CN116087498A

CN116087498A - DNA dendritic immunodetection reagent and method

Info

Publication number: CN116087498A
Application number: CN202111308240.4A
Authority: CN
Inventors: 张伯伦; 陈传品; 朱曲波; 孙若为; 丁建东
Original assignee: Hunan Zaochen Nano Robot Co ltd
Current assignee: Hunan Zaochen Nano Robot Co ltd
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2023-05-09

Abstract

The invention relates to the technical field of biology, in particular to a DNA dendritic immunodetection reagent and a method. In the detection reagent provided by the invention, the design of the auxiliary B can reduce the molecular chain decomposition speed, so that the stability is improved, the detection reagent is more suitable for clinical instant detection of biomarkers, and the background signal is lower, so that the detection is more sensitive and accurate.

Description

DNA dendritic immunodetection reagent and method

Technical Field

The invention relates to the technical field of biology, in particular to a DNA dendritic immunodetection reagent and a method.

Background

Point-of-care-testing (POCT), which is a test performed at the bedside of a patient, is mainly used in the departments of physical examination center, emergency, intensive care, clinic, etc. The main inspection items include: tumor marker detection, cardiac marker detection, inflammatory marker detection, kidney marker detection, blood glucose marker detection, and the like. The detection method mainly comprises isotope Radio Immunity (RIA), colloidal gold, enzyme-linked immunity (ELISA), time-resolved fluorescence (TRFIA) and chemical development (CLIA). The core steps of the detection methods mainly comprise two parts, namely one part is to realize the identification of an object to be detected through immune reaction, and the other part is to enable immune reaction products to generate signals and identify and report the signals. It can be seen that detection and reporting of signals generated by immune responses can have a critical impact on the accuracy and sensitivity of the detection results.

The nonlinear hybridization chain reaction (BHCR) can easily achieve secondary or exponential amplification. The nonlinear hybridization chain reaction is a hairpin-free nonlinear HCR system. The system mainly comprises double-chain subarate-A, substrate-B marked by fluorophores and quenching groups, and corresponding auxiliary agents A and B. The secondary or exponential amplification is achieved by controlling the growth of chain branches through quenched substrate kinetics to produce fluorescent DNA dendrimers. The method has the advantages of mild condition, no need of enzyme, simple scheme and excellent amplification efficiency. Thus, BHCR can be combined with a variety of detection methods to establish biosensing for ultrasensitive detection of various low levels of analytes. However, the time and detection efficiency of the BHCR reaction reagent in the prior art for nonlinear hybridization chain reaction are required to be improved.

Disclosure of Invention

In view of the above, the present invention aims to provide an instant test reagent and method for improving the detection efficiency.

The BHCR reaction reagent provided by the invention,

comprising the following steps: a trigger probe, an FA probe, a QA probe, a booster A, FB probe, a QB probe, and a booster B;

the sequence of the trigger probe consists of an isolation region and a trigger identification region; the trigger recognition area consists of a segment a, a segment b and a segment c which are connected in sequence,

the sequence of the FA probe consists of 2 repeated units and 1 reverse complementary sequence of the trigger recognition region; each of the repeating units consists of a segment x, a segment y and a segment z connected in sequence;

the sequence of the auxiliary A consists of reverse complementary sequences of 2 fragments y and 1 fragment c;

the sequence of the QA probe consists of reverse complementary sequences of a fragment b, a fragment c and 2 fragments y;

the sequence of the FB probe consists of a reverse complementary sequence of 1 repeating unit and the trigger recognition region sequence;

the sequence of the auxiliary B is a reverse complementary sequence of 18bp at the 5' end of the QB probe;

the sequence of the QB probe consists of a reverse complementary sequence of a trigger recognition region with 7bp of a deletion 5 'end and 1 repeated unit sequence with 5bp of a deletion 3' end.

In the invention, the isolation region sequence of the trigger probe is ployA; the nucleic acid sequence of the trigger recognition region is TGACGAACTAGTTGATGAAGCTG, wherein the fragment a is TGACG, the fragment b is AACTAGTTGATG, and the fragment c is AAGCTG. In some embodiments, the quarantine area is AAAAA.

In the present invention, the sequence of the repeating unit is GTGTGCCTATTATGTCTCCTCCT, wherein the fragment x is GT, the fragment y is GTGCCTATTATGTC, and the fragment z is tcctctct.

In some embodiments, the trigger probe is biotin-labeled at the 5' end, fluorescent groups are labeled at the 5' ends of FA and QB, and quenching groups are labeled at the 3' ends of FB and QA. In some embodiments, the fluorophore is 6-FAM and the quencher is BHQ1.

