the micro-droplets, and monitoring the fluorescent signals of the micro-droplets in real time in the LAMP reaction process. The digital detection mode based on the micro-droplets can effectively improve the sensitivity and the accuracy of the nucleic acid detection of the new coronavirus, and the new coronavirus can still be rapidly detected under the condition that the virus concentration of a sample to be detected is very low.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a micro-droplet-based real-time digital LAMP nucleic acid detection method.
Background
The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method and is characterized in that 4 specific amplification products are designed for 6 regions of a target genePrimer, under the action of strand displacement DNA polymerase (Bst DNAPmerase), constant temperature amplification is carried out at 60-65 ℃, and 10 can be realized within about 15-60 minutes 9 ～10 10 Amplification of nucleic acids.
LAMP is simple to operate, has low requirements on instruments and equipment, and can realize reaction by one water bath pot or thermostat; and the detection of the result is simple, and the judgment is carried out by observing the generation of white turbidity or DNA dye fluorescence by naked eyes, so the method is simple, convenient and quick, and is very suitable for quick diagnosis.
However, the traditional LAMP is a qualitative or relatively quantitative detection, and when the content of microorganisms in a sample is too low, the LAMP detection has a false negative result, and the sensitivity and the accuracy of the LAMP still need to be further improved.
Disclosure of Invention
The invention aims to provide a real-time digital LAMP nucleic acid detection method based on micro-droplets, which can realize absolute quantitative detection and has high detection sensitivity and accuracy.
The invention provides a real-time digital LAMP nucleic acid detection method based on micro-droplets, which comprises the following steps:
1) Mixing the oil phase and the LAMP reaction solution in a microchannel, and cutting into a plurality of water-in-oil micro-droplets; the LAMP reaction solution comprises a reagent for LAMP detection and a sample to be detected;
2) After the micro-droplets are laid into a single layer, performing LAMP reaction, monitoring the fluorescent signal of the micro-droplets in real time, and counting the proportion of the fluorescent micro-droplets; and quantitatively calculating the copy number of the target microorganism nucleic acid molecule according to the proportion of the fluorescent micro-droplets.
Preferably, the LAMP detection reagent includes a primer for a conserved segment of the nucleic acid genome of the novel coronavirus.
Preferably, the conserved segments of the novel coronavirus nucleic acid genome comprise a novel coronavirus E gene.
Preferably, the LAMP detection reagent comprises a primer group for amplifying a novel coronavirus E gene; the primer group comprises F3, B3, FIP, BIP, LF and LB; the nucleotide sequences of F3, B3, FIP, BIP, LF and LB are respectively shown in SEQ ID NO. 1-SEQ ID NO. 6.
Preferably, the LAMP detection reagent further comprises a DNA dye, deoxyuridine 5' -triphosphate, uracil-DNA glycosylase, phosphate and a protein denaturant.
Preferably, the oil phase is HFE 7500 or FC40.
Preferably, the tiling is to tile the microdroplets in a closable glass chamber; the diameter of the micro-droplet is equal to the height of the cavity of the glass chamber; and the step of carrying out LAMP reaction on the micro-droplets comprises the steps of heating the glass chamber to the temperature required by the LAMP reaction, then carrying out heat preservation, and triggering the LAMP reaction.
Preferably, the diameter of the micro-droplets is 100 to 2000 μm.
Preferably, the real-time monitoring of the fluorescent signal of the microdroplet comprises: and tracking the micro-droplets in real time by adopting imageJ, and quantifying the change of the fluorescence signals in the micro-droplets along with time.
