CN114410640B

CN114410640B - Aptamer for detecting measles virus, kit and application

Info

Publication number: CN114410640B
Application number: CN202210179393.1A
Authority: CN
Inventors: 周代志; 罗建刚; 庄杨; 谢明铎; 刘光存
Original assignee: Shitong Landa Shenzhen Biotechnology Development Co ltd
Current assignee: Shitong Landa Shenzhen Biotechnology Development Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2023-07-11
Anticipated expiration: 2042-02-25
Also published as: CN114410640A

Abstract

The application discloses a nucleic acid aptamer for detecting measles virus, a kit and application. The aptamer for detecting measles virus is a nucleic acid fragment of the sequence shown in Seq ID No.1 or the complementary sequence thereof. The nucleic acid aptamer for detecting measles virus has the advantages of high specificity and affinity, small molecule, easiness in synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like, and can be modified; overcomes the problems that measles virus specific antibody is not easy to express and synthesize and is easy to degrade, nonspecific immunogenicity exists, and the like. The nucleic acid aptamer is used for measles virus detection, and has the advantages of high sensitivity, good specificity, wide measurement range, simplicity in operation and capability of rapidly and accurately detecting measles virus.

Description

Aptamer for detecting measles virus, kit and application

Technical Field

The application relates to the technical field of measles virus detection, in particular to a nucleic acid aptamer for detecting measles virus, a kit and application.

Background

Measles virus (measles virus) is the causative agent of measles, a single negative strand RNA virus belonging to the Paramyxoviridae family in classification. Measles virus has only one serotype, but recent studies have shown that there is also a small variation in the antigen of measles virus. Measles is a common acute infectious disease in children, which is very contagious. The clinical characteristics are fever, runny nose, cough, conjunctivitis, eruption and the like. Humans are the only natural host of measles virus, acute patients are the source of infection and patients are infectious six days before to 3 days after eruption. Can be spread by means of spray and intimate contact, etc. Current laboratory diagnostic mode of measles virus infection: virus separation, antibody detection and antigen detection.

Sample detection of measles virus is important in clinical detection and experimental research. In the prior art, specific antibodies are mostly adopted for identification of virus infection or virus protein detection, so that the preparation is complex, the steps are complex, the cost is high, and certain defects are caused in large-scale popularization. In addition, the specific antibody as a protein component has the defects of difficult expression and synthesis, easy degradation, nonspecific immunogenicity and the like.

Therefore, how to develop a new measles virus detection technology with strong specificity and high sensitivity is a problem to be solved urgently at present.

Disclosure of Invention

The object of the present application is to provide a novel aptamer, kit and application for detecting measles virus.

The application adopts the following technical scheme:

in one aspect, the application discloses a nucleic acid aptamer for detecting measles virus, which is a nucleic acid fragment of the sequence shown in Seq ID No.1 or the complementary sequence thereof.

Seq ID No.1：

5’-TTCAGCACTCCACGCATAGCCATCTTACCATCCTTTACGATAAAGTCTGGGGCGTCGGCTACCTATGCGTGCTACCGTGAA-3’。

It should be noted that, the aptamer of the present application is a aptamer against an antigen epitope of a measles virus target protein, which is screened by an artificial screening technology-exponential enrichment ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX), and has higher specificity and affinity. Compared with the existing measles virus specific antibody, the nucleic acid aptamer disclosed by the application overcomes the problems that the antibody is not easy to express and synthesize, is easy to degrade, has non-specific immunogenicity and the like; in addition, the aptamer has the advantages of small molecule, easiness in modification and synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like. The nucleic acid aptamer is used for measles virus detection, has the advantages of high sensitivity, good specificity, wide measurement range and simple operation, can rapidly and accurately detect measles virus target protein, is suitable for large-scale popularization, and has good market prospect. In one implementation mode of the application, the detection results of measles virus infected patient samples and healthy human samples show that the positive detection rate, namely the sensitivity, of the nucleic acid aptamer is 98% for measles virus infected patients, and the positive detection rate for healthy people is 0%; the nucleic acid aptamer can effectively detect measles virus infected patients without false positive.

