CN112575065A

CN112575065A - Detection method based on hybrid chain reaction amplified output signal and detection kit thereof

Info

Publication number: CN112575065A
Application number: CN202011444938.4A
Authority: CN
Inventors: 邓玉林; 于世永; 徐建栋
Original assignee: Beijing Ligong Genshu Technology Co ltd
Current assignee: Beijing Ligong Genshu Technology Co ltd
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-30
Anticipated expiration: 2040-12-08
Also published as: CN112575065B

Abstract

The invention provides a detection method and a detection kit thereof based on a hybrid chain reaction amplified output signal. The detection method comprises the following steps of selecting a target sequence: selecting at least one specific target sequence and at least one non-specific target sequence of a single-stranded nucleic acid molecule to be amplified as target sequences; designing a hairpin probe: designing corresponding sets of probe pairs which respectively generate hybridization chain reactions with the selected specific target sequence(s) and the selected non-specific target sequence(s), so that the specific target sequence(s) and the non-specific target sequence(s) can both generate hybridization chain reactions under the action of the corresponding probe pairs so as to be amplified and amplify output signals; and detecting the colloidal gold test strip based on the hybrid chain reaction. The invention also provides a detection kit corresponding to the method, which has the technical effects of convenient and sensitive detection.

Description

Detection method based on hybrid chain reaction amplified output signal and detection kit thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a detection method for amplifying an output signal based on a hybrid chain reaction and a single-stranded nucleic acid molecule detection kit thereof.

Background

Principle of hybrid chain reaction

The relevant principles of Hybridization Chain Reaction (HCR) were first proposed in 2004 by Dirks and Pierce (see: Triggered amplification by Hybridization Chain Reaction R.M. Dirks and N.A. Pierce, Proc. Natl. Acad. Sci. U.S.A.,2004,101, 15275-15278). The hybridization chain reaction is an in vitro isothermal signal amplification technology based on no enzyme assistance, and belongs to the self-assembly signal amplification technology. Compared with thermal cycle amplification techniques, including Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR), hybrid Chain Reaction has the advantages of simple conditions (constant temperature), simple operation (no need of expensive professional instruments), low cost (no need of enzyme mediation), controllable amplification Reaction kinetics, high sensitivity and the like (see Lidong, Chen Xingham, Wu hong Xiang, et al., application of hybrid Chain Reaction in biosensing detection technology [ J ] Anhui medical university journal, 2018,053(012): 1976-. Based on the foregoing advantages, the hybridization chain reaction has drawn extensive attention in the detection of DNA, miRNA, protein and metal ions in the biosensing field (see liu yong xin, yuan dan, chen, etc. the research progress of hybridization chain reaction technology combined with nanomaterials for nucleic acid detection [ J ] the progress of chinese materials, 2020,039(004): 325-.

As shown in FIG. 1, the principle of the hybridization chain reaction proposed by Dirks et al mainly utilizes two single-stranded DNA fragments with hairpin-type probes H1 and H2 as partial complementarity, and after adding primer I, because the primer I is complementary with ab segment of the first hairpin probe H1, the formed double-stranded aa bb is more stable than the hairpin structure, so as to initiate the complementary reaction. Meanwhile, because the cb segment that becomes single-stranded due to the hairpin opening is complementary to the cb segment of the second hairpin probe H2, the opening of the second hairpin probe H2 is triggered in the same way, and thus the reaction continues continuously, and a chain reaction occurs, so that a long chain is formed. Although the hybrid chain reaction does not need enzyme participation and does not need a temperature rise and drop process, the energy source can be obtained by competing hybridization in a single-stranded nucleic acid probe molecule based on a chain displacement cyclic amplification method to form an assembly structure.

However, because the long chain of the hybridization chain reaction product is different from the PCR product, no chemical bond is formed between the probe and the probe under the hybridization chain reaction, but the probe and the probe are mutually connected by the action of hydrogen bonds between nucleotides, so that after the chain is extended to a certain degree, the steric hindrance is more and more great, and the molecules are more and more subjected to external force, so that the single-chain nucleic acid chain becomes unstable and cannot be sufficiently amplified. If the strand is not long enough, the amplification effect of the hybridization chain reaction is relatively small compared to the huge molecular weight of the single-stranded nucleic acid molecule, so that the method has no advantage in amplification effect compared with PCR.

Test strip detection based on hybrid chain reaction

An ok important application of the hybrid chain reaction amplification technology is the analytical detection of exosomes, drugs/biomolecules, DNA, RNA, metal ions in organisms, antigens, antibodies, enzymes and cells in liquid biopsies. For example, Na Ying et al propose a test strip protocol for nucleic acids. As shown in fig. 2, the H1 probe and the H2 probe were modified with biotin (biotin). Wherein the thermostable double-strand specific nuclease (DSN) is capable of selectively degrading DNA in deoxyribonucleic acid double strands (double-stranded DNA) and single-stranded DNA and single-stranded RNA hybrids. The microRNA is combined with a Report primer (Report Probe) to form a double strand, and the DSN cuts a complementary part of the double strand, so that a complementary strand marked with a fluorescent group (FAM) is cut off. After further purification by magnetic bead method, the complementary strand labeled with a Fluorophore (FAM) is complementarily bound to the hybridization chain reaction product, and the product is made fluorescent. The specific detection scheme is that a nucleic acid Test strip (LFNAB) is combined with a pad coated with nanogold (AuNPs) of Streptavidin (SA), a detection Line (Test Line, namely T Line) is provided with a monoclonal antibody (anti-FAM mAb) of anti-FAM, and when the structure of FAM and Biotin (Biotin) is contained, the complex is captured on the detection Line through specific reaction between the anti-FAM mAb (in a Test area) and a FAM-labeled complementary chain sequence of the complex. The AuNPs accumulation in the T line can show red on the T line, and further the detection of the target is realized. The excess SA-AuNP conjugate continues to move through the Control Line (C-Line) of reaction between biotin and streptavidin SA on the AuNPs surface, forming a second red band.

However, in the aforementioned conventional detection technology based on the hybrid chain reaction, since the complementary sequence fixed on the detection line of the test strip is generally tens of bases, which is shorter than the nucleic acid molecule in the actual sample, the target sequence may be wrapped inside due to the influence of the spatial structure of the nucleic acid molecule, and cannot be bound to the complementary sequence, so that the target sequence cannot be captured by the T-line, thereby reducing the detection effect. Therefore, although the scheme of Na Ying is adopted, the detection of the specific signal of the anti-FAM mAb and the biotin signal can be realized, thereby improving the detection sensitivity. However, the problems that the amplification effect of the hybridization chain reaction is not good, the reaction still needs to be purified by using double-strand specific nuclease (DSN) and a magnetic bead method, and the steps are complex still cannot be solved under the scheme.

Fluorescence detection

The current commonly used nucleic acid amplification detection is a fluorescent quantitative method. For example, the detection methods of mirnas mainly include quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Northern blot analysis, clone sequencing, real-time fluorescent quantitative PCR, Microarray analysis, and the like. However, in these methods, qRT-PCR has high requirements on temperature and instruments, and professional techniques are high; northern blot analysis generally requires 3 working days, is time-consuming, complex in process and low in detection sensitivity, a clone sequencing method is time-consuming and labor-consuming, and real-time fluorescent quantitative PCR and Microarray (Microarray) analysis require the help of a fluorescent instrument.

