KR20210145972A - A method of digital detecting using crispr diagnositcs and an apparatus having the same - Google Patents

Abstract

The present invention relates to a digital detection method and an apparatus thereof. According to an embodiment of the present invention, the digital detection method of the present invention comprises the following steps of: mixing a CRISPR-Cas protein that specifically binds to a target gene with a reporter capable of confirming whether the CRISPR-Cas protein is activated in the sample to be analyzed; loading the mixed sample into a well array including a plurality of micro-wells of a cartridge for digital detection; and detecting the target gene in the mixed sample by detecting a change in the reporter in each micro-well by using the digital detection cartridge.

Description

Digital detection method using CRISPR diagnostic method and device thereof

The present invention relates to a method for digitally detecting a target material in a sample, and more particularly, to a method and apparatus for digitally detecting a target material using CRISPR diagnostics.

Gene amplification technology is an essential process in molecular diagnosis, and is a technology that repeatedly replicates and amplifies a specific nucleotide sequence of a trace amount of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample. Among them, polymerase chain reaction (PCR) is a representative gene amplification technology and consists of three steps: DNA denaturation, primer bonding, and DNA extension. Since the step is dependent on the temperature of the sample, DNA can be amplified by repeatedly changing the temperature of the sample.

Previously, PCR was generally performed in 96 or 384-well microplates. When higher throughput is required, conventional PCR methods in microplatelets are not cost effective or efficient. However, if the PCR reaction volume is reduced, it is possible to reduce the consumption of reagents and shorten the amplification time at the reduced thermal mass of the reaction volume. This strategy can also be implemented in an array format (m x n), resulting in many smaller reaction volumes. The use of any array also allows for scalable high-throughput assays with increased quantitation sensitivity, dynamic range and specificity.

In order to solve these existing disadvantages, research on digital polymerase chain reaction (digital PCR) using sequence formats has been actively conducted. Results from digital PCR can be used to detect and quantify concentrations of rare alleles, provide absolute quantification of nucleic acid samples, and also measure fold changes with low nucleic acid concentrations. In general, increasing the number of replicates increases the accuracy and reproducibility of digital PCR results.

In digital PCR, a solution containing a relatively small number of target polynucleotides or nucleic acid sequences may be divided into small test samples, whereby each sample generally contains one molecule of the target nucleotide sequence or target nucleotides does not contain the molecules of the sequence. When the samples are then thermally cycled in a PCR protocol, procedure, or experiment, the sample comprising the target nucleotide sequence is amplified to produce a positive detection signal, while samples containing no target nucleotide sequence are not amplified and the detection signal does not create

The main existing digital PCR method uses end-point PCR in which the process of injecting the sample into the reaction space and the process of amplifying the nucleotide sequence are divided, so the reaction in which the nucleotide sequence is amplified in real time in each reaction space is performed. can't measure

In addition, genome editing based on the recently developed RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease Cas protein is capable of targeting knock-out, transcriptional activation and single guide RNA. (sgRNA) (ie, crRNA-tracrRNA fusion transcripts) provides a groundbreaking technique for inhibition, which has demonstrated scalability by targeting numerous gene loci. The CRISPR-Cas system consists of a guide RNA (gRNA) having a sequence complementary to a target gene or nucleic acid and CRISPR enzyme, a nuclease capable of cleaving a target gene or nucleic acid, and the gRNA and CRISPR enzyme form a CRISPR complex. and cleaving or modifying a target gene or nucleic acid by the formed CRISPR complex. The CRISPR system is an immune system of prokaryotes and archaea, and research on its utility is rapidly increasing as one of the recent gene scissors.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a digital detection method and apparatus for improving diagnosis or analysis efficiency capable of accurately analyzing a target material from a small amount of sample.

Another object to be solved by the present invention is to provide a digital detection method and device capable of improving the competitiveness of reagent prices and reducing the production cost of analysis and diagnostic devices by shortening the time for analysis and simplifying the testing process. will be.

Another problem to be solved by the present invention is to provide a digital detection method and apparatus for absolutely quantifying a target substance in a sample.