It should be noted that the key point of the present application is to find out that the nucleic acid fragment of the sequence shown in Seq ID No.1 can be used as a nucleic acid aptamer for specifically detecting measles virus; it will be appreciated that the nucleic acid fragments of the complementary sequences of the sequences shown in Seq ID No.1 have the same specificity and affinity as well.

In one implementation of the present application, the nucleic acid sequence of the aptamer of the present application is conjugated with at least one of a fluorescent substance, a nano luminescent material, biotin, digoxin and an enzyme label.

Preferably, the fluorescent substance is a fluorescent group, which includes a FAM fluorescent group or a Cy5 fluorescent group. It is understood that FAM fluorophores or Cy5 fluorophores are merely fluorophores employed in one implementation of the present application, and that other fluorophores are not excluded.

Preferably, the nano luminescent material is a quantum dot or an up-conversion luminescent material.

Preferably, the enzyme label is horseradish peroxidase, sucrase or a functional polypeptide.

In one implementation of the present application, the nucleic acid sequence of the aptamer of the present application has at least one modification of phosphorylation, methylation, demethylation, amination, sulfhydrylation, isotopication, carboxylation, thio-modification, 2-methoxy modification, 3' -end inversion dT/dG modification.

It should be noted that the key point of the present application is to develop and obtain aptamer capable of specifically and sensitively detecting measles virus, and for specific labeling or modification of aptamer, reference may be made to the existing labeling or modification scheme of aptamer.

Another aspect of the present application discloses a peptide nucleic acid engineered from the nucleic acid aptamer of the present application.

It should be noted that peptide nucleic acids are a class of DNA analogues in which the backbone of the sugar phosphate is replaced by a polypeptide backbone, i.e. the backbone of the pentose phosphate diester linkage in DNA is replaced by a neutral peptide chain amide 2-aminoethylglycine linkage, the remainder being identical to DNA, and peptide nucleic acids can recognize and bind to DNA or RNA sequences in the form of Watson-Crick base pairing to form a stable duplex structure. It will be appreciated that the peptide nucleic acids of the present application are engineered with the nucleic acid aptamers of the present application and thus have similar specificity and sensitivity to the nucleic acid aptamers of the present application.

It should be noted that, the key of the present application is to specifically detect nucleic acid aptamers of measles virus, and as to how to specifically modify the nucleic acid aptamers into peptide nucleic acids, reference may be made to the prior art, and no specific limitation is made herein.

In a further aspect, a kit for the detection of measles virus is disclosed, comprising the nucleic acid aptamer of the present application, or the peptide nucleic acid of the present application.

In one implementation of the present application, the kit of the present application further comprises at least one of the following:

(A) A protein-coated plate for immobilizing the sample solution;

(B) A protein coating liquid;

(C) A sealing liquid;

(D) A rinsing liquid;

(E) Sample pretreatment liquid.

The kit is characterized by comprising the nucleic acid aptamer, and can specifically detect measles virus; as for other consumable materials or reagents to be used, for example, (a) a protein-coated plate for fixing a sample solution, (B) a protein-coated liquid, (C) a blocking liquid, (D) a rinsing liquid, and (E) a sample pretreatment liquid, etc., may be assembled into a kit according to the need, or may be prepared by themselves or purchased separately.

In one implementation of the present application, the formulation of the protein coating solution is: 35mM NaHCO ₃ 、17mM Na ₂ CO ₃ pH9.3-pH9.9; the formula of the sealing liquid is as follows: 136mM NaCl, 2.6mM KCl, 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 0.05% Tween-20, 1% BSA, pH7.1-pH7.5; the formula of the rinsing liquid is as follows: 136mM NaCl, 2.6mM KCl, 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 0.05% Tween-20, pH7.1-pH7.5; the formula of the sample pretreatment liquid is as follows: 150mM NaCl, 1% Triton X-100, 50mM Tris, pH7.8-pH8.2.

In a further aspect, the application discloses the use of the aptamer of the application, or the peptide nucleic acid of the application, or the kit of the application, for preparing a medicament or a tool for diagnosing or treating diseases caused by measles virus.

It will be appreciated that the nucleic acid aptamers, peptide nucleic acids and kits of the present application are capable of specifically detecting measles virus; therefore, it can also be used for diagnosing diseases caused by measles virus. For example, whether the subject has a disease is determined by detecting whether measles virus is contained in an isolated sample of the subject, and thereby determining whether the subject is infected with measles virus. Thus, the nucleic acid aptamer, peptide nucleic acid and kit can be used for preparing medicines or tools for diagnosing or treating measles virus-caused diseases.

