CN114214461B - Isothermal HIV nucleic acid detection kit and detection method - Google Patents

Isothermal HIV nucleic acid detection kit and detection method Download PDF

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CN114214461B
CN114214461B CN202111606719.6A CN202111606719A CN114214461B CN 114214461 B CN114214461 B CN 114214461B CN 202111606719 A CN202111606719 A CN 202111606719A CN 114214461 B CN114214461 B CN 114214461B
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reaction
tube
gold
buffer system
dna
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CN114214461A (en
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田野
杨思畅
闵乾昊
徐子棋
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Nanjing University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Abstract

The invention discloses an isothermal HIV nucleic acid detection kit and a detection method, wherein the kit comprises a tube A reaction reagent and a tube B reaction reagent; the A tube reaction reagent comprises a buffer system containing NaCl, a walker and a protective chain, a track single chain and streptavidin magnetic beads; the B tube reactant comprises gold a, gold B and Mg 2+ A buffer system. The detection process of the kit does not need complex reagents and equipment, can be separated from the laboratory environment operation as far as possible, and the result is presented in a visual mode, so that the existence of a detection object and the approximate concentration range are intuitively and rapidly reflected, the instant detection is realized, and the practical application is convenient.

Description

Isothermal HIV nucleic acid detection kit and detection method
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a rapid and convenient isothermal HIV nucleic acid detection kit and a detection method.
Background
Detection of Human Immunodeficiency Virus (HIV) is a key component of aids control program, as a retrovirus, which when invaded into cells, reverse transcribes its own RNA into DNA, integrating into the host cell genome. Currently, detection of HIV is largely divided into nucleic acid detection and serological detection. The serological targets are antigens or antibodies, and common methods include enzyme-linked immunosorbent assay (De la Rica and Stevens, 2012), immunofluorescence assay (Pandori et al, 2013) and western blotting (Zhou et al, 2015 b), and the existing test papers for personal detection generally use ELISA to detect HIV antibodies, but because HIV antibodies are low in level, antibodies can be detected in blood for about six weeks. In contrast, HIV nucleic acid detection will reduce the detection window period to around one week, so viral nucleic acid detection is of great importance for early disease control. The usual detection method for HIV nucleic acid is the polymerase chain reaction (PCR, ding et al 2010), which has high sensitivity and specificity, can detect RNA and DNA, adds fluorescent groups into the PCR reaction system, monitors the whole PCR process in real time by accumulation of fluorescent signals, and finally can obtain quantitative results by comparing standard curves. Although the PCR method is mature and reliable as a common method for detecting nucleic acid, the PCR method has the defects of high equipment requirement, high detection cost and long time, and needs thermal cycle and complex primer design. Thus, there is an urgent need to develop simple, rapid and sensitive techniques for early diagnosis of HIV infection.
The currently commonly used nucleic acid amplification technology includes Strand Displacement Amplification (SDA), hybridization Chain Reaction (HCR), rolling Circle Amplification (RCA), DNA nanomachines and the like, wherein the DNA walker is one of the DNA nanomachines, can perform autonomous and highly directional movement on linear, two-dimensional and three-dimensional tracks through dynamic interaction, can be applied to directional transmission and amplification of detection signals, and has good application prospects in the aspect of nucleic acid detection. For example, team Li Feng constructed 3D-DNA walker on gold spheres, released walker by strand displacement reaction when target DNA was present, and driven with endonucleases to walk the walker along orbital molecules on three-dimensional surfaces and release a large number of fluorescently labeled substrate molecules, thereby rapidly detecting tubercle bacillus DNA fragments in serum samples (article: ACS Nano 2016,10,2,2324-2330). He Zhike et al devised an HIV nucleic acid detection method based on the RCA reaction and DNA walker, with the HIV target as the primer for the RCA reaction, and the product of RCA containing hundreds of repeats for priming walking of the DNA walker, releasing fluorescent signals (article: nanoscales, 2018,10,17206-17211). Double-layer DNA walker is designed for HIV nucleic acid detection by using steel and the like, when an object exists, an entropy-driven DNA molecular hybridization reaction is activated, so that the outer layer walker walks, the inner layer walker releases a chain, the inner layer walker is driven by enzyme, and a fluorescence signal is generated by cutting a rail molecule through an enzymatic reaction (article: biosensors and Bioelectronics, volume 133,2019,243-249). Liang Guoxi et al designed a simple magnetic DNA walker for detecting mirnas, triggered walker cleavage of substrate strands with the target, after magnetic separation, a large number of substrate strands were incubated with nanogold, which was not aggregated due to the presence of large amounts of single-stranded DNA in high-salt solution, so that the concentration of the target could be visualized by a color change from grey black to purple (article: sensors and Actuators B: chemical, volume 337,2021,129813).