In the invention, the BHCR reaction reagent is applied to the preparation of an immediate test reagent.

The invention also provides a reagent for instant test, which comprises a trigger probe, a reagent A, a reagent B, a reagent C and a reagent D;

the reagent A comprises an FA probe, a QA probe and a co-A;

the reagent B comprises a FB probe, a QB probe and a B assisting probe;

the reagent C comprises magnetic beads coated with capture antibodies;

the complex formed by the trigger probe and the avidin-labeled detection antibody is marked as a reagent D;

or the detection antibody marked by avidin is marked as a reagent D, and the independent existence of a trigger probe is marked as a reagent E.

In the invention, the preparation of the reagent A comprises the following steps: and cooling the FA probe and the QA probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary A to obtain the reagent A.

In the invention, the preparation of the reagent B comprises the following steps: and cooling the FB probe and the QB probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary B to obtain the reagent B.

In the invention, the preparation of the complex formed by the trigger probe and the avidin-labeled detection antibody comprises the following steps: after labeling the detection antibody with avidin, the detection antibody was mixed with a trigger probe in PBS containing 0.5% BSA, and incubated to obtain a complex.

In some embodiments, the capture antibody is selected from the group consisting of a tumor marker antibody, a cardiac marker antibody, an inflammatory marker antibody, a renal marker antibody, or a blood glucose marker antibody,

the detection antibody is an anti-antibody. In a specific embodiment, the detection antibody is an anti-human antibody. For example, a rabbit anti-human antibody, a murine anti-human antibody, or a sheep anti-human antibody.

In some embodiments, the tumor marker antibody comprises AFP, PSA, carcinoembryonic antigen (CEA), cancer antigen 242 (CA 242), neuronal specific enolase NSE, serum Ferritin (SF), gastrin precursor release peptide (PROGRP), or squamous cell carcinoma antigen (SCC).

The invention also provides an instant detection method which utilizes the reagent provided by the invention to detect the object to be detected.

In some embodiments, the detection method comprises:

incubating a carrier of the capture antibody with an object to be detected, and then reacting with an avidin-labeled detection antibody to obtain a product I; incubating the trigger probe, the reagent A and the reagent B together to obtain a product II; after incubation of product I and product II, the signal was detected.

In other embodiments, the detection method comprises:

reacting a trigger probe with avidin and a detection antibody to obtain a product i; incubating a carrier of the capture antibody with an analyte, and then reacting with the product i to obtain a product ii; incubating the reagent A and the reagent B together to obtain a product iii; product ii was reacted with product iii and the signal was detected.

In some embodiments, the test agent is a sample from blood, tissue, fecal matter, or secretions. In an embodiment of the present invention, the sample is a blood sample.

In the detection reagent provided by the invention, the design of the auxiliary B can reduce the molecular chain decomposition speed, so that the stability is improved, the detection reagent is more suitable for clinical instant detection of biomarkers, and the background signal is lower, so that the detection is more sensitive and accurate.

Drawings

FIG. 1 shows the reaction principle of the reagent of the present invention.

Detailed Description

The present invention provides instant test reagents and methods, and one skilled in the art can, in light of the disclosure herein, suitably modify the process parameters to achieve. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The BHCR reaction reagent provided by the invention comprises: a trigger probe, an FA probe, a QA probe, a booster A, FB probe, a QB probe, and a booster B.

The various probes in the reagents of the present invention are all DNA, more preferably single-stranded DNA. The sequence is not strictly limited, and the detection effect can be realized as long as the conditions described in the present invention are satisfied.

The BHCR reaction reagent provided by the invention is used for detecting products after antigen-antibody reaction so as to amplify detection signals. In the antigen-antibody reaction, the capture antibody coated on the substrate can capture the antigen in the object to be detected, and the detection antibody with the biomarker recognizes the antigen, so that the biomarker is connected with the antigen through the detection antibody.

In the invention, the sequence of the trigger probe consists of an isolation region and a trigger recognition region, and is further modified with biotin or avidin. When detecting the biotin-labeled antibody, the labeled avidin of the trigger probe. And when detecting the antibody labeled avidin, the trigger probe labeled biotin. In the invention, the marker of the trigger probe is positioned at the 5' end of the trigger probe. In some embodiments, the trigger probe is structured as a biotin-isolation region-trigger recognition region. In some embodiments, the sequence of the isolation region is polyA, and the trigger recognition region sequence is not limited and is complementary to a portion of the FA. The length of the trigger recognition area is 25-30, and in the invention, the length is 28bp. In some embodiments, the trigger probe has a structure of: biotin-AAAAATGACGAACTAGTTGATGAAGCTG. The trigger recognition area is divided into three fragments for conveniently describing the relation between the structures of other probes in the reagent and the trigger probes. Thus, the trigger recognition region consists of a segment a, a segment b, and a segment c, which are sequentially connected, wherein the segment a is TGACG, the segment b is AACTAGTTGATG, and the segment c is AAGCTG.