In a further aspect the present application discloses a method for the detection of measles virus for non-diagnostic therapeutic purposes comprising the use of a nucleic acid aptamer of the present application, or a peptide nucleic acid of the present application, or a kit of the present application, for the detection of measles virus in an environment, food, tool, ex vivo sample or biological source.

The detection method of the present application can be used for detecting measles virus and diseases caused by measles virus; however, in addition to this, measles virus in the environment, food, tools, ex vivo samples or material of biological origin can be detected to avoid spreading or transmission of measles virus. For example, measles virus tests are performed in hospitals or in particular environments, it being possible to determine whether these environments are at risk of measles virus infection; for another example, measles virus input can be avoided by performing measles virus detection on incoming foods, tools, ex vivo samples or biogenic material.

The beneficial effects of this application lie in:

the nucleic acid aptamer for detecting measles virus has the advantages of high specificity and affinity, small molecule, easiness in synthesis, difficulty in degradation, no toxicity, no immunogenicity and the like, and can be modified; overcomes the problems that measles virus specific antibody is not easy to express and synthesize and is easy to degrade, nonspecific immunogenicity exists, and the like. The nucleic acid aptamer is used for measles virus detection, and has the advantages of high sensitivity, good specificity, wide measurement range, simplicity in operation and capability of rapidly and accurately detecting measles virus.

Drawings

FIG. 1 is a secondary structure of an aptamer for use in detecting measles virus in the examples of the present application.

Detailed Description

The aptamer is a single-stranded nucleic acid, generally about 20-60 bases in size, capable of forming a specific three-dimensional structure by folding and performing high-affinity and high-specificity binding with a corresponding target molecule. The nucleic acid aptamer can be obtained by screening through an artificial screening technology simulating a natural evolution process, namely an exponential enrichment ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX), and is a novel nucleic acid molecule with a recognition function. Specific nucleic acid aptamers highly compatible with a target substance can be screened from a random single-stranded nucleic acid sequence library by using the SELEX technology, which has been successfully applied to screening of a number of target substances including metal ions, organic dyes, drugs, proteins, amino acids, and various cytokines, etc. The technology has the characteristics of large storage capacity, wide application range, high resolution, high affinity, relatively simple, rapid and economical screening process, high practicability and the like. The nucleic acid aptamer has the characteristics of high specificity and high affinity corresponding to the target substance, and is similar to the functions of the antibody, but the target range of the nucleic acid aptamer is wider, and the nucleic acid aptamer can be well applied to various aspects such as medical detection, diagnosis, treatment and the like.

The reason for the high specificity and affinity of the aptamer is that single-stranded nucleic acids, when encountering a target molecule, can form unique three-dimensional structures such as pockets, hairpin, G-tetramers, etc., which when binding to the target molecule are based on the principle that antibodies specifically recognize the corresponding epitope, e.g. by hydrogen bonding, hydrophobic structures, ionic bonds, etc., specifically bind to the target molecule. Based on the biological characteristics that the aptamer has higher specificity for identifying pathogenic microorganisms or pathological cells, the aptamer can be widely applied to detection technology and biosensor construction, and can realize accurate diagnosis of pathogens or diseases.

However, there are no specific measles virus detection nucleic acid aptamers and related studies and reports. Thus, if nucleic acid aptamers for detecting measles virus can be developed, not only the specificity and sensitivity of measles virus detection can be improved; moreover, the problems of difficult expression and synthesis, easy degradation and nonspecific immunogenicity of the antibody can be avoided.

Therefore, the SELEX technology is creatively adopted to screen nucleic acid aptamer aiming at measles virus target protein epitope, so as to obtain measles virus detection nucleic acid aptamer with higher specificity and affinity.

Based on the above studies and knowledge, the present application developed a nucleic acid aptamer for detecting measles virus, which is a nucleic acid fragment of the sequence shown in Seq ID No.1 or the complementary sequence thereof;

Seq ID No.1：

the nucleic acid aptamer has the following advantages:

the aptamer aiming at measles virus target protein antigen epitope, which is screened by the SELEX technology, has higher specificity and affinity; and has the advantages of no immunogenicity, small molecule, modification, easy synthesis, difficult degradation, no toxicity and the like. The aptamer sequence used in the application is obtained by screening through a SELEX technology, and has complete originality.

