Method, kit and application for detecting RNA based on non-nucleic acid amplification of Cas13

Abstract

The present invention provides a method, a kit and an application of non-nucleic acid amplification detection RNA based on Cas13, and relates to the field of biotechnology. The reaction system of the method includes Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, a fuel chain, an RNA reporter molecule and a metal ion. When the target RNA is present, the Cas13 protein cuts the stem-loop DNA to release the stem-loop single-stranded B that can displace DS1-b in DS1, and DS1-b further displaces the DNAzyme that can cut RNA reporter molecules in DS2, while the fuel chain displaces the stem-loop single-stranded B in the intermediate ID so that it continues to participate in the reaction, achieving the purpose of amplifying the signal. The detection method has the advantages of fast detection, simple detection conditions, and no need for complex precision equipment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method, a kit and an application for detecting RNA based on non-nucleic acid amplification of Cas13. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] At present, the detection of RNA mainly uses reverse transcription real-time fluorescence quantitative PCR. RNA is converted into cDNA under the action of reverse transcriptase, and then cDNA is detected by real-time fluorescence quantitative PCR. It requires repeated denaturation and annealing processes, which are cumbersome and time-consuming. There are also some RNA detection methods based on isothermal amplification, such as EXPAR, NASBA, etc.; but the signal amplification strategy system based on nucleic acid amplification is complex and time-consuming, and the nucleic acid amplification products produced are prone to aerosol pollution.

[0004] Existing DNA cascade amplification based on strand displacement reaction for RNA detection can only detect short-sequence RNA due to the limitations of the strand displacement reaction itself. Moreover, most of the detection signal output of DNA cascade amplification based on strand displacement reaction is achieved by adding fluorescent groups or other modifications to a stem-loop structure or double-stranded DNA component in the strand displacement reaction, which is complex to prepare and difficult to preserve, and is prone to false positive results.

[0005] However, regardless of the method used and despite the technical improvements made, the current methods for RNA detection usually have many disadvantages in their application, especially: (1) reverse transcription plus real-time fluorescence quantitative PCR requires two-tube operation, which can easily cause RNA degradation; (2) a dedicated and precise temperature control instrument is required; (3) it is time-consuming and not conducive to rapid detection; (4) the amplified nucleic acid is prone to aerosol contamination, which is difficult to eliminate; (5) DNA cascade amplification based on chain displacement reaction can only detect short-sequence RNA; (6) The signal amplification strategy based on chain displacement reaction uses a stem-loop or double-stranded DNA component in the reaction to label a fluorescent group or other signal-generating substances, which is complex to produce and difficult to preserve.

[0006] The above shortcomings are caused by the following reasons: (1) PCR-based amplification methods cannot directly amplify RNA, but require reverse transcription and a PCR amplification reaction with repeated heating and cooling. (2) Amplification methods based on constant temperature amplification are time-consuming and prone to aerosol contamination of nucleic acid amplification products. (3) Strand displacement reactions can only detect short-sequence RNA. (4) The output form of the signal of the DNA cascade amplification signal reaction is often complex and difficult to operate. Therefore, how to improve the RNA detection method is an urgent problem to be solved.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a method for detecting RNA by non-nucleic acid amplification based on Cas13 to improve the defects of RNA detection in the prior art. Based on this method, the object of the present invention is also to provide a kit for RNA detection and their applications.

[0009] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0010] In the first aspect, a method for detecting RNA by non-nucleic acid amplification based on Cas13 is provided, wherein the reaction system of the method comprises Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, a fuel chain, an RNA reporter molecule and a metal ion;

[0011] The crRNA targets a target RNA;

[0012] The stem-loop DNA is composed of folded single-stranded DNA, and at least two uracil ribonucleotides are present near the stem structure of the ring structure on one side of the stem-loop DNA; the uracil ribonucleotides separate the stem-loop DNA into two parts, a stem-loop single strand A and a stem-loop single strand B;

[0013] DS1 is a double-stranded DNA, which is composed of complementary DS1-t and DS1-b through complementary pairing. The DS1 has a single-stranded DNA segment DS1A composed of DS1-t, and the stem-loop single-stranded B is at least partially complementary to the single-stranded DNA segment DS1A.

[0014] The fuel chain is a single-stranded DNA, and the fuel chain is partially complementary to the DS1-t; the partially complementary pairing position is not located at the single-stranded DNA segment DS1A, and the DNA at the partially complementary pairing position does not bind to the stem-loop single-stranded B;

[0015] DS2 is a double-stranded DNA, which is composed of complementary DNAzyme and blocker through complementary pairing. The DS2 has a single-stranded DNA segment DS2A composed of blocker, and the DS1-b is at least partially complementary to the single-stranded DNA segment DS2A.