The invention provides a real-time digital LAMP nucleic acid detection method based on micro-droplets, which comprises the following steps: mixing the oil phase and the LAMP reaction solution in a microchannel, and cutting into a plurality of water-in-oil micro-droplets; the micro-droplets are tiled into a single layer; and carrying out LAMP reaction on the micro-droplets, and monitoring the fluorescent signals of the micro-droplets in real time. According to the invention, the LAMP reaction solution is divided into the micro-droplets, so that the local template concentration is effectively improved, the amplification efficiency is improved, and the absolute nucleic acid template content can be deduced by counting the proportion of the positive micro-droplets to the fluorescent marker. The invention can effectively improve the sensitivity and the accuracy of the detection of the new coronavirus nucleic acid by a digital detection mode based on micro-droplets, and can still quickly detect the new coronavirus under the condition that the virus concentration of a sample to be detected is very low.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic diagram of the process for detecting a novel coronavirus nucleic acid according to the present invention;
FIG. 2 is a real shot of the resulting microdroplets;
FIG. 3 is a photograph showing real-time images of the E gene of the novel coronavirus nucleic acid;
FIG. 4 is a real-time detection curve of real-time digital LAMP on the basis of microdroplets for the E gene of the novel coronavirus;
FIG. 5 shows the detection time of the E gene of a new coronavirus at different concentrations; the filled bars represent the detection time in the EP tube and the open bars represent the detection time in the micro-droplets.
Detailed Description
The invention provides a real-time digital LAMP nucleic acid detection method based on micro-droplets, which comprises the following steps:
1) Mixing the oil phase and the LAMP reaction solution in a microchannel, and cutting into a plurality of water-in-oil micro-droplets; the LAMP reaction solution comprises a reagent for LAMP detection and a sample to be detected;
2) After the micro-droplets are laid into a single layer, performing LAMP reaction, monitoring the fluorescent signal of the micro-droplets in real time, and counting the proportion of the fluorescent micro-droplets; and quantitatively calculating the copy number of the target microorganism nucleic acid molecules according to the proportion of the fluorescent micro-droplets.
Firstly, mixing an oil phase and an LAMP reaction solution in a microchannel, and cutting the mixture into a plurality of water-in-oil micro-droplets; the LAMP reaction solution comprises a reagent for LAMP detection and a sample to be detected.
In the invention, the LAMP detection reagent is preferably used for quickly amplifying and detecting the new coronavirus nucleic acid in a sample to be detected; the LAMP detection reagent preferably includes primers for conserved segments of the nucleic acid genome of the novel coronavirus. In the present invention, the conserved segments of the novel coronavirus nucleic acid genome preferably include the novel coronavirus E gene.
In the present invention, the reagent for LAMP detection preferably comprises a primer set for amplifying a novel coronavirus E gene; the primer group comprises F3, B3, FIP, BIP, LF and LB; the nucleotide sequence of the F3 is shown as SEQ ID NO.1, and specifically comprises the following components: tgagtacaactatgtactcat; the nucleotide sequence of B3 is shown as SEQ ID NO.2, and specifically comprises the following steps: ttcagattttaacacgagagt; the nucleotide sequence of the FIP is shown as SEQ ID NO.3, and specifically comprises the following steps: ACCACGAAAGCAAGAAAAGTTCGTTTCGGAAGAGACAG; the nucleotide sequence of the BIP is shown as SEQ ID NO.4, and specifically comprises the following steps: TTGCTAGTTACACATGCCATCCTTAGGTTTTTACAAGACTACTCACGT; the nucleotide sequence of the LF is shown as SEQ ID NO.5, and specifically comprises the following components: CGCTATTAACTATTAACG; the nucleotide sequence of the LB is shown in SEQ ID NO.6, and specifically comprises the following steps: GCGCTTCGATTGTGTGCGT.
In the present invention, the concentration of each primer used is preferably 0.3X 10 -6 And M. When the novel coronavirus exists in a sample to be detected, the primer group disclosed by the invention can be used for quickly amplifying the virus nucleic acid.
In the present invention, the reagent for LAMP detection preferably further comprises a DNA dye, dUTP (deoxyuridine 5' -triphosphate), UDG (uracil-DNA glycosylase), TCEP (phosphate salt), and Guanadine HCl (protein denaturant).
In the invention, TCEP and Guanadine HCL in the reagent for LAMP detection are used for inhibiting the activity of RNase, preventing the RNase from degrading RNA nucleic acid of virus and reducing the false negative rate of detection.
In the present invention, the LAMP reaction solution is preferably composed of, in 25. Mu.l, the following components: reactions 12.5. Mu.l of Water Start 2X MasterMix, 0.175. Mu.l of 100mM dUTP, 0.5. Mu.l of 1000U/ml UDG, 2.5. Mu.l of 2M Guanadine HCl, 2.325. Mu.l of5 mM TCEP, 0.5. Mu.l of 25. Mu.M Syto82, 2. Mu.l of nucleic-free water, 2.5. Mu.l of 10X primer pairs and 2. Mu.l of the sample to be tested.
