CN114752657A - Polydisperse liquid drop digital nucleic acid detection method and application thereof - Google Patents

Abstract

The invention discloses a polydisperse droplet digital nucleic acid detection method and application thereof, comprising the following steps: mixing an oil phase solution, a detection sample and a detection reagent, generating droplets by a simple method and incubating, according to the number of positive droplets With volume, the target nucleic acid molecule in the sample is quantitatively detected. The method rapidly divides the reaction reagent into independent droplets through a simple dispersion operation, thereby reducing the requirements on the environment and instruments, and at the same time, the demand for samples and detection reagents is also significantly reduced. Moreover, through rapid emulsion splitting, nucleic acid amplification or Cas protein cleavage of the target before reagent splitting can be effectively avoided, and the target nucleic acid concentration is calculated based on the droplet size, and the detection sensitivity can reach aM level.

Description

A kind of polydisperse droplet digital nucleic acid detection method and its application

technical field

The invention belongs to the field of gene detection, and in particular relates to a polydisperse droplet digital nucleic acid detection method and application thereof.

Background technique

Nucleic acid detection is an important means of infectious disease prevention, diagnosis and monitoring. In the related art, methods for nucleic acid detection include real-time quantitative polymerase chain reaction (qPCR) and nucleic acid isothermal amplification technology. Among them, real-time quantitative polymerase chain reaction (qPCR) is the gold standard for clinical nucleic acid detection and quantification. Especially with the development of microfluidic technology, digital PCR has gradually become a new generation of absolute quantitative PCR technology. In addition, nucleic acid isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), sequence-dependent amplification Nucleic acid amplification (NASBA), helicase-dependent amplification (HDA), etc. are also gradually applied in various fields because they do not require temperature cycling, have low dependence on instruments, high sensitivity, and good selectivity. Other techniques, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR) (eg SHERLOCK, DETECTR), have also attracted much attention due to their fast and sensitive properties. However, in practice, although CRISPR-based methods are used in combination with nucleic acid amplification technology to achieve high-sensitivity detection, due to differences in reaction temperature and buffer component concentrations, this combination is often divided into multiple sections, which cannot be The realization of one-step detection not only increases the difficulty of experimental operation, but also increases the possibility of cross-contamination. Moreover, this segmented operation also makes these methods unsuitable for digital nucleic acid detection, limiting their application. Moreover, the existing methods for preparing monodisperse droplets are complicated in operation, time-consuming, prone to dead volume, and waste of reagents. It is not conducive to the organic combination of digital nucleic acid detection technology.

Therefore, developing a one-step nucleic acid detection technology that can be combined with digital nucleic acid detection will provide strong technical support for the stability, high throughput and high sensitivity of nucleic acid detection.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a polydisperse droplet digital nucleic acid detection method. The detection method in the present invention can quickly divide the reaction reagent into independent droplets through simple operations, thereby reducing the requirements for complex instruments. Furthermore, nucleic acid amplification or cleavage of the target by Cas protein prior to reagent splitting is effectively avoided by this rapid emulsion fractionation. Compared with monodisperse droplets, under the same target molecule concentration, polydisperse droplets require less sample volume, and the detection process is convenient, fast and highly sensitive. The polydisperse droplet-based CRISPR assay can achieve aM-level detection sensitivity even without incorporating a pre-amplification process.

A first aspect of the present invention provides a polydisperse droplet digital nucleic acid detection method, comprising the following steps: mixing an oil phase solution, a detection sample and a detection reagent, dispersing into droplets and incubating, according to the number of positive droplets As well as volume, quantitatively detect target nucleic acid molecules in a sample.

The digital nucleic acid detection method is to disperse the reaction solution containing the target molecule into thousands of small chambers, so that each chamber contains only one or zero target molecules. After the reaction, positive and negative chambers can be distinguished according to the fluorescence intensity, and accurate quantification can be achieved by the total number of chambers and the total number and volume of positive chambers. Due to the confinement effect, the concentration of target molecules in the chamber increases, making digital detection methods more sensitive than bulk detection. And its counting method makes it possible to achieve absolute quantification of target nucleic acid.