In the invention, the sequence of the FA probe consists of n repeated units and 1 reverse complementary sequence of the trigger recognition region; to facilitate description of the relationship between the structure of other probes in the reagent and the FA probe, each of the repeat units is divided into 3 fragments, each of the repeat units consisting of fragment x, fragment y and fragment z, which are sequentially linked. The structure of the FA probe of the present invention can be expressed as: (fragment x-fragment y-fragment z) the reverse complement of the n-trigger recognition region.

In the detection process of the reaction reagent provided by the invention, each repeating unit in the FA probe can be combined with one FB probe, and each FA comprises more than 3 repeating units, so that each FA can be combined with at least 3 FBs. Thereby improving the efficiency and sensitivity of detection. According to the invention, through designing FA and 2 FB combinations, FA and 3 FB combinations and FA and 4 FB combinations, reaction is carried out for 5 minutes respectively, and the result shows that the fluorescence intensity obtained in the same time is increased along with the increase of the number of the repeated units. However, in theory, the fluorescence intensity increases with the number of repeating units, but the length of FA is not more than 300bp in view of improving the reaction stability.

In some embodiments of the invention, the sequence of the repeat unit is GTGTGCCTATTATGTCTCCTCCT, wherein the fragment x is GT, the fragment y is GTGCCTATTATGTC, and the fragment z is tcctct. In some embodiments, the number of repeating units is 3 or 4. In the present invention, the probe detection with more than 3 repeating units gives higher sensitivity and shorter period than the FA probe with more than two repeating units.

In the invention, the 5' end of the FA probe is modified with a fluorescent group. The fluorescent group is selected from Alexa350, alexa488, alexa 532, alexa 549, alexa 647, alexa 680, CF350, CF488, CF532, CF594, CF647, CF680, 6-FAM, FITC, TRITC, 6-TET HEX, VIC, ROX, cal, texas Red, CY5, quasar670 or Quasar705;

in some embodiments, n=3, and the FA probe has the structure:

6-FAM-GTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTCAGCTTCATCAACTAGTTCGTCA；

in other embodiments, n=4, and the FA probe has the structure:

6-FAM-GTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTGTGTGCCTATTATGTCTCCTCCTCAGCTTCA TCAACTAGTTCGTCA。

in the invention, the sequence of the QA probe consists of reverse complementary sequences of a fragment b, a fragment c and n fragments y in a trigger probe. The structure can be expressed as: fragment b-fragment c- (reverse complement of fragment y) n. In QA, the value of n is equal to the value of n in FA. Wherein the sequence of the reverse complement of fragment y is GACATAATAGGCAC AC.

The 3' end of QA is modified with a quenching group. The quenching group on the QA probe is capable of quenching the fluorescent group on the FA probe. The quenching group is selected from BHQ-0, BHQ-1, BHQ-2, BHQ-3, MGB, dabCry or Eclip se lambda.

In some embodiments, n=3, and the structure of QA is:

AACTAGTTGATGAAGCTGGACATAATAGGCACACGACATAATAGGCACAC/GACATAATAGGCACAC-BHQ1

in other embodiments, n=4, and the structure of QA is:

AACTAGTTGATGAAGCTGGACATAATAGGCACACGACATAATAGGCACAC/GACATAATAGGCACACGACATAATAGGCACAC-BHQ1

the sequence of the auxiliary A consists of n fragments y and 1 reverse complementary sequences of the fragments c; the structure can be expressed as the reverse complement of (fragment y) n-fragment c. Wherein the value of n is equal to the value of n in the FA. The reverse complement of the fragment c is CAGCTT. No fluorophores or quenching groups are modified on co-a.

In some embodiments, n=3, and the structure of the auxiliary a is:

GTGCCTATTATGTCGTGCCTATTATGTCGTGTGCCTATTATGTCCAGCTT

in other embodiments, n=4, and the structure of the auxiliary a is:

GTGCCTATTATGTCGTGCCTATTATGTCGTGCCTATTATGTCGTGTGCCTATTATGTCCAGCTT

the preparation of the reagent A comprises the following steps: and cooling the FA probe and the QA probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary A to obtain the reagent A.