Although the prior art, such as CN111778315A and CN111172243A, has attempted to use fluorescence kit in hybridization chain reaction, the detection is carried out by using quenching effect labeled with fluorescent group. However, these solutions still need to use instruments for fluorescence detection, and do not meet the dual requirements of the current market for rapid, sensitive and convenient detection. For example, if everyone in a high disease area is screened by fluorescence detection, it will consume much time and effort, and it will also cause cross infection of people. Therefore, how to provide a portable/sick population to carry out rapid preliminary nucleic acid detection at home becomes important and valuable for application, and a scheme for further re-examination based on the preliminary detection result after preliminary diagnosis becomes important.

Based on the background, the core technical problems to be solved by the invention are as follows: how to provide a detection method for amplifying an output signal based on a multi-site hybridization chain reaction under the advantages of no enzyme, constant temperature and simple operation of the hybridization chain reaction amplification technology, so that the amplification effect of the hybridization chain reaction is further ensured.

Disclosure of Invention

In order to solve the above-mentioned core technical problems, the applicant has succeeded in designing a detection method for amplifying an output signal based on a hybridization chain reaction, which has a good amplification effect and is easy and convenient to operate.

Specifically, the invention provides a detection method for amplifying an output signal based on a hybridization chain reaction, which comprises the following steps: (1) selecting a target sequence; (2) designing a hairpin probe; and (3) detection based on hybridization chain reaction;

the (1) target sequence selection comprises: selecting X specific target sequences and Y non-specific target sequences of a single-stranded nucleic acid molecule to be amplified as target sequences together, wherein X and Y are integers which are more than or equal to 1;

the hairpin probe design (2) comprises: designing X groups of probe pairs for respectively carrying out hybridization chain reaction on the selected X specific target sequences, wherein the X groups of probe pairs comprise H1a_mProbe and H2a_m2X probes in total, wherein m is respectively equal to any integer from 1 to X, and Y groups of probe pairs which are selected from Y non-specific target sequences and respectively subjected to hybridization chain reaction comprise H1b_nProbe and H2b_n2Y probes are counted, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by a hybridization chain reaction under the action of the corresponding probe pairs;

the (3) is based on a hybrid strandThe detection of the reaction of formula (la) comprises: adding the single-stranded nucleic acid molecule to be amplified and the DNA comprising H1a on a colloidal gold test strip_mProbe and H2a_mThe total number of the probes is 2X and the probes comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, the probes react at room temperature to form hybrid chain reaction products, and then the hybrid chain reaction products are added to the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip.

The invention also provides a single-stranded nucleic acid molecule detection kit based on the hybrid chain reaction amplified output signal. The detection kit at least comprises:

(1) hairpin probes comprising (a) X sets of probe pairs capable of priming the respective hybridization chain reaction of X specific target sequences of a single-stranded nucleic acid molecule to be amplified, said X sets of probe pairs further comprising H1a_mProbe and H2a_m2X probes are counted, and m is respectively equal to any integer from 1 to X; and (b) Y group probe pairs which can trigger the hybridization chain reaction of Y non-specific target sequences of the single-stranded nucleic acid molecule to be amplified respectively, wherein the Y group probe pairs further comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by hybridization chain reaction under the action of the corresponding probe pairs; and (2) a colloidal gold test strip capable of capturing the specific target sequence and the non-specific target sequence.

Drawings

FIG. 1 is a schematic diagram of the principle of the hybridization chain reaction in the prior art.

Fig. 2 is a schematic diagram of a test strip detection principle in the prior art.

FIG. 3 is a schematic diagram of the two-site hybridization chain reaction principle of the present invention.

FIG. 4 is a schematic diagram of the principle of the three-site hybridization chain reaction of the present invention.

Figure 5 is a schematic of a bispecific capture of the present invention.

Fig. 6 is a colloidal gold visual inspection chart according to example 1 of the present invention.

FIG. 7 is a fluorescence visualization test chart of example 1 of the present invention.

Fig. 8 is a colloidal gold visual inspection chart according to example 2 of the present invention.

FIG. 9 is a fluorescence visualization test chart of example 2 of the present invention.

Fig. 10 is a colloidal gold visualization assay of the bispecific capture assay of example 2 of the invention.

FIG. 11 is a fluorescence visualization graph of the hybridization chain reaction at different reaction times in example 2 of the present invention.

FIG. 12 is a fluorescence visualization graph of the hybridization chain reaction at different reaction temperatures in example 2 of the present invention.

FIG. 13 is a fluorescence visualization graph of hybridization chain reaction at different ion concentrations in example 2 of the present invention.

Detailed Description

The following explains specific embodiments of the present invention. It is to be understood that the following examples are merely illustrative of particular embodiments, uses, and so forth of the invention in order to facilitate a better understanding of the relevant art and are not intended to limit the scope of the invention in any way. Various changes or modifications may be effected therein by one skilled in the art, and equivalents may be made thereto without departing from the scope of the invention as defined in the claims appended hereto.

Principle of the invention

Multiple site hybridization chain reaction

In the common hybridization chain reaction, a target sequence in a target gene opens a hairpin structure of an H1 probe, and carries out base complementary pairing with a part corresponding to an H1 probe to form a double chain, an unpaired sequence in an H1 probe opens the hairpin structure of H2 and forms the double chain through base complementary pairing, an unpaired sequence of an H2 probe continues to open the hairpin structure of H1 for complementary pairing, and the steps are repeated in sequence to carry out chain reaction, so that the double chain structure is continuously prolonged. However, in the conventional hybrid chain reaction, the amplified target sequence only includes the specific sequence, and the specific sequence is generally short, so the amplification amount is limited and the effect is not obvious.

The multi-site hybridization chain reaction is based on the principle of common hybridization chain reaction, but is characterized in that at least one non-specific target sequence and at least one specific target sequence of a single-stranded nucleic acid molecule to be amplified are selected as target sequences by the target sequences for simultaneous amplification, and the probe design comprises the steps of respectively designing a probe pair with the selected non-specific target sequence or sequences for the hybridization chain reaction; and designing a probe pair for the selected one or more specific target sequences to perform hybridization chain reaction with the selected one or more specific target sequences. Finally, one or more non-specific target sequences and one or more specific target sequences of the single-stranded nucleic acid molecules to be amplified can be subjected to hybridization chain reaction so as to be amplified, so that amplification based on the multi-site hybridization chain reaction is realized.