The nucleic acid aptamer has the advantages of high sensitivity, good specificity, wide measurement range, simple operation, capability of rapidly detecting target proteins, suitability for large-scale popularization and good market prospect. In one implementation of the application, in the detection of measles virus infected patient samples and healthy human samples, the positive detection rate, namely the sensitivity of measles virus infected patients is 98%; the positive detection rate of healthy people is 0%.

The nucleic acid aptamer can be used as a detection object, so that the problems that an antibody is not easy to express and synthesize, is easy to degrade, has nonspecific immunogenicity and the like can be effectively solved.

The present application is described in further detail below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.

Example 1 nucleic acid aptamer for detection of measles virus

1. Determination of measles virus target protein epitope

According to literature review and bioinformatic analysis, specific epitope regions on target proteins suitable as detection targets for nucleic acid aptamers are determined as follows:

(1) Selecting measles virus H protein as an aptamer to screen a target protein, wherein the target protein has a sequence shown as a Seq ID No. 2;

Seq ID No.2：

MSPQRDRINAFYKDNPHPKGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNATLLEARATNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLSRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKGSELSQLSMHRVFEVGVIRNPGLGAPVFHMTNYFEQSVSNDFSNCMVALGELKFAALCHREDSITIPYQGSGKSVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVNLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFRVSPNLFTVPIKEAGEDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNSR。

(2) Based on the determined amino acid sequence, nucleotide sequence synthesis and prokaryotic codon optimization are directly carried out in biological company, and the synthesized gene fragment is inserted into the BamHI/HindIII cleavage site of prokaryotic expression vector pET32 a. Nucleic acid sequence synthesis and insertion into prokaryotic expression vector pET32a were all accomplished by the company Shanghai, inc. of biological engineering.

After obtaining the cloning vector, the target protein for aptamer screening is induced to be expressed, which specifically comprises the following steps: (1) firstly, inoculating the preactivated bacterial liquid into a fresh LB culture medium according to a ratio of 1:100, and culturing at 37 ℃ for 2-2.5 h to ensure that the OD value of the bacterial liquid is about 0.6-0.8; (2) cooling the bacterial liquid in an ice-water mixture; (3) the final concentration was added with 0.1mM IPTG and the reaction was induced at 16℃for 15h.

2. Construction of aptamer libraries and primer design

(1) Single-stranded DNA messages containing 41 random sequences, which include 10, were synthesized in vitro by the Gene Synthesis service of the biological engineering (Shanghai) Co., ltd ¹⁴ The nucleotide sequences of the ssDNA, i.e., the initial single-stranded DNA random library, are as follows:

5’-TTCAGCACTCCACGCATAGC-(N41)-CCTATGCGTGCTACCGTGAA-3’

of these, N41 is a single-stranded DNA of 41 random sequences.

(2) Aiming at the 5' -end fixed sequence of the aptamer, an upstream primer P1 is designed and synthesized, wherein the upstream primer P1 is specifically a sequence shown by a Seq ID No.3, and is synthesized by a biological engineering (Shanghai) stock company; seq ID No.3:5'-TTCAGCACTCCACGCATAGC-3'.

(3) Aiming at the fixed sequence of the 3 'end of the aptamer, a downstream primer P2 is designed and synthesized, the downstream primer P2 is specifically a sequence shown by a Seq ID No.4, and simultaneously, a biotin group (biotin) is connected to the 5' end of the primer and is synthesized by a division company of biological engineering (Shanghai);

Seq ID No.4：5’-TTCACGGTAGCACGCATAGG-3’。

3. screening of nucleic acid aptamers from libraries using SELEX technology

(1) Positive screening: 10nmol of the initial single-stranded DNA random library was dissolved in 500. Mu.L of PBS solution, water-bathed with a constant temperature water bath at 92℃for 5min, followed by rapid insertion into ice for 10min, after which the treated initial single-stranded DNA random library was incubated with the target protein of the sequence shown by Seq ID No.2 on ice for 1h, ni-NTAMagnetic Agarose Beads was added and incubation was continued for 1h. After incubation was completed, the supernatant was removed by adsorption using a magnetic separator and the Beads were washed with 2mL of PBS. Finally, 10mL of precooled PBS is used for resuspension of the Beads, the Beads are fully blown and evenly mixed, then the Beads are centrifuged in a constant-temperature water bath at 92 ℃ for 10min and 13000g, and the supernatant is collected, thus obtaining the single-stranded DNA library for specifically recognizing the target protein after the first round of screening.