[0016] The RNA reporter molecule is a single-stranded RNA modified with a luminescent group and a quenching group;

[0017] The binding arm of the DNAzyme is complementary to the RNA reporter molecule;

[0018] The detection method comprises: when the target RNA is present, the Cas13 protein binds to the target RNA under the guidance of crRNA, cuts the stem-loop DNA in the reaction system so that the stem-loop DNA is separated into two parts: a stem-loop single strand A and a stem-loop single strand B; the stem-loop single strand B undergoes a strand displacement reaction with the DS1 to obtain DS1-b in a single-stranded DNA state and an intermediate ID formed by the combination of the stem-loop single strand B and DS1-t;

[0019] The fuel chain undergoes a chain displacement reaction with the intermediate ID to obtain a stem-loop single-stranded DNA state B, and the obtained stem-loop single-stranded DNA state B continuously undergoes a chain displacement reaction with DS1 in the presence of the fuel chain;

[0020] The DS1-b in the single-stranded DNA state undergoes a strand displacement reaction with the DS2 to obtain a DNAzyme in the single-stranded DNA state. The DNAzyme cuts the RNA reporter molecule in the presence of metal ions to release a fluorescent signal in the reaction system.

[0021] In a second aspect, a kit for RNA detection is provided, the kit comprising reagents for constituting the reaction system described in the first aspect.

[0022] In the third aspect, a method for detecting RNA by non-nucleic acid amplification based on Cas13 described in the first aspect, or the use of the kit described in the second aspect in detecting pathogenic microorganisms for non-diagnostic and therapeutic purposes, or in preparing products for detecting pathogenic microorganisms is provided.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Cas13-based non-nucleic acid amplification method for detecting RNA provided by the present invention utilizes the Cas13 protein to recognize the detection RNA, triggers a cascade amplification reaction of the DNA loop to amplify the detection signal, and DNAzyme outputs the signal.

[0025] This method does not require nucleic acid pre-amplification of the RNA target to be detected, and can directly detect RNA. The stem-loop structure is used to release the target that triggers the DNA cascade amplification signal. The strand displacement reaction between DNA chains is used to form a signal cycle amplification system for DNA cascade amplification, thereby releasing a large amount of DNAzyme. The RNA substrate is fluorescently labeled by using the cutting characteristics of DNAzyme on the RNA substrate in the presence of metal ions, and the results are output.

[0026] The detection method provided by the present invention can realize the reaction in a single container and keep the temperature constant throughout the process. The reaction can be carried out at a temperature from room temperature to 42 degrees, but preferably at about 37 degrees. In the preferred embodiment, a significant difference between a positive result and a negative result can be observed within about 10 minutes.

[0027] The method for detecting RNA provided by the present invention or a kit based on the method can be applied to detecting pathogenic microorganisms, or preparing products for detecting pathogenic microorganisms, and has the advantages of rapid detection, simple detection conditions, and no need for complex and precise equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 Schematic diagram of the principle of the detection method of Example 1;

[0030] Figure 2 The fluorescence intensity of samples with different concentrations is detected by the detection method of Example 1;

[0031] Figure 3 It is a line graph of sample molar concentration and fluorescence value of the detection method of Example 1;

[0032] Figure 4 The specificity test results of the detection method in Example 1;

[0033] Figure 5 The fluorescence intensity of samples with different concentrations is detected by the detection method of Example 2;

[0034] Figure 6 It is a line graph of sample molar concentration and fluorescence value of the detection method of Example 2;

[0035] Figure 7 The specificity test results of the detection method in Example 2 are as follows;

[0036] Figure 8 is the fluorescence intensity of the reaction system containing different concentrations of DS1 in Example 3;

[0037] Fig. 9 is the fluorescence intensity of the reaction system containing different concentrations of DS2 in Example 3;

[0038] Fig.10 is the fluorescence intensity of the reaction system containing fuel chains of different concentrations in Example 3;

[0039] Fig.11 The fluorescence intensity variation curves of the reaction systems containing fuel chains and not containing fuel chains in Example 3 over time;

[0040] Fig.12is the fluorescence intensity of the reaction system containing different stem-loop DNA in Example 3. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] In the first aspect, a method for detecting RNA by non-nucleic acid amplification based on Cas13 is provided, wherein the reaction system of the method includes Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, a fuel chain, an RNA reporter molecule and metal ions.

[0043] The crRNA targets the target RNA, and the crRNA can recognize a specific RNA nucleic acid sequence and activate the Cas13 protein. Those skilled in the art can design the sequence of the crRNA according to the target RNA to be detected, and the present invention is not limited to this.

[0044] After being activated, the Cas13 protein can cut the uracil base in the stem-loop DNA to dissociate it and release the stem-loop single-stranded B.