FIG. 3 provides the basic principle of the two-site hybridization chain reaction amplification of the present invention. The long chain represents a single-stranded nucleic acid molecule to be amplified, and the single-stranded nucleic acid molecule to be amplified comprises 1 specific target sequence and 1 non-specific target sequence which are jointly used as a target sequence to be amplified, wherein the sequences of the non-specific target sequence in the 5 'to 3' direction are c and d respectively, and the sequences of the specific target sequence in the 5 'to 3' direction are f and h respectively; to which 1 set of probe pairs designed for specific target sequences and capable of hybridizing chain reaction therewith was added, including H1a₁Probe and H2a₁Probes, and 1 group of probes which are designed aiming at non-specific target sequences and can be hybridized with the non-specific target sequences and are added to the probes, wherein the probes comprise H1b₁Probe and H2b₁And (3) a probe. The H1a₁The sequence of the probe can be divided into four sequences of d, e, d and c from the 5 'to the 3' direction, and the H2a₁The sequence of the probe is divided into e, d, c and d sequences from the 5 'to the 3' direction, wherein the c and c sequences, the d and d sequences and the e and e sequences can be respectively complementary; the H1b₁The sequences of the probes can be divided into four sequences of h, k, h and f from the 5 'direction to the 3' direction; the H2b₁The sequence of the probe is divided into k, h, f and h four sequences from 5 'to 3', wherein f and f sequences, h and h sequences, and k sequences can be respectively complementary; the specific targetThe sequence may open H1a₁Hairpin structure of probe, and H1a₁Base-complementary pairing of the probe-corresponding portions to form a double strand, H1a₁Unpaired sequence in the probe opens H2a₁Hairpin structure of probe and formation of double strand by base complementary pairing, H2a₁The unpaired sequence of the probe again continues to open H1a₁The hairpin structures are subjected to complementary pairing, and are sequentially reciprocated to generate chain reaction, so that the double-chain structure is continuously prolonged; similarly, the non-specific target sequence may open H1b₁Hairpin structure of probe, and H1b₁Base-complementary pairing of the probe-corresponding portions to form a double strand, H1b₁Unpaired sequence in the probe opens H2b₁Hairpin structure of probe and formation of double strand by base complementary pairing, H2b₁The unpaired sequence of the probe again continues to open H1b₁The hairpin structures are subjected to complementary pairing, and are sequentially reciprocated to generate chain reaction, so that the double-chain structure is continuously prolonged.

FIG. 4 provides the basic principle of the amplification of the three-site hybridization chain reaction of the present invention. The content consistent with that of FIG. 3 is not repeated, and only the main difference is that 1 specific target sequence is selected, and the probe pair for the specific target sequence to perform the hybridization chain reaction is H1a₁And H2a₁2 nonspecific target sequences are selected, each nonspecific target sequence comprises 1 nonspecific target sequence (1) and 1 nonspecific target sequence (2), and the probe pair which performs hybridization chain reaction with the nonspecific target sequence (1) is H1b₁And H2b₁The probe pair which undergoes a hybridization chain reaction with the nonspecific target sequence (2) is H1b₂And H1b₂(ii) a Wherein the non-specific target sequence (2) is divided into two sections of p and t sequences from the 5 'end to the 3' end. The H1b₂The sequence of the probe can be divided into four sequences of t, w, t and p from the 5 'to the 3' direction, and the H2b₂The sequences of the probes are divided into w, t, p and t sequences from 5 'to 3', wherein p and p sequences, t and t sequences, w and w sequences can be complementary respectively. The non-specific target sequence can open H1b₂Hairpin structure of probe, and H1b₂Base complementary pairing of probe-corresponding portionsForming a double strand, H1b₂Unpaired sequence in the probe opens H2b₂Hairpin structure of probe and formation of double strand by base complementary pairing, H2b₂The unpaired sequence of the probe again continues to open H1b₂The hairpin structures are subjected to complementary pairing, and are sequentially reciprocated to generate chain reaction, so that the double-chain structure is continuously prolonged.

There are many examples of the amplification of the multi-site hybridization chain reaction of the present invention, and one skilled in the art can further perform (1) target sequence selection and (2) hairpin probe design based on the principles of the present invention in combination with different sequences of the single-stranded nucleic acid molecule to be amplified.

Test strip detection based on hybrid chain reaction

The colloidal gold test strip adopted by the invention has a structure similar to that in the prior art shown in figure 2. The technology in the field can completely adopt the conventional colloidal gold test strip detection scheme in the prior art to perform detection, that is, after the steps of target sequence selection and hairpin probe design of the invention are completed, the detection based on the hybrid chain reaction comprises the following steps: adding the single-stranded nucleic acid molecule to be amplified and the DNA comprising H1a on a colloidal gold test strip_mProbe and H2a_mThe total number of the probes is 2X and the probes comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, the probes react at room temperature to form hybrid chain reaction products, and then the hybrid chain reaction products are added to the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip.

FIG. 5 sets forth the principle of a preferred embodiment of the present invention, a dual specific capture assay, which utilizes antigen-antibody specific binding to further capture H2a_mA probe; on the other hand, a complementary strand is arranged on a detection line on the colloidal gold test strip, and by utilizing the specific complementation of nucleic acid sequences, the complementary strand can further capture one or more specific sequences R in a long-chain open reading frame of the single-chain nucleic acid molecule to be amplified_n. Said one or more sequences R_nPreferably, it is not selected as a unique sequence of the specific target sequence participating in the hybridization chain reaction, since the hybrid chainThe long chain of the product of the reaction of formula (I) has a spatial structure, which may prevent complementary binding between the complementary strand capture specific target sequence and the single-stranded sequence S, and thus the capture effect may be influenced.

It should be noted that the present invention does not limit the necessity of using the above-mentioned bispecific capturing, and the capturing effect can be further enhanced by using any of the above-mentioned specific capturing methods based on the conventional protocols. See the detailed description of the invention.

Detailed description of the invention

Based on the above, the invention provides the following detection method based on the amplified output signal of the hybridization chain reaction, comprising the following steps: (1) selecting a target sequence; (2) designing a hairpin probe; and (3) detection based on hybridization chain reaction:

(1) the target sequence selection comprises: selecting X specific target sequences and Y non-specific target sequences of a single-stranded nucleic acid molecule to be amplified as target sequences together, wherein X and Y are integers which are more than or equal to 1;

(2) hairpin probe design includes: designing X groups of probe pairs for respectively carrying out hybridization chain reaction on the selected X specific target sequences, wherein the X groups of probe pairs comprise H1a_mProbe and H2a_m2X probes in total, wherein m is respectively equal to any integer from 1 to X, and Y groups of probe pairs which are selected from Y non-specific target sequences and respectively subjected to hybridization chain reaction comprise H1b_nProbe and H2b_n2Y probes are counted, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by a hybridization chain reaction under the action of the corresponding probe pairs; and

(3) detection based on the hybrid chain reaction includes: adding the single-stranded nucleic acid molecule to be amplified and the DNA comprising H1a on a colloidal gold test strip_mProbe and H2a_mThe total number of the probes is 2X and the probes comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, the probes react at room temperature to form hybrid chain reaction products, and then the hybrid chain reaction products are added to the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip.

Further preferably, the invention also provides a double visual detection scheme, and the specific embodiment is the H2b_nThe 5' end of the probe is also marked with a fluorescent group; the H1a_mProbe and H1b_nThe 3' ends of the probes are all provided with biotin labels, the detection based on the hybrid chain reaction is detected by adopting a colloidal gold test strip, and the colloidal gold on a sample pad on the colloidal gold test strip is provided with streptavidin capable of identifying the biotin labels. So that the crowd to be detected can automatically finish quick preliminary nucleic acid detection, and the preliminary nucleic acid detection result is further rechecked after the preliminary diagnosis is confirmed.

Preferably, said H2a of the present invention_mThe 5' end of the probe can also be provided with an antigen marker, and the detection line on the colloidal gold test strip is also provided with an antibody which can be specifically combined with the antigen marker, so that the capture effect of the colloidal gold test strip on the final hybrid chain reaction product is enhanced.

