CN114381360A

CN114381360A - Nucleic acid detection system and application thereof

Info

Publication number: CN114381360A
Application number: CN202011137316.7A
Authority: CN
Inventors: 谈畅; 黄昊; 许奇齐
Original assignee: Canon Medical Systems Corp
Current assignee: Canon Medical Systems Corp
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2022-04-22
Also published as: JP2022068846A

Abstract

The present invention provides a nucleic acid detection system for detecting a target nucleic acid in a sample, comprising 1 st to 4 th chambers forming a liquid flow path, liquid flowing from the 1 st chamber to the 4 th chamber, wherein the 1 st chamber comprises a swab inlet for inserting a swab to which the sample is attached, the 2 nd chamber comprises a reagent for thermally inactivating and lysing the sample, the 3 rd chamber comprises a reagent for isothermal amplification of the target nucleic acid, and the 4 th chamber comprises a test strip for CAS enzymatic reaction and lateral flow detection with the target nucleic acid, and uses thereof.

Description

Nucleic acid detection system and application thereof

Technical Field

The invention relates to a nucleic acid detection system and application thereof.

Background

The current nucleic acid detection technology is mainly based on PCR, which is a gold standard in the field of nucleic acid detection, and a specific nucleic acid detection process is shown in FIG. 1 and comprises the steps of sampling, nucleic acid extraction, amplification, detection, result reporting and the like. This technique requires expensive PCR instruments, requires appropriate polymerases, primers and/or probes, has long overall operation times and high requirements on the degree of expertise of the skilled person, and has specific requirements on the experimental environment (certified PCR laboratories). Numerous attempts have been made by those skilled in the art to simplify the nucleic acid detection procedure and obtain the detection result quickly. As an integrated detection technique in which multiple steps are integrated, patent document 1 describes a system capable of amplifying a plurality of target nucleic acids in one reaction chamber, and this system can achieve the objective of "sample input and result output", but it still has a problem that false positives cannot be avoided because it is a PCR-based technique, and a detection device used therefor is high in cost and requires a specific laboratory instrument for detection, and thus POCT (point-of-care testing) cannot be achieved.

The SHERLOCK technique (Specific High Sensitivity Enzymatic UnLOCKing technique, Specific High Sensitivity Enzymatic Reporter un locking) is a novel rapid, High Sensitivity, low cost diagnostic tool developed by chinese scientist with new CRISPR-Cas system, and can be used to detect diseases such as zika virus infection and dengue infection, which uses Cas13a protein with "associated cleavage" property and uses special amplification technique to detect RNA in a sample, thereby forming a detection system capable of accurately detecting individual nucleic acid molecules in serum, urine and saliva (see non-patent document 1). However, as shown In fig. 1 or fig. 2, the existing SHERLOCK technology and other CRISPR-based IVD (In Vitro Diagnosis) detection technologies require multiple steps of sampling, nucleic acid extraction, amplification, Cas enzyme reaction, detection, etc., and between the steps, there are risks of contamination due to decapping operations, and In addition, there are problems that specific requirements for experimental environments are required, and storage and transportation conditions of liquid reaction systems are required, and thus improvements In integration and automation are needed.

Documents of the prior art

Patent document

Patent document 1: US79680406A

Non-patent document

Non-patent document 1: gootenberg, Jonathan S., et al, "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6," Science 360.6387(2018): 439-.

Disclosure of Invention

Problems to be solved by the invention

In view of the above-described state of the art, it is an object of the present invention to provide a highly integrated, automated and portable nucleic acid detecting system which can be stored and transported at room temperature without requiring expensive equipment, without requiring a professional technician, without requiring a special laboratory, and which can ensure sequence specificity of detection results, and which is free from contamination to the inside and outside.

The invention utilizes the editable sequence specificity and the associated shearing activity of enzymes such as CAS12/13 and the like to detect the target nucleic acid in a sample, forms a continuous liquid flow path by connecting specific 1 st chamber to 4 th chamber in sequence, reasonably deploys a reagent compound on a paper base material for example, and reports the nucleic acid detection result through a color reaction on a lateral flow test strip, thereby integrating multiple steps such as sampling, nucleic acid extraction, amplification, Cas enzyme reaction, detection color development and the like in the nucleic acid detection, and constituting an automatic and portable nucleic acid detection system which can realize sample input and result output and can be applied to POCT and can be used for detecting the target nucleic acid in the sample.