[0045] In an optional embodiment, the Cas13 protein includes a Cas13a protein or a Cas13b protein. Both the Cas13a protein and the Cas13b protein have the property of recognizing and detecting RNA, and preferentially cutting the phosphodiester bond between uracil bases (U) and uracil bases (U) after activation.

[0046] In an optional embodiment, the Cas13 protein is a Cas13a protein.

[0047] The stem-loop DNA is composed of folded single-stranded DNA, and at least two uracil ribonucleotides (U) are present near the stem structure of the ring structure on one side of the stem-loop DNA; the two uracil ribonucleotides separate the stem-loop DNA into two parts, a stem-loop single strand A and a stem-loop single strand B.

[0048] In an optional embodiment, the side of the two uracil ribonucleotides away from the stem structure is the stem-loop single strand B. Since the uracil ribonucleotide is located at the position of the loop structure close to the stem structure, this modification site divides the stem-loop DNA into a side with most of the loop structure and a side that is mainly the stem structure (see Figure 1 ), in this embodiment, the portion on one side having most of the loop structure is the stem-loop single strand B.

[0049] In an optional embodiment, the number of the uracil ribonucleotides is 2 to 7, for example, but not limited to 2, 3, 4, 5, 6 or 7.

[0050] In an optional embodiment, the length of the stem structure in the stem-loop DNA is 10 to 20 bp, for example, but not limited to, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bp, preferably 15 bp.

[0051] DS1 is a double-stranded DNA, which is used to bind to the stem-loop single-stranded B to release DS1-b and make it single-stranded. DS1 is composed of complementary DS1-t (single-stranded DNA) and DS1-b (single-stranded DNA) through complementary pairing. DS1-t has a part that is not complementary to DS1-b at one end, so that the DS1 has a single-stranded DNA segment DS1A composed of DS1-t, and the stem-loop single-stranded B is at least partially complementary to the single-stranded DNA segment DS1A for a strand displacement reaction.

[0052] In an optional embodiment, the length of DS1A is at least 2 bp, optionally 6 to 10 bp, for example, but not limited to 6, 7, 8, 9 or 10 bp.

[0053] The fuel strand is a single-stranded DNA, which is used to bind to the intermediate ID (double-stranded DNA after the stem-loop single-stranded B and DS1-t are combined) and replace the stem-loop single-stranded B, so that the stem-loop single-stranded B enters the reaction system again, so that it continues to enter the cycle. The fuel strand is partially complementary to the DS1-t, and the partially complementary pairing position is not located in the single-stranded DNA segment DS1A to avoid the fuel chain and DS1 from undergoing a chain displacement reaction in the reaction system. And the DNA at the partially complementary pairing position does not bind to the stem-loop single-stranded B to avoid the formation of the intermediate ID, and the stem-loop single-stranded B binds to DS1-t, resulting in the occupation of the position where the fuel chain binds to DS1-t during a chain displacement reaction.

[0054] DS2 is a double-stranded DNA, which is used to bind to the single-stranded DS1-b to release DNAzyme. The DS2 is composed of complementary DNAzyme (single-stranded DNA) and blocker (single-stranded DNA) through complementary pairing. The blocker has a part at one end that is not complementary to the DNAzyme, so that the DS2 has a single-stranded DNA segment DS2A composed of the blocker. The DS1-b is at least partially complementary to the single-stranded DNA segment DS2A for a strand displacement reaction.

[0055] In an optional embodiment, the length of DS2A is at least 2 bp, optionally 6 to 10 bp, for example, but not limited to 6, 7, 8, 9 or 10 bp.

[0056] DNAzyme (deoxyribozyme) is a type of deoxynucleotide that specifically cuts the substrate RNA chain in the presence of metal ions, and has the characteristics of unlimited substrate cutting and cyclic target recovery. The binding arms (left binding arm and right binding arm) of the DNAzyme are complementary to the RNA reporter molecule. In the presence of metal ions, the DNAzyme binds to the RNA reporter molecule and cuts it to produce a fluorescent signal, indicating the presence of the target RNA. Those skilled in the art can design the corresponding left and right binding arm sequences of the DNAzyme according to the nucleotide sequence of the RNA reporter molecule, or design the nucleotide sequence of the RNA reporter molecule according to the selected DNAzyme, and the present invention is not limited to this.

[0057] In an alternative embodiment, the DNAzyme is 10-23 DNAzyme or 8-17 DNAzyme.

[0058] The RNA reporter molecule is a single-stranded RNA modified with a luminescent group and a quenching group, wherein the luminescent group and the quenching group are located on both sides of the DNAzyme cleavage site. When it is cleaved by the DNAzyme, the luminescent group and the quenching group are separated, so that a light signal appears in the reaction system.