Preferably, the detection line on the colloidal gold test strip of the present invention further comprises a single-stranded sequence S, wherein the single-stranded sequence S can be associated with one or more sequences R specific to the long-chain open reading frame of the single-stranded nucleic acid molecule to be amplified_nComplementation; with the proviso that said one or more sequences R_nThe test strip is not selected as the specific target sequence and participates in the hybrid chain reaction, so that the capture effect of the colloidal gold test strip on the final hybrid chain reaction product is enhanced.

Preferably, the detection method based on the amplified output signal of the hybridization chain reaction provided by the invention comprises the following specific steps:

(1) and (3) selecting a target sequence:

selecting specific X sequences in a long-chain open reading frame of a single-chain nucleic acid molecule to be amplified from the specific target sequence; the nonspecific target sequence randomly selects Y from the long chain of the single-stranded nucleic acid molecule to be amplified; the sequences of the non-specific target sequence in the 5 'to 3' direction are c and d respectively, and the sequences of the specific target sequence in the 5 'to 3' direction are f and h respectively;

(2) designing a hairpin probe:

the H1a_mThe sequence of the probe can be divided into four sequences of d, e, d and c from the 5 'to the 3' direction, and the H2a_mThe sequence of the probe is divided into e, d, c and d sequences from the 5 'to the 3' direction, wherein the c and c sequences, the d and d sequences and the e and e sequences can be respectively complementary, the length of the c and c sequences is 4-10 bases, the length of the d and d sequences is 12-24 bases, and the length of the e and e sequences is 4-10 bases;

the H1b_nThe sequences of the probes can be divided into four sequences of h, k, h and f from the 5 'direction to the 3' direction;

the H2b_nThe sequence of the probe is divided into k, h, f and h four sequences from 5 'to 3', wherein f and f sequences, h and h sequences and k sequences can be respectively complementary, the length of f and f sequences is 4-10 bases, the length of h and h sequences is 12-24 bases, and the length of k and k sequences is 4-10 bases;

(3) detection based on hybridization chain reaction:

adding the H1a to the solution of the single-stranded nucleic acid molecule to be amplified at a concentration of at least 10 times that of the solution_mProbe and H2a_m2X kinds of probes in total and a probe pair H1b with the concentration at least 10 times of the solution of the single-stranded nucleic acid molecules to be amplified_nProbe and H2b_nAnd 2Y probes are added, a buffer system is formed by adding buffer solution, a hybrid chain reaction product is formed after reaction at room temperature, then the hybrid chain reaction product, the colloidal gold and the loading buffer solution are added on the colloidal gold test strip, and the signal mark change on the colloidal gold test strip is analyzed.

It should be noted that the concentration limitation of at least 10 times is not necessarily performed, but it may enhance the amplification effect. Those skilled in the art will appreciate that a certain reduction in the concentration range does not affect the practice of the present invention.

Further preferably, the non-specific target sequence is 24 bases in length and the specific target sequence is 24 bases in length; the H1a_mThe length of the probe is 48 bases, the first 18 bases are the same as the last 18 bases of the specific target sequence from the 5 'end to the 3' end, and the 19 th to 24 th bases contain the non-reversible sequencePairing to form a loop portion of 6 bases of a duplex, the 25 th to 42 th bases being paired with the first 18 bases in reverse order, the 43 th to 48 th bases being paired with the first 6 bases of a specific target sequence in reverse order; the H2a_mThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1a_mThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1a_mThe 25 th to 42 th base sequences of the probe are identical, the 25 th to 30 th bases are identical to the first 6 base sequences of the specific target sequence, and the 31 th to 48 th bases are identical to H1a_mThe first 18 base sequences of the probes are identical; the H1b_nThe probe is 48 bases in length, the first 18 bases are the same as the last 18 bases of the nonspecific target sequence in the direction from the 5 'end to the 3' end, the 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be reverse-sequence-paired into a double strand, the 25 th to 42 th bases are reverse-sequence-paired with the first 18 bases, and the 43 th to 48 th bases are reverse-sequence-paired with the first 6 bases of the nonspecific target sequence; the H2b_nThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1b_nThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1b_nThe 25 th to 42 th base sequences of the probe are the same, the 25 th to 30 th bases are the same as the first 6 base sequences of the nonspecific target sequence, and the 31 th to 48 th bases are the same as H1b_nThe first 18 base sequences of the probes are identical.

The invention provides a preferred embodiment, when the single-stranded nucleic acid molecule to be amplified is the single-stranded ribonucleic acid molecule of the novel coronavirus, the specific target sequence can be selected from 1, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 1 nonspecific target sequence can be selected, the sequence is shown as SEQ ID NO.4, and the probe pair which has hybridization chain reaction with the nonspecific target sequence is H1b₁And H2b1, said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO. 6. Further preferablyThe detection line on the colloidal gold test strip is also provided with a single-chain sequence S which can be complementary with the specific target sequence, and the sequence of the single-chain sequence S is shown in SEQ ID NO.7, so that the detection line on the colloidal gold test strip can further capture a hybridization reaction product.

In another preferred embodiment, when the single-stranded nucleic acid molecule to be amplified is a single-stranded ribonucleic acid molecule of a novel coronavirus, the specific target sequence is selected from 1, the sequence is shown as SEQ ID NO.1, and the probe pair which performs hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 2 nonspecific target sequences are selected, and comprise 1 nonspecific target sequence (1) and 1 nonspecific target sequence (2), the sequence of the nonspecific target sequence (1) is shown as SEQ ID NO.4, the sequence of the nonspecific target sequence (2) is shown as SEQ ID NO.8, and the probe pair which has hybridization chain reaction with the nonspecific target sequence (1) is H1b₁And H2b₁Said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO.6, and the probe pair which has hybridization chain reaction with the non-specific target sequence (2) is H1b₂And H1b₂Said H1b₂The sequence is shown as SEQ ID NO.9, and the H2b₂The sequence is shown as SEQ ID NO. 10. Further preferably, the detection line on the colloidal gold test strip is further provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown in SEQ ID NO.7, so that the detection line on the colloidal gold test strip can further capture a hybridization reaction product. It is to be noted that other preferred embodiments of the present invention, such as antigen-antibody specific capture, and dual visualization of biotin and fluorescent labels, can be further adapted by those skilled in the art to further refine the detection process. And will not be described in detail herein.

The invention also provides a single-stranded nucleic acid molecule detection kit based on the hybrid chain reaction amplified output signal, which at least comprises:

(1) hairpin probes, thereforThe hairpin probe comprises (a) X groups of probe pairs which can initiate the hybridization chain reaction of X specific target sequences of the single-stranded nucleic acid molecule to be amplified respectively, and the X groups of probe pairs further comprise H1a_mProbe and H2a_m2X probes are counted, and m is respectively equal to any integer from 1 to X; and (b) Y group probe pairs which can trigger the hybridization chain reaction of Y non-specific target sequences of the single-stranded nucleic acid molecule to be amplified respectively, wherein the Y group probe pairs further comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by hybridization chain reaction under the action of the corresponding probe pairs;

(2) a colloidal gold test strip capable of capturing the specific target sequence and the non-specific target sequence.