(2) And (3) PCR amplification: the single-stranded DNA library specifically recognizing the target protein after the first round of screening is subjected to PCR amplification to prepare a secondary library. The PCR reaction system is as follows: 25. Mu.L of Premix Tap Mix, 8. Mu.L of single-stranded DNA library after screening, 2.5. Mu.L of upstream primer P1, 2.5. Mu. L, ddH of downstream primer P2 ₂ O12. Mu.L, total volume 50. Mu.L. PCR cycle amplification was performed according to the following procedure: denaturation at 95℃for 3min, followed by 35 cycles of amplification: after the circulation is finished, the reaction is terminated by extending at 72 ℃ for 10min and finally maintaining at 16 ℃ for 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃. Agarose gel recovery is carried out on the library amplified by PCR to obtain pure library fragments, and a recovery kit is adopted

Gel Extraction Kit。

(3) Preparing a secondary single-stranded DNA library: further preparation of amplified secondary library: the agarose gel recovered library fragment and 100 u L streptavidin labeled magnetic beads were incubated for 20min at normal temperature, the double-stranded DNA library was bound to the surface of the magnetic beads by utilizing the affinity of biotin on the double-stranded DNA library with streptavidin, then the magnetic separator was used for adsorption, the supernatant was removed, and the magnetic beads were washed with 2mL PBS. The double-stranded DNA library was denatured into single strands by adding NaOH solution having a final concentration of 200mM at room temperature for 10min, wherein one strand with biotin-tag was bound to the magnetic beads and the one strand without biotin-tag was detached from the supernatant. Collecting supernatant, removing NaOH by a desalting column, adding 500 mu L of PBS, and collecting the solution to obtain the single-stranded DNA library required for the next round of screening.

(4) Repeating the steps (1) positive screening, (2) PCR amplification and (3) preparation of the secondary single-stranded DNA library for 15 times, wherein the single-stranded DNA library obtained by the final screening has the strongest recognition capability on target proteins.

Sequencing the single-stranded DNA obtained by the final screening by the Optimum in the Optimum of Pachyrhizus.

Sequencing results show that the single-stranded DNA obtained by the final screening in this example is the sequence shown in Seq ID No.1, namely the nucleic acid aptamer for detecting measles virus in this example.

Seq ID No.1：

(5) In order to sensitively detect the reaction product in the reaction system, the FAM fluorescent group is labeled at the 5' -end of the aptamer of the sequence shown in Seq ID No. 1. The aptamer labeled with FAM fluorophore was synthesized by the company limited of biological engineering (Shanghai).

Example 2 verification of nucleic acid aptamer

1. Two-level structure prediction

In this example, the secondary structure prediction was performed on the aptamer of the sequence shown in Seq ID No.1 obtained by the screening in example 1 using structure prediction software RNA Structure Program, and the results are shown in FIG. 1.

The results in FIG. 1 show that the nucleic acid aptamer of the sequence shown in Seq ID No.1 can form a specific stem-loop structure and hairpin structure.

2. Binding force test of aptamer and target protein

Detecting the binding force of the aptamer and the target protein by using a ForteBio Octet RED96 interaction analyzer, setting PBS as a control test, and referring to the following webpage according to experimental principles and operation steps:

http://hzaml.hzau.edu.cn/info/1107/3266.htm#/

the results of the ForteBio Octet RED96 interaction analyzer are shown in table 1.

TABLE 1 results of binding force test of aptamer to target protein

Aptamer	Dissociation constant Kd (nM) with target protein
		Measles virus-aptamer	7.8
PBS control	No combination

The results in Table 1 show that the aptamer of the sequence shown in Seq ID No.1 is able to bind specifically to the H protein of measles virus.