[0059] In an optional embodiment, the metal ions in the reaction system include Mg 2+ 、Na + , K + , Pb 2+ , Mn 2+ , Hg 2+ 、Cd 2+ , Cu 2+ 、UO2 2+ and Ca 2+ At least one of, preferably including Mg 2+ .

[0060] In an optional embodiment, the nucleotide sequence of the stem-loop single strand B is shown in SEQ DI NO.3, the nucleotide sequence of DS1-t is shown in SEQ DI NO.4, the nucleotide sequence of DS1-b is shown in SEQ DI NO.5, the nucleotide sequence of the fuel chain is shown in SEQ DI NO.6, the nucleotide sequence of the blocker is shown in SEQ DI NO.7, the nucleotide sequence of the DNAzyme is shown in SEQDI NO.8, and the nucleotide sequence of the RNA reporter molecule is shown in SEQ DI NO.9.

[0061] In an optional embodiment, the nucleotide sequence of the stem-loop DNA is shown as SEQ DI NO. 2, 11, 12 or 13.

[0062] In an optional embodiment, the crRNA targets Neisseria gonorrhoeae, and the nucleotide sequence is shown in SEQ ID NO.1.

[0063] In an optional embodiment, the crRNA targets Chlamydia trachomatis, and the nucleotide sequence is shown in SEQ ID NO.10.

[0064] In an optional embodiment, the reaction system includes: Cas13 protein 10~200 nM (for example, it can be but not limited to 10, 20, 50, 80, 100, 120, 150, 180 or 200 nM), crRNA 200~300 nM (for example, it can be but not limited to 200, 220, 250, 280 or 300 nM), stem-loop DNA 100~1000 nM (for example, it can be but not limited to 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nM), DS1 100~500 nM (for example, it can be but not limited to 100, 200, 300, 400 or 500 nM), DS2 50~500 nM (e.g., but not limited to 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM), fuel chain 10-200 nM (e.g., but not limited to 10, 20, 50, 80, 100, 120, 150, 180, or 200 nM), RNA reporter 100-1000 nM (e.g., but not limited to 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM), metal ion 1-200 mM (e.g., but not limited to 1, 5, 10, 20, 50, 80, 100, 120, 150, 180, or 200 mM), and RNase inhibitor 100 U / mL-5000 U / mL (for example, but not limited to, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 U / mL).

[0065] In an optional embodiment, the reaction system includes: Cas13 protein 50nM, crRNA 225nM, stem-loop DNA 250nM, DS1 200nM, DS2 100nM, fuel chain 50nM, RNA reporter molecule 250nM, Mg 2+ 10 mM and 2000 U / mL RNase inhibitors.

[0066] The method for detecting RNA based on non-nucleic acid amplification of Cas13 provided by the present invention includes: when the target RNA exists, the Cas13 protein binds to the target RNA under the guidance of crRNA and activates the Cas13 protein to cut the target RNA, and the activated Cas13 protein has a side cutting effect, that is, the Cas13 protein not only cuts the target RNA, but also cuts the RNA around the target RNA in the reaction system, especially preferring to cut the phosphodiester bond between uracil bases (U) and uracil bases (U). Therefore, the activated Cas13 protein cuts the phosphodiester bond between adjacent uracil ribonucleotides in the stem-loop structure, so that the stem-loop DNA is divided into two parts: a stem-loop single-stranded state A and a stem-loop single-stranded state B. Stem-loop DNA is similar to a "switch effect". When the target RNA exists, the uracil base in the stem-loop DNA is cut by the activated Cas13 protein and dissociated into a stem-loop single-stranded A and a stem-loop single-stranded B; when the target RNA does not exist, the stem-loop DNA "locks" the stem-loop single-stranded B and cannot be released.

[0067] Since the stem-loop single-stranded B can complementarily pair with the single-stranded DNA segment DS1A, the stem-loop single-stranded B can undergo a strand displacement reaction with DS1 to obtain DS1-b in a single-stranded DNA state and an intermediate ID formed by the combination of the stem-loop single-stranded B and DS1-t.

[0068] Since the fuel chain is partially complementary to the DS1-t, and the position of the partial complementary pairing is not located in the single-stranded DNA segment DS1A, and the DNA at this position does not bind to the stem-loop single-stranded B, the intermediate exposes a single-stranded DNA segment that can complementarily pair with the fuel chain, so that the fuel chain and the intermediate ID undergo a chain displacement reaction to obtain the stem-loop single-stranded B in a single-stranded DNA state, thereby releasing and displacing the stem-loop single-stranded B again. The stem-loop single-stranded B can be continuously added to the next cycle to form a DNA cascade amplification reaction, continuously displacing the DS1-b chain, and participating in the next step of displacing the DNAzyme in DS2.