It should be noted that the aforementioned colloidal gold test strip only represents a device for detecting single-stranded nucleic acid molecules by using the principle of colloidal gold test strip, and therefore any detection device adopting the hairpin probe design of the present invention and amplifying single-stranded nucleic acid molecules to be detected based on multi-site hybridization chain reaction shall belong to the colloidal gold test strip mentioned herein, including but not limited to the test strip itself, the related array device, etc.

Preferably, said H2b_nThe 5' end of the probe is also marked with a fluorescent group; the H1a_mProbe and H1b_nThe 3' ends of the probes are all provided with biotin labels, the detection based on the hybrid chain reaction is detected by adopting a colloidal gold test strip, and the colloidal gold on a sample pad on the colloidal gold test strip is provided with streptavidin capable of identifying the biotin labels.

Preferably, said H2a_mThe 5' end of the probe is also provided with an antigen marker, and the detection line on the colloidal gold test strip is also provided with an antibody which can be specifically combined with the antigen marker.

Preferably, the detection line on the colloidal gold test strip also carries a single-stranded sequence S, and the single-stranded sequence S can be in open reading with the long chain of the single-stranded nucleic acid molecule to be amplifiedOne or more sequences R specific for the frame_nComplementation; with the proviso that said one or more sequences R_nAre not selected as the specific target sequence and participate in the hybridization chain reaction.

The following provides a specific target sequence and probe selection condition suitable for the single-stranded nucleic acid molecule detection kit based on the hybridization chain reaction amplified output signal:

further preferably, the non-specific target sequence is 24 bases in length and the specific target sequence is 24 bases in length; the H1a_mThe probe is 48 bases in length, the first 18 bases are identical to the last 18 bases of the specific target sequence in the direction from the 5 'end to the 3' end, the 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be paired into a double strand in reverse order, and the 25 th to 42 th basesThe first 18 bases are subjected to reverse sequence pairing, and the 43 th to 48 th bases are subjected to reverse sequence pairing with the first 6 bases of the specific target sequence; the H2a_mThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1a_mThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1a_mThe 25 th to 42 th base sequences of the probe are identical, the 25 th to 30 th bases are identical to the first 6 base sequences of the specific target sequence, and the 31 th to 48 th bases are identical to H1a_mThe first 18 base sequences of the probes are identical; the H1b_nThe probe is 48 bases in length, the first 18 bases are the same as the last 18 bases of the nonspecific target sequence in the direction from the 5 'end to the 3' end, the 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be reverse-sequence-paired into a double strand, the 25 th to 42 th bases are reverse-sequence-paired with the first 18 bases, and the 43 th to 48 th bases are reverse-sequence-paired with the first 6 bases of the nonspecific target sequence; the H2b_nThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1b_nThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1b_nThe 25 th to 42 th base sequences of the probe are the same, the 25 th to 30 th bases are the same as the first 6 base sequences of the nonspecific target sequence, and the 31 th to 48 th bases are the same as H1b_nThe first 18 base sequences of the probes are identical.

The invention provides a novel coronavirus detection kit (a specific preparation method is shown in example 1), wherein 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which generates a hybrid chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 1 nonspecific target sequence is selected, the sequence is shown as SEQ ID NO.4, and the probe pair which has hybridization chain reaction with the nonspecific target sequence is H1b₁And H2b1, said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO. 6. Preferably, the detection line on the colloidal gold test strip is also provided with a single element which can be complementary with the specific target sequenceThe chain sequence S is shown as SEQ ID NO. 7.

Based on the principle of the invention, the invention further provides another specific preparation mode of a novel coronavirus detection kit, which is shown in example 2), wherein 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which generates hybrid chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 2 nonspecific target sequences are selected, and comprise 1 nonspecific target sequence (1) and 1 nonspecific target sequence (2), the sequence of the nonspecific target sequence (1) is shown as SEQ ID NO.1, the sequence of the nonspecific target sequence (2) is shown as SEQ ID NO.8, and the probe pair which has hybridization chain reaction with the nonspecific target sequence (1) is H1b₁And H2b₁Said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO.6, and the probe pair which has hybridization chain reaction with the non-specific target sequence (2) is H1b₂And H1b₂Said H1b₂The sequence is shown as SEQ ID NO.9, and the H2b₂The sequence is shown as SEQ ID NO. 10. Preferably, the detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7.

It should be noted that, it is fully within the skill of the art to further select more specific target sequences or non-specific target sequences according to the principles and content of the present invention, so as to complete the design of the corresponding probe pairs, implement the detection method based on the hybridization chain reaction to amplify the signal output signal and the preparation of the detection kit of the present invention. For example, X is 2, i.e., 2 segments of the specific target sequence selected for amplification; the probe pairs respectively designed for the 2 specific target sequences are H1a₁Probe and H2a₁A probe; h1a₂Probe and H2a₂The total number of the probes is 4; designing Y as 3, and selecting 3 sections of nonspecific target sequences for amplification; the probe pairs respectively designed for the 3 specific target sequences are H1b₁Probe and H2b₁A probe; h1b₂Probe and H2b₂A probe; h1b₃Probe and H2b₃And 6 probes are counted, then the probes react at room temperature to form hybrid chain reaction products, and then the hybrid chain reaction products are added to the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip. In view of the limited accessibility of the number of test operations, the present invention provides only 2 example test details, but it should not be construed as limiting the applicability of the invention. The person skilled in the art can implement more amplification schemes based on the technical schemes disclosed in the present invention, and so on.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1: two-site hybridization chain reaction amplification detection method aiming at novel coronavirus single-stranded nucleotide molecules Kit thereof

As described above, example 1 of the present invention further provides a detection method and kit for amplifying an output signal based on a hybridization chain reaction, wherein the single-stranded nucleic acid molecule to be amplified is a single-stranded ribonucleic acid of a novel coronavirus or a corresponding deoxyribonucleotide thereof (the difference is that uracil replaces thymine), and in this test, the applicant performed a test using the corresponding deoxyribonucleotide in consideration of safety, but those skilled in the art know that the same protocol can be applied to the single-stranded ribonucleic acid of the novel coronavirus. The following are specific embodiments:

(1) and (3) selecting a target sequence:

selecting 1 specific target sequence in the deoxynucleotide corresponding to the single-stranded ribonucleic acid of which the single-stranded nucleic acid molecule to be amplified is the novel coronavirus, wherein the sequence is shown as SEQ ID NO.1, the non-specific target sequence is selected from 1, and the sequence is shown as SEQ ID NO.4,

(2) sequence design:

the probe pair which generates hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁Having a sequence shown in SEQ ID NO.3, andthe probe pair for the non-specific target sequence to perform hybridization chain reaction is H1b₁And H2b1, said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO. 6. The sequence information mentioned in this example 1 is shown in table 1 below. In the H2b₁A fluorescent group (FAM) is added at the 5' end of the probe; the H1a₁Probe and H1b₁The 3' ends of the probes are all added with Biotin labels (Biotin).

(3) And (3) detection:

adding the single-stranded nucleic acid molecule to be amplified and the H1a on a colloidal gold test strip_mProbe and H2a_mA probe and the H1b_nProbe and H2b_nAnd (3) reacting the probe at room temperature to form a hybrid chain reaction product, and then adding the hybrid chain reaction product on the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip.