In addition, the nucleic acid detecting system of the present invention may further comprise a plurality of 4 th chambers simultaneously in one system, thereby enabling simultaneous detection of a plurality of target nucleic acids, and thus can be applied to genotyping such as HPV genotyping or MTB/RIF, and simultaneous detection of DNA and RNA, and the like.

Means for solving the problems

The present invention provides the following technical means.

1. A nucleic acid detecting system for detecting a target nucleic acid in a sample, comprising 1 st to 4 th chambers forming a liquid flow path, a liquid flowing from the 1 st chamber to the 4 th chamber, wherein,

the 1 st chamber contains a swab inlet for insertion of a swab to which a sample is attached,

the 2 nd chamber contains reagents for heat inactivation and lysis of the sample,

the 3 rd chamber contains reagents for amplifying the target nucleic acid, and

the 4 th chamber contains a test strip for Cas enzyme reaction and lateral flow detection with the target nucleic acid.

2. The nucleic acid detecting system according to [1], wherein a filter for purifying a sample is provided between the 1 st chamber and the 2 nd chamber.

3. The nucleic acid detecting system according to [1], wherein the 1 st chamber further comprises an extrusion channel.

4. The nucleic acid detecting system according to [1], wherein a wax valve for separating chambers is provided between the 2 nd chamber and the 3 rd chamber.

5. The nucleic acid detecting system according to [1], wherein a wax valve for separating chambers is provided between the 3 rd chamber and the 4 th chamber.

6. The nucleic acid detecting system according to [4] or [5], wherein the wax valve is composed of a separate wax layer or is adsorbed on a support material.

7. The nucleic acid detecting system according to any one of [1] to [5], wherein the system has a temperature control unit that controls the temperature of the 3 rd chamber.

8. The nucleic acid detecting system according to [4] or [5], wherein the wax valve is opened by heating of a temperature control unit.

9. The nucleic acid detecting system according to any one of [1] to [5], wherein the reagents in the 2 nd chamber and the 3 rd chamber are lyophilized reagents.

10. The nucleic acid detecting system according to [9], wherein the lyophilized reagent is a lyophilized reagent ball or is adsorbed on a scaffold material.

11. The nucleic acid detection system according to any one of [1] to [5], wherein the reagent for the Cas enzyme reaction comprises a Cas enzyme complex,

the Cas enzyme complex comprises a Cas enzyme, a guide nucleic acid, and a probe,

the guide nucleic acid comprising a guide sequence capable of binding to the target nucleic acid and capable of forming a complex with the Cas enzyme,

the probe is a DNA or RNA based molecule comprising a non-target nucleic acid sequence, which when cleaved by the Cas enzyme is capable of producing the molecule required for the subsequent reaction.

12. The nucleic acid detection system according to any one of [1] to [5], wherein a reagent that performs the Cas enzyme reaction is a lyophilized reagent.

13. The nucleic acid detecting system according to [12], wherein the lyophilized reagent is a lyophilized reagent ball or is adsorbed on a scaffold material.

14. The nucleic acid detection system according to any one of [1] to [5], wherein the amplification is one selected from nucleic acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, and nickase amplification.

15. The nucleic acid detection system according to any one of [1] to [5], wherein the 4 th chamber comprises a sample pad, a Cas enzyme reaction region, a gold-labeled anti-FAM antibody region, a streptavidin region, and an antibody capture region in this order along a liquid flow path.

16. The nucleic acid detection system of [15], wherein the 4 th chamber further comprises a Cas enzyme reaction termination region comprising a Cas enzyme antibody or inhibitor after the Cas enzyme reaction region and before the gold-labeled anti-FAM antibody region.

17. The nucleic acid detection system according to [15] or [16], wherein the 4 th chamber further comprises an anhydrous copper sulfate strip after the antibody capture zone.

18. A nucleic acid detecting system for simultaneously detecting a plurality of target nucleic acids in a sample, comprising the nucleic acid detecting system according to any one of [1] to [17], wherein the 4 th chamber contains 2 or more test strips.

19. A method for detecting a target nucleic acid in a sample using the nucleic acid detection system according to any one of [1] to [17], the method comprising:

sampling, inserting the swab into the swab inlet of the nucleic acid detection system;

and

observing a color change of the test strip in the 4 th chamber.

20. The method of [19], wherein the sample is urine, blood, serum, cerebrospinal fluid or saliva.

21. A method for genotyping a target nucleic acid in a sample using the nucleic acid detection system of [18], the Cas complexes contained in the 2 or more test strips being designed for different target sequences, respectively.