[0069] Since the DS1-b is at least partially complementary to the single-stranded DNA segment DS2A, the DS1-b in the single-stranded DNA state can undergo a strand displacement reaction with DS2 to displace a DNAzyme in the single-stranded DNA state. When metal ions are present, the released DNAzyme single strand will bind to the corresponding RNA fluorescent reporter molecule and cut the RNA reporter molecule, and the luminescent group and the quenching group in the cut RNA reporter molecule will separate, so that the fluorescent signal is released in the reaction system.

[0070] In an optional embodiment, the reaction conditions of the method include a reaction temperature of 25 to 42° C., such as but not limited to 25, 30, 35, 37, 40 or 42° C., and a reaction time of 5 to 30 min, such as but not limited to 5, 10, 15, 20, 25 or 30 min.

[0071] In an optional embodiment, the reaction system further comprises a buffer, such as but not limited to 400 mM Tris-HCl.

[0072] In an optional embodiment, the luminescent group includes but is not limited to FAM, FITC, TET, HEX, JOE20, rhodamine (Rhodamine dyes such as R110, TA MRA, TexasRed, etc.), ROX, AlexaFluor dyes and cyanine dyes (such as Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5).

[0073] In an optional embodiment, the quenching group includes but is not limited to DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2 and BHQ-3.

[0074] The method for detecting RNA by non-nucleic acid amplification based on Cas13 provided by the present invention is based on the chain displacement reaction principle of toehold: different nucleic acid molecules compete for hybridization with complementary chains to obtain a more thermodynamically stable double-stranded chain. The single-stranded nucleic acid combines with the toehold end in the double-stranded complex, and a branch migration of base exchange occurs to form a new double-stranded complex and a new single-stranded nucleic acid. The entire reaction process is mainly controlled by the Gibbs free energy change, and no enzyme participation is required. Toehold refers to a single-stranded DNA fragment consisting of 6-10 bases at the end of double-stranded DNA.

[0075] When existing technologies use RNA as a target for detection, most use reverse transcription real-time fluorescence quantitative PCR, which requires nucleic acid amplification and a process that requires strict and precise temperature control equipment; and the chain displacement reaction is restricted by the length limit of RNA nucleic acids and cannot detect long-sequence RNA.

[0076] The present invention does not require pre-amplification of nucleic acid and can be a constant temperature reaction throughout the process, and the length of the RNA to be detected or the nucleic acid sequence is not limited. When the RNA target to be detected exists, the Cas13 protein can be activated to cut the stem-loop structure to produce a single strand. Even if only a small amount of single strands are produced, the DNAzyme single strand can be continuously replaced in the DNA cascade reaction, and the RNA reporter is cut to produce a fluorescent signal. The reaction is fast, and a significant difference between the positive sample and the negative control can be observed within 10 minutes.

[0077] In the method provided by the present invention, except for the RNA target gene, crRNA, RNA reporter molecule and uracil bases in the stem-loop DNA, the remaining nucleic acid components of the reaction system are all composed of DNA chains, which can effectively avoid being cut by the Cas13 protein.

[0078] In a second aspect, a kit for RNA detection is also provided, wherein the kit comprises reagents for constituting the reaction system described in the first aspect.

[0079] In an optional embodiment, the kit includes Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, fuel chain, RNA reporter molecule and metal ion, and the working concentration of each component is: Cas13 protein 10~200 nM (for example, it can be but not limited to 10, 20, 50, 80, 100, 120, 150, 180 or 200nM), crRNA 200~300 nM (for example, it can be but not limited to 200, 220, 250, 280 or 300mM), stem-loop DNA 100~1000 nM (for example, it can be but not limited to 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000nM), DS1 100~500 nM (for example, it can be but not limited to 100, 200, 300, 400 or 500nM), DS2 50~500 nM (e.g., but not limited to 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM), fuel chain 10-200 nM (e.g., but not limited to 10, 20, 50, 80, 100, 120, 150, 180, or 200 nM), RNA reporter 100-1000 nM (e.g., but not limited to 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM), metal ion 1-200 mM (e.g., but not limited to 1, 5, 10, 20, 50, 80, 100, 120, 150, 180, or 200 mM), and RNase inhibitor 100 U / mL-5000 U / mL (for example, but not limited to, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 U / mL).

[0080] In an optional embodiment, the kit includes Cas13a protein, crRNA, stem-loop DNA, DS1, DS2, fuel chain, RNA reporter molecule and Mg 2+ions, the working concentrations of the components were: Cas13 protein 50 nM, crRNA 225 nM, stem-loop DNA 250 nM, DS1 200 nM, DS2 100 nM, fuel chain 50 nM, RNA reporter 250 nM, Mg 2+ 10 mM and RNase inhibitor 2000U / mL.