In the preparation process of the reagent A, the FA and the QA are denatured at 95 ℃ for 5min, then slowly cooled to room temperature within 30min, and the FA strand and the QA strand form complementary double chains according to the base complementary pairing principle. Fragment z, however, forms a stem-loop structure because it is not complementary to the fragment on QA. In the double strand formed by FA and QA, the distance between the fluorophore and the quencher is very close, resulting in the fluorescence on the FA probe assuming a "quenched" state.

The sequence of the FB probe consists of a reverse complementary sequence of 1 repeating unit and a trigger recognition region sequence identical to that of the trigger probe. I.e., the structure of the FB probe can be expressed as: reverse complement of the repeat unit-trigger recognition region sequence. The number of the repetitive unit reverse complementary sequences on the FB probe is only 1, and the number of the repetitive units in the FA is not changed due to the change of the number of the repetitive units in the FA. Wherein the reverse complement of the repeat unit is AGGAGGAGACATAATAGGCACAC.

The 3' end of the FB probe is modified with a quenching group selected from BHQ-0, BHQ-1, BHQ-2, BHQ-3, MGB, dabCry or Eclip se lambda.

In some embodiments, the FB probe has a structure of:

AGGAGGAGACATAATAGGCACACTGACGAACTAGTTGATGAAGCTG-FAM。

the QB probe sequence consists of a reverse complementary sequence of a trigger recognition region with 7bp of a deletion 5 'end and 1 repeated unit sequence with 5bp of a deletion 3' end. I.e. the structure of the QB probe can be expressed as: reverse complement (trigger recognition region 5 '-7 bp) - (repeat unit 3' -5 bp). Wherein, 5' -7bp of the trigger recognition region is CTAGTTGATGAAGCTG, and the reverse complementary sequence is CAGCTTCATCAACTAG. The 3' -end-5 bp of the repeating unit is GTGTGCCTATTATGTCTC. In the invention, the 5' end of the QB probe is modified with a fluorescent group. The modified quenching group on the FB probe is capable of quenching the fluorescent group on the QB probe. The modified quenching groups on the QB probe are Alexa350, alexa488, alexa 532, alexa 549, alexa 647, alexa 680, CF350, CF488, CF532, CF594, CF647, CF680, 6-FAM, FITC, TRITC, 6-TET HEX, VIC, ROX, cal, texas Red, CY5, quasar670 or Quasar705.

The sequences of the auxiliary B and the QB probes are reversely complementary. Specifically, the auxiliary B is complementary with the reverse direction of 18-20 bp of the 5' end of the QB probe. Wherein, 20bp sequence at the 5' end of the QB probe is: CAGCTTCATCAACTAGGTGTGC, the sequence of helper B is GCACACCTAGTTGATGAAGC, or the sequence of helper B is CACACCTAGTTGATG AAGC, or the sequence of helper B is ACACCTAGTTGATG AAGC. The invention tries the length of the auxiliary B, and the result shows that the auxiliary B with the length of 18bp is more beneficial to reducing the decomposition speed, thereby improving the stability of the reaction. Therefore, when the length of the auxiliary B is 18bp, the effect is better than the detection effect of 19bp or 20bp, and the fluorescent signal obtained by detecting the auxiliary B by 18bp within 35min is more remarkable.

The preparation of the reagent B comprises the following steps: and cooling the FB probe and the QB probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary B to obtain the reagent B.

In the preparation process of the reagent B, the FB and the QB are denatured for 5min at 95 ℃, then slowly cooled to room temperature within 30min, and the FB strand and the QB strand form complementary double chains according to the base complementary pairing principle. In the double strand formed by FB and QB, the distance of the fluorophore and the quencher is very close, resulting in the fluorescence on the FB probe assuming a "quenched" state.

The biotin mark on the trigger probe contained in the reaction reagent can identify the product with the streptomycin mark after antigen-antibody reaction. Also, when reagents a and B are added to the system, the trigger hybridizes to and interfaces with the exposed footholds on the FA probe, resulting in a branching migration reaction that displaces part of the Q strand from the F strand and opens the first loop, then helps a displace the Q strand by docking to the newly exposed footholds and produces byproducts. Fluorescence in the F chain is restored by dissociating the Q chain with the quenching group from the F chain. The F strand in substrate strand A exposes two identical sequences connected in series, which can be hybridized to both substrate strands B simultaneously by their foot points. The Q strand is displaced with the aid of the auxiliary strand Ass B or Ass B-2nt and by-product B is produced, more fluorescent reporter groups being released; and two single stranded regions consisting of the same sequence as the trigger strand DNA are exposed to enter a new round of reaction. The tree-like nanocomposite is finally formed by the cyclic reaction, and more by-products a and B are generated (fig. 1).