Compared with the existing detection methods based on monodisperse droplets, the detection method in the present invention has the advantages of simple operation, short time, no dead volume, effectively avoiding waste of reagents, and improving detection sensitivity and quantitative accuracy.

According to the first aspect of the present invention, in some embodiments of the present invention, the detection reagent is an isothermal amplification detection reagent.

In some embodiments of the present invention, the isothermal amplification detection reagent includes primers, enzymes, chromogenic solutions, and dNTPs.

Of course, those skilled in the art can reasonably select specific isothermal amplification detection reagents according to actual use requirements, including but not limited to isothermal amplification reactions such as LAMP, RPA, RCA, NASBA, SDA, etc., in order to cooperate with the detection method in the present invention. The purpose of nucleic acid detection.

According to the first aspect of the present invention, in some embodiments of the present invention, the detection reagent is a CRISPR-Cas reaction system reagent.

In some embodiments of the present invention, the CRISPR-Cas reaction system reagents include Cas protein and crRNA.

Of course, those skilled in the art can reasonably select specific CRISPR-Cas reaction system reagents according to actual use requirements, so as to cooperate with the detection method in the present invention to achieve the purpose of nucleic acid detection.

In some embodiments of the present invention, the CRISPR-Cas reaction system includes a CRISPR-Cas12a reaction system and a CRISPR-Cas13a reaction system.

Of course, those skilled in the art can choose other CRISPR-Cas reaction systems for alternative use, including but not limited to CRISPR-Cas12a, CRISPR-Cas13a, and CRISPR-Cas14, according to actual use requirements.

In some embodiments of the present invention, the oil phase solution includes fluorine oil and carbon oil.

Of course, those skilled in the art can reasonably select the applicable oil phase solution according to actual use requirements, including but not limited to the above-mentioned fluorine oil and carbon oil.

In some embodiments of the present invention, the oil phase solution further includes a surfactant.

In some embodiments of the present invention, the surfactant includes Span 80, Tween 20, AbilEM90, Abil EM180.

Of course, those skilled in the art can reasonably select appropriate surfactants for compounding with corresponding oils according to actual use requirements, including but not limited to the above-mentioned Span 80, Tween 20, Abil EM90, and Abil EM180.

In some embodiments of the present invention, the oil phase solution is specifically a mixed solution of 90% (v/v) isopropyl palmitate and 10% Abil EM180.

In some embodiments of the present invention, the target nucleic acid molecule is quantitatively calculated by at least one of the following formulas (1) to (4),

where n represents the total number of droplets, b represents the total number of negative droplets, v represents the volume of the droplet, λ represents the concentration of the sample to be tested, P represents the probability of positive droplets, and m represents the type of droplet size (> 20000), x represents the type of positive droplet size, _ni represents the total number of droplets in a certain volume, _bi represents the total number of negative droplets in a certain volume, _{vi represents the volume of a certain droplet, and vj represents} the unique volume The volume of the droplet after liquefaction.

In some embodiments of the present invention, formula (1) is a formula for calculating the probability of positive droplets when the droplets are of uniform volume. Equation (2) is a formula for calculating the probability of positive droplets in the case of polydispersity derived from Equation (1). And for formula (2), if the mediation is equal to zero, the concentration at which the probability of positive droplets is maximized can be obtained, and this calculation formula is the above formula (3). Further assuming that under polydisperse droplets, the volume of each droplet is unique, equation (3) can be simplified to equation (4).

In some embodiments of the present invention, the detection method further comprises dispersing after the oil phase solution, the detection sample and the detection reagent are mixed.

In some embodiments of the present invention, the dispersion includes vibration dispersion, blowing and suction dispersion, stirring dispersion, and ultrasonic dispersion.

