DIGITAL NUCLEIC ACID AMPLIFICATION TESTING METHOD AND INTEGRATED DETECTION SYSTEM BASED ON CRISPR-CAS TECHNOLOGY

Abstract

Disclosed in the present invention are a digital nucleic acid amplification testing method and an integrated detection system based on CRISPR-Cas technology. The integrated detection system comprises an integrated reaction chip, a temperature control module, a light source and an optical signal detector. The method comprises: uniformly dividing a nucleic acid amplification reagent into amplification micro-droplets, then mixing the amplification micro-droplets after digital nucleic acid amplification with detection micro-droplets containing CRISPR-Cas detection reagent to perform a CRISPR reaction, and when the reaction is finished, detecting an optical signal to realize high-specificity testing of a target object, and the concentration or copy number of nucleic acid molecules in a sample to be tested is also obtained, and high-sensitivity absolute quantitative testing of a target object is realized.

Claims

1. An integrated detection system based on droplet digital nucleic acid amplification and CRISPR-Cas technology, comprising an integrated reaction chip (3), a temperature control module, a light source and an optical signal detector; wherein the integrated reaction chip (3) is distributed with a droplet generating area for amplification reagents (4), a nucleic acid amplification area (6), a droplet generating area for detection reagents (7), a droplet fusion area (8) and an optical detection area (9); there are microchannels (5) used for connecting the droplet generating area for amplification reagents (4) and the nucleic acid amplification area (6), connecting the nucleic acid amplification area (6) and the droplet fusion area (8), connecting the droplet generating area for detection reagents (7) and the droplet fusion area (8), and connecting the droplet fusion area (8) and the optical detection area (9); the light source and the optical signal detector are respectively located on upper and lower sides of the optical detection area (9), and the temperature control module is placed below or above the nucleic acid amplification area (6) to heat the nucleic acid amplification area (6), the droplet fusion area (8) is a Y-shaped or T-shaped microchannel, and three ends of the Y-shaped or T-shaped microchannel are respectively connected with the nucleic acid amplification area (6), the droplet generating area for detection reagents (7) and the optical detection area (9) through respective microchannels (5); a width of the microchannel (5) is the same as a size of a single droplet or slightly smaller than the diameter of the droplet, so that only the one single droplet passes through the microchannel (5) in sequence; the nucleic acid amplification area (6) contains the solution of nucleic acid amplification reagent, and the solution of nucleic acid amplification system is generated into amplification microdroplets (12); the droplet generating area for detection reagents (7) contains the solution of CRISPR-Cas detection reagent, and the solution of CRISPR-Cas detection reagent is generated into detection microdroplets (13); the amplification microdroplets (12) after undergoing amplification in the nucleic acid amplification area (6) enter the droplet fusion area (8) along with the detection microdroplets (13) at a fixed flow rate, collision and aggregation of droplets are performed to carry out fusion of one of the amplification microdroplets (12) and one of the detection microdroplets (13) respectively to form mixed microdroplets (14), and the mixed microdroplets (14) are subjected to the CRISPR reaction.

2. The integrated detection system based on droplet digital nucleic acid amplification and CRISPR-Cas technology according to claim 1, wherein the integrated reaction chip (3) is provided with a quick connection structure (10), and the temperature control module is connected to the nucleic acid amplification area (6) on the integrated reaction chip (3) through the quick connection structure (10).

3. The integrated detection system based on droplet digital nucleic acid amplification and CRISPR-Cas technology according to claim 1, wherein a cooling device is placed in the testing system droplet generating area (7), or a cooling channel (11) is arranged around the droplet generating area for detection reagents (7), and a coolant is added to the cooling channel (11).

4. (canceled)