According to an embodiment of the present invention, mixing a CRISPR-Cas protein specifically binding to a target gene with a sample to be analyzed and a reporter capable of confirming whether the CRISPR-Cas protein is activated; loading the mixed sample into a well array including a plurality of micro-wells of a cartridge for digital detection; and detecting the target gene in the mixed sample by detecting a change in the reporter in each micro-well using the digital detection cartridge.

In one embodiment, the CRISPR-Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus avicellas (Staphylococcus avicellas9), Cas9 , Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9) Prevotella, and Francisella 1 (Cpf1) may be any one or more.

The pitch of the micro-wells may be less than 150 μm, and the target gene in the mixed sample may be quantified by mounting the digital detection cartridge in a reader system.

In an embodiment, the reporter may include a fluorescent material, and the fluorescent material may be displayed by being cleaved by the activated CRISPR-Cas protein.

In the detecting of the target gene, fluorescence displayed in each well may be measured in real time.

In an embodiment, the digital detection method may not require an amplification step of the sample including the polymerase chain reaction (PCR).

In addition, the diagnosis target sample may be obtained by performing only the step of extracting the nucleic acid from the sample collected without heating, or may be the sample itself collected without performing the heating and nucleic acid extraction step.

According to an embodiment of the present invention, a digital detection method and apparatus for improving analysis efficiency and analysis accuracy of a target material from a small amount of a diagnostic target sample using a digital device for quantitative analysis of CRISPR-Cas protein and target material is to provide

According to another embodiment of the present invention, by not performing the step of amplifying the sample, it is possible to shorten the time for sample analysis and simplify the test process, thereby improving the competitiveness of the reagent price and reducing the production cost of the analysis and diagnosis device. To provide a digital detection method and apparatus therefor.

In addition, according to another embodiment of the present invention, a digital detection method capable of absolutely quantifying a target material in a sample by analyzing the sample using a digital detection cartridge including a CMOS sensor and a digital detection device, and an apparatus thereof is to provide

1 shows the activation of CRISPR-Cas protein in a sample according to an embodiment of the present invention.
2 is a flowchart illustrating a digital detection method according to an embodiment of the present invention.
3 to 5 are diagrams illustrating a digital detection method according to an embodiment of the present invention.
6 is a perspective view of a cartridge for digital detection according to an embodiment of the present invention, and FIG. 7 is an exploded perspective view of the cartridge for digital detection shown in FIG. 6 .
8 is a perspective view schematically illustrating an example of the shape of the well array shown in FIG. 7 .
9 is a diagram illustrating a digital detection method according to another embodiment of the present invention.
10 shows a method for detecting a target substance in a sample using a general CRISPR-Cas protein.
11 is a diagram illustrating a digital detection method of a target material in a sample using a CRISPR-Cas protein according to an embodiment of the present invention.
12 is a diagram illustrating a digital detection method of a target material in a sample using a CRISPR-Cas protein according to an embodiment of the present invention.
13 to 15 are diagrams illustrating a method for preparing a sample to be analyzed according to various embodiments of the present disclosure.
16 is a schematic diagram of a step of collecting a sample to be analyzed according to an embodiment of the present invention.
17 illustrates a step of preparing a sample to be analyzed according to an embodiment of the present invention.
18 illustrates steps for digital detection of a mixed sample according to an embodiment of the present invention.
19 is a diagram illustrating an apparatus for preparing an analysis target sample according to an embodiment of the present invention, and FIGS. 20A to 20C are diagrams illustrating a method for preparing an analysis target sample using the device.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art, and the following examples may be modified in various other forms, and the scope of the present invention is as follows It is not limited to an Example. Rather, these examples are provided so as to more fully and complete the present invention, and to fully convey the spirit of the present invention to those skilled in the art.

In addition, in the following drawings, the thickness or size of each layer is exaggerated for convenience and clarity of description, and the same reference numerals in the drawings refer to the same elements. As used herein, the term “and/or” includes any one and combination of one or more of those enumerated items.

The terminology used herein is used to describe specific embodiments, not to limit the present invention. "comprise" and/or "comprising" as used herein specify the presence of the recited shapes, numbers, steps, operations, members, elements, and/or groups thereof; It does not exclude the presence or addition of one or more other shapes, numbers, movements, members, elements and/or groups.

In addition, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, preferred embodiments of the present invention will be further described with reference to the accompanying drawings. The same reference numerals are used for the same components in the drawings, and repeated descriptions of the same components are omitted.