3. Specific detection

In the embodiment, the human serum albumin, the immune serum globulin, the measles virus H protein and the aptamer are respectively adopted for specific detection, and the experimental principle and the operation steps can be referred to the following webpage:

http://hzaml.hzau.edu.cn/info/1107/3266.htm#/

the experimental results show that the aptamer of the example 1 has no binding with human serum albumin and immune serum globulin, and only binds with measles virus H protein; the nucleic acid aptamer of example 1 was shown to have better specificity.

4. Stability test

At room temperature, 0.2. Mu.g of the aptamer of example 1 was placed in 1mL of serum, and 0.2. Mu.g of the aptamer of example 1 was placed in an aqueous solution. After 4 weeks at normal temperature, the sample was subjected to RT-PCR. The sequence shown by the upstream primer P1, namely the sequence shown by the Seq ID No.3, the sequence shown by the downstream primer P2, namely the sequence shown by the Seq ID No.4, and the PCR reaction system is as follows: 25. Mu.L of Premix Tap Mix, 8. Mu.L of aptamer dissolved in serum or aqueous solution, 2.5. Mu.L of upstream primer P1, 2.5. Mu. L, ddH of downstream primer P2 ₂ O12. Mu.L, total volume 50. Mu.L. PCR cycle amplification was performed according to the following procedure: denaturation at 95℃for 3min, followed by 35 cycles of amplification: after the circulation is finished, the reaction is terminated by extending at 72 ℃ for 10min and finally maintaining at 16 ℃ for 30s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃.

The detection result shows that the nucleic acid aptamer placed at normal temperature has stable structure and does not degrade.

5. Virus infection experiments

The sensitive host cells were infected with measles virus, while a negative control group without infection was set. Cells were plated in 6-well plates and incubated at MOI of 0.1 at infection in a 37℃cell incubator for 36h. Cells were digested with pancreatin 36h after infection, 200 μl of pancreatin was added to each well, centrifuged at 2000rpm to obtain cell pellet and supernatant removed, and resuspended in 500 μl of PBS. Cells were fixed and permeabilized, specifically with reference to pages https:// www.novopro.cn/arotics/2016201601627111 #/; and then mixing the nucleic acid aptamer with the fluorescent group label corresponding to measles virus with the cell suspension of the infected virus and the cell suspension of the uninfected control group respectively for 1h to enable the final concentration to reach the concentration of about 1 mug/mL, and analyzing and calculating the fluorescence value of the FAM channel by adopting a flow cytometry.

The results showed a significant increase in fluorescence values of aptamer markers corresponding to measles virus infected cell samples, indicating that the aptamer selected in example 1 was useful as a detector of measles virus infection.

Example 3 kit for detection of measles Virus

The kit for detecting measles virus comprises the following formula:

(A) A protein-coated plate for immobilizing the sample solution; the example uses a 96-well protein coated plate;

(B) A protein coating liquid;

(C) A sealing liquid;

(D) A rinsing liquid;

(E) Sample pretreatment liquid;

(F) The aptamer screened in example 1 was labeled with FAM fluorophore.

Wherein, (a) 96-well protein-coated plate for fixing sample solution: ELISA plates with protein adsorption capacity are used directly on the market. And (B) protein coating liquid: belongs to a conventional molecular detection reagent, and has a formula of 35mM NaHCO ₃ 、17mM Na ₂ CO ₃ pH9.3-pH9.9, a double distilled water configuration was used. (C) a blocking solution: belongs to a conventional molecular detection reagent, and has the formula of 136mM NaCl, 2.6mM KCl and 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 0.05% Tween-20, 1% BSA, pH7.1-pH7.5, double distilled water was used. (D) rinsing liquid: belongs to a conventional molecular detection reagent, and has the formula of 136mM NaCl, 2.6mM KCl and 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 0.05% Tween-20, pH7.1-pH7.5, using a double distilled water configuration. (E) sample pretreatment liquid: belongs to a conventional molecular detection reagent, and has the formula of 150mM NaCl, 1% Triton X-100 and 50mM Tris, pH7.8-pH8.2. (F) a fluorescent group-labeled aptamer: the FAM fluorophore-labeled aptamer synthesized in example 1 was dissolved in TE buffer and used at a working concentration of 200nM.