Most of the existing methods for detecting HIV nucleic acid by using DNA walker show fluorescence, but the observation of fluorescence signals needs a certain device, which cannot be visually displayed, and the method is more difficult to operate outside a laboratory, has higher cost of fluorescent groups, and is difficult to reflect the detection result rapidly on site. The colorimetric analysis method based on the nano gold is simple and low in cost, can be used for on-site detection, but has low sensitivity, so that the clinical application of the colorimetric analysis method in nucleic acid detection is limited.
Disclosure of Invention
The invention aims to provide a rapid and convenient isothermal HIV nucleic acid detection kit and a detection method aiming at the technical problems in the background art. The detection process of the kit does not need complex reagents and equipment, can be separated from the laboratory environment operation as far as possible, and the result is presented in a visual mode, so that the existence of a detection object and the approximate concentration range are intuitively and rapidly reflected, the instant detection is realized, and the practical application is convenient.
The aim of the invention can be achieved by the following technical scheme:
an isothermal HIV nucleic acid detection kit comprising a tube a reagent and a tube B reagent; the A tube reaction reagent comprises a buffer system containing NaCl, a walker and a protective chain, a track single chain and streptavidin magnetic beads; the B tube reactant comprises gold a, gold B and Mg 2+ And the buffer system is characterized in that gold a and gold b are respectively prepared by modifying DNA molecules of a sequence and a sequence b on the surface of nano gold.
The nano gold (with the diameter of about 13 nm) is prepared by a sodium citrate reduction method, and then DNA molecules of a sequence and a sequence of b sequence are respectively modified on the surfaces of the nano gold, so that the nano gold is named as gold a and gold b in the specification, and the preparation method and the modification method of the nano gold are conventional methods known to a person skilled in the art.
As a preferable technical scheme, the preparation method of the A pipe reactant comprises the following steps:
(1) Hybridizing the walker to the protective strand to form a partially complementary hybridized duplex;
(2) Dispersing streptavidin magnetic beads and the hybridization double chains and the track single chains prepared in the step (1) in a buffer system containing NaCl together to obtain a reaction system;
(3) Reacting the reaction system obtained in the step (2) in a rotary mixer for 12-18 hours to obtain magnetic beads modified with DNA;
(4) Washing the supernatant, and re-dispersing the magnetic beads modified with DNA in a buffer system containing NaCl to obtain a tube A reaction reagent;
the preparation method of the B pipe reactant comprises the following steps:
dispersing gold a and gold b into Mg 2+ And (3) in the buffer system, obtaining the B-tube reaction reagent.
It is further preferred that the walker to Protect chains described in step (1) are in a molar ratio of 1:2 hybridization at 37 ℃; the hybridization duplex of step (2): track single strand (molar ratio) =1:8, the concentration of hybridized double strand in the reaction system was 0.5 μm.
Further preferably, the reaction conditions in step (3) are: the reaction was spun at 20rpm at room temperature.
Further preferably, gold a and gold B are dispersed into Mg in the preparation of the B-tube reagent 2+ The final concentration of each gold in the buffer system was 16-20nM.
As a preferred technical scheme, the nucleotide sequences of the walker, the protective chain and the track single chain are respectively as follows:
the DNA molecules of the two sequences a and b are respectively:
Sequence(5’-3’)
a DNA molecule of sequence a TATGAAGTGATGGAT-SH
b sequence DNA molecules TAGTGCATTTAACAT-SH
Further preferably, the streptavidin magnetic beads are streptavidin magnetic beads with the diameter of 1 mu M; the buffer system containing NaCl is 1 xTAE 0.1M NaCl; said Mg 2+ The buffer system was 1×TAE12.5mM Mg (A C ) 2 A buffer system.
A method of detecting an isothermal HIV nucleic acid detection kit, the method comprising the steps of:
(1) Adding a DNA solution of a sample to be detected into a tube A reaction reagent for reaction;
(2) The reaction solution was sucked to dry the supernatant, and the beads were redispersed in Mg 2+ The reaction is carried out in a buffer system of the Mg 2 + The buffer system was 1 xTAE 12.5mM Mg (A C ) 2 A buffer system;
(3) Separating the supernatant from the reaction liquid, taking the supernatant to react in a B tube reaction reagent, and after the reaction is finished, reflecting the result by contrasting the color of the B tube nano-gold, wherein the concentration of the target is increased, and the color of the B tube is deepened from orange red to deep red or even purple red. For colorimetry, the lowest concentration distinguishable by naked eyes is 0.1nM, and for detection range analysis, there is a linear relationship in the interval of 0.1-500nM, and at concentrations above 500nM, the purple color of the B tube is deeper and more pronounced sedimentation occurs.