Of course, those skilled in the art can also use other simple dispersion methods for dispersion according to actual use requirements, including but not limited to the above-mentioned vibration dispersion, blowing and suction dispersion, stirring dispersion, and ultrasonic dispersion.

In some embodiments of the present invention, the oscillation frequency is 2000-2500 rpm, and the oscillation time is 1-5 s.

In some embodiments of the present invention, the oscillation frequency is 2500 rpm, and the oscillation time is 1 s.

In some embodiments of the present invention, the number of blows and suctions is 4-7 times.

In some embodiments of the present invention, the number of blows and suctions is 4 times.

The duration of the oscillation is related to the oscillation frequency, and the number of blows and suctions is related to the different ranges of tips. Therefore, those skilled in the art can reasonably adjust the length of the oscillation time and the number of blows and suctions according to actual use requirements to obtain similar dispersion effects.

In some embodiments of the present invention, the specific dispersion method is: after mixing the oil phase solution with the target molecule and the detection reaction reagent, oscillate on a vortex shaker for 1-5 seconds or blow and suck with a pipette 4-7 times, The reagent can thus be rapidly divided into polydisperse droplets.

The segmentation of samples in the prior art is mainly achieved by techniques such as microfluidic chips, capillary tubes, and immiscible phase cutting. However, these methods all have some unavoidable problems. For example, the fabrication of microfluidic chips requires complex and sophisticated instruments and relies on experienced staff. The size and uniformity of the droplets are related to the stability of the instrument, the size of the channel, the precision of the microfabrication, and the viscosity of the continuous phase. These methods require specialized instruments and complex operating steps, which will result in increased detection time and cost. And the long-term droplet preparation process will lead to early amplification of nucleic acids and undesired enzymatic reactions, resulting in inaccurate quantification of the final results. In the present invention, the vortex oscillator is used to oscillate for several seconds or the pipette is repeatedly sucked and sucked, so that the reaction reagent quickly forms polydisperse droplets with different volumes in the oil phase, which is also applicable to various nucleic acid detection methods.

In some embodiments of the present invention, when a polydispersed droplet-based isothermal amplification nucleic acid detection method is used, the detection step is: after mixing the detection sample with the LAMP reaction system, quickly add an appropriate amount of oil phase solution to cover. After oscillating at the maximum oscillation frequency of 2500 rpm for 1-2 s, perform isothermal amplification, collect fluorescent signals, calculate the volume of positive droplets, and quantify the concentration of target molecules in the sample.

In some embodiments of the present invention, when the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection technology is used, the detection step is: after mixing the detection sample with the CRISPR-Cas reaction system, quickly adding an appropriate amount of oil phase solution to cover. After shaking at 2500rpm for 1-2s, the fluorescence signal was collected, the number and volume of positive droplets were calculated, and the concentration of target molecules in the sample was quantified.

A second aspect of the present invention provides a computer-readable storage medium, where the computer-readable storage medium stores computer-executable instructions for executing the detection method described in the first aspect of the present invention.

A third aspect of the present invention provides a nucleic acid detection system, into which the detection method described in the first aspect of the present invention is input.

The beneficial effects of the present invention are:

1. The present invention no longer uses complex microfluidic systems, chips or instruments to generate uniform droplets as in the prior art in the segmentation of samples, and does not require complex operations, and has a low degree of dependence on equipment, which can be achieved within a few seconds. The rapid segmentation of the generated reagents is completed, and the droplets are concentrated below 50 μm.

2. The sample demand and reagent consumption in the present invention are low, the total amount of reagents required is less than 5 μL, there is no dead volume problem in traditional digital nucleic acid detection methods, the sensitivity is higher, the quantification is more accurate, and it will not cause reagents. waste, reducing the cost of experiments.

3. The present invention effectively avoids nucleic acid amplification or Cas protein cleavage of the target through rapid emulsion splitting, and calculates the target nucleic acid concentration based on the number and size of droplets, and the detection sensitivity can reach aM level.