The invention provides an application of a BHCR reaction reagent in preparing an immediate test reagent.

The invention also provides a reagent for the instant test, which comprises the BHCR reaction reagent, magnetic beads coated with capture antibodies and an avidin-labeled detection antibody. In the prior art, it is generally thought that the detection effect is lowered because the probes or enzymes cannot be stored in a mixed state. Experiments show that the invention can shorten the time of clinical detection and obtain good detection stability by mixing and storing part of reagents or forming a compound for storage.

For example: the instant detection reagent provided by the invention comprises:

reagent A, FA probe+QA probe+auxiliary A. Wherein FA and QA form double strand and then add co-A, reagent A is stored at 4deg.C.

Reagent B, FB probe+QB probe+auxiliary B. Wherein FB and QB form double chain, and then assist B is added, and reagent B is stored at 4deg.C.

Reagent C, magnetic beads and antibodies. It is prepared by coating magnetic beads with antibodies. The online mode between the magnetic beads and the antibodies is covalent coupling. The magnetic beads are magnetic beads with surface layers modified by carboxyl, amino or sulfhydryl groups. The grain diameter is 100 nm-5 μm. The preferred particle size is 1 μm to 3. Mu.m. Preserving at 4 ℃.

The reagent D is a detection antibody and streptavidin; wherein, the streptomycin marked detection antibody plays a role in the up-down, and the detection antibody can be monoclonal antibody or polyclonal antibody, and is connected with streptavidin.

Reagent E, trigger probe.

In the reagent, experiments prove that the reagent A, the reagent B and the reagent D can be kept stable within 3 months, and good detection effect can be obtained after the reagent A, the reagent B and the reagent D are placed at 4 ℃ for 3 months. Reagent C can be stored at 4deg.C for 2 years, and reagent E and 4deg.C can be stored for 12 months without affecting detection result.

Alternatively, the instant detection reagents provided by the present invention include:

The reagent D is a detection antibody, streptavidin and a trigger probe; wherein, the streptomycin marked detection antibody plays a role in the up-down, the detection antibody can be monoclonal antibody or polyclonal antibody, and the mark is mainly connected with streptavidin. And incubating the streptavidin-modified detection antibody and the trigger probe for 0-0.1 h at the temperature of 30-37 ℃ to form a compound A. Based on the structural features of streptavidin and trigger probes, streptavidin can be linked to 4 trigger dnas. Preserving at 4 ℃.

In the reagent, experiments prove that the reagent A, the reagent B and the reagent D can be kept stable within 3 months, and good detection effect can be obtained after the reagent A, the reagent B and the reagent D are placed at 4 ℃ for 3 months. Reagent C can be stored at 4℃for 2 years without affecting the detection result.

The capture antibody in the detection reagent of the invention can be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred. If the antibody contains other amino and sulfhydryl impurity molecules, the impurity molecules need to be removed by means of dialysis, G-25 and the like so as not to cause competitive reactions (such as Tris, glycine, BSA and the like) of the antibody. The preparation method of the antibody-coated magnetic bead mainly comprises the following steps: putting magnetic beads into a coupling reaction bottle, adding buffer solution, then adding an antibody (the magnetic bead amount mg: the antibody amount mg=20:1), screwing the coupling reaction bottle, putting the coupling reaction bottle into a constant temperature shaking table, shaking at the constant temperature of 25 ℃ for 1-6 hours, guaranteeing shaking and mixing uniformly, adding buffer solution into the coupling reaction bottle for cleaning (magnetic separation by a method, removing supernatant), closing active groups (3% -5% BSA) which do not react, and storing in a refrigerator at the temperature of 4 ℃.

The invention also provides a method for instant detection, which comprises the following steps: incubating a carrier of the capture antibody with an object to be detected, and then reacting with an avidin-labeled detection antibody to obtain a product I; incubating the trigger probe, the reagent A and the reagent B together to obtain a product II; after incubation of product I and product II, the signal was detected.

Alternatively, the method for instant detection includes: reacting a trigger probe with avidin and a detection antibody to obtain a product i; incubating a carrier of the capture antibody with an analyte, and then reacting with the product i to obtain a product ii; incubating the reagent A and the reagent B together to obtain a product iii; product ii was reacted with product iii and the signal was detected.

In the method of the invention, a part of the detection reagent is reacted before clinical detection to form a compound, so that the compound does not need to be prepared in situ in clinical use, thereby greatly shortening the detection time and not affecting the detection accuracy.