Further preferably, the reaction conditions in step (1) are: reacting for 1-2 hours at 30-37 ℃; the reaction conditions in step (2) are: reacting for 1-2 hours at 30-37 ℃; the reaction conditions in step (3) are: the reaction was carried out at 30℃for 1 hour. Still more preferably, the reaction conditions in step (1) are: reacting for 2 hours at 32 ℃; the reaction conditions in step (2) are: reacting for 2 hours at 32 ℃; the reaction conditions in step (3) are: the reaction was carried out at 30℃for 1 hour.
The working principle of the kit is as follows: according to the scheme, two reactions of A, B tubes are designed, isothermal amplification is realized by using DNA walker in the tube A reaction, and aggregation of nano gold is initiated by using amplification products in the tube B reaction to different degrees, so that quick visual detection of HIV DNA is realized. Specifically, a small amount of DNA molecules as walker and a large amount of DNA orbital molecules are jointly loaded on a magnetic bead with the diameter of 1 mu M, wherein the DNA walker comprises a recognition sequence for a deoxyribozyme (DNAzyme), track chain track contains the cleavage sequence of DNAzyme, at Mg 2+ When present, DNAzyme can act to break the substrate strand, resulting in weaker binding of DNA walker to the orbitals, with the result that DNA walker can "walk" along the orbital molecules on the three-dimensional surface and cleave and release a large number of substrate molecules. On this basis, the sequence of the target nucleic acid molecule is introduced into a DNA walker and DNAzyme is inactivated by hybridization with a partially complementary protective sequence, the protective strand. When the target molecule is present, the protective sequence is removed by a toehold mediated strand displacement reaction, releasing the DNA walker, and DNAzyme is activated to cleave the substrate allowing signal amplification. The supernatant is sucked out by utilizing magnetic separation and transferred to a B tube, the B tube reaction realizes visual presentation of detection results, the aggregation of nano gold is utilized, specifically, DNA is modified on a 13nm gold ball, the originally dispersed nano gold is aggregated by utilizing complementary pairing of the amplified product of the previous step and the DNA base on the gold ball, different concentrations of the amplified product can induce aggregation of different degrees, gradient change colors are presented, and the gradient is directly corresponding to the concentration gradient of a target object (the schematic diagram of the A, B tube reaction is shown in figure 1).
The technical scheme of the invention mainly comprises two invention points: (1) Firstly, the DNAzyme, DNAzyme driven by metal ions is a nucleic acid sequence in the design of the DNA walker, can be directly contained in the sequence design of the walker and the track, does not need to be additionally added, and compared with the traditional protease, DNAzyme is not easily influenced by environmental factors such as temperature, pH and the like, has higher catalytic activity and is more stable. (2) Secondly, DNA walker is assembled on three-dimensional magnetic beads, so that walking efficiency can be improved, and magnetic separation can be utilized to simply and rapidly separate the magnetic beads from supernatant (washing magnetic beads) after each step of reaction, so that the influence of the previous reaction on a subsequent reaction system is small, and the product added with the nano gold system is purer.
The detailed process of the most preferable technical scheme of the invention is as follows: the rapid and convenient isothermal HIV nucleic acid detection kit comprises a tube A reaction reagent and a tube B reaction reagent, wherein the specific steps for preparing the tube A reaction reagent and the tube B reaction reagent are as follows:
preparation of tube A reagent:
(1) walker to Protect chain in a molar ratio of 1:2 hybridize at 37 ℃ to form a partially complementary double-stranded structure.
(2) 0.1mg of streptavidin magnetic beads with a diameter of 1. Mu.M, the hybridization duplex prepared in step (1), the track single strand were co-dispersed in 50. Mu.l of a buffer system containing 0.1M NaCl, wherein the hybridization duplex: track single strand (molar ratio) =1:8, hybridization duplex concentration in the system 0.5 μm.
(3) The system is rotated on a rotary mixer at 20rpm for reaction at room temperature for 12-18.
(4) The supernatant was washed off, and the DNA-modified beads were redispersed in 40. Mu.l of a buffer system containing 0.1M NaCl to obtain a tube A reagent.
Preparation of tube B reagent: gold a and gold b (modified with different DNA, diameter about 13 nm) were dispersed in 1 XTAE 12.5mM Mg (A C ) 2 The final concentration of each gold was about 16-20nM in the buffer system, yielding a B-tube reagent.