In the present invention, the DNA dye preferably includes SYTO82, cyber Green or EverGreen. When the novel coronavirus exists in a sample to be detected, the LAMP detection reagent can emit a fluorescent signal after amplifying the virus nucleic acid, and the signal can be gradually enhanced along with the nucleic acid amplification.
In the invention, dUTP and UDG in the reagent for LAMP detection are used for preventing cross contamination and reducing the false positive rate of detection.
In the present invention, the oil phase is preferably HFE 7500 or FC40; both HFE 7500 and FC40 were purchased from 3M company.
In the present invention, the volume ratio of the oil phase to the LAMP reaction solution is preferably 2 to 7:1.
the method for wrapping the LAMP reaction solution by the oil phase is not particularly limited. In the specific implementation process of the invention, the operation of wrapping the LAMP reaction solution by adopting the oil phase is preferably carried out in a co-flow microfluidic chip, and the droplet size is determined by the chip structure; and (3) inputting an oil phase into the outermost layer of the co-flow microfluidic chip, inputting an LAMP reaction solution into the middle layer, and outputting the generated micro liquid drops from an output port. Connecting a PE2 pipe with an output port and collecting the micro-droplets in a small pipe to wrap the oil relative to the LAMP reaction solution.
In the present invention, the diameter of the micro-droplets is preferably 100 to 2000 μm, more preferably 100, 150, 200, 300, 400, 500, 800, 1000, 1200 or 2000 μm.
In the present invention, the micro droplets are preferably micro droplets having the same diameter. In the invention, the tiling is to tile the micro-droplets in a sealable glass chamber for spreading the micro-droplets into a single layer so as to monitor the amplification reaction in the micro-droplets in real time; the diameter of the micro-droplets is equal to the cavity height of the glass chamber. In the present invention, laying down the microdroplets into a monolayer preferably comprises injecting the microdroplets into the glass chamber to form a monolayer of microdroplets. In the laboratory, the glass chamber is preferably formed by separating two glass sheets by using an adhesive tape; the height of the glass cavity is equal to the thickness of the adhesive tape; the thickness of the tape is preferably equal to the diameter of the microdroplets, for example 150 μm when the diameter of the microdroplets is 150 μm. If the height of the glass chamber is too low, the micro-droplets are stressed and are easy to be unstable, and the problem of droplet coalescence occurs; if the glass chamber height is too high, a monolayer of droplet spread will not be guaranteed, thereby making imaging and tracking of individual droplets cumbersome.
After the micro-droplets are injected into the glass chamber, the glass chamber is completely sealed, so that the evaporation of the micro-droplets is prevented; the invention preferably uses epoxy glue to fully seal the glass chamber.
In the invention, the LAMP reaction of the micro-droplets comprises heating the glass chamber to the temperature required by the LAMP reaction, and then carrying out heat preservation to trigger the LAMP reaction.
In the invention, the step of heating the glass chamber to the temperature required by the LAMP reaction and then preserving the heat comprises the step of placing the glass chamber on a heating table preheated to the temperature required by the LAMP reaction, heating and preserving the heat, and triggering the constant temperature condition of the LAMP reaction.
In the present invention, the temperature of the LAMP reaction is preferably 58 to 65 ℃, more preferably 58, 59, 60, 61, 62, 64, or 65 ℃. In the present invention, the LAMP reaction time is preferably 15 to 20min.
In the present invention, the real-time monitoring of the fluorescence signal of the microdroplet comprises: and tracking the micro-droplets in real time by adopting imageJ, and quantifying the change of the fluorescence signals in the micro-droplets along with time.
In the invention, the real-time monitoring of the fluorescent signal of the micro-droplet comprises the following steps: and shooting the micro-droplets in real time, wherein the shooting frequency is preferably one frame every 30 s.