[0081] In the third aspect, there is also provided a method for detecting RNA by non-nucleic acid amplification based on Cas13 of the first aspect, or the use of the kit of the second aspect in detecting pathogenic microorganisms for non-diagnostic and therapeutic purposes, or in preparing products for detecting pathogenic microorganisms. The pathogenic microorganisms include but are not limited to viruses, bacteria or fungi.

[0082] In an optional embodiment, the pathogenic microorganisms include Neisseria gonorrhoeae and / or Chlamydia trachomatis.

[0083] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0084] Example 1

[0085] This embodiment provides a method for detecting the 16S rRNA gene of Neisseria gonorrhoeae (N.gonorrhoeae).

[0086] 1. Detection method:

[0087] Step 1:

[0088] The stem-loop DNA, DS1, and DS2 were prepared into double-stranded structures by annealing operation, and the amount prepared once could be used multiple times. The specific annealing system was 5 μM single-stranded stem-loop DNA, 5 μM single-stranded DS1-b and DS1-t, and 5 μM single-stranded blocker and DNAzyme. The annealing operation was performed separately, and the reaction sequencing was 95 °C for 5 min, then cooled to room temperature at a rate of -1 °C / min, and stored at -20 °C;

[0089] Step 2:

[0090] The reaction system includes Cas13 protein 50 mM, crRNA 225 mM, stem-loop DNA 250 mM, DS1 200 mM, DS2 100 mM, fuel chain 50 mM, RNA reporter 250 mM, Mg 2+ 10 mM and 2000 U / mL RNase inhibitor. The reaction temperature was 37°C for 30 min.

[0091] The nucleotide sequences of each component are shown in the following table:

[0092] Table 1

[0093]

[0094] 2. Sensitivity:

[0095] Total RNA of Neisseria gonorrhoeae was extracted, and the template concentration was diluted to 500aM, 1fM, 2fM, 3fM, 4fM, 5fM, 10fM and 20fM, respectively. The samples to be tested were reacted using the reaction system and conditions of step 2. The curve of fluorescence intensity changing with time is shown in FIG. Figure 2 As shown, it can be seen that the sensitivity of the detection method of Example 1 in detecting Neisseria gonorrhoeae 16S rRNA can reach 2fM.

[0096] The linear graph of template RNA concentration and corresponding fluorescence value is shown in Figure 3 As shown, it can be seen that the method provided by this embodiment has good linearity, and the linear correlation coefficient R 2 =0.9715, which can be used for quantitative detection of Neisseria gonorrhoeae.

[0097] 3. Specificity:

[0098] The total RNA of Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Ureaplasma urealyticum, Escherichia coli, herpes simplex virus HSV-1 and HSV-1 were extracted respectively, and the samples to be tested were reacted according to the reaction system and conditions of step 2. The test results are as follows: Figure 4 As shown, it can be seen that the detection method provided in Example 1 has good specificity and can only detect Neisseria gonorrhoeae.

[0099] Example 2

[0100] This embodiment provides a method for detecting the 16S rRNA gene of Chlamydia trachomatis (C. trachomatis).

[0101] 1. Detection method:

[0102] Steps 1 and 2 are the same as in Example 1, the crRNA nucleotide sequence is shown in the following table, and the nucleotide sequences of the remaining components are the same as in Example 1.

[0103] Table 2

[0104]

[0105] 2. Sensitivity:

[0106] Total RNA of Chlamydia trachomatis was extracted, and the template concentration was diluted to 1 fM, 5 fM, 10 fM, 50 fM, 100 fM, 500 fM and 1 pM, respectively. The sample to be tested was reacted with the reaction system and conditions of Example 1. The curve of fluorescence intensity change over time is shown in FIG. Figure 5 As shown in FIG. 1 , it can be seen that the sensitivity of the detection method of Example 2 for detecting Chlamydia trachomatis 16S rRNA can reach 10 fM. The linear graph of template RNA concentration and corresponding fluorescence value is shown in FIG. Figure 6 As shown, it can be seen that the method provided by this embodiment has good linearity, and the linear correlation coefficient R 2 =0.9527, which can be used for quantitative detection of Chlamydia trachomatis.

[0107] 3. Specificity:

[0108] The total RNA of Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N.gonorrhoeae), Treponema pallidum (T. Pallidum), Ureaplasma urealyticum (U.urealyticum), Escherichia coli (E.coli), herpes simplex virus HSV-1 and HSV-1 were extracted respectively, and the samples to be tested were reacted according to the reaction system and conditions of Example 1. The test results are as follows: Figure 7 As shown, it can be seen that the detection method provided in Example 2 has good specificity and can only detect Chlamydia trachomatis.