The cartridge for digital detection used for digital detection of the present invention has a structure for mounting samples, sample volumes, or reaction volumes in a predetermined arrangement on a substrate, and mounting the samples in a predetermined arrangement of respective reaction positions in the substrate include structure.

In various embodiments, apparatus, apparatus, systems, and methods for loading samples in articles used to detect target substances in large, small volume samples are provided. Such target substances may be any suitable biological target, but include DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (eg, circulating tumor cells), or other suitable target biomolecules. In various embodiments, these biological components are used in applications such as fetal diagnosis, multiplex dPCR, virus detection, and quantitation standardization, genotyping, sequence validation, mutation detection, genetic biology, rare allele detection, and copy number change detection. It can also be used in conjunction with various analysis methods and systems in various fields.

In addition, detailed information on the components of the digital PCR cartridge, digital PCR module or system, definition, and operation method is the applicant's Republic of Korea Patent Nos. 10-0822672, 10-0825087, 10-0801448, No. 10-0808114, No. 10-1803497, No. 10-1753644, and No. 10-1904506 are disclosed, and the registered patent documents may be incorporated herein by reference in their entirety.

1 shows the activation of CRIPSPR-Cas protein in a sample according to an embodiment of the present invention.

Referring to FIG. 1 , a CRISPR-Cas protein specifically binding to a target material and a reporter capable of confirming whether the CRISPR-Cas protein is activated may be included in the tube. When the CRISPR-Cas protein is activated, it has a characteristic of cutting a single-stranded material around it. In addition, the reporter may include a fluorescent material, and the fluorescent material may exhibit fluorescence when the reporter is cleaved.

If a target material is present in a sample to be analyzed in a tube, the CRISPR-Cas protein in the tube may be activated (Activated CRISPR CAS) by binding to the target material. The activated CRISPR-Cas protein is shown in red in FIG. 1 , and the activated CRISPR-Cas protein can cleave reporters (shown in blue) around it. Since the cleaved reporters (shown in red) can show fluorescence, the target substance in the tube can be detected by detecting the fluorescence.

However, as shown in FIG. 1 , the activated CRISPR-Cas protein has a limit in its cleavage ability to cleave surrounding reporters. can only be In this case, it is difficult to accurately detect a target substance in a diagnostic sample.

A method for solving the above problems in a digital detection method and an apparatus thereof according to an embodiment of the present invention will be proposed with reference to FIGS. 2 to 16C below.

2 to FIG. 2 are flowcharts illustrating a digital detection method according to an embodiment of the present invention, and FIGS. 3 to 5 are diagrams illustrating a digital detection method according to an embodiment of the present invention.

Referring to FIG. 2 , a CRISPR-Cas protein including an enzyme binding to the target material and a reporter including a fluorescent material may be mixed with an analysis target sample to be analyzed for the presence of a target material first ( S10). In this case, when the target material is present in the sample to be analyzed, the CRISPR-Cas protein is activated to cut the surrounding reporter, so that the fluorescent material can be detected. On the other hand, when the target material is not present in the sample to be analyzed, the CRISPR-Cas protein is not activated and thus the fluorescent material will not be detected.

In general, since the target material is present in a small amount in the sample to be analyzed, the amount of the activated CRISPR-Cas protein is inevitably small and the amount of the fluorescent material detected by the reporter cannot be small. Therefore, in order to accurately and quickly detect a target material, there is a method of amplifying the analyte sample (increasing the sample) or dividing the analyte sample into small amount units to increase the amount of the detected fluorescent material. may be needed

The digital detection method according to an embodiment of the present invention includes a method of analyzing a sample to be analyzed by dividing it into small amount units. The sample mixed in step S10 may be loaded into a plurality of microwells included in the well array of the digital detection cartridge (S20), and by detecting a change in the reporter in each microwell loaded with the sample to be analyzed A target material may be detected (S30). Steps S20 and S30 will be described with reference to the drawings below in FIG. 3 .

3 to 5 are diagrams illustrating a digital detection method according to an embodiment of the present invention.