Effects of the invention

The present invention can provide a nucleic acid detection system that is small in size and has high detection sensitivity.

Drawings

FIG. 1 is a schematic diagram showing a comparison between the present invention and a nucleic acid detection technique of the prior art.

Fig. 2 is a schematic diagram showing the detection flow of CRISPR-based IVD detection techniques.

FIG. 3 is a schematic view showing a nucleic acid detecting system according to a first embodiment of the present invention.

FIG. 4 is a schematic diagram showing a specific configuration of each chamber in the disposable detecting section of the nucleic acid detecting system of the present invention.

FIG. 5 is a schematic diagram showing a sampling step performed by the nucleic acid detecting system of the present invention.

FIG. 6 is a schematic diagram showing the structure of the 1 st chamber of the nucleic acid detecting system of the present invention.

FIG. 7 is a schematic diagram showing the structure of a wax valve in the nucleic acid detecting system of the present invention.

FIG. 8 is a schematic diagram showing a specific configuration of the 4 th chamber of the nucleic acid detecting system of the present invention. From left to right are: sample pad, Cas enzyme reaction zone, Cas enzyme reaction termination zone (Cas enzyme antibody or inhibitor), gold-labeled anti-FAM antibody zone, streptavidin zone (control band 1), antibody capture zone (test band), anhydrous copper sulfate band (control band 2), absorbent pad.

FIG. 9 is a diagram showing reactions performed in respective regions when a positive sample and a negative sample pass through the nucleic acid detecting system of the present invention.

FIG. 10A is a schematic view showing a color development judged to be effective when a positive sample and a negative sample pass through the nucleic acid detecting system of the present invention, and FIG. 10B is a schematic view showing various color developments judged to be ineffective.

FIG. 11 is a schematic view showing a nucleic acid detecting system according to a second embodiment of the present invention.

FIG. 12 is a photograph showing the results of detection in example 2.

Description of the symbols

10 nucleic acid detection system

100 disposable test part

200 temperature control unit

1 st Chamber 1

2 nd chamber

3 rd chamber

4 th Chamber 4

5 swab entrance

6 extrusion channel

7 extruding funnel

8 extruding protrusion

9 Filter

W wax valve

R freeze-drying reagent ball

P stent material

S-absorbing material

H heating block

11 sample pad

12 Cas enzyme reaction zone

13 Cas enzyme reaction termination region

14 gold-labeled anti-FAM antibody region

15 streptavidin region

16 antibody capture zone

17 anhydrous copper sulfate strip

18 absorbent pad

Detailed Description

Embodiments of the present invention will be described in detail with reference to the drawings, but the present invention is not limited to these embodiments, and includes all embodiments satisfying the features described in the claims of the present application.

First embodiment

The first embodiment of the present invention relates to a detection system for detecting a target nucleic acid in a sample, comprising 1 st to 4 th chambers forming a liquid flow path, a liquid flowing from the 1 st chamber to the 4 th chamber, wherein,

the 3 rd chamber contains reagents for amplifying the target nucleic acid, and

FIG. 3 is a schematic view showing a nucleic acid detecting system according to a first embodiment of the present invention. As shown in FIG. 3, the nucleic acid detecting system 10 can be divided into a disposable detecting part 100 and a temperature control unit 200, wherein in the disposable detecting part 100, the 1 st chamber 1 to the 4 th chamber 4 are connected in order from the upper side to the lower side in the figure to form a continuous liquid flow path. When the disposable sensing portion is, for example, a paper-based material, liquid may flow from the 1 st chamber to the 4 th chamber by a siphon effect or capillary phenomenon.

For the above-mentioned paper-based material, there is no particular limitation, and various materials commonly used in the art for making disposable test strips can be used.

The disposable detecting part 100 may be in a cylindrical or rectangular parallelepiped shape, for example, a ball-point pen core shape, and has a length of, for example, 5 to 15cm, an inner diameter of, for example, 1 to 5mm, and any of the above-mentioned ranges of the length and the inner diameter, for example, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 11cm, 12cm, 13cm, 14cm, 15cm, and the like, and an inner diameter of, for example, 1mm, 2mm, 3mm, 4mm, 5mm, and the like, without particular limitation.