Further describing (3) the specific steps of the hybridization chain reaction-based detection and the preparation manner of the related detection kit are as follows, but those skilled in the art will know that any design scheme of the target sequence and the probe sequence of the present invention, which can capture the specific target sequence and the non-specific target sequence, can be used to realize the example 1 of the present invention, and the selection of the sample preparation and the buffer solution, etc. is not the main inventive point of the present invention.

1) Preparation of a detection kit:

a) the sample pad was completely soaked in the sample pad pre-treatment buffer and left for 30 min. The pretreatment buffer was then discarded and washed with copious amounts of PBS buffer and dried.

b) The conjugate pad was completely soaked in the conjugate pad pre-treatment buffer and left for 30 min. The pretreatment buffer was then discarded and washed with copious amounts of PBS buffer and dried.

c) And taking a proper amount of AuNPs-SA, diluting the AuNPs-SA to be 0.02mg/ml by using PBS, spotting the AuNPs-SA on the pretreated bonding pad by using a pipette, and drying the AuNPs-SA for later use.

d) And (3) scribing a T line: taking 5OD S sequence which can be complementary with the specific target sequence and is shown as SEQ ID NO.7, centrifuging at 12000r/min at 4 ℃ for 30S, and preparing 100 mu M stock solution (the volume of the stock solution to be added is packed) by using TE buffer solution. After 1mg of streptavidin was dissolved in 1ml of LTE buffer, a defined amount of streptavidin was mixed with the T-line sequence in a volume ratio of 2: 1. After mixing, the mixture was left at room temperature for 3 hours. Centrifuging the mixed solution at 12000rpm at 4 deg.C for 10min with 30KD ultrafilter tube, discarding the lower layer solution, and centrifuging at 1000g for 30min with inverted ultrafilter tube at 4 deg.C to obtain about 30 μ L solution. When the membrane is scratched, the solution is scratched on the nitrocellulose membrane at about 1/3 points on the side close to the conjugate pad at a flow rate of 1. mu.L/cm.

e) C, line drawing: BSA-biotin was diluted to 1.2mg/mL with PBS and drawn at a rate of 1. mu.L/cm onto the nitrocellulose membrane at about 1/3 on the side near the absorbent pad. After two membrane scribes (T line and C line), the nitrocellulose membrane is dried at room temperature in a dark place for standby.

f) Assembling the test strip: the detection line and quality control line detection method comprises the steps of sequentially overlapping and bonding a pretreated sample pad (made of a glass cellulose film), a prepared binding pad (made of a glass cellulose film) coated with a prepared colloidal gold-labeled aptamer, a nitrocellulose film labeled with a detection line and a quality control line and a water absorption pad (made of water absorption filter paper) on a PVC (polyvinyl chloride) base plate, wherein the nitrocellulose film labeled with the detection line and the quality control line is fixed on the base plate at first, one end of the nitrocellulose film is pressed for 2mm by the water absorption pad, the other end of the nitrocellulose film is pressed for 2mm by the binding pad, and the binding pad is pressed for 2mm by the sample pad.

g) Preparing a probe solution: probes were designed according to the probe sequence design procedure in example 1 above, and then formulated with Tris-HCl buffer (pH 8.0, NaCl 0.75M) to a concentration of 1 μ M.

h) Preparation of a loading buffer: 17.53g of NaCl and 8.82g of sodium citrate dihydrate were weighed, dissolved in 800mL of ultrapure water, adjusted to pH 7.0 with 1M hydrochloric acid, and the volume was adjusted to 1L. The solution was diluted 80-fold with ultrapure water to prepare a loading buffer.

2) Sample treatment:

the positive control is a new coronavirus solution reaching 4000 copies/mL; negative control is no target sequence buffer solution; adding the synthesized single-stranded nucleic acid molecules to be amplified into a 170 mu LTE buffer solution to prepare a solution of the single-stranded nucleic acid molecules to be amplified with the concentration of 0.1 mu M (6 mu g), centrifuging at the rotating speed of 7000 rpm for 2 minutes, and shaking and uniformly mixing.

3) And (3) hybridization chain reaction:

prepare 15 μ L system according to the following ratio: mu.L of long-chain DNA, 2. mu. L H1, 2. mu. L H2 and 9. mu.L of Tris-HCl buffer were added thereto, and the mixture was allowed to stand at room temperature for 15 min.

4) Sample loading and colloidal gold visual detection:

after the reaction, 85. mu.L of the sample buffer solution was added to the reaction solution to prepare 100. mu.L of the solution, and the test strip sample pad was immersed in the solution to carry out the test strip reaction for 10 min.

a) Visual detection of colloidal gold: and after the test strip reaction, photographing the test strip by using a mobile phone. As shown in FIG. 6, a red band appeared on the T line, which is a positive result, and a red band did not appear, which is a negative result.

b) Fluorescence visualization detection: and (3) placing the test strip under a body type fluorescence microscope, adjusting blue light excitation, and carrying out fluorescence observation. As shown in FIG. 7, the result was positive when there was fluorescence at the line T, and the result was negative when there was no fluorescence.

Table 1 sequence information involved in this example 1

Example 2: three-position hybridization chain reaction amplification detection aiming at novel coronavirus single-stranded nucleotide molecule Kit thereof

All steps in example 2 are identical to those in example 1, and are not repeated, and the core difference lies in the selection of target sequences and probe design. Specifically, the method comprises the following steps:

the specific target sequence is selected from 1, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 2 nonspecific target sequences are selected,comprises 1 each of a non-specific target sequence (1) and a non-specific target sequence (2), the sequence of the non-specific target sequence (1) is shown as SEQ ID NO.4, the sequence of the non-specific target sequence (2) is shown as SEQ ID NO.8, and the probe pair which has hybridization chain reaction with the non-specific target sequence (1) is H1b₁And H2b₁Said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO.6, and the probe pair which has hybridization chain reaction with the non-specific target sequence (2) is H1b₂And H1b₂Said H1b₂The sequence is shown as SEQ ID NO.9, and the H2b₂The sequence is shown as SEQ ID NO. 10. The detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7. The sequence information mentioned in this example 2 is shown in table 2 below. In the H2a₁The 5' end of the probe is added with digoxin antigen markers, and when the test strip is manufactured, 1mg/mL digoxin antibody is used for marking at the T line of the test strip.

Table 2 sequence information involved in this example 2

The results of the dual visualization test of example 2 are as follows:

a) visual detection of colloidal gold: and after the test strip reaction, photographing the test strip by using a mobile phone. As shown in FIG. 8, three probe pairs were added, including H1a₁Probe and H2a₁Probe, H1b₁Probe and H2b₁Probe, H1b₂Probe and H2b₂The test strip for reaction detection of the probes and the test strip after reaction of one group of the probes are independently added have red strips at the T line, and the red strips are positive results. Only long chain and no probe is added, and no red appears at the test strip detection lineColor band, negative result.

b) Fluorescence visualization detection: and (3) placing the test strip under a body type fluorescence microscope, adjusting blue light excitation, and carrying out fluorescence observation. As shown in FIG. 9, three probe pairs were added, including H1a₁Probe and H2a₁Probe, H1b₁Probe and H2b₁Probe, H1b₂Probe and H2b₂The probe, the test strip for reaction detection and the test strip for detection after the probe is independently added into one group of probes for reaction all show green fluorescent bands at the T line, and the result is positive. And only the test strip detection line with the long chain and without the added probe has no green fluorescent band, and the test strip is a negative result.