[0109] Example 3

[0110] 1. Using the reaction system and reaction conditions of Example 1, the concentrations of DS1 in the reaction system were adjusted to 100 nM, 200 nM, 300 nM, 400 nM and 500 nM respectively to detect the 16S rRNA gene of Neisseria gonorrhoeae. The fluorescence intensities of different reaction systems were as follows: Figure 8 As shown, it can be seen that the target gene can be detected when the working concentration of DS1 is between 100 and 500 nM, and the fluorescence intensity is optimal when the working concentration of DS1 is 200 nM.

[0111] Second, using the reaction system and reaction conditions of Example 1, the concentration of DS2 in the reaction system was adjusted to 100mM, 200mM, 300mM, 400mM and 500mM respectively to detect the 16S rRNA gene of Neisseria gonorrhoeae. The fluorescence intensity of different reaction systems was as follows: Fig. 9 As shown, it can be seen that the target gene can be detected when the working concentration of DS2 is between 100 and 500 mM, and the fluorescence intensity is optimal when the working concentration of DS2 is 100 mM.

[0112] 3. Using the reaction system and reaction conditions of Example 1, the fuel chain concentrations in the reaction system were adjusted to 0 nM, 10 nM, 50 nM, 100 nM, 150 nM and 200 nM, respectively, to detect the 16S rRNA gene of Neisseria gonorrhoeae. The fluorescence intensities of different reaction systems (fuel chain content 10 nM, 50 nM, 100 nM, 150 nM and 200 nM) were as follows: Fig.10 As shown, it can be seen that the target gene can be detected when the working concentration of the fuel chain is 10~200nM, and the fluorescence intensity is optimal when the working concentration of the fuel chain is 50nM.

[0113] Comparison of the fluorescence intensity changes over time between the reaction system without the fuel chain (fuel chain content 0 nM) and the reaction system (fuel chain content 50 nM) shows that Fig.11 As shown, it can be seen that the presence of the fuel chain can amplify the fluorescence intensity.

[0114] 4. Using the reaction system and reaction conditions of Example 1, the stem-loop DNAs in the following table were used to detect the 16S rRNA gene of Neisseria gonorrhoeae. The fluorescence intensities of the reaction systems with different stem-loop DNAs were as follows: Fig.12 As shown, it can be seen that the use of stem-loop DNA B (hairpinB) has the best effect.

[0115] Table 3

[0116]

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting RNA by non-nucleic acid amplification based on Cas13 for non-diagnostic and therapeutic purposes, characterized in that: The reaction system includes Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, fuel chain, RNA reporter molecule and metal ions; The crRNA targets a target RNA; The stem-loop DNA is composed of folded single-stranded DNA, and at least two uracil ribonucleotides are present near the stem structure of the ring structure on one side of the stem-loop DNA; the uracil ribonucleotides separate the stem-loop DNA into two parts, a stem-loop single strand A and a stem-loop single strand B; DS1 is a double-stranded DNA, which is composed of complementary DS1-t and DS1-b through complementary pairing. The DS1 has a single-stranded DNA segment DS1A composed of DS1-t, and the stem-loop single-stranded B is at least partially complementary to the single-stranded DNA segment DS1A. The fuel chain is a single-stranded DNA, and the fuel chain is partially complementary to the DS1-t; the partially complementary pairing position is not located at the single-stranded DNA segment DS1A, and the DNA at the partially complementary pairing position does not bind to the stem-loop single-stranded B; DS2 is a double-stranded DNA, which is composed of complementary DNAzyme and blocker through complementary pairing. The DS2 has a single-stranded DNA segment DS2A composed of blocker, and the DS1-b is at least partially complementary to the single-stranded DNA segment DS2A. The RNA reporter molecule is a single-stranded RNA modified with a luminescent group and a quenching group; The binding arm of the DNAzyme is complementary to the RNA reporter molecule; The nucleotide sequence of the stem-loop single strand B is shown in SEQ ID NO.3, the nucleotide sequence of DS1-t is shown in SEQ ID NO.4, the nucleotide sequence of DS1-b is shown in SEQ ID NO.5, the nucleotide sequence of the fuel chain is shown in SEQ ID NO.6, the nucleotide sequence of the blocker is shown in SEQ ID NO.7, the nucleotide sequence of the DNAzyme is shown in SEQ ID NO.8, and the nucleotide sequence of the RNA reporter molecule is shown in SEQ ID NO.9; The nucleotide sequence of the stem-loop DNA is shown in SEQ ID NO. 2, 11, 12 or 13; The method for detecting RNA comprises: when the target RNA is present, the Cas13 protein binds to the target RNA under the guidance of crRNA, cuts the stem-loop DNA in the reaction system so that the stem-loop DNA is separated into two parts: a stem-loop single strand A and a stem-loop single strand B; the stem-loop single strand B undergoes a strand displacement reaction with the DS1 to obtain DS1-b in a single-stranded DNA state and an intermediate ID formed by the combination of the stem-loop single strand B and DS1-t; The fuel chain undergoes a chain displacement reaction with the intermediate ID to obtain a stem-loop single-stranded DNA state B, and the stem-loop single-stranded DNA state B continuously undergoes a chain displacement reaction with DS1 in the presence of the fuel chain; The DS1-b in the single-stranded DNA state undergoes a strand displacement reaction with the DS2 to obtain a DNAzyme in the single-stranded DNA state, and the DNAzyme cuts the RNA reporter molecule in the presence of metal ions, so that a fluorescent signal is released in the reaction system; The reaction conditions include a reaction temperature of 25-42° C. and a reaction time of 5-30 min.