The temperature control unit 200 may be non-disposable, for example, may be made disposable integral with the disposable sensing portion 100, but is preferably non-disposable from a cost standpoint. The temperature control unit 200 may be a nested hollow shell or a semi-open groove shape that fits the disposable detection portion 100, for example, a ball-point pen-like hollow shell. The temperature control unit 200 may perform temperature-set heating of the corresponding chamber and wax valve of the disposable sensing part 100 through the heating block H. In use, after the swab after sampling is inserted into the disposable detection portion 100, the disposable detection portion 100 is inserted or embedded in the temperature control unit 200 in its entirety or in part, and then the temperature control unit 200 is started to perform a set temperature control program. However, the temperature control means is not essential, and the nucleic acid detecting system of the present invention may not include the temperature control means when the reaction is a reaction at normal temperature.

< Chamber 1 >

FIG. 4 is a schematic diagram showing a specific configuration of each chamber in the disposable detecting section of the nucleic acid detecting system shown in FIG. 3. As shown in fig. 4, the 1 st chamber 1 contains a swab inlet 5 for inserting a swab to which a sample is attached.

FIG. 5 is a schematic diagram showing a sampling step performed by the nucleic acid detecting system of the present invention. As shown in fig. 5, after sampling the swab, the swab is inserted into a swab inlet 5 of the nucleic acid detection system, the swab is pressed against the system wall or the channel 6 is squeezed to allow a liquid sample containing the target nucleic acid to enter the liquid flow path, and then the swab is broken, and the disposable detection portion 100 is inserted into the temperature control unit 200, so that the liquid sample entering the liquid flow path can flow from the 1 st chamber 1 to the 4 th chamber 4 by a siphon effect or a capillary phenomenon.

FIG. 6 is a schematic diagram showing the structure of the 1 st chamber of the nucleic acid detecting system of the present invention. The structure of the 1 st chamber 1 is not particularly limited as long as the swab can be inserted therein, and may be, for example, a configuration of the pressing funnel 7, specifically, a funnel shape having the pressing protrusion 8 on the inner wall as shown in fig. 6. Between the 1 st chamber 1 and the 2 nd chamber 2 there is preferably also a filter 9, which allows a primary filtration treatment of the sample to purify it.

< Chamber 2 >

As shown in fig. 4, the 2 nd chamber 2 contains a reagent for heat-inactivating and lysing the sample, and the liquid sample is activated by hydration after passing through the filter 9 into the 2 nd chamber, and is heat-inactivated and lysed by the reagent therein. The agent for heat inactivation and lysis is, for example, an enzyme-containing buffer or the like for inactivation and lysis.

Examples of the enzyme include protease K, RNase nuclease and/or digestive peptidase. These enzymes degrade membrane proteins, proteins binding to DNA and RNA, and the like, and dissolve viral capsids, thereby releasing nucleic acids such as DNA and RNA into a sample. The enzyme may be reacted at room temperature, but may be reacted at a high temperature by selecting a type of enzyme resistant to high temperature. At this time, the target nucleic acid in the sample can be further inactivated by heating the chamber.

In addition to the above-mentioned enzymes, the buffer may contain conventional components which may be contained in the preparation of buffers in the art, for example, EDTA, Tris-HCl, etc., and is not particularly limited.

The reagent contained in the 2 nd chamber 2 of the nucleic acid detecting system of the present invention is preferably a freeze-dried reagent (hereinafter, the reagents are similar) which is a freeze-dried reagent ball R or is adsorbed on the support material P.

FIG. 7 is a schematic diagram showing the structure of a wax valve in the nucleic acid detecting system of the present invention. Between the 2 nd chamber 2 and the 3 rd chamber 3 there may be a thermally unstable valve, e.g. a wax valve W, as shown in fig. 7, separating the chambers. The wax valve may be formed of a separate wax layer or may be attached to the support material P, and an absorbent material S may be disposed around the wax valve W to be absorbed by the absorbent material S when the wax valve is melted. The wax valve W (and the wax valve W between the 3 rd chamber and the 4 th chamber described below) is opened when heated, thereby enabling the liquid sample to flow from the 2 nd chamber to the 3 rd chamber.

< chamber 3>

As shown in FIG. 4, the 3 rd chamber 3 contains reagents for amplifying a target nucleic acid, and a liquid sample is amplified in the chamber, for example, isothermally. The amplification may be any one selected from nucleic acid sequence based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nickase amplification. In order to smoothly perform amplification, the temperature of the 3 rd chamber 3 may be controlled by the temperature control unit 200, and the wax valve W may be heated by the temperature control unit 200. Thereby controlling the flow of the liquid sample between the chambers.