Example 3: dual target capture detection for novel coronaviruses

Example 3 provides the results of differential capture effect assays using complementary strand and/or antibody labels on colloidal gold test strips. Wherein the operation of the group (1) is to scribe the complementary strand S only on the detection line on the colloidal gold test strip; group (2) was conducted by streaking only the detection line on the colloidal gold test strip with digoxin antibody, which was allowed to react with H2a₁Digoxin label binding is carried at the 5' end of the probe; the operation of group (3) is to simultaneously use the antibody and the complementary strand S to scribe on the detection line of the colloidal gold test strip, and other specific operations are completely consistent with those of example 2 except the above description, including target sequence selection, probe design and detection scheme. The results of the alignment test in example 3 are shown in FIG. 10.

According to the detection result, the T line capture effect of simultaneously adopting the complementary chain and the antibody for labeling is obviously better than the capture effect of only adopting one of the complementary chain or the antibody for labeling.

Example 4: optimized design of detection method for novel coronavirus

During the pre-step test process of the above embodiments, the applicant further designs various optimized designs which may affect the detection result, so as to search for suitable reaction conditions. Specifically, the application makes the following designs for reaction time, reaction temperature and ion concentration:

(1) reaction time: 4 lanes are set, 2 of which are experimental groups, and target sequences and probe groups are added; 1 is a control group, and a probe group is added, and a target group is not added; 1 is blank, no probe set and no target set. The hybridization chain reaction time was set at 0min, 5min, 10min, 15min, 20 min, 30 min. From the fluorescence visualization electrophoresis results of FIG. 11, the fluorescence coloration effect was more significant at 15 minutes between the hybridization chain reactions.

Based on the rapid reaction, the technical scheme of the invention can enable detection people to obtain a detection result within half an hour through a test strip technology, the detection time is short (sampling: 5 min; adding a reaction reagent: 15 min; loading a test strip for 5 min; visual detection: 5 min; the experiment can be completed within 30 min), and the technical advantages of rapid and convenient detection are ensured.

(2) Reaction temperature: and 13 lanes are arranged, wherein 1 group is a standard sample, the other groups are test groups, the hybridization chain reaction temperature is respectively set to be 0 ℃, 25 ℃, 50 ℃ and 70 ℃, and 2 groups of samples are added at each temperature, and from the fluorescence visualization electrophoresis result of fig. 12, when the hybridization chain reaction temperature is about 25 ℃, the reaction product is the longest, and the hybridization chain reaction is more suitable for being performed.

(3) Ion concentration: 20 lanes are set, wherein 1 group is a standard sample, and the other groups are test groups, and NaCl with different concentrations is added into the hybridization chain reaction system as follows: 0M, 0.25M,0.5M, 0.75M, 1M, 1.5M, from the fluorescence visualization electrophoresis result of FIG. 13, the ion concentration has certain influence on the hybridization chain reaction efficiency, the hybridization chain reaction does not occur when the concentration is 0M NaCl, and the reaction is more thorough when the concentration is higher in a certain range. The optimum concentration was 0.75M, as judged from the remaining amount of the reactant.

To facilitate the practice by those skilled in the art, Table 1 lists the individual ingredient purchasers and their specific trade names in the examples.

TABLE 3 purchasers of the major ingredients and their specific trade names

For the avoidance of doubt, the aforementioned multi-site hybridization chain reaction, bispecific capture, dual visualization detection schemes are not interdependent or exclusive, but the combined use will maximize the technical effects of the present invention.

Sequence listing

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Claims

1. A detection method based on hybrid chain reaction amplified output signals is characterized by comprising the following steps: (1) selecting a target sequence; (2) designing a hairpin probe; and (3) detection based on hybridization chain reaction;

the target sequence selection comprises: selecting X specific target sequences and Y non-specific target sequences of a single-stranded nucleic acid molecule to be amplified as target sequences together, wherein X and Y are integers which are more than or equal to 1;

the hairpin probe design comprises: designing X groups of probe pairs for respectively carrying out hybridization chain reaction on the selected X specific target sequences, wherein the X groups of probe pairs comprise H1a_mProbe and H2a_m2X probes in total, wherein m is respectively equal to any integer from 1 to X, and Y groups of probe pairs which are selected from Y non-specific target sequences and respectively subjected to hybridization chain reaction comprise H1b_nProbe and H2b_n2Y probes are counted, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by a hybridization chain reaction under the action of the corresponding probe pairs;

the hybridization chain reaction-based assay comprises: adding the single-stranded nucleic acid molecule to be amplified and the DNA comprising H1a on a colloidal gold test strip_mProbe and H2a_mThe total number of the probes is 2X and the probes comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, the probes react at room temperature to form hybrid chain reaction products, and then the hybrid chain reaction products are added to the colloidal gold test strip to analyze the signal mark change on the colloidal gold test strip.

2. The method of claim 1 for detecting amplified output signals based on hybridization chain reaction, wherein: the H2b_nThe 5' end of the probe is also marked with a fluorescent group; the H1a_mProbe and H1b_nThe 3' ends of the probes are all provided with biotin labels, the detection based on the hybrid chain reaction is detected by adopting a colloidal gold test strip, and the colloidal gold on a sample pad on the colloidal gold test strip is provided with streptavidin capable of identifying the biotin labels.

3. The method of claim 1 for detecting amplified output signals based on hybridization chain reaction, wherein: the H2a_mThe 5' end of the probe is also provided with an antigen marker, and the detection line on the colloidal gold test strip is also provided with an antibody which can be specifically combined with the antigen marker.

4. The method of claim 1 for detecting amplified output signals based on hybridization chain reaction, wherein: the detection line on the colloidal gold test strip is also provided with a single-chain sequence S, and the single-chain sequence S can be matched with one or more sections of sequences R which are specific in the long-chain open reading frame of the single-chain nucleic acid molecule to be amplified_nComplementation; with the proviso that said one or more sequences R_nAre not selected as the specific target sequence and participate in the hybridization chain reaction.

5. The hybridization chain reaction based detection method for amplified output signals according to any one of claims 1 to 4, characterized in that the steps are in particular:

(1) and (3) selecting a target sequence:

(2) designing a hairpin probe:

the H1b_nThe sequence of the probe can be divided into 5 'to 3' directionsh. k, h, f four sequences;

(3) detection based on hybridization chain reaction:

6. The method of claim 5, wherein the amplified output signal is detected by a hybridization chain reaction:

the length of the non-specific target sequence is 24 bases, and the length of the specific target sequence is 24 bases;

the H1a_mThe probe is 48 bases in length, the first 18 bases are the same as the last 18 bases of the specific target sequence from the 5 'end to the 3' end, the 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be reversely paired into a double strand, the 25 th to 42 th bases are reversely paired with the first 18 bases, and the 43 th to 48 th bases are reversely paired with the first 6 bases of the specific target sequence;

the H2a_mThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1a_mThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1a_mThe 25 th to 42 th base sequences of the probe are identical, the 25 th to 30 th bases are identical to the first 6 base sequences of the specific target sequence, and the 31 th to 48 th bases are identical to H1a_mThe first 18 base sequences of the probes are identical;

the H1b_nThe probe is 48 bases in length, the first 18 bases are the same as the last 18 bases of the nonspecific target sequence in the direction from the 5 'end to the 3' end, the 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be reverse-sequence-paired into a double strand, the 25 th to 42 th bases are reverse-sequence-paired with the first 18 bases, and the 43 th to 48 th bases are reverse-sequence-paired with the first 6 bases of the nonspecific target sequence;

the H2b_nThe length of the probe is 48 bases, and from the 5 'end to the 3' end, the first 6 bases are connected with H1b_nThe 25 th to 42 th bases of the probe are subjected to reverse sequence pairing, and the 7 th to 24 th bases of the probe are subjected to H1b_nThe 25 th to 42 th base sequences of the probe are the same, the 25 th to 30 th bases are the same as the first 6 base sequences of the nonspecific target sequence, and the 31 th to 48 th bases are the same as H1b_nThe first 18 base sequences of the probes are identical.