Description of drawings

FIG. 1 is a schematic flowchart of a method for detecting polydisperse droplet nucleic acid in an embodiment of the present invention.

Fig. 2 is the droplet size distribution generated by the polydisperse droplet nucleic acid detection method in the embodiment of the present invention, wherein A is oscillation dispersion, and B is blowing and suction dispersion.

FIG. 3 is a droplet bright field (A) and a fluorescence field (B) image of the polydisperse droplet-based isothermal amplification nucleic acid detection method in the embodiment of the present invention.

4 is an image of a blank control droplet (A) and a positive droplet (B) containing target molecules of the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method in the embodiment of the present invention.

Fig. 5 is the sensitivity test of the polydisperse droplet-based CRISPR-Cas12a amplification-free DNA detection method in the embodiment of the present invention, A is a comparison diagram under different concentrations, and B is a comparison diagram under the same concentration.

Fig. 6 is the sensitivity test of the polydisperse droplet-based CRISPR-Cas13a amplification-free RNA detection method in the embodiment of the present invention, A is a comparison diagram under different concentrations, and B is a comparison diagram under the same concentration.

7 is the test of the polydisperse droplet-based CRISPR-Cas12a/Cas13a dual gene (DNA and RNA) simultaneous detection method in the embodiment of the present invention, A is the FAM and HEX fluorescence when there are DNA and RNA templates in the system The droplet image of the channel, B is the statistical result of positive droplets in each channel when DNA and RNA templates exist in the system.

Detailed ways

In order to make the invention purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, rather than for limiting the present invention.

The experimental materials and reagents used, unless otherwise specified, are conventional consumables and reagents that can be obtained from commercial sources.

A kind of polydisperse droplet digital nucleic acid detection method

The detection process is shown in Figure 1. The detection principle of the polydisperse droplet digital nucleic acid detection method in the embodiment of the present invention is as follows: the nucleic acid detection method in the embodiment of the present invention is mainly based on the nucleic acid detection realized by the combination of polydisperse droplet and nucleic acid isothermal amplification technology.

Among them, polydisperse droplets are obtained by coating target molecules and detection reagents with a specific oil phase solution.

Detection reaction reagents include conventional nucleic acid detection reagents in the art, such as dNTPs, specific primers, probes, polymerases and other components, as well as non-amplification technology-related reagents based on the CRISPR-Cas system, such as Cas12a or Cas13a-related proteins , crRNA, detection buffer. Of course, those skilled in the art can reasonably select other nucleic acid detection reagents according to actual use requirements, including but not limited to the above-mentioned dNTPs, specific primers, probes, polymerases, Cas12a or Cas13a-related proteins, crRNA, and detection buffers.

As for the oil phase solution, a single oil phase solution in the art, such as fluorine oil, carbon oil, etc., can be used, or it can be obtained by compounding it with a surfactant. Of course, those skilled in the art can reasonably select the applicable oil phase solution according to actual use requirements, including but not limited to the above-mentioned fluorine oil and carbon oil. As for the selection of surfactants, in the embodiments of the present invention, non-ionic surfactants are specifically used, including span80, tween20, Abil EM90, Abil EM180 and the like. Of course, those skilled in the art can reasonably select appropriate surfactants for compounding with corresponding oils according to actual use requirements, including but not limited to the above span80, tween20, Abil EM90, and Abil EM 180.

Among them, for the specific preparation method of polydisperse droplets, the specific method is: after mixing the oil phase solution with the target molecule and the detection reaction reagent, oscillate on a vortex shaker for 1-5 seconds or blow and suck with a pipette 4-7 times , so that the reagent can be rapidly divided into polydisperse droplets. However, it should be noted that the length of the oscillation is related to the oscillation frequency, and the number of blows and suctions is related to the tips of different ranges. Therefore, those skilled in the art can reasonably adjust the length of the oscillation time and the number of blows and suctions according to the actual use requirements. A similar dispersion effect is obtained.