In the present invention, the analyte is a sample derived from blood, tissue, excrement or secretion. In an embodiment of the present invention, the sample is a blood sample. In particular, the blood sample is serum or plasma.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1

The PSA standard with specific concentration is used for detecting objects, the effects of different probes are verified, and the experiments are divided into the following groups:

the probe sequences in the reagents of group 1 are:

the probe sequences in the reagents of group 2 are:

the probe sequences in the reagents of group 3 are:

the BHCR reaction solution includes reagent A, reagent B and buffer solution.

1.1, reagent A is prepared by the following method: f-strand of substrate-A (FA) and Q-strand of substrate-A (QA) are put into a PCR instrument with the temperature of 90-95 ℃ and the temperature is reduced to 55 ℃ for 1 min/DEG C, then the mixture is taken out and reduced to the room temperature, and auxiliary A is added, so that the reagent A is obtained. FA and QA become double-stranded first, and double-stranded is formed by taking the group 1 probe design as an example. Stored at 4℃for 3 months.

1.2, reagent B is prepared by the following method: f-strand of substrate-B (FB) and Q-strand of substrate-B (QB) are placed into a PCR instrument at 90-95 ℃, the temperature is reduced to 55 ℃ for 1 min/DEG C, then the mixture is taken out and reduced to room temperature, and B is added to obtain the reagent B. FB and QB become double-stranded, and double-stranded is formed by taking the group 1 probe design as an example. Stored at 4℃for 3 months.

1.3, reagent C from magnetic beads+PSA antibody

And (3) after activating the surface of the superparamagnetic carboxyl magnetic bead, coupling with a PSA antibody, and sealing to prepare the reagent C. The buffer solution of reagent C is PBS, pH 7.4, 0.09% sodium azide and 0.01% Tween 20. Wherein the density of the magnetic beads is 50mg/ml. Stored at 4 ℃ for 2 years.

1.4: reagent D is prepared from detection antibody +streptavidin +trigger dnas:

firstly preparing a streptavidin marked detection antibody, then adding a trigger probe, and reacting for 1h at 30-37 ℃ to prepare a reagent D. The buffer for reagent D was 0.5% PBS. Stored at 4℃for 12 months.

2. PSA standards (concentration 4 ng/ml) were tested with the reagents described above, comprising the steps of:

2.1, mixing the reagent C with a standard at 25 ℃, incubating for 30 minutes, and collecting magnetic beads;

2.2, mixing the magnetic beads collected in the step 2.1 with the reagent D at 25 ℃ for reaction for 40 minutes;

2.3, mixing the reagent A and the reagent B to react for 20 minutes at 37 ℃;

2.4, pouring the solution in the step 2.3 into the step 2.2, and reacting for 15-30 minutes at 37 ℃;

2.5 removing the supernatant in the step 2.4 by using a magnetic separation method, washing the magnetic beads once by using a PBST or PBS solution, and using

Ultra-high sensitivity polychromatic flow cytometer for detecting fluorescence intensity

The results showed that groups 1 to 3 were each repeated 5 times, and the average value of the obtained fluorescence values was in turn: group 1:11000, group 2:12000, group 3:13800. It can be seen that: with the increase of the number of the repeating units, the fluorescence intensity detected within a certain reaction time is increased, and the fluorescence intensity of the group 2 and the group 3 is obviously stronger than that of the group 1. However, since the reaction is unstable by further increasing the number of repeating units, the number of repeating units is preferably 3 to 4.

Example 2

1. Several sets of primers were designed as follows: wherein each group Trigger, FA, QA, FB, QB remained unchanged, its sequence was as follows:

2. the reagents A to D were prepared in the same manner as in example 1, and the PSA standard (concentration: 4 ng/ml) was tested with each set of reagents, comprising the steps of:

2.3, mixing the reagent A and the reagent B to react for 20 minutes at 37 ℃;

The results showed that groups 1 to 3 were each repeated 5 times, and the average value of the obtained fluorescence values was in that order: group 1:11000, group 2:11600, group 3:12800. It can be seen that: the structure is not easy to open spontaneously by reducing the length of the assistant B, so that the aim of reducing the background is fulfilled. When the length of the auxiliary B is 18bp, the fluorescence value is higher, the linearity of the fluorescence increasing curve along with time is better, and the background is lower.

Example 3

In order to shorten the clinical testing time, attempts have been made to react the reagents to form complexes prior to testing, to avoid "on-the-fly" in clinical testing.