Example 4 use of the kit

A virus infection experiment was performed using the kit of example 4 with 50 clinical throat swab samples confirmed to be positive for measles virus and 50 clinical throat swab samples confirmed to be free of measles virus as controls.

Meanwhile, three commercial measles virus antigen detection kits were used as controls, and the three commercial measles virus antigen detection kits were labeled as kit a, kit B, and kit C, respectively.

The specific experimental method comprises the following steps:

1. before use, all reagents and samples are returned to room temperature, and the samples to be tested are diluted to proper concentration by coating liquid.

2. To the corresponding wells, 100. Mu.L of the sample to be tested was added and incubated for 2 hours at room temperature. The coating time can be optimized and adjusted according to actual conditions.

3. The solution was discarded and washed 2 times with rinse solution, 200 μl of rinse solution was added to each sample well. After the final wash is completed, the remaining rinse liquid is removed by pipetting or pouring.

4. 200. Mu.L of blocking solution was added to the sample wells and incubated for 2 hours at room temperature.

5. The solution was discarded. The washing step of step 3 was repeated.

6. To the sample wells, 100. Mu.L of the aptamer with fluorescent group label was added and incubated for 2 hours at room temperature in the dark. The incubation time can be optimally adjusted according to the actually detected fluorescence signal intensity.

7. The solution was discarded. The washing step of step 3 was repeated for a total of 4 washes.

8. The full-band laser enzyme-labeled instrument is used for detecting the sample hole, and the following steps are set: the excitation wavelength was 494nm and the detection wavelength was 522nm.

The result judging mode is as follows: and the positive result is obtained when the fluorescence value is greater than 50.

Statistical example 4 kit and as controls kit a, kit B and kit C the results of the detection of 50 measles virus positive samples and 50 measles virus free control samples are shown in table 2.

Table 2 detection results of aptamer and antigen detection kit on positive sample and control sample

The data in Table 2 are positive results and the number of samples, respectively, and for example, "49/50" means that the number of positive results detected in 50 measles virus positive samples is 49 parts, and "0/50" means that the number of positive results detected in 50 measles virus-free control samples is 0 part.

The results in table 2 show that the kit of example 4 is effective in detecting measles virus positive samples with a sensitivity of 98% and no false positive detection for the control samples without measles virus; the sensitivity of the kit A, the kit B and the kit C to measles virus positive samples is lower than that of the aptamer screened in the embodiment 1, and the kit B also has false positive detection.

The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.

SEQUENCE LISTING

<110> Shitonglanda (Shenzhen) Biotechnology development Co., ltd

<120> aptamer for detection of measles virus, kit and use thereof

<130> 21I33071

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 81

<212> DNA

<213> measles virus nucleic acid aptamer

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ttcagcactc cacgcatagc catcttacca tcctttacga taaagtctgg ggcgtcggct 60

acctatgcgt gctaccgtga a 81

<210> 2

<211> 617

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<213> immunogenic region of measles virus H protein

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Met Ser Pro Gln Arg Asp Arg Ile Asn Ala Phe Tyr Lys Asp Asn Pro