When the polydisperse droplets are formed, they can be sent to a temperature control device for incubation and amplification. The temperature control devices used include but are not limited to PCR instruments, thermostats, constant temperature water baths, and constant temperature incubators. After the incubation, the positive droplets were read by a fluorescence microscope or a simple mobile phone-based device and their brightfield positions were recorded, and the volume and number of positive droplets were calculated.

Among them, the calculation method for the original concentration of the detected target molecule is:

When the droplet is of uniform volume, the probability of a positive droplet is:

Among them, in formula (1), n represents the total number of droplets, b represents the total number of negative droplets, v represents the volume of the droplet, λ represents the concentration of the sample to be tested, and P represents the probability of positive droplets when the volume is uniform.

It is derived from this that the probability of positive droplets in the case of polydispersity is:

Among them, in formula (2), n _i represents the total number of droplets in a certain volume, b _i represents the total number of negatives in a certain volume of droplets, v _i represents the volume of a certain droplet, λ represents the concentration of the sample to be tested, P represents the probability of positive droplets in the case of polydispersity.

For formula (2), if the medium is equal to zero, the concentration that maximizes the probability of positive droplets can be obtained. The specific formula is:

Among them, in formula (3), n _i represents the total number of droplets in a certain volume, b _i represents the total number of negatives in a certain volume of droplets, vi _represents the volume of a certain droplet, λ represents the concentration of the sample to be tested, m represents the droplet size category.

Further assuming that under polydisperse droplets, the volume of each droplet is unique, then the above formula can be simplified to:

Among them, in formula (4), v _j represents the volume of the droplet after the volume is unique, λ represents the concentration of the sample to be tested, and x represents the size of the positive droplet.

Finally, according to the total volume of the sample and the volume of each positive droplet read by the microscope, the concentration of the sample can be calculated.

The polydisperse droplet isothermal amplification or CRISPR amplification-free nucleic acid detection technology obtained based on the above theory, wherein, when the polydisperse droplet CRISPR amplification-free nucleic acid detection technology is used, it not only does not require pre-nucleic acid amplification to realize the Cas protein The mediated nucleic acid detection also effectively avoids the problem of false positives or false negatives caused by amplification. Moreover, when the polydisperse droplet CRISPR amplification-free nucleic acid detection technology is used, its advantage over the polydisperse droplet constant temperature amplification nucleic acid detection technology is that since the target molecule does not undergo an amplification reaction to amplify the signal, it cannot be guaranteed that each Nucleic acid templates all generate signals, so the standard curve is used for quantification. The detection linear range of this method is 0.1-100 fM, so it can relatively sensitively detect various nucleic acid molecules.

Example 1 Detection method of isothermal amplification nucleic acid based on polydisperse droplets

In this embodiment, the target molecule is detected using a polydisperse droplet-based isothermal amplification nucleic acid detection method.

The specific detection steps are as follows:

1) A total of 3 μL of the LAMP reaction system containing the target molecule was transferred to an EP tube, and an appropriate amount of oil phase solution was quickly added to the EP tube to cover.

Wherein, in this embodiment, the target molecule is specifically the pEF-nsp125-3HA plasmid containing the new coronavirus N gene DNA (the construction method of the plasmid is carried out with reference to the conventional technical manual in the field, wherein the new coronavirus N gene DNA is obtained through the new coronavirus N Gene RNA (database No. NC-045512) was obtained by reverse transcription); the specific LAMP reaction system is shown in Table 1.