Referring to FIG. 3 , the mixed sample may be loaded into a plurality of micro-wells of the cartridge for digital detection, and in the micro-wells where the target material is present, the CRISPR-CAS protein is activated by binding to the target material (Positive Reaction). Fluorescent substances can be detected by cleaved reporters by Activated CRISPR CAS in According to an embodiment of the present invention, since fluorescence can be detected from a very small amount of sample, it is possible to accurately digitally detect a target material without amplifying the sample to be analyzed. In addition, in an embodiment, the presence or absence of the target material may be checked in real time, or the presence or absence of the target material may be determined by end-point detection.

As shown in the lower right of FIG. 4 , when the target material is checked by the end-point detection method, it is difficult to accurately determine the intermediate value, so whether the target material is actually present (positive/negative) is checked. It can be difficult to distinguish with certainty. If there is a lot of initial background fluorescence in the sample, even if it is determined as positive in endpoint detection, it may be negative rather than positive. Therefore, the accuracy of detecting the target substance in the analyte sample is lowered.

As shown in the lower right of FIG. 5 , a target material in a sample to be analyzed may be identified by a real-time detection method. In this case, since a difference in the signal for each time is detected and a change in the magnitude thereof is displayed, positive or negative can be clearly distinguished. In an embodiment, even if the initial background fluorescence signal is large, it may be accurately determined as negative if there is no change in the signal intensity thereafter, and positive if the change continues to occur. In addition, since the signal change in each microwell can be checked in real time, quantitative analysis and analysis of the presence or absence of the target material can be performed, and it is possible to know which microwell the target material is present in, so that the target material can be extracted It may be possible to use it for various experiments in the future.

6 is a perspective view schematically illustrating a cartridge for digital detection according to an embodiment of the present invention, and FIG. 7 is an exploded perspective view of the cartridge for digital detection shown in FIG. 6 .

6 and 7, the cartridge for digital detection according to an embodiment of the present invention is a cartridge-type inspection module, including an upper case 110, a bottom case 120, a microfluidic chamber 130, and a well array ( 140 ), a CMOS photosensor array 150 , and a PCB 160 . In one embodiment, the cartridge for digital detection may be detachably coupled to a reader system of a digital real-time detection equipment such as, for example, a digital real-time PCR equipment.

The upper case 110 may include a donut-shaped cover body including an upper hole formed in a central region and a window member disposed in the upper hole. The upper case 110 is fastened to the bottom case 120 . In this embodiment, although the upper case 110 and the bottom case 120 are shown to have a flat cylindrical shape, various shapes are possible. The window member may include a transparent material. The window member may include a PDMS material, but is not limited thereto.

The bottom case 120 may accommodate the microfluidic chamber 130 , the well array 140 , the CMOS photosensor array 150 , and the PCB 160 , and may be coupled to the upper case 110 . For example, the bottom case 120 and the upper case 110 may be fastened by a hook method, but is not limited thereto, and the fastening method is not limited thereto.

The microfluidic chamber 130 may include a membrane switch protruding upward, and an inlet formed on one side of the membrane switch for injection of a liquid sample. The lower edge region of the microfluidic chamber 130 may contact the edge region of the PCB 160 . In the lower central region of the microfluidic chamber 130 , a space with a retracted shape may be formed to accommodate the CMOS photosensor array 150 and the well array 140 mounted on the PCB 160 . The microfluidic chamber 130 may be made of a material such as PDMS. The microfluidic chamber 130 may be formed using any material as long as it has flexibility, transparency, PCR compatibility, and low autofluorescence, and can be injection molded.

The well array 140 may be attached to the lower surface of the microfluidic chamber 130 and disposed on the CMOS photosensor array 150 . The well array 140 may be formed of an etched silicon material. In one embodiment, well array 140 includes a plurality of micro wells 142 . The shape, size, number, etc. of the micro-wells 142 may vary. In one embodiment, the well array 140 may include a plurality of micro-wells (approximately 1,000 to 100,000) serving as a digital detection reaction site using the CRISPR-Cas protein.

An analysis sample such as a powder sample or a liquid sample may be accommodated in each of the micro-wells 142 . The analysis sample is a specific component for the analysis of a biological material. That is, the analysis sample is a component for quantitative or qualitative analysis of a specific biological material, for example, protein, DNA, RNA, etc., and refers to a primer, a probe, an antibody, an aptamer, a DNA or RNA polymerase, etc. It refers to a component necessary to perform a polymerase chain reaction, a constant temperature enzymatic reaction, or LCR (Ligase Chain Reaction). In an embodiment, the mixed sample prepared in step S10 of FIG. 2 may be loaded into each of the micro-wells 142 .