Reagents and methods for amplification can be performed by referring to the descriptions in the prior art, for example, those described in https:// www.neb.com/protocols/2014/06/17/loop-mediated-isothermal-amplification-lamp or https:// patents.

< Chamber 4 >

When the wax valve W between the 3 rd chamber 3 and the 4 th chamber 4 is heated and melted to be opened, the liquid sample flows into the 4 th chamber 4.

As shown in fig. 4, the 4 th chamber 4 contains a test strip for Cas enzyme reaction and lateral flow detection with a target nucleic acid in a sample.

The reagents for performing the Cas enzyme reaction comprise a Cas enzyme complex, in particular, a Cas enzyme complex may comprise a Cas enzyme, a guide nucleic acid which may comprise a guide sequence capable of binding to a target nucleic acid in a sample and capable of forming a complex with the Cas enzyme, and a probe which may be a DNA or RNA based molecule comprising a non-target nucleic acid sequence which when cleaved by the Cas enzyme is capable of producing the molecule required for a subsequent reaction.

The Cas enzyme is, for example, Cas12, Cas13, or Cas 14. They may or may not have target nucleic acid cleavage activity, for example at least one selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b and Cas14c, for example at least one selected from dCas12a, dCas13a, dCas13b, dCas14a, dCas14b and dCas14 c. For example, for a nucleic acid detection system of the invention, where it is only necessary to detect the presence or absence of a target nucleic acid, the Cas enzyme may not be required to be active, but may also be active.

FIG. 8 is a schematic diagram showing a specific configuration of the 4 th chamber of the nucleic acid detecting system of the present invention. From left to right are: sample pad 11, Cas enzyme reaction region 12(Cas enzyme complex), Cas enzyme reaction termination region 13(Cas enzyme antibody or inhibitor), gold-labeled anti-FAM antibody region 14, streptavidin region 15 (control band 1), antibody capture region 16 (test band), anhydrous copper sulfate band 17 (control band 2), absorbent pad 18.

FIG. 9 is a diagram showing reactions in the respective regions when a positive sample and a negative sample pass through the nucleic acid detecting system of the present invention.

The detection and color development of the positive and negative samples in the 4 th chamber 4 will be described with reference to FIGS. 8 and 9:

1. after entering the sample pad 11, the liquid sample (hereinafter also referred to simply as "liquid") will first pass through the Cas enzyme reaction zone where the positive sample activates the Cas enzyme complex, cleaving the probes (reporter sequence) and thereby producing separated FAM and biotin; while negative samples did not cleave the probe.

2. After the product after the Cas enzyme reaction passes through the Cas enzyme reaction termination region, the Cas enzyme reaction is terminated and the Cas enzyme is captured and does not continue to flow forward. Thereby, the reaction efficiency and time for detection can be ensured.

3. The liquid continues to flow forward and the cleaved and uncleaved reporter sequence binds to the anti-FITC, FAM antibodies in the gold labeled FAM antibody region.

4. When the liquid continues to reach the streptavidin area, biotin is captured, gold-labeled particle aggregation is formed, and color development is performed (control band 1); in this region, the reporter sequence will be visualized, whether cleaved or not.

5. When the liquid continues to reach the antibody capture area, the cut FAM end + FITC, FAM antibody + gold-labeled particles of the report sequence are combined with the antibody capture area to form gold-labeled particle aggregation, so that the color development (namely, double-line color development) is realized. If the sample is negative, the reporter sequence is not cleaved and all reporter sequences have been bound by the streptavidin region, the antibody capture region will not be colored.

6. The liquid continuously flows forward through the anhydrous copper sulfate strip to reach the absorption pad, no matter whether the sample has a target sequence or not, the color of the aqueous solution is changed into blue when the aqueous solution flows through the anhydrous copper sulfate strip, the liquid volume is indicated to be sufficient when the color of the aqueous solution is changed into blue, the test strip flows completely, and the detection is judged to be invalid when the anhydrous copper sulfate strip (the control strip 2) does not develop the color. Therefore, the quality control of the detection effectiveness of the whole test strip can be realized.

As is clear from the above description, the present detection result can be judged to be valid only when the control band 1 and the control band 2 are simultaneously developed. According to the invention, the design that the anhydrous copper sulfate strip (the control strip 2) is arranged outside the control strip 1 is adopted, so that the problem that false negative or invalid detection possibly occurs due to insufficient sample volume quantity when the control strip of the test paper strip is arranged in front of the test strip in the prior art and the test strip is arranged behind the test strip is solved. Thus, the accuracy of nucleic acid detection can be improved by the present invention.