1 5 10 15

His Pro Lys Gly Ser Arg Ile Val Ile Asn Arg Glu His Leu Met Ile

20 25 30

Asp Arg Pro Tyr Val Leu Leu Ala Val Leu Phe Val Met Phe Leu Ser

35 40 45

Leu Ile Gly Leu Leu Ala Ile Ala Gly Ile Arg Leu His Arg Ala Ala

50 55 60

Ile Tyr Thr Ala Glu Ile His Lys Ser Leu Ser Thr Asn Leu Asp Val

65 70 75 80

Thr Asn Ser Ile Glu His Gln Val Lys Asp Val Leu Thr Pro Leu Phe

85 90 95

Lys Ile Ile Gly Asp Glu Val Gly Leu Arg Thr Pro Gln Arg Phe Thr

100 105 110

Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys Phe Leu Asn Pro Asp

115 120 125

Arg Glu Tyr Asp Phe Arg Asp Leu Thr Trp Cys Ile Asn Pro Pro Glu

130 135 140

Arg Ile Lys Leu Asp Tyr Asp Gln Tyr Cys Ala Asp Val Ala Ala Glu

145 150 155 160

Glu Leu Met Asn Ala Leu Val Asn Ala Thr Leu Leu Glu Ala Arg Ala

165 170 175

Thr Asn Gln Phe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr

180 185 190

Thr Ile Arg Gly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu

195 200 205

Tyr Leu Ser Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser

210 215 220

Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser

225 230 235 240

Ser Lys Gly Ser Glu Leu Ser Gln Leu Ser Met His Arg Val Phe Glu

245 250 255

Val Gly Val Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met

260 265 270

Thr Asn Tyr Phe Glu Gln Ser Val Ser Asn Asp Phe Ser Asn Cys Met

275 280 285

Val Ala Leu Gly Glu Leu Lys Phe Ala Ala Leu Cys His Arg Glu Asp

290 295 300

Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys Ser Val Ser Phe Gln

305 310 315 320

Leu Val Lys Leu Gly Val Trp Lys Ser Pro Thr Asp Met Gln Ser Trp

325 330 335

Val Pro Leu Ser Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser

340 345 350

Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro

355 360 365

Thr Thr Arg Thr Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln

370 375 380

Ala Cys Lys Gly Lys Ile Gln Ala Leu Cys Glu Asn Pro Glu Trp Ala

385 390 395 400

Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asn

405 410 415

Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly

420 425 430

Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His

435 440 445

Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu

450 455 460

Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg Phe Arg Val Ser Pro

465 470 475 480

Asn Leu Phe Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala

485 490 495

Pro Thr Tyr Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser

500 505 510

Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr

515 520 525

Tyr Asp Thr Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser

530 535 540

Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys

545 550 555 560

Gly Val Pro Ile Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys

565 570 575

Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly

580 585 590

His Ile Thr His Ser Gly Met Val Gly Met Gly Val Ser Cys Thr Val

595 600 605

Thr Arg Glu Asp Gly Thr Asn Ser Arg

610 615

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<400> 3

ttcagcactc cacgcatagc 20

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

ttcacggtag cacgcatagg 20

Claims

1. A nucleic acid aptamer for use in the detection of measles virus, characterized in that: the nucleic acid aptamer is a nucleic acid fragment of a sequence shown in a Seq ID No. 1;

Seq ID No.1：

2. the nucleic acid aptamer of claim 1, wherein: the nucleic acid sequence of the nucleic acid aptamer is combined with at least one of fluorescent substances, nano luminescent materials, biotin, digoxin and enzyme labels.

3. The nucleic acid aptamer of claim 2, wherein: the fluorescent substance is a fluorescent group, and the fluorescent group comprises a FAM fluorescent group or a Cy5 fluorescent group;

the nano luminescent material is a quantum dot or up-conversion luminescent material;

the enzyme label is horseradish peroxidase, sucrase or functional polypeptide.

4. A kit for the detection of measles virus, characterized in that: the kit comprises the aptamer of any one of claims 1-3.

5. The kit of claim 4, wherein: also included is at least one of the following,

(A) A protein-coated plate for immobilizing the sample solution;

(B) A protein coating liquid;

(C) A sealing liquid;

(D) A rinsing liquid;

(E) Sample pretreatment liquid.

6. The kit of claim 5, wherein: the formula of the protein coating liquid is 35mM NaHCO ₃ 、17mM Na ₂ CO ₃ ，pH9.3-pH9.9；

The blocking solution has the formula of 136mM NaCl, 2.6mM KCl and 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 、0.05% Tween-20、1% BSA，pH7.1-pH7.7；

The formula of the rinsing liquid comprises 136mM NaCl, 2.6mM KCl and 2mM KH ₂ PO ₄ 、9mM Na ₂ HPO ₄ 、0.05% Tween-20，pH7.1-pH7.5；

The sample pretreatment solution has a formula of 150mM NaCl, 1% Triton X-100, 50mM Tris, pH7.8-pH8.2.

7. Use of a nucleic acid aptamer according to any one of claims 1 to 3 or a kit according to any one of claims 4 to 6 for the preparation of a medicament or a tool for diagnosing a disease caused by measles virus.

8. A method for detecting measles virus for non-diagnostic therapeutic purposes, characterized by: comprising measles virus detection of an environment, food, tool, ex vivo sample or material of biological origin using a nucleic acid aptamer according to any one of claims 1 to 3, or a kit according to any one of claims 4 to 6.