The CMOS photosensor array 150 is disposed under the well array 140 , and captures an image of a CRISPR-Cas reaction product performed in the micro wells 142 of the well array 140 in real time. The CMOS photosensor array 150 is disposed by being inserted into a substrate hole formed in the PCB 150 . The CMOS photosensor array 150 detects fluorescence (emission light) generated from a plurality of probes by excitation light. The detection of the fluorescence may be performed by a time separation method or a wavelength separation method.

In the case of the time separation method described above, as the fluorescent material expresses the emitted light in response to the excitation light, the fluorescent sensor array or a single sensor constituting the array detects the emitted light passing through the emission filter, and the time constant of the detected emitted light to detect the fluorescence.

In the case of the above-described wavelength separation method, as the fluorescent material expresses the emitted light in response to the excitation light, the fluorescent sensor array or a single sensor constituting the array detects the emitted light passing through the emission filter, and the spectrum of the detected emitted light Fluorescence is detected by analysis.

The PCB 150 may be disposed under the microfluidic chamber 130 and accommodate the mounted CMOS photosensor array 150 . The PCB 150 may be disposed to contact the bottom edge region of the microfluidic chamber 130 . A vent hole 152 may be formed in a portion of the PCB 150 in contact with the bottom edge region of the microfluidic chamber 130, and the vent hole 152 includes the CMOS photosensor array 150 and the well array ( 140) can be connected to the interstitial space.

8 is a perspective view schematically illustrating an example of the shape of the well array shown in FIG. 7 . 8 illustrates that the micro-well has a circular shape, various shapes such as a hexagon and a square are possible.

Referring to FIG. 8 , the shape, size, and volume of the micro-wells can be adjusted. The micro-wells have a shape through which upper and lower portions are penetrated. In this embodiment, the micro-well may have a thickness of less than 700 μm and a pitch of less than 150 μm. The micro-well may have various shapes, such as a hexagonal shape or a circular shape. Well array 140 may be treated with a hydrophilic coating. The well array 140 may be attached to the microfluidic chamber 130 by plasma or thermal or UV curing adhesives.

Referring back to FIGS. 6 and 7 , the CMOS photosensor array 150 is disposed under the well array 140 , and fluorescence generated by CRISPR protein activation in micro-wells of the well array 140 is captured in real time. . Preferably, the CMOS photosensor array 150 is disposed to correspond to the substrate hole formed in the PCB 160 to measure fluorescence emitted from the well array 140 . In this case, the entire well array 140 is disposed. The emitted fluorescence may be measured, and the fluorescence emitted from each microwell may be measured separately. The measured fluorescence may be analyzed in a manner such as a graph shown in the lower right of FIGS. 4 and 5 .

9 is a diagram illustrating a digital detection method according to another embodiment of the present invention. The digital detection method of the present invention can detect a target substance from a trace amount of a sample in a simple way without amplifying the analyte sample by using the CRISPR-Cas protein and a digital detection cartridge. Referring to FIG. 9 , the digital detection method of the present invention may detect a target material in real time, or selectively detect a target material at an end-point. In addition, in order to prepare a sample for analysis, nucleic acid extraction (Nucleic Acid Extraction) and heating (Heating) steps may be performed as in a general case, and either the nucleic acid extraction step and the heating step are not selectively performed, or both steps The sample itself collected without performing the test may be used as a sample to be analyzed without pre-treatment.

As described above, since digital detection according to an embodiment of the present invention does not require performing isothermal PCR, the time for testing is shortened and work is simplified, and since reagents necessary for PCR are not used, reagents for detecting target substances The price can be reduced, and the production cost of the product can be reduced. In addition, the digital detection method can more accurately quantify the target material.

10 is a diagram illustrating a digital detection method according to another embodiment of the present invention. Referring to FIG. 10 , PCR may be performed prior to detecting a target material using the CRISPR-Cas protein in the digital detection method of FIG. 9 . The PCR may be performed before or after CRISPR-Cas protein treatment, and may be isothermal PCR.