5. A digital nucleic acid amplification testing method based on CRISPR-Cas technology applied to the integrated detection system claimed in claim 1, comprising: uniformly dividing a solution of the nucleic acid amplification reagent (1) is into tens of thousands of amplification microdroplets (12), and selecting an operating environment to realize a nucleic acid amplification in the amplification microdroplets (12); at the same time, uniformly dividing the solution of CRISPR-Cas detection reagents (2) into tens of thousands of detection microdroplets (13); fusing the detection microdroplets (13) and the amplification microdroplets (12) with each other one by one, afterwards, performing a CRISPR reaction, and realizing a high-specificity testing of a target object by detecting an optical signal; the solution of the nucleic acid amplification reagent (1) enters and passes through the droplet generating area for amplification reagents (4), and is uniformly divided into tens of thousands of the amplification microdroplets (12), and the nucleic acid amplification area (6) is heated by the temperature control module; afterwards, the amplification microdroplets (12) are driven to pass through microchannels (5) and enter the nucleic acid amplification area (6) at a fixed flow rate to realize digital nucleic acid amplification; the solution of the CRISPR-Cas detection reagent (2) is uniformly divided into tens of thousands of the detection microdroplets (13) in the droplet generating area for detection reagents (7), and the detection microdroplets (13) contain a single-stranded oligonucleotides probe labeled with fluorescence groups as a fluorescent probe; the amplification microdroplets (12) after undergoing amplification in the nucleic acid amplification area (6) enter the droplet fusion area (8) along with the detection microdroplets (13) at the fixed flow rate, collision and aggregation of droplets are performed to carry out fusion of one of the amplification microdroplets (12) and one of the detection microdroplets (13) respectively to form mixed microdroplets (14), and the mixed microdroplets (14) are subjected to the CRISPR reaction; when the CRISPR reaction is completed, the mixed microdroplets (14) enter the optical detection area (9), and a target strand in the mixed microdroplets (14) is identified and captured through gRNA, and a DNA enzyme cleavage activity is activated; if the mixed microdroplet (14) contains the target strand, the fluorescent probe is cleaved, so that the fluorescence group and a quencher group are separated, and a fluorescent signal is emitted under excitation of the light source, the fluorescent signal of the single mixed microdroplet (14) is analyzed through the optical signal detector to obtain a ratio of positive droplets, and then a concentration or a copy number of nucleic acid molecules in the nucleic acid amplification reagent is calculated, so as to obtain a detection result.

6. (canceled)

7. (canceled)

8. The digital nucleic acid amplification testing method based on CRISPR-Cas technology according to claim 5, wherein one of the following two methods is adopted to obtain the ratio of positive droplets: (A) the mixed microdroplets (14) are detected one by one: the mixed microdroplets (14) are driven to flow through the optical detection area (9) in sequence, the optical signal detector detects an optical signal and converts the optical signal into an electric signal, and a ratio p of the positive droplets is obtained through processing of the electrical signal; (B) all microdroplets are detected simultaneously: all of the mixed microdroplets (14) converge in the optical detection area (9) and are arranged dispersedly, a camera or a mobile phone with a photographing function is utilized to take pictures of the optical detection area (9) to obtain an optical image, and the ratio p of the positive droplets is obtained through image processing; the method finally calculates an average number of nucleic acid molecules λ in each of the mixed microdroplets (14) according to the ratio p of the positive droplets according to the following formula, so as to obtain a concentration or a copy number of the nucleic acid molecules in a sample to be tested:
λ=−ln(1−p).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a testing flow chart of the present disclosure.

[0043] FIG. 2 is a testing principle diagram of the present disclosure.

[0044] FIG. 3 is a top view of an integrated reaction chip in the present disclosure.

[0045] FIG. 4 is a top view of a cooling channel in the integrated reaction chip of the present disclosure.

[0046] FIG. 5 is a top view of a droplet fusion area (Y-shaped microchannel) in the integrated reaction chip of the present disclosure.

[0047] In the figure: nucleic acid amplification reagent 1, CRISPR-Cas detection reagent 2, integrated reaction chip 3, droplet generating area for amplification reagents 4, microchannel 5, nucleic acid amplification area 6, droplet generating area for detection reagents 7, droplet fusion area 8, optical detection area 9, quick connection structure 10, cooling channel 11, amplification microdroplet 12, detection microdroplet 13, mixed microdroplet 14.

DESCRIPTION OF EMBODIMENTS

[0048] The present disclosure will be further described below with reference to the accompanying drawings, but the present disclosure is not limited to the following embodiments.

[0049] Specifically, the embodiment includes an integrated reaction chip 3, a temperature control module, a light source and an optical signal detector. As shown in FIG. 3, the integrated reaction chip 3 is distributed with a droplet generating area for amplification reagents 5, a nucleic acid amplification area 6, a droplet generating area for detection reagents 7, a droplet fusion area 8 and an optical detection area 9. There are microchannels 5 used for connecting the droplet generating area for amplification reagents 4 and the nucleic acid amplification area 6, connecting the nucleic acid amplification area 6 and the droplet fusion area 8, connecting the droplet generating area for detection reagents 7 and the droplet fusion area 8, and connecting the droplet fusion area 8 and the optical detection area 9.