The isothermal HIV nucleic acid detection kit comprises the following detection procedures:
(1) Mu.l of HIV DNA solution with unknown concentration is added into the tube A reaction reagent and reacted for 1 to 2 hours at the temperature of 30 to 37 ℃.
(2) The supernatant was blotted and the beads were redispersed in 50. Mu.l, 1 XTAE 12.5mM Mg (A C ) 2 In the buffer system of (2) and reacting for 1-2 hours at 30-37 ℃.
(3) The supernatant was separated, and 10. Mu.l of the supernatant was reacted in 10. Mu.l of the B-tube reagent at 30℃for 1 hour to obtain the results.
The room temperature of the invention is 30+/-10 ℃.
The invention has the beneficial effects that:
the kit designed by the invention is used for detecting HIV, the whole process is carried out in a low-temperature environment of about 30 ℃, and complex reagents and equipment are not needed (the reagents needed by detection are only pure water, 0.1M NaCl buffer solution and 12.5mM Mg) 2+ Buffer solution), the detection process is not longer than 5 hours, the operation is simple, and the detection process can be operated outside a laboratory. The colorimetric method relies on the nano gold agglomeration of modified DNA, the principle is that base complementation is more stable than the agglomeration of bare gold by relying on the change of salt concentration, and meanwhile, the naked eye signal reading can be realized without equipment and operation for detecting fluorescent signals.
Drawings
FIG. 1 is a schematic diagram of a A, B tube reaction.
FIG. 2 shows the result of colorimetric experiments; the test tube number is from small to large, the concentration of the target object is from small to large, the color of the test tube is gradually deepened from orange red of the nano gold, the test tube gradually becomes dark red when the concentration is 1nM, the test tube basically presents purple when the concentration is increased to 100nM, the concentration is further increased to purple deepen, and meanwhile, a more obvious sedimentation phenomenon occurs.
FIG. 3 is a graph showing the ultraviolet absorption spectrum of the B tube after adding different concentrations of the target.
FIG. 4 is a graph (R) of a linear fit of absorption values of a B-tube with different concentrations of the target 2 ＝0.9897)。
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the embodiments described herein are only for illustrating the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Example 1
A rapid and convenient isothermal HIV nucleic acid detection kitThe kit comprises a tube A reactant and a tube A reactant, wherein the tube A reactant comprises a walker and Protect chain, a track single chain, streptavidin magnetic beads and a 1 xTAE 0.1M NaCl buffer system; the B-tube reagent comprises gold a, gold B and 1 xTAE 12.5mM Mg (A C ) 2 A buffer system.
Wherein the specific steps for preparing the A-tube reactant and the A-tube reactant are as follows:
preparation of tube A reagent:
(1) walker to Protect chain in a molar ratio of 1:2 at 37 ℃ to form a partially complementary hybridized double strand structure.
(2) 0.1mg of streptavidin magnetic beads with a diameter of 1. Mu.M, the hybridization duplex prepared in step (1), the track single strand were co-dispersed in 50. Mu.l of a buffer system of 1 xTAE 0.1M NaCl, wherein the hybridization duplex: track single strand (molar ratio) =1:8, hybridization duplex concentration in the system 0.5 μm.
The nucleotide sequences of the walker and the protective chain and the track single chain are respectively as follows:
(3) The above system was subjected to a rotary reaction on a rotary mixer at 20rpm at room temperature for about 12 hours.
(4) The supernatant was washed off, and the DNA-modified beads were redispersed in 40. Mu.l of a 1 XTAE 0.1M NaCl buffer system to give a tube A reagent.
Preparation of tube B reactant: gold a and gold b are prepared by respectively modifying DNA molecules of two sequences a and b on the surface of nano gold. The preparation method and modification method of the nano-gold are conventional methods known to those skilled in the art (nano-gold is prepared and modified by a standard sodium citrate method, nat Protoc 1,246-252 (2006)) and the preparation method and modification method of the nano-gold are known to those skilled in the art, and then DNA molecules of two sequences, namely a and b, are modified on the surface of the nano-gold by a sodium citrate reduction method, respectively, so that the nano-gold is named gold a and gold b.
Gold a and gold b were dispersed in 1×TAE12.5mM Mg (A C ) 2 Buffer systemThe final concentration of each gold was about 20nM, giving a B-tube reagent.
The DNA molecules of the two sequences a and b are respectively:
the isothermal HIV nucleic acid detection kit comprises the following detection procedures:
(1) Mu.l of HIV DNA solution with different concentrations was added to the tube A and reacted at 32℃for 2 hours.