Second embodiment

A second embodiment of the present invention relates to a detection system for simultaneously detecting a plurality of target nucleic acids in a sample, comprising the nucleic acid detection system of the first embodiment, wherein the 4 th chamber comprises 2 or more test strips.

Similarly to the principle and operation in the nucleic acid detecting system of the first embodiment, the liquid flows from the 1 st chamber 1 to the 4 th chamber 4. In this embodiment, however, the 4 th chamber bifurcates into two or more channels to detect multiple target nucleic acids in parallel, and the editable Cas complex on each channel can be different to detect different target nucleic acids.

FIG. 11 is a schematic view showing a nucleic acid detecting system according to a second embodiment of the present invention. The nucleic acid detection system is provided with 2 4 th chambers (lateral flow test strips), so that the simultaneous detection of two target nucleic acids can be realized.

The detection system according to the second embodiment of the present invention can also be used for genotyping, such as HPV genotyping or MTB/RIF, and can be adapted for simultaneous detection of DNA and RNA, for example, when Cas complexes contained in more than 2 test strips are designed for different target sequences, respectively, target nucleic acids in a sample can be genotyped.

Thus, the present invention also relates to a method of genotyping a target nucleic acid in a sample using the nucleic acid detection system of the second embodiment, wherein the Cas complexes contained in the 2 or more test strips are designed for different target sequences, respectively.

Furthermore, the present invention relates to a method for measuring a target nucleic acid in a sample using the detection system of the first or second embodiment described above, the method comprising: sampling, inserting the swab into the swab inlet of the detection system; turning on the temperature control unit; and observing a color change of the test strip in the 4 th chamber. Wherein, the sample can be urine, blood, serum, cerebrospinal fluid or saliva, etc.

Examples

The nucleic acid detecting system and the nucleic acid detecting method of the present invention are specifically exemplified below by taking a novel coronavirus (hereinafter also referred to as "COVID 19") as an example, but the present invention is not limited to these examples.

Example 1: production of nucleic acid detecting System

According to the genome sequence of the novel coronavirus, an LAMP primer for LAMP isothermal amplification and a crRNA sequence for detection by using a Cas12 enzyme are designed aiming at the N gene.

A plasmid containing an N gene fragment with a template of COVID19, wherein the N gene is as follows (SEQ ID NO: 1):

CCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAA

the LAMP primer sequences used in this example are shown in Table 1 below.

TABLE 1

The designed crRNA sequence (SEQ ID NO: 8) was as follows:

UAAUUUCUACUAAGUGUAGAUUUGAACUGUUGCGACUACGU Probe sequence: FITC-T12-Biotin

The above materials are all synthesized and purchased from Nanjing Kingsry.

LAMP amplification method is referable

Instructions for the LAMP kit.

Test strips for COVID19 were prepared as follows:

1. glass fiber filter paper (Whatman, 1827-021) was cut to the desired size.

2. Autoclaved for 90 minutes (autoclave, MKII).

3. Blocking was performed in 5% nuclease-free BSA (EM Millipore, 126609-10GM) for 12 hours.

4. Washing was performed 3 times with nuclease-free water (Life Technologies, AM 9932).

5. 4% RNAscope (Life Technologies, AM7006) was added and left at 60 ℃ for 20 minutes, followed by 3 washes with nuclease-free water.

6. The treated paper was dried in an oven (DHG-9140A, Shanghai-Heng) at 80 ℃ for 15 minutes.

7. According to design, the components of each chamber are disposed on the above treated test strips, wherein:

a) and a 2 nd chamber:

TABLE 2

Reagent	Volume (microliter)
		50mM TCEP (tris (2-carboxyethyl) phosphine, ab142040, Abcam)	1
2mM EDTA(T9191,TaKaRa)	1
		RNase inhibitor (M0314S, NEB)	1
10mM Tris-HCl (pH 8.0)	10
		500mM trehalose (TS1M-100, Life Sciences Advanced Technologies)	1
Digestive peptidase (ACP, A3547, Sigma Aldrich)	5
		H₂O	1
Total volume	20

b) And a3 rd chamber: the amplification mixtures are shown in Table 3.