When the DNA dye is SYTO82, the LAMP detection reagent can emit a red fluorescent signal after amplifying the virus nucleic acid, the excitation light source adopts a 540nm optical filter, and the emission light source adopts a 560nm optical filter;
when the DNA dye is EverGreen or Cyber Green, the LAMP detection reagent amplifies virus nucleic acid and then emits a Green fluorescent signal, the excitation light source adopts a 488nm optical filter, and the emission light source adopts a 520nm optical filter;
in the process of monitoring the fluorescent signal of the micro-droplet in real time, the proportion of the fluorescent micro-droplet is counted; the copy number of the target microorganism nucleic acid molecule is quantitatively calculated according to the proportion of the fluorescent micro-droplets, and the severity of infection can be reflected.
The following poisson distribution equation describes the theoretical probability P (λ, k) of a droplet containing a given number of viral nucleic acid molecules (k, k =0,1,2,3 \ 8230;) as a function of the average number of nucleic acid molecules (λ) in the microdroplet:
specifically, λ is the number of viral nucleic acid molecules in the premix divided by the total number of microdroplets, which is the total volume of the LAMP reaction solution divided by the average microdroplet volume. The sum of the probabilities of all positive microdroplets including 1,2,3 and the like is equal to the proportion of the fluorescent microdroplets counted in the experiment, lambda can be further calculated through iterative calculation, and finally the number of virus nucleic acid molecules in the LAMP reaction solution is obtained, wherein P is the probability, e is a constant of natural logarithm, and e =2.718281828.
In order to further illustrate the present invention, the method for real-time digital LAMP nucleic acid detection based on micro-droplet provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
And 1, premixing the LAMP detection reagent and a sample to be detected in an EP tube. Performing amplification detection aiming at the E gene segment of the new coronavirus nucleic acid, wherein the LAMP primer combinations are as follows:
primer pair 1:
F3:TGAGTACGAACTTATGTACTCAT(SEQ ID NO.1)；
B3:TTCAGATTTTTAACACGAGAGT(SEQ ID NO.2)；
and (3) primer pair 2:
FIP:ACCACGAAAGCAAGAAAAAGAAGTTCGTTTCGGAAGAGACAG(SEQ ID NO.3)；
BIP:TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT(SEQ ID NO.4)；
and (3) primer pair:
LF:CGCTATTAACTATTAACG(SEQ ID NO.5)；
LB:GCGCTTCGATTGTGTGCGT(SEQ ID NO.6)。
2. and (3) subpackaging the premixed solution of the LAMP reaction into the micro-droplets. As shown in fig. 1, a vacuum column is generated by using an injector and a fixing clamp, and then suction is realized through the vacuum column, so that a reactant and an oil phase are sucked into a chip to be converged to form a micro-droplet; in a co-flow microfluidic chip, an oil phase HFE 7500 is input into the outermost layer, LAMP reaction solution is input into the middle layer, and the rightmost side is an output port, namely, the generated micro liquid drops are output from the port. The output port was connected with a PE2 pipe and the droplets were collected in a small tube. To this end, the LAMP reaction solution (premix) has been dispensed into microdroplets (fig. 2). The 25 μ l of LAMP was converted into microdroplets entirely, consuming 2-7 times the volume of the outer phase oil phase.
The LAMP reaction solution is prepared from the following components in percentage by weight: each 25. Mu.l reaction was 12.5. Mu.l of Water Start 2X Master Mix, 0.175. Mu.l of 100mM dUTP, 0.5. Mu.l of 1000U/ml UDG, 2.5. Mu.l of 2M Guanadine HCl, 2.325. Mu.l of5 mM TCEP, 0.5. Mu.l of 25. Mu.M Syto82, 2. Mu.l of nucleic-free water, 2.5. Mu.l of 10X primer pair, and 2. Mu.l of E gene template.
3. Two glass sheets were separated by a 150 μm thick tape to form a chamber with a height of 150 μm, and the collected micro-droplets were slowly injected into the glass chamber by a pipette gun to spread and fix the micro-droplets (fig. 1).
4. The glass chamber with the micro-droplets inside is placed on a heating table preheated to 64 ℃ in advance, and the light source and the camera are turned on to observe the occurrence of reaction in each droplet in real time (figure 1). The light source was set to match the DNA dye selected as SYTO82, and the light emission wavelength was 540nm and 560nm.