The experiment included the following, each component was stored separately, prepared before detection, and the detection was performed with a PSA standard (concentration 4 ng/mL) as a test substance for 12 hours, and fluorescence intensity was recorded. And then after each reagent is prepared and stored for a period of time (1 month, 2 months, 3 months, 6 months, 12 months, 18 months and 24 months), detection is carried out, and the difference of the detection effect of the reagent is analyzed compared with that of the reagent which is not stored. The difference is within 3%, and the reaction effect is not considered to be affected.

1. And cooling the FA probe and the QA probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary A to obtain the reagent A. The reagent does not affect the detection effect after 3 months of storage at 4 ℃, and the detection effect is reduced thereafter.

2. And cooling the FB probe and the QB probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary B to obtain the reagent B. The reagent does not affect the detection effect after 3 months of storage at 4 ℃, and the detection effect is reduced thereafter.

3. The preparation method of the magnetic beads modified by the antibody comprises the steps of putting the magnetic beads into a coupling reaction bottle, adding a buffer solution, then adding the antibody (the magnetic bead amount mg: the antibody amount mg=20:1), screwing the coupling reaction bottle into a constant temperature shaking table, shaking at the constant temperature of 25 ℃ for 1-6 hours, ensuring shaking and mixing uniformly, adding the buffer solution into the coupling reaction bottle for cleaning (magnetic separation and supernatant removal), closing active groups (3% -5% BSA) which do not react, preserving in a refrigerator at the temperature of 4 ℃, and keeping stable for 2 years.

4. The detection antibodies are marked by streptavidin, PSA standard products with the concentration of 0.003ng/ml, 0.1ng/ml, 4ng/ml and 10ng/ml are respectively detected, the fluorescence values are 1100, 6800, 12100 and 42500 in sequence, and the linear relation between the fluorescence values and the standard product concentration is good. Its lowest detection limit can be up to 1X 10 ^-12 ng/ml, the reagent has good accuracy.

5. the trigger probe is independently stored at 4 ℃, and the detection result is not affected within 12 months.

6. The detection antibody, streptavidin and trigger probe react to form a compound, the preparation method comprises that PBS containing 0.5% BSA is directly mixed according to a certain proportion, the pH is 7.4-8, the mixture can be stored for 3 months at the temperature of 4 ℃ without influencing the detection result, and the detection effect is reduced.

Example 4

1. The PSA standard is used as a detection object and is detected by adopting the following probes:

reagent A, B, C, D was prepared in the same manner as in group 1 of example 1.

PSA standards (concentration 4 ng/ml) were tested with the reagents described above, comprising the steps of:

2.3, mixing the reagent A and the reagent B to react for 20 minutes at 37 ℃;

The fluorescence intensity is detected by an ultra-high sensitivity polychromatic flow cytometer.

The results show that the reagent has good repeatability when the detection is repeated 5 times, the average value of fluorescence values is 12910, and the RSD is 2.3%. Its lowest detection limit can be up to 1X 10 ^-12 ng/ml, the reagent has good accuracy.

Example 5

1. PSA is used as a detection object, serum is used as a sample, and the following probes are used for detection:

reagent A, B, C was prepared in the same manner as in group 1 of example 1.

Reagent D: preparing a streptavidin-labeled detection antibody.

Reagent E: a trigger probe.

2. The PSA standard is tested with the above reagents, comprising the steps of:

2.3, mixing the reagent A, the reagent B and the reagent E, and reacting for 20 minutes at 37 ℃;

The results showed that the fluorescence value averaged 12900RSD was 1.6% for 3 replicates, indicating good reproducibility of the reagent. Its lowest detection limit can be up to 1X 10 ^-12 ng/ml, the reagent has good accuracy.

Example 6

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reagent A, B, C was prepared in the same manner as in group 1 of example 1.

Reagent D: preparing a streptavidin-labeled detection antibody.

Reagent E: a trigger probe.

2. The PSA standard is tested with the above reagents, comprising the steps of:

The results showed that the repeated detection was 3 times, the average fluorescence value was 12000 and the rsd was 1.5%, indicating that the reagent had good reproducibility. Its lowest detection limit can be up to 1X 10 ^-12 ng/ml, the reagent has good accuracy.

Example 7

PSA standard products with different concentrations are used as detection objects, and the following probes are adopted for detection:

2. the PSA standard is tested with the above reagents, comprising the steps of:

2.5, removing the supernatant in the step 2.4 by using a magnetic separation method, and magnetizingThe beads were washed once with PBST or PBS solution

The results show that，PSA standard products with the concentration of 0.003ng/ml, 0.1ng/ml, 4ng/ml and 10ng/ml are detected respectively, the fluorescence values are 1100, 6800, 12100 and 42500 in sequence, and the linear relation between the fluorescence values and the standard product concentration is good. Indicating that the reagent has good reproducibility. Its lowest detection limit can be up to 1X 10 ^-12 ng/ml, the reagent has good accuracy.