As such, when PCR is performed in addition to CRISPR-Cas protein treatment, sensitivity can be increased by amplifying a target material through PCR, and specificity can be increased due to specific binding of gRNA through CRISPR fluorescence signal amplification. It is possible to sensitively detect a target substance from a sample to be analyzed. In addition, absolute quantification of the target material is possible by analyzing the target material using a digital detection method.

11 is a flowchart illustrating a method for analyzing a target material using a general CRISPR-Cas protein. Referring to FIG. 11 , RNA is extracted from a sample according to a general protocol, and the sample containing the extracted RNA is amplified isothermal at about 62° C. for about 20 minutes. Preferably, the PCR may be performed by RT-PCR (Step 1). Also, it is possible to form a gRNA complex with Cas12a protein at about 37° C. for about 30 minutes (Step 1).

Thereafter, the reporter cleavage step through activation of the Cas 12 protein by mixing the Cas 12a+gRNA complex with the amplified analyte sample may be performed at about 37° C. for about 10 minutes (Step 2). Thereafter, the presence or absence of the target material may be analyzed by performing lateral flow analysis of the fluorescent material due to the reporter cleavage for about 2 minutes. In this case, the accuracy of target material detection can be improved because the sample to be analyzed is amplified by performing PCR and the amount of the sample is increased. increase, and the disadvantages of low accuracy of lateral flow analysis should be addressed.

12 is a flowchart illustrating a method for analyzing a target material using a CRISPR-Cas protein according to an embodiment of the present invention. Referring to FIG. 12 , RNA is extracted according to a general protocol, and additional processing of the sample including the extracted RNA is not required. In addition, the Cas12a protein and gRNA complex can be formed at about 37° C. for about 30 minutes (Step 0), and the complex is prepared in advance before starting the analysis of the target material, thereby reducing the time required for the analysis of the target material. can do it

By mixing the Cas 12a+gRNA complex with the prepared RNA extraction sample, the reporter cleavage step through activation of the Cas 12 protein can be performed at about 37° C. for about 10 minutes (Step 2). Thereafter, the presence or absence of the target material may be analyzed using the cartridge and apparatus for digital detection according to an embodiment of the present invention without using the lateral flow analysis method. In this case, since PCR is unnecessary, the disadvantages of the PCR described with reference to FIG. 11 can be eliminated, and the effect of accurately analyzing the target material in real time can be provided by performing digital analysis using a digital detection cartridge. .

13 to 15 are diagrams illustrating a method for preparing a sample to be analyzed according to various embodiments of the present disclosure.

Referring to FIG. 13 , the sample to be analyzed may be used by diluting the sample itself collected without nucleic acid extraction and heating process in distilled water or by diluting it in phosphate buffered physiological saline (PBS). In order to obtain the sample to be analyzed, the general nucleic acid extraction and heating steps may not be performed. Thereafter, the sample to be analyzed may be mixed with Cas13a or Cas12a protein, gRNA, reporter material, distilled water, and a buffer.

The mixed sample is loaded into the aforementioned cartridge for digital detection and heated at about 80° C. for about 1 to 15 minutes, and then the target material in the mixed sample can be analyzed at about 30 to 70° C. for 5 to 60 minutes. This digital detection method does not require a heat treatment cycle and can simply check fluorescence detection indicating the presence or absence of a target material at intervals of 10 seconds to 10 minutes. In this case, since the nucleic acid extraction and heating process is unnecessary in preparing the sample to be analyzed, the disadvantage that the nucleic acid extraction process has low efficiency and the problem of contamination can be solved.

On the other hand, in the general digital PCR method, real-time detection and target material are generated because a droplet is generated to confirm the target material in the sample to be analyzed, and the presence or absence of the target material is analyzed using the end-point detection method. extraction can be difficult.

Referring to FIG. 14 , a sample to be analyzed may be prepared by incubating a sample collected without a nucleic acid extraction process at about 60 to 100° C. for 1 to 15 minutes. The nucleic acid extraction step may not be performed to obtain the sample to be analyzed. Thereafter, the sample to be analyzed may be mixed with Cas13a or Cas12a protein, gRNA, reporter material, distilled water, and a buffer.