5. When the glass chamber is placed on the heating stage, the imaging focus should be adjusted as soon as possible to start imaging. The arrangement was such that a photograph was taken every 30 seconds and stopped after 1h of continuous shooting, and FIG. 3 shows real-time imaging of the E gene of the new coronavirus nucleic acid.
6. The fluorescent signal in each micro-droplet of the shot image is quantitatively analyzed by using ImageJ, so that a real-time amplification S-shaped curve of the E gene can be obtained; and the detection time, i.e., the time corresponding to the first inflection point (the time when the reaction starts to progress to exponential amplification) obtained by performing the second derivation on the sigmoid curve can be obtained (fig. 4).
7. Repeating steps 1 to 6, when detecting the E gene concentration of different new coronaviruses (100, 10 and 1 copy per microliter), the detection time of the E gene is not dependent on the concentration of the virus nucleic acid in LAMP reaction in the micro-droplet. It can be seen that the Cq values of the E genes when amplified in the microdroplets were around 15min, regardless of the concentration of 100 copies per microliter or 10 or 1 copy per microliter (fig. 5); in the LAMP reaction in the EP tube, the detection time of the E gene is obviously prolonged along with the reduction of the concentration of the E gene. Therefore, the efficiency of detecting the novel coronavirus nucleic acid is ensured by the micro-droplet-based real-time digital LAMP.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. The real-time digital LAMP nucleic acid detection method based on the micro-droplets comprises the following steps:
1) Mixing the oil phase and the LAMP reaction solution in a microchannel, and cutting into a plurality of water-in-oil micro-droplets; the LAMP reaction solution comprises a reagent for LAMP detection and a sample to be detected;
2) After the micro-droplets are flatly laid into a single layer, performing LAMP reaction, monitoring the fluorescent signal of the micro-droplets in real time, and counting the proportion of the fluorescent micro-droplets; and quantitatively calculating the copy number of the target microorganism nucleic acid molecules according to the proportion of the fluorescent micro-droplets.
2. The real-time digital LAMP nucleic acid detection method according to claim 1, characterized in that the LAMP detection reagent comprises primers for conserved segments of the novel coronavirus nucleic acid genome.
3. The real-time digital LAMP nucleic acid detection method according to claim 2, characterized in that the conserved segment of the novel coronavirus nucleic acid genome comprises novel coronavirus E gene.
4. The real-time digital LAMP nucleic acid detection method according to claim 3, wherein the LAMP detection reagent comprises a primer set for amplifying a novel coronavirus E gene; the primer group comprises F3, B3, FIP, BIP, LF and LB; the nucleotide sequences of F3, B3, FIP, BIP, LF and LB are respectively shown in SEQ ID NO. 1-SEQ ID NO. 6.
5. The real-time digital LAMP nucleic acid detection method according to any one of claims 1 to 4, characterized in that the LAMP detection reagent further comprises DNA dyes, deoxyuridine 5' -triphosphate, uracil-DNA glycosylase, phosphate salts and protein denaturants.
6. The real-time digital LAMP nucleic acid detection method according to claim 5, wherein the DNA dye comprises SYTO82, cyber Green or Ever Green.
7. The real-time digital LAMP nucleic acid detection method according to claim 1, wherein the oil phase is HFE 7500 or FC40.
8. The real-time digital LAMP nucleic acid detection method according to claim 1, characterized in that the tiling is to tile the micro-droplets in a sealable glass chamber; the diameter of the micro-droplet is equal to the height of the cavity of the glass chamber;
and performing LAMP reaction on the micro-droplets, namely heating the glass chamber to the temperature required by the LAMP reaction, then preserving the heat, and triggering the LAMP reaction.
9. The real-time digital LAMP nucleic acid detection method according to claim 1, wherein the diameter of the micro-droplet is 100 to 2000 μm.
10. The real-time digital LAMP nucleic acid detection method according to claim 1, wherein the real-time monitoring of the micro-droplet fluorescence signal comprises: and tracking the micro-droplets in real time by adopting imageJ, and quantifying the change of the fluorescence signals in the micro-droplets along with time.
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