Table 1

component Volume (μL) final concentration dNTPs 1.5 1.2mM primer set 0.75 in total 1× LAMP dyes 0.25 1× Mg²⁺ 1.25 10mM Bst 2.0 0.75 480U/mL Isothermal buffer 1.25 1× plasmid template 1 30.3pg/μL H₂O 5.5

Wherein, the specific nucleotide sequence of the primer is:

FIP: 5'-GCGGCCAATGTTTGTAATCAGTAGACGTGGTCCAGAACAA-3' (SEQ ID NO: 1);

BIP: 5'-TCAGCGTTCTTCGGAATGTCGCTGTGTAGGTCAACCACG-3' (SEQ ID NO: 2);

F3: 5'-GCTGCTGAGGCTTCTAAG-3' (SEQ ID NO:3);

B3: 5'-GCGTCAATATGCTTATTCAGC-3' (SEQ ID NO: 4);

Loop F: 5'-CCTTGTCTGATTAGTTCCTGGT-3' (SEQ ID NO:5);

Loop B: 5'-TGGCATGGAAGTCACACC-3' (SEQ ID NO: 6).

In this embodiment, the oil phase solution is specifically a mixed solution of 90% (v/v) isopropyl palmitate and 10% Abil EM180.

2) Shaking at 2500 rpm for 1 s, the LAMP system was divided into droplets of different sizes. Observation using a microscope revealed that the liquid size was mainly distributed in the 10-50 μm range (as shown in Figure 2).

3) Transfer the generated droplets to a thermostat for amplification (the temperature is controlled at 60-65° C., and the reaction time is 30-60 minutes). The droplet signal was then collected using a fluorescence microscope and brightfield recorded in the same field of view to determine the volume of the positive droplet. The concentration of the target molecule in the sample is calculated according to the formulas (1) to (4) and the positive droplet volume in the above-mentioned embodiment.

Among them, the determination method of positive droplets is: the sum of the average fluorescence intensity of the droplets in the blank control group and 3 times the standard deviation is set as the threshold value of positive droplets. droplets.

The results are shown in Figure 3.

When the target molecule is the new coronavirus N gene DNA (plasmid concentration is 30.3pg/μL) as an example, using the polydisperse droplet-based isothermal amplification nucleic acid detection method in Example 1 for detection, it can be found that in bright field and fluorescence The formation of droplets and the characterization of positive droplets were clearly visible under the field, demonstrating the excellent resolution and detection sensitivity of the above method at template plasmid concentrations of at least 30.3 pg/μL.

Example 2 CRISPR-Cas amplification-free nucleic acid detection method based on polydisperse droplets

In this example, the target molecule was detected using the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method.

In this example, the CRISPR-Cas system used is selected as CRISPR-Cas12a. Of course, those skilled in the art can also choose other CRISPR-Cas systems to be substituted according to actual usage requirements.

The specific detection steps are as follows:

1) After mixing the target molecule to be tested and the CRISPR-Cas12a detection reagent in the EP tube according to the composition shown in Table 2, quickly add an appropriate amount of oil phase solution to the EP tube to cover.

Among them, in this embodiment, the target molecule is specifically the DNA of the new crown N gene; the specific CRISPR-Cas reaction system is shown in Table 2.

Table 2

component Volume (μL) final concentration Cas12a 0.5 200nM crRNA 1.25 250nM Reporter 1.25 1μM Buffer 1.25 1× DNA template 1.25 10fM H₂O 7

Wherein, in this embodiment, the specific nucleotide sequence of crRNA is: 5'-UAAUUUCUACUAAAGUGUAGAUCCCCCAGCGCUUCAGCGUUC-3' (SEQ ID NO:7). Template DNA sequence: 5'-ATGCGCGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAAT-3' (SEQ ID NO: 8).

2) Shake at 2500rpm for 1s, the CRISPR-Cas reaction system is divided into droplets of different sizes. Observation using a microscope revealed that the liquid size was mainly distributed in the 10-50 μm range.

3) Transfer the resulting droplets to a thermostat for CRISPR-Cas cleavage (37-45°C, reaction for 60-120 minutes). The signal from the droplets was then collected using a fluorescence microscope, and brightfield recorded in the same field of view to determine the number and volume of positive droplets. According to the number of positive droplets at different concentrations, draw a standard curve. Quantify unknown sample concentrations based on the standard curve.