7. The method for detecting amplified output signal based on hybridization chain reaction according to any of claims 2 to 3 and claims 5 to 6, characterized in that: when the single-stranded nucleic acid molecule to be amplified is the single-stranded ribonucleic acid molecule of the novel coronavirus, 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 1 nonspecific target sequence is selected, the sequence is shown as SEQ ID NO.4, and the probe pair which has hybridization chain reaction with the nonspecific target sequence is H1b₁And H2b1, said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO. 6.

8. The method of claim 7, wherein the amplified output signal is detected by a hybridization chain reaction: the detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7.

9. The method for detecting amplified output signal based on hybridization chain reaction according to any of claims 2 to 3 and claim 5, wherein: when the single-stranded nucleic acid molecule to be amplified is the single-stranded ribonucleic acid molecule of the novel coronavirus, 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 2 nonspecific target sequences are selected, and comprise 1 nonspecific target sequence (1) and 1 nonspecific target sequence (2), the sequence of the nonspecific target sequence (1) is shown as SEQ ID NO.4, the sequence of the nonspecific target sequence (2) is shown as SEQ ID NO.8, and the probe pair which has hybridization chain reaction with the nonspecific target sequence (1) is H1b₁And H2b₁Said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO.6, and the probe pair which has hybridization chain reaction with the non-specific target sequence (2) is H1b₂And H1b₂Said H1b₂The sequence is shown as SEQ ID NO.9, and the H2b₂The sequence is shown as SEQ ID NO. 10.

10. The method of claim 9 for detecting amplified output signals based on hybridization chain reaction, wherein: the detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7.

11. A single-stranded nucleic acid molecule detection kit based on a hybridization chain reaction amplified output signal is characterized by at least comprising:

(1) hairpin probes comprising (a) X sets of probe pairs capable of priming the respective hybridization chain reaction of X specific target sequences of a single-stranded nucleic acid molecule to be amplified, said X sets of probe pairs further comprising H1a_mProbe and H2a_m2X probes are counted, and m is respectively equal to any integer from 1 to X; and (b) Y group probe pairs which can trigger the hybridization chain reaction of Y non-specific target sequences of the single-stranded nucleic acid molecule to be amplified respectively, wherein the Y group probe pairs further comprise H1b_nProbe and H2b_nThe probes are 2Y probes in total, and n is respectively equal to any integer from 1 to Y; the X specific target sequences and the Y non-specific target sequences can be amplified by hybridization chain reaction under the action of the corresponding probe pairs;

12. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 11, wherein: the H2b_nThe 5' end of the probe is also marked with a fluorescent group; the H1a_mProbe and H1b_nThe 3' ends of the probes are all provided with biotin labels, the detection based on the hybrid chain reaction is detected by adopting a colloidal gold test strip, and the colloidal gold on a sample pad on the colloidal gold test strip is provided with streptavidin capable of identifying the biotin labels.

13. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 11, wherein: the H2a_mThe 5' end of the probe is also provided with an antigen marker, and the detection line on the colloidal gold test strip is also provided with an antibody which can be specifically combined with the antigen marker.

14. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 11, wherein: the detection line on the colloidal gold test strip is also provided with a single-chain sequence S, and the single-chain sequence S can be matched with one or more sections of sequences R which are specific in the long-chain open reading frame of the single-chain nucleic acid molecule to be amplified_nComplementation; provided however thatOne or more sequences R_nAre not selected as the specific target sequence and participate in the hybridization chain reaction.

15. The kit for detecting a single-stranded nucleic acid molecule based on amplification output signal of hybridization chain reaction according to claims 11 to 14, wherein:

the H2b_nThe sequence of the probe is divided into k, h, f and h four sequences from 5 'to 3', wherein f and f sequences, h and h sequences and k sequences can be respectively complementary, the length of f and f sequences is 4-10 bases, the length of h and h sequences is 12-24 bases, and the length of k and k sequences is 4-10 bases.

16. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 15,

the H1a_mThe probe is 48 bases in length, from 5 'end to 3' end, onwards18 bases are identical to the last 18 bases of the specific target sequence, 19 th to 24 th bases are a loop forming part containing 6 bases which cannot be reverse sequence paired into a double strand, 25 th to 42 th bases are reverse sequence paired with the first 18 bases, and 43 th to 48 th bases are reverse sequence paired with the first 6 bases of the specific target sequence;

17. The kit for detecting a single-stranded nucleic acid molecule based on amplification output signal of hybridization chain reaction according to claims 12 to 13, wherein: when the single-stranded nucleic acid molecule to be amplified is the single-stranded ribonucleic acid molecule of the novel coronavirus, 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 1 nonspecific target sequence is selected, the sequence is shown as SEQ ID NO.4, and the probe pair which has hybridization chain reaction with the nonspecific target sequence is H1b₁And H2b1, said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO. 6.

18. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 17, wherein: the detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7.

19. The kit for detecting a single-stranded nucleic acid molecule based on amplification output signal of hybridization chain reaction according to any one of claims 12 to 13, wherein: when the single-stranded nucleic acid molecule to be amplified is the single-stranded ribonucleic acid molecule of the novel coronavirus, 1 specific target sequence is selected, the sequence is shown as SEQ ID NO.1, and the probe pair which has hybridization chain reaction with the specific target sequence is H1a₁And H2a₁Said H1a₁The sequence is shown as SEQ ID NO.2, and the H2a₁The sequence is shown as SEQ ID NO.3, 2 nonspecific target sequences are selected, and comprise 1 nonspecific target sequence (1) and 1 nonspecific target sequence (2), the sequence of the nonspecific target sequence (1) is shown as SEQ ID NO.1, the sequence of the nonspecific target sequence (2) is shown as SEQ ID NO.8, and the probe pair which has hybridization chain reaction with the nonspecific target sequence (1) is H1b₁And H2b₁Said H1b₁The sequence is shown as SEQ ID NO.5, and the H2b₁The sequence is shown as SEQ ID NO.6, and the probe pair which has hybridization chain reaction with the non-specific target sequence (2) is H1b₂And H1b₂Said H1b₂The sequence is shown as SEQ ID NO.9, and the H2b₂The sequence is shown as SEQ ID NO. 10.

20. The kit for detecting a single-stranded nucleic acid molecule based on an amplified output signal of a hybridization chain reaction according to claim 19, wherein: the detection line on the colloidal gold test strip is also provided with a single-stranded sequence S which can be complementary with the specific target sequence, and the sequence of the single-stranded sequence S is shown as SEQ ID NO. 7.