Since the mixed sample has undergone a heating process when preparing the sample to be analyzed, the target material in the mixed sample can be analyzed at about 30 to 70° C. for 5 to 60 minutes without a subsequent heating step after being loaded into the digital detection cartridge described above. This digital detection method does not require a heat treatment cycle and can simply check fluorescence detection indicating the presence or absence of a target material at intervals of 10 seconds to 10 minutes.

On the other hand, as described above, in the general digital PCR method, real-time detection and target material are generated because droplets are created to identify the target material in the analyte sample, and the presence or absence of the target material is analyzed using the endpoint detection method. extraction can be difficult.

Referring to FIG. 15 , an analysis target sample may be prepared by extracting RNA from a sample sample collected by a general method, and mixing Cas13a or Cas12a protein, gRNA, and a reporter material with the extracted RNA sample. The mixed sample may be loaded into the aforementioned cartridge for digital detection, and the target material in the mixed sample may be analyzed at about 30 to 70° C. for 5 to 60 minutes without a heating step. Such a digital detection method does not require a heat treatment cycle, and fluorescence detection indicating the presence or absence of a target material can be simply checked at intervals of 10 seconds to 10 minutes.

16 is a schematic diagram of a step of collecting a sample to be analyzed according to an embodiment of the present invention.

Referring to Figure 16, the sample in the present invention is a nasopharyngeal sample (Nasopharyngeal swab), an oropharyngeal swab (Oropharyngeal swab), sputum (Sputum) 100 to 1000 μl, saliva (Saliva) 100 to 1000 μl, blood serum / plasma (Blood serum/plasma) 100 to 1000 μl, urine 100 to 1000 μl, cerebrospinal fluid (CSF) 100 to 1000 μl, or stool (Stool) 100 to 1000 μl, but is not limited thereto. Anything you want is possible. The sample collected using the swab rod is included in a sample tube, and 100 μl to 3 ml of the sample in the tube is diluted with 100 to 1000 μl of distilled water or 100 to 1000 μl of phosphate buffered saline (PBS) for 5 to Vortexing can be performed for 60 seconds.

17 illustrates a step of preparing a sample to be analyzed according to an embodiment of the present invention.

Referring to FIG. 17, 1 to 100 μl of a sample taken from the sample prepared with reference to FIG. 16 may be added to a PCR tube, and heat may be applied to 60 to 100° C. for 1 to 15 minutes, which is added to the PCR tube It is a step for promoting sample mixing, activation of CRISPR-Cas protein, and cleavage of a reporter substance, which is distinct from performing PCR. In addition, the preparing step (heating process) of the sample to be analyzed may be selectively performed.

18 illustrates steps for digital detection of a target substance in a mixed sample according to an embodiment of the present invention.

Referring to FIG. 18 , about 30 μl of the sample mixed with reference to FIGS. 16 and 17 may be injected into the cartridge for digital detection described above with reference to FIGS. 6 to 8 . The cartridge for digital detection into which the mixed sample is injected may detect a target material in the mixed sample using a digital detection system such as a digital PCR system. In this case, the target material can be detected in real time from a much smaller amount of sample compared to the detection method of the target material using the general CRISPR-Cas protein, and since PCR is not used, it is not affected by the PCR inhibitor. it can't be Accordingly, the digital detection method according to an embodiment of the present invention may have high sensitivity capable of detecting even when a single copy of the target material is included in the microwell.

19 is a diagram illustrating an apparatus for preparing an analysis target sample according to an embodiment of the present invention, and FIGS. 20A to 20C are diagrams illustrating a method for preparing a diagnostic target sample using the device.

19 is a sample preparation device for analysis is a plunger (10), a vent hole (vent hole, 20), PCR tubes (30a, 30b), a paraffin plug (Paraffin plug, 40), a body portion (50), the upper cover (60) ), and a microchannel layer 70 .

One end of the plunger 10 may be connected to one 30b of the PCR tube, and a vent hole 20 may be formed in the cylinder sidewall of the plunger 10 . The PCR tube 30b connected to the plunger 10 may be connected to one end of the microchannel layer 70 . The microchannel layer 70 may be a fluid passage through which the microfluid may move. A reaction solution 80 may be loaded into the microchannel layer 70 , and the reaction solution 80 may be of any kind as long as it is a material capable of reacting with the sample contained in the PCR tube 30a.