TABLE 3

Reagent	Volume (microliter)
		WarmStart LAMP 2X Master Mix(E1700S，NEB)	12.5
Primer Mix (LAMP Primer sequence shown in Table 1)	2.5
		H₂O	10
Total volume	25

c) The wax valve used was palm wax (C804522, MACKLIN).

d) And a 4 th chamber:

the reagents and other components contained in the Cas enzyme reaction zone are shown in tables 4 and 5.

TABLE 4

Reagent	Final concentration	Volume (microliter)
			LbaCas12a(M0653S，NEB)	1μM	1
crRNA (SEQ ID NO. 8)	1μM	1
			Probe needle	10μM	1
H₂O		17
			Total volume		20

TABLE 5

Other Components	Volume (microliter)
		Cas12 inhibitor, AcrVA (Kinrei)	1
Streptavidin (N7021S, NEB)	1
		Gold-labeled anti-FAM antibody (ab19491, abcam)	1
Protein A (ab84187, abcam)	1
		Copper sulfate (C805358, MACKLIN)	1

8. Liquid nitrogen chilling and freeze-drying were performed overnight.

9. The finished test strip is assembled into a sleeve in a dry environment.

Example 2: detection of COVID19 Using the nucleic acid detection System of example 1

The COVID19 from example 1 was combined with a temperature control unit using a test strip, and 50. mu.L of a positive plasmid solution (positive sample) and a solution without COVID19 (negative sample) were added dropwise to chamber 1. The temperature control unit works according to a preset program, and the sample enters a No. 2 chamber from a No. 1 chamber; since this embodiment is a simulated sample, the sample continues into chamber 3 without lysis and thermal inactivation in chamber 2. The temperature of the 3 rd chamber is controlled at 65 ℃, LAMP amplification is carried out in the chamber, and after 10 minutes, the wax valve is opened to enter the 4 th chamber. Controlling the temperature of the 4 th chamber at 37 ℃, firstly enabling the liquid to enter a Cas enzyme reaction zone, hydrating a freeze-dried reagent in a relevant zone, recognizing a target by the pre-designed Cas enzyme, and cutting the probe, wherein the reaction time is 5 minutes; the cut probe continues to move towards the water absorption pad of the test strip, according to the designed principle, the control band 1 is firstly changed into red, then the test band is changed into red, finally the anhydrous copper sulfate band (control band 2) is changed into blue, the three bands of the positive sample are all colored, and the test band of the negative sample is not colored. FIG. 12 is a photograph showing the test strip (4 th chamber) showing the above-mentioned detection results;

it can be seen that the present invention can provide a highly integrated, automated and portable nucleic acid detecting system which does not require expensive equipment, does not require a professional technician, does not require a special laboratory, can be stored and transported at room temperature and can ensure sequence specificity of detection results, is free from contamination to the inside and outside, and can realize "sample entry, result exit" and can be applied to POCT. In addition, the nucleic acid detection system of the present invention can simultaneously detect a plurality of target nucleic acids by including a plurality of 4 th chambers, and thus can be applied to genotyping, simultaneous detection of DNA and RNA, and the like.

Sequence listing

<110> Canon medical systems corporation

<120> nucleic acid detection system and use thereof

<130> PTMA-20079-CN(98G81000985-CN-A)

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1000

<212> DNA

<213> COVID-19

<400> 1

ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa atgaaagatc tcagtccaag 60

atggtatttc tactacctag gaactgggcc agaagctgga cttccctatg gtgctaacaa 120

agacggcatc atatgggttg caactgaggg agccttgaat acaccaaaag atcacattgg 180

cacccgcaat cctgctaaca atgctgcaat cgtgctacaa cttcctcaag gaacaacatt 240

gccaaaaggc ttctacgcag aagggagcag aggcggcagt caagcctctt ctcgttcctc 300

atcacgtagt cgcaacagtt caagaaattc aactccaggc agcagtaggg gaacttctcc 360

tgctagaatg gctggcaatg gcggtgatgc tgctcttgct ttgctgctgc ttgacagatt 420

gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa caacaaggcc aaactgtcac 480

taagaaatct gctgctgagg cttctaagaa gcctcggcaa aaacgtactg ccactaaagc 540

atacaatgta acacaagctt tcggcagacg tggtccagaa caaacccaag gaaattttgg 600

ggaccaggaa ctaatcagac aaggaactga ttacaaacat tggccgcaaa ttgcacaatt 660

tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt ggcatggaag tcacaccttc 720