[0050] The light source and the optical signal detector are respectively located on the upper and lower sides of the optical detection area 9, and the temperature control module is placed above or below the nucleic acid amplification area 6 to heat the nucleic acid amplification area 6. When the digital nucleic acid amplification is finished, the temperature control module may be turned off or removed.

[0051] The temperature control module is suitable for a variable temperature environment of PCR, and may also realize the constant temperature environment suitable for constant temperature amplification technologies such as LAMP, RPA, and NASBA.

[0052] The integrated reaction chip 3 is provided with a quick connection structure 10, and the temperature control module is connected to the nucleic acid amplification area 6 on the integrated reaction chip (3) through the quick connection structure 10, so that the temperature control module and the nucleic acid amplification area 6 are positioned and fixed through the quick connection structure 10. In a specific implementation, the quick connection structure 10 can be a connection structure based on magnet attraction, a snap structure based on simple rotation, a snap structure based on push-type self-locking, etc.

[0053] A cooling device is placed in the droplet generating area for detection reagents 7 in the amplification process, or a cooling channel 11 is arranged around the droplet generating area for detection reagents 7. As shown in FIG. 4, a coolant is added to the cooling channel 11, so that the droplet generating area for detection reagents 7 is cooled during the amplification process.

[0054] The width of the microchannel 5 is the same as the size of a single droplet, so that only a single droplet can pass through the microchannel 5 in sequence.

[0055] The light source may be a light emitting diode LED, a laser diode, or the like.

[0056] The optical signal detector may be a photomultiplier, a PMT, a photodiode, a CCD (Charge Coupled Device), a mobile phone with a camera function, an optical microscope, and the like.

[0057] The cooling device may be a cool fan, a heat sink, coolant, and so on for cooling temperature.

[0058] As shown in FIG. 5, the droplet fusion area 8 is a Y-shaped microchannel, and the three ends of the Y-shaped microchannel are respectively connected with the nucleic acid amplification area 6, the droplet generating area for detection reagents 7 and the optical detection area 9 through the respective microchannels 5.

[0059] The droplet generating area for amplification reagents 4 and the droplet generating area for detection reagents 7 are both droplet generating areas, and they can control the generation and size of droplets by adjusting the structure and the two-phase flow rate ratio through the micro-pipe structure based on T-channel method, flow focusing method or coaxial flow focusing method.

[0060] The nucleic acid amplification area 6 and the temperature control module are positioned and fixed through a quick connection structure, for example, a connection structure based on magnet attraction. The temperature control module may be selected according to the amplification technology adopted. If the polymerase chain reaction (PCR) is adopted, the temperature control module may be selected from the variable temperature control module or the multi-temperature zone control module; if the constant temperature amplification technology such as LAMP is adopted, the temperature control module may be selected from a single temperature zone control module.

[0061] In order to prevent the temperature of the nucleic acid amplification area 6 from affecting the activity of the reagents in the CRISPR-Cas system, the temperature control module only regulates the temperature of the nucleic acid amplification area on the integrated reaction chip 3. When the digital nucleic acid amplification is finished, the temperature control module may be turned off or removed.

[0062] When amplification is completed, the amplified microdroplet 12 and the detection microdroplet 13 enter the droplet fusion area 8 at a fixed flow rate. Through the appropriate flow channel design, the two microdroplets are brought into contact and fused together, and then subjected to the CRISPR reaction. The detection of target objects is realized by detecting the optical signals. For example, the CRISPR-Cas detection reagent includes a single-stranded oligonucleotide probe labeled with fluorescence groups. After the CRISPR reaction is completed, the target strand can be identified and captured through gRNA, and the DNA enzyme cleavage activity thereof is activated. If the mixed microdroplet 14 contains a target strand, the fluorescent probe is cleaved, so that the fluorescence group and the quencher fluorescence group are separated, and a fluorescent signal is emitted when excited by a light source of a certain wavelength.

[0063] To perform optical signal detection, two different methods may be adopted for implementation:

[0064] (1) Mixed microdroplets 14 are detected one by one: In this method, the mixed microdroplets 14 flow through the optical detection area one by one in sequence at a fixed rate. Under the excitation of the light source with a certain wavelength (if there are no fluorescent materials in the mixed microdroplet, the light source for excitation is not required), the optical signal detector, such as PMT and photodetector, converts the optical signal into an electric signal. The optical signal of the mixed microdroplet 14 is continuously recorded, and a ratio of positive droplets can be obtained by filtering the waveform of the electrical signal, removing the baseline, and segmenting the threshold value.