2. The method according to claim 1, characterized in that: The side of the uracil ribonucleotide away from the stem structure is the stem-loop single strand B.

3. The method according to claim 1, characterized in that The metal ions include Mg 2+ 、Na + , K + , Pb 2+ , Mn 2+ , Hg 2+ 、Cd 2+ , Cu 2+ 、UO2 2+ and Ca 2+ At least one of .

4. The method according to claim 1, characterized in that The crRNA targets Neisseria gonorrhoeae, and the nucleotide sequence is shown in SEQ ID NO.1; or, the crRNA targets Chlamydia trachomatis, and the nucleotide sequence is shown in SEQ ID NO.

10.

5. The method according to any one of claims 1 to 4, characterized in that The reaction system includes: Cas13 protein 10-200 nM, crRNA 200-300 nM, stem-loop DNA 100-1000 nM, DS1 100-500 nM, DS2 50-500 nM, fuel chain 10-200 nM, RNA reporter molecule 100-1000 nM, metal ion 1-200 mM and RNase inhibitor 100 U / mL-5000 U / mL.

6. A kit for RNA detection, characterized in that: Includes Cas13 protein, crRNA, stem-loop DNA, DS1, DS2, fuel chain, RNA reporter molecule and metal ions; The crRNA targets a target RNA; The stem-loop DNA is composed of folded single-stranded DNA, and at least two uracil ribonucleotides are present near the stem structure of the ring structure on one side of the stem-loop DNA; the uracil ribonucleotides separate the stem-loop DNA into two parts, a stem-loop single strand A and a stem-loop single strand B; DS1 is a double-stranded DNA, which is composed of complementary DS1-t and DS1-b through complementary pairing. The DS1 has a single-stranded DNA segment DS1A composed of DS1-t, and the stem-loop single-stranded B is at least partially complementary to the single-stranded DNA segment DS1A. The fuel chain is a single-stranded DNA, and the fuel chain is partially complementary to the DS1-t; the partially complementary pairing position is not located at the single-stranded DNA segment DS1A, and the DNA at the partially complementary pairing position does not bind to the stem-loop single-stranded B; DS2 is a double-stranded DNA, which is composed of complementary DNAzyme and blocker through complementary pairing. The DS2 has a single-stranded DNA segment DS2A composed of blocker, and the DS1-b is at least partially complementary to the single-stranded DNA segment DS2A. The RNA reporter molecule is a single-stranded RNA modified with a luminescent group and a quenching group; The binding arm of the DNAzyme is complementary to the RNA reporter molecule; The nucleotide sequence of the stem-loop single strand B is shown in SEQ ID NO.3, the nucleotide sequence of DS1-t is shown in SEQ ID NO.4, the nucleotide sequence of DS1-b is shown in SEQ ID NO.5, the nucleotide sequence of the fuel chain is shown in SEQ ID NO.6, the nucleotide sequence of the blocker is shown in SEQ ID NO.7, the nucleotide sequence of the DNAzyme is shown in SEQ ID NO.8, and the nucleotide sequence of the RNA reporter molecule is shown in SEQ ID NO.9; The nucleotide sequence of the stem-loop DNA is shown in SEQ ID NO. 2, 11, 12 or 13.

7. The kit according to claim 6, characterized in that The crRNA targets Neisseria gonorrhoeae, and the nucleotide sequence is shown in SEQ ID NO.1; or, the crRNA targets Chlamydia trachomatis, and the nucleotide sequence is shown in SEQ ID NO.

10.

8. Use of the kit according to claim 7 for detecting pathogenic microorganisms for non-diagnostic and non-therapeutic purposes, or for preparing products for detecting pathogenic microorganisms, wherein the pathogenic microorganism is Neisseria gonorrhoeae or Chlamydia trachomatis.

Images