gggaacgtgg ttgacctaca caggtgccat caaattggat gacaaagatc caaatttcaa 780

agatcaagtc attttgctga ataagcatat tgacgcatac aaaacattcc caccaacaga 840

gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa gccttaccgc agagacagaa 900

gaaacagcaa actgtgactc ttcttcctgc tgcagatttg gatgatttct ccaaacaatt 960

gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1000

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

gctgcaatcg tgctacaact 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

tctgtcaagc agcagcaaag 20

<210> 4

<211> 42

<212> DNA

<213> Artificial sequence

<400> 4

tgcgactacg tgatgaggaa cgttgccaaa aggcttctac gc 42

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<400> 5

ttcaactcca ggcagcagta ggcaagagca gcatcaccgc 40

<210> 6

<211> 17

<212> DNA

<213> Artificial sequence

<400> 6

ttgactgccg cctctgc 17

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

<400> 7

ggaacttctc ctgctagaat ggc 23

<210> 8

<211> 41

<212> RNA

<213> Artificial sequence

<400> 8

uaauuucuac uaaguguaga uuugaacugu ugcgacuacg u 41

Claims

the 3 rd chamber contains reagents for amplifying the target nucleic acid, and

2. The nucleic acid detection system according to claim 1, wherein a filter for purifying a sample is provided between the 1 st chamber and the 2 nd chamber.

3. The nucleic acid detection system of claim 1, wherein the 1 st chamber further comprises an extrusion channel.

4. The nucleic acid detecting system according to claim 1, wherein a wax valve for separating chambers is provided between the 2 nd chamber and the 3 rd chamber.

5. The nucleic acid detecting system according to claim 1, wherein a wax valve for separating chambers is provided between the 3 rd chamber and the 4 th chamber.

6. A nucleic acid detection system according to claim 4 or 5 wherein the wax valve is comprised of a separate wax layer or adsorbed on a scaffold material.

7. The nucleic acid detecting system according to any one of claims 1 to 5, which has a temperature control unit that controls the temperature of the 3 rd chamber.

8. The nucleic acid detecting system according to claim 4 or 5, wherein the wax valve is opened by heating of a temperature control unit.

9. The nucleic acid detecting system according to any one of claims 1 to 5, wherein the reagents in the 2 nd chamber and the 3 rd chamber are lyophilized reagents.

10. The nucleic acid detecting system according to claim 9, wherein the lyophilized reagent is a lyophilized reagent ball or is adsorbed on a scaffold material.

11. The nucleic acid detection system of any one of claims 1-5, wherein the reagent for the Cas enzyme reaction comprises a Cas enzyme complex,

12. The nucleic acid detection system of any one of claims 1-5, wherein the reagent that performs the Cas enzyme reaction is a lyophilized reagent.

13. The nucleic acid detecting system according to claim 12, wherein the lyophilized reagent is a lyophilized reagent ball or is adsorbed on a scaffold material.

14. The nucleic acid detection system according to any one of claims 1 to 5, wherein the amplification is one selected from nucleic acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, or nickase amplification.

15. The nucleic acid detection system of any one of claims 1 to 5, wherein the 4 th chamber comprises a sample pad, a Cas enzyme reaction region, a gold-labeled anti-FAM antibody region, a streptavidin region, and an antibody capture region in that order along a liquid flow path.

16. A nucleic acid detection system according to claim 15, wherein the 4 th chamber further comprises a Cas enzyme reaction termination region comprising a Cas enzyme antibody or inhibitor after the Cas enzyme reaction region and before the gold-labeled anti-FAM antibody region.

17. The nucleic acid detection system of claim 15 or 16, wherein the 4 th chamber further comprises a strip of anhydrous copper sulfate after the antibody capture zone.

18. A nucleic acid detection system for simultaneously detecting a plurality of target nucleic acids in a sample, comprising the nucleic acid detection system of any one of claims 1-17, wherein the 4 th chamber comprises 2 or more test strips.

19. A method for detecting a target nucleic acid in a sample using the nucleic acid detection system according to any one of claims 1 to 17, the method comprising:

sampling, inserting the swab into the swab inlet of the nucleic acid detection system; and

observing a color change of the test strip in the 4 th chamber.

20. The method of claim 19, wherein the sample is urine, blood, serum, cerebrospinal fluid, or saliva.

21. A method of genotyping a target nucleic acid in a sample using the detection system of claim 18, the Cas complexes contained in the 2 or more test strips being designed for different target sequences, respectively.