[0065] (2) All mixed microdroplets are detected simultaneously: In this method, all mixed microdroplets 14 converge in the optical detection area. Under the excitation of the light source with a certain wavelength (if there are no fluorescent materials in the mixed microdroplet, the light source for excitation is not required), the optical inspection area is photographed using a camera or a mobile phone with a photo function to obtain an optical image, and the ratio of positive droplets can be obtained through image processing, including acquisition of regions of interest, filtering, threshold segmenting, and counting.

[0066] No matter in the method (1) or the method (2), the proportion p of positive droplets may be obtained. According to the Poisson distribution principle, the average number of nucleic acid molecules λ in each mixed microdroplet 14 may be calculated according to the following formula, so as to obtain the concentration or copy number of nucleic acid molecules in a sample to be tested.

λ=−ln(1−p).

[0067] Below is a combination of the method of the present disclosure and the integrated detection system, the content of the present disclosure and the implementation process are further elaborated:

[0068] 1) Preparation of liquid to be tested:

[0069] {circle around (1)} The required nucleic acid amplification reagent 1 is prepared according to the actual amplification technology adopted.

[0070] {circle around (2)} A CRISPR-Cas detection reagent 2 with suitable concentration is prepared.

[0071] 2) The prepared nucleic acid amplification reagent 1 is added to the droplet generating area for amplification reagents 4 of the integrated reaction chip 3 to generate the amplification microdroplet 12 required for digital nucleic acid amplification.

[0072] 3) A suitable temperature control module is selected according to the adopted amplification technology, the nucleic acid amplification area 6 on the integrated reaction chip 3 is fixed above the temperature control module through the quick connection structure 10, and the power of the temperature control module is turned on for heating, thus achieving nucleic acid amplification.

[0073] 4) The prepared CRISPR-Cas detection reagent 2 is added to the droplet generating area for detection reagents 7 of the integrated reaction chip 3 to generate the detection microdroplet 13 required for optical detection. In order to reduce the influence of the amplification heating process on the activity of the reagents in the CRISPR-Cas detection reagent 2, a coolant may be added in the cooling channel 11.

[0074] 5) After the digital nucleic acid amplification is performed, the temperature control module is turned off, the amplification microdroplet 12 and the detection microdroplet 13 flow into the droplet fusion area 8 at a certain flow rate, as shown in FIG. 5. After passing through the Y-shaped microchannel, the two droplets are fused one by one, followed by a CRISPR reaction in the mixed microdroplet 14.

[0075] 6) When the CRISPR reaction is completed, the optical signal of a single mixed microdroplet 14 can be obtained. There are two methods to implement the process:

[0076] {circle around (1)} Mixed microdroplets 14 are detected one by one: In this method, the mixed microdroplets 14 flow through the optical detection area one by one in sequence at a fixed rate. Under the excitation of the light source with a certain wavelength, a PMT or a photodetector can be used to convert the optical signal into an electric signal. The optical signal of the mixed microdroplet 14 is recorded.

[0077] {circle around (2)} All microdroplets are detected simultaneously: In this method, all mixed microdroplets 14 converge in the optical detection area. Under the excitation of the light source with a certain wavelength, a camera or a mobile phone with a photographing function is utilized to take pictures of the optical detection area to obtain an optical image.

[0078] 7) The two optical signal acquisition methods described in step 6) respectively correspond to two different optical signal processing and result analysis methods:

[0079] {circle around (1)} After obtaining the optical signal of each droplet in the mixed microdroplet 14, a ratio p of positive droplets can be obtained by filtering the waveform of the electrical signal, removing the baseline, and segmenting the threshold value.

[0080] {circle around (2)} After obtaining the optical image of the mixed microdroplet 14, the ratio p of positive droplets can be obtained through image processing for acquisition of regions of interest, filtering, threshold segmenting, and counting.

[0081] According to method {circle around (1)} or method {circle around (2)}, the proportion of positive droplets can be obtained, and the concentration or copy number of nucleic acid molecules in the sample to be tested can be calculated according to the Poisson distribution principle, and the detection result can be obtained.

[0082] 8) The integrated reaction chip 3 is removed and replaced with a new integrated reaction chip or the detection is terminated.

[0083] The above-mentioned specific embodiments are used to explain the present disclosure, rather than limit the present disclosure. Within the spirit of the present disclosure and the scope to be protected by the claims, any modifications and revisions made to the present disclosure all fall into the scope to be protected by the present disclosure.