One end of the microchannel layer 70 is connected to the PCR tube 30b connected to the plunger 10 , and the other end may be combined with the paraffin plug 40 . The paraffin plug 40 may be combined with the PCR tube 30a not connected to the plunger 10 . The plunger 10 , the PCR tubes 30a and 30b , and the paraffin plug 40 may be fastened to each other in the body portion 50 . The body portion 50 may have an upper cover 60 formed on the upper side. In one embodiment, the microchannel layer 70 may be formed by molding the body portion 50, or may be formed as a separate layer.

Referring to FIGS. 20A to 20C , first, the PCR tube 30a connected to the paraffin plug 40 is separated and the collected sample is filled in the PCR tube 30a ( FIG. 20A ). After that, the PCR tube 30a filled with the sample is coupled to the body part 50 again, and when heat is applied to the PCR tube 30a for about 2 minutes, the paraffin of the paraffin plug 40 in the PCR tube 30a can be melted. (Fig. 20b).

When paraffin is melted, the paraffin plug 40 may move the sample in the PCR tube 30a as a passage for the fluid. When the piston of the plunger 10 is pulled, a negative pressure by vacuum is formed inside the plunger 10, and the sample in the PCR tube 30a is transferred through the paraffin plug 40 by the formed negative pressure to the microchannel layer 70 After the reaction solution 80 in the microchannel layer 70 flows into the PCR tube 30b connected to the plunger 10, the sample that has moved to the microchannel layer 70 is also plunged (10). It can flow into the PCR tube (30b) connected to.

In this case, the volume of the sample solution flowing into the PCR tube 30b may be controlled by the position of the vent hole 20 formed in the sidewall of the plunger 10 . Thereafter, the PCR reaction may be performed by separating the PCR tube 30b containing the sample and the reaction solution. However, the sample to be analyzed in the PCR tube 30b prepared as described above may be used for various reactions for detecting a target material, not a PCR reaction. For example, when the reaction solution 80 preloaded on the microchannel layer 70 is a reporter containing a CRISPR-Cas protein and a fluorescent material cut by the activated CRISPR-Cas protein, the PCR tube 30b is It is not used to perform PCR, but real-time digital detection can be performed using a cartridge for digital detection.

It is common in the art to which the present invention pertains that the present invention described above is not limited to the above-described embodiments and the accompanying drawings, and that various substitutions, modifications and changes are possible within the scope without departing from the technical spirit of the present invention. It will be clear to those who have knowledge.

Claims (9)

mixing a CRISPR-Cas protein that specifically binds to a target material and a reporter capable of confirming whether the CRISPR-Cas protein is activated in a sample to be analyzed;
loading the mixed sample into a well array including a plurality of micro-wells of a cartridge for digital detection; and
and detecting the target substance in the mixed sample by detecting a change in the reporter in each micro-well using the digital detection cartridge. The method of claim 1,
The CRISPR-Cas protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus, Francisella novicida Cas9 (FnCaureus, Francisella Casiss9) (SaCas9) NcCas9), Neisseria meningitis Cas9 (NmCas9) Digital detection method, characterized in that any one or more of Prevotella and Francisella 1 (Cpf1). The method of claim 1,
The digital detection method, characterized in that the pitch of the micro-wells is less than 150um. The method of claim 1,
The digital detection method, characterized in that the target gene in the mixed sample can be quantified by mounting the digital detection cartridge to a reader system. The method of claim 1,
The reporter includes a fluorescent material, and the digital detection method, characterized in that the fluorescent material is displayed by being cleaved by the activated CRISPR-Cas protein. 6. The method of claim 5,
The step of detecting the target gene is a digital detection method, characterized in that the fluorescence displayed in each well is measured in real time. The method of claim 1,
The digital detection method, characterized in that it does not require the step of amplifying the sample including the polymerase chain reaction (PCR). The method of claim 1,
The diagnostic target sample is a digital detection method, characterized in that obtained by performing only the step of extracting the nucleic acid from the sample collected without heating. The method of claim 1,
The diagnostic target sample is a digital detection method, characterized in that the sample itself is collected without performing heating and nucleic acid extraction steps.

Images