The results are shown in Figures 4-5.

As shown in Fig. 4, it can be found that at the template concentration of 10 fM, its positive droplets can be clearly found from microscopy and can be accurately determined.

As shown in Figure 5. The inventors further adjusted the concentration range of the template (0(NTC), 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ aM), and at the same time carried out repeated experiments for 10 ² aM to verify the stability of the detection technology (as shown in Figure 5B ) , it can be found that the detection sensitivity of the detection method in the present invention can reach 10 ² aM and the results are stable after multiple measurements, which are significantly different from the control group, indicating that the detection method in this embodiment has high detection sensitivity and stability. it is good.

Example 3 CRISPR-Cas amplification-free nucleic acid detection method based on polydisperse droplets

In this example, the target molecule was detected using the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method.

In this embodiment, the CRISPR-Cas system used is selected as CRISPR-Cas13a. Of course, those skilled in the art can also choose other CRISPR-Cas systems to be substituted according to actual usage requirements. CRISPR-Cas systems useful in the present invention include, but are not limited to, CRISPR-Cas12a, CRISPR-Cas13a.

The specific detection steps are as follows:

1) After mixing the target molecule to be tested and the CRISPR-Cas13a detection reagent in the EP tube according to the composition shown in Table 2, quickly add an appropriate amount of oil phase solution to the EP tube to cover.

Among them, in this embodiment, the target molecule is specifically the single-stranded RNA molecule of the new crown N gene (NC-045512); the specific CRISPR-Cas reaction system is shown in Table 3.

table 3

component Volume (μL) final concentration Cas13a 1.25 100nM crRNA 1.25 100nM Reporter 1.25 500nM Buffer 1.25 1× Template 1.25 500nM H₂O 6.25

Wherein, in this embodiment, the specific nucleotide sequence of the crRNA is: 5'-GACCACCCCAAAAAUGAAGGGGACUAAAACUUUGCGGCCAAUGUUUGUAA-3' (SEQ ID NO: 9). The template RNA sequence is: 5'-AACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCCCCAGCGCUUCAGCGUUCUUCGGAAUGUCGCGCAUUGGCAUGGAAGUC-3' (SEQ ID NO: 10).

2) Shake at 2500rpm for 1s, the CRISPR-Cas reaction system is divided into droplets of different sizes. Observation using a microscope revealed that the liquid size was mainly distributed in the 10-50 μm range.

3) Transfer the resulting droplets to a thermostat for CRISPR-Cas cleavage (37-45°C, reaction for 60-120 minutes). The droplet signal was then collected using a fluorescence microscope, and brightfield recorded in the same field of view to determine the number and volume of positive droplets. According to the number of positive droplets at different concentrations, draw a standard curve. Quantify unknown sample concentrations based on the standard curve.

The results are shown in Figure 6.

As shown in Figure 6. The inventors further adjusted the concentration range of the template (0(NTC), 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ aM), and at the same time carried out repeated experiments for 10 ¹ aM to verify the stability of the detection technology (such as Figure 6B), it can be found that the detection sensitivity of the detection method in the present invention can reach 10 ¹ aM and the result is stable after multiple measurements, all have significant differences with the control group, illustrating that the detection sensitivity of the detection method in the present embodiment is high , Good stability.

Example 4 CRISPR-Cas amplification-free dual-gene detection method based on polydisperse droplets

In this example, the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method is used to simultaneously detect DNA and RNA.

In this embodiment, the CRISPR-Cas systems used are selected as CRISPR-Cas12a and CRISPR-Cas13a. Of course, those skilled in the art can also choose other CRISPR-Cas systems for substitution according to actual usage requirements. CRISPR-Cas systems useful in the present invention include, but are not limited to, CRISPR-Cas12a, CRISPR-Cas13a.