Example 8

Detection of serum samples: under the experimental conditions of example 7, the immune BHCR detection platform was used to detect the concentration of AFP in serum of liver cancer patients obtained from a certain hospital, and the concentration was close to the value of AFP measured by the hospital. In addition, the measured value of each sample fluctuates little, and the detection result is as follows:

sample of	Example 7 reagent detection data (ng/mL)
		1	43.70±0.56
2	17.34±0.73
		3	72.80±3.35

Specificity examination under the conditions of example 7, the immunization BHCR detection platform was used to detect 1ng/mL of AFP antigen and 100ng/mL of non-target antigen (CEA, PSA, igG), and as a result, the fluorescence intensity of AFP was significantly stronger than that of non-target antigen. The method has good specificity.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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Claims

A bhcr reagent comprising: a trigger probe, an FA probe, a QA probe, a booster A, FB probe, a QB probe, and a booster B;

the sequence of the trigger probe consists of an isolation region and a trigger identification region; the trigger recognition area consists of a segment a, a segment b and a segment c which are connected in sequence,

the sequence of the FA probe consists of 2 repeated units and 1 reverse complementary sequence of the trigger recognition region; each of the repeating units consists of a segment x, a segment y and a segment z connected in sequence;

the sequence of the auxiliary A consists of reverse complementary sequences of 2 fragments y and 1 fragment c;

the sequence of the QA probe consists of reverse complementary sequences of a fragment b, a fragment c and 2 fragments y;

the sequence of the FB probe consists of a reverse complementary sequence of 1 repeating unit and the trigger recognition region sequence;

the sequence of the auxiliary B is a reverse complementary sequence of 18bp at the 5' end of the QB probe;

the sequence of the QB probe consists of a reverse complementary sequence of a trigger recognition region with 7bp of a deletion 5 'end and 1 repeated unit sequence with 5bp of a deletion 3' end.
2. The BHCR reagent according to claim 1, wherein the isolation region sequence of the trigger probe is ployA; the nucleic acid sequence of the trigger recognition region is TGACGAACTAGTTGATGAAGCTG, wherein fragment a is TGACG, fragment b is AACTAGTTGATG, and fragment c is AAGCTG.
3. The BHCR reagent according to claim 1, wherein the repeat unit has a sequence of GTGTGCCTATTATGTCTCCTCCT, wherein the fragment x is GT, the fragment y is GTGCCTATTATGTC, and the fragment z is tcctct.
4. The BHCR reagent according to any one of claims 1 to 3, wherein the trigger probe is biotin-labeled at the 5' end, fluorescent groups are labeled at the 5' ends of FA and QB, and quenching groups are labeled at the 3' ends of FB and QA.
5. The BHCR reagent according to claim 4, wherein the fluorescent moiety is 6-FAM and the quenching moiety is BHQ1.
6. Use of the BHCR reagent as set forth in any one of claims 1 to 5 in the preparation of a point-of-care reagent.
7. A reagent for on-the-fly testing, comprising: trigger probe, reagent A, reagent B, reagent C and reagent D;

the reagent A comprises the FA probe, QA probe and auxiliary A according to any one of claims 1 to 5;

the reagent B comprises the FB probe, the QB probe and the B-aid according to any one of claims 1 to 5;

the reagent C comprises magnetic beads coated with capture antibodies;

the complex formed by the trigger probe and the avidin-labeled detection antibody is marked as a reagent D;

or the detection antibody marked by avidin is marked as a reagent D, and the independent existence of a trigger probe is marked as a reagent E.
8. The reagent according to claim 7, wherein:

the preparation of the reagent A comprises the following steps: cooling the FA probe and the QA probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, then naturally cooling to the room temperature, and adding the auxiliary A to obtain a reagent A;

the preparation of the reagent B comprises the following steps: and cooling the FB probe and the QB probe to 55 ℃ at the temperature of 90-95 ℃ for 1 min/DEG C, naturally cooling to the room temperature, and adding the auxiliary B to obtain the reagent B.
9. The reagent according to claim 7, wherein: the preparation of the complex formed by the trigger probe and the avidin-labeled detection antibody comprises the following steps: after labeling the detection antibody with avidin, the detection antibody was mixed with a trigger probe in PBS containing 0.5% BSA, and incubated to obtain a complex.
10. An instant detection method, comprising: detection of an analyte using the reagent of claim 8 or 9.