(2) The supernatant was blotted and the beads were redispersed in 50. Mu.l, 1 XTAE 12.5mM Mg (A C ) 2 In the buffer system of (2) were reacted at 32℃for 2 hours.
(3) The supernatant was separated, and 10. Mu.l of the supernatant was reacted in 10. Mu.l of the B-tube reagent at 30℃for 1 hour to obtain the results.
TABLE 1 comparison of target concentration and numbering for reaction systems
Experimental results: the ultraviolet absorption value reflects that the absorption value curve moves upwards along with the increase of the concentration of the target object, the agglomeration degree becomes high, the color of the corresponding gold is changed from orange red to deep red to purple, and finally aggregation and sedimentation are generated (as shown in figure 2); the absorbance linear fit shows that the concentration of the target varies substantially linearly between 0.1nM and 500nM, and that more than 500nM is gentle, so that the tube A prepared as described above can detect the target DNA fragment of more than 0.1nM to 500nM in tube B (see FIGS. 3 and 4). The lowest concentration distinguishable by naked eyes was 0.1nM, and above 500nM, the purple color of the B tube was deeper and more pronounced sedimentation occurred.
Sequence listing
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Claims (7)

1. An isothermal HIV nucleic acid detection kit, characterized in that: the kit comprises a tube A reaction reagent and a tube B reaction reagent; the A tube reaction reagent comprises a buffer system containing NaCl, a walker and a protective chain, a track single chain and streptavidin magnetic beads; the B pipe reaction reagent packageInclude gold a, gold b and Mg 2+ The buffer system is characterized in that gold a and gold b are respectively prepared by modifying DNA molecules of a sequence and a sequence b on the surface of nano gold;
the sequences of the walker and the protective chain and track single chains are respectively as follows:
walker：biotin-T-40-T-TTCCACACTGACTCTCTTCTCCGAGCCGGTCGAAATAGTC
GCGC；
Protect：GAAGAGAGTCAGTGTGGAAAATCTCTAGC；
track：biotin-T-10-T-GCGCGACTAT(rA)GGAAGAGAATCCATCACTTCATATTATGTT
AAATGCACTA；
the DNA molecules of the two sequences a and b are respectively:
a DNA molecule of sequence a: TATGAAGTGATGGAT-SH;
b DNA molecule of sequence: TAGTGCATTTAACAT-SH.
2. The isothermal HIV nucleic acid detection kit according to claim 1, wherein:
the preparation method of the A-tube reactant comprises the following steps:
(1) Walker and Protect chains were combined in a molar ratio of 1:2 at 37 ℃ to form a partially complementary hybridization duplex;
(2) Dispersing streptavidin magnetic beads and the hybridization double chains and the track single chains prepared in the step (1) in a buffer system containing NaCl together to obtain a reaction system; the molar ratio of the hybridization double strand to the track single strand is 1:8, and the concentration of the hybridization double strand in the reaction system is 0.5 mu M;
(3) Reacting the reaction system obtained in the step (2) in a rotary mixer for 12-18 hours to obtain magnetic beads modified with DNA;
(4) Washing the supernatant, and re-dispersing the magnetic beads modified with DNA in a buffer system containing NaCl to obtain a tube A reaction reagent;
the preparation method of the B pipe reactant comprises the following steps:
dispersing gold a and gold b into Mg 2+ And (3) in the buffer system, obtaining the B-tube reaction reagent.
3. The isothermal HIV nucleic acid detection kit according to claim 2, wherein: in preparing the B-tube reagent, gold a and gold B are dispersed into Mg 2+ The final concentration of each gold in the buffer system was 16-20nM.
4. Isothermal HIV nucleic acid detection kit according to claim 1 or 2, characterized in that: the streptavidin magnetic beads are streptavidin magnetic beads with the diameter of 1 mu M; the buffer system containing NaCl is 1 xTAE 0.1M NaCl; said Mg 2+ The buffer system was 1 xTAE 12.5mM Mg (A C ) 2 A buffer system.
5. The detection method of isothermal HIV nucleic acid detection kit according to any one of claims 1-3, wherein: the detection method comprises the following steps:
(1) Adding a DNA solution of a sample to be detected into a tube A reaction reagent for reaction;
(2) The reaction solution was sucked to dry the supernatant, and the beads were redispersed in Mg 2+ The reaction is carried out in a buffer system, and the Mg 2+ The buffer system was 1 xTAE 12.5mM Mg (A C ) 2 A buffer system;
(3) Separating the supernatant from the reaction liquid, taking the supernatant to react in a B tube reaction reagent, and after the reaction is finished, reflecting the detection result by contrasting the color of the B tube nano-gold, wherein the concentration of the