The specific detection steps are as follows:

1) After mixing the target DNA/RNA and CRISPR-Cas12a/Cas13a detection reagent in the EP tube according to the composition shown in Table 3, quickly add an appropriate amount of oil phase solution to the EP tube to cover.

Wherein, in this embodiment, the DNA target molecule is specifically a synthetic double-stranded DNA sequence, and the RNA target molecule is a synthetic single-stranded RNA sequence; the specific CRISPR-Cas reaction system is shown in Table 4.

Table 4

Wherein, in this example, Cas12a-crRNA and Cas13a-crRNA are crRNAs in Example 2 and Example 3.

2) Shake at 2500rpm for 1s, the CRISPR-Cas reaction system is divided into droplets of different sizes. Observation using a microscope revealed that the liquid size was mainly distributed in the 10-50 μm range.

3) Transfer the resulting droplets to a thermostat for CRISPR-Cas cleavage (37-45°C, reaction for 60-120 minutes).

The results are shown in Figure 7.

As shown in Figure 7. The template DNA and RNA concentrations were 100 fM, and repeated tests were carried out to verify the stability of the detection technology. It can be found that when the detection method in the present invention contains a template in the system, there are significant differences with the control group. The detection method can realize the double gene detection of DNA and RNA.

The detection methods in the embodiments of the present invention can also be used in actual detection operations in the form of computer-readable storage media or detection systems, and the detection methods in the above-mentioned embodiments are executed by executing relevant instructions to complete the detection of specific nucleic acid molecules. .

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Sun Yat-Sen University

<120> A polydisperse droplet digital nucleic acid detection method and its application

<130>

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Claims (10)

1. A polydisperse droplet digital nucleic acid detection method, comprising the steps of: and mixing and incubating the oil phase solution, the detection sample and the detection reagent, and quantitatively detecting the target nucleic acid molecules in the sample according to the quantity and the volume of the positive liquid drops.

2. The detection method according to claim 1, wherein the detection reagent is a constant-temperature amplification detection reagent, and the constant-temperature amplification detection reagent comprises a primer, an enzyme, a color developing solution and dNTP.

3. The detection method as claimed in claim 1, wherein the detection reagent is a CRISPR-Cas reaction system reagent comprising Cas protein and crRNA.

4. The detection method according to any one of claims 1 to 3, wherein the oil phase solution comprises fluorine oil and carbon oil.

5. The detection method as claimed in any one of claims 1 to 3, wherein the oil phase solution further comprises a surfactant, and the surfactant comprises Span 80, Tween 20, Abil EM90 and Abil EM 180.

6. The detection method according to claim 1 or 2, wherein the target nucleic acid molecule is quantitatively calculated by at least one of the following formulas (1) to (4),

wherein in each formula, n represents the total number of droplets, b represents the total number of negative droplets, v represents the volume of the droplets, λ represents the concentration of the sample to be tested, P represents the probability of positive droplets, m represents the droplet size category, x represents the positive droplet size category, n represents the concentration of the sample to be tested, and _iRepresents the total number of droplets of a certain volume, b_iRepresents the total number of negatives in a volume of droplets, v_iRepresents the volume of a certain drop, v_jRepresenting the volume of the drop after the volume normalization.

7. The detection method according to any one of claims 1 to 3, further comprising dispersing after mixing the oil phase solution, the detection sample and the detection reagent, wherein the dispersing comprises oscillation dispersing, suction dispersing, stirring dispersing and ultrasonic dispersing.

8. The detection method according to claim 7, wherein the oscillation frequency is 2000-2500rpm, the oscillation time is 1-5s, and the number of blowing and sucking times is 4-7.

9. A computer-readable storage medium having stored thereon computer-executable instructions for performing the detection method of claims 1-8.

10. A nucleic acid detecting system, wherein the detecting method according to claim 1 to 8 is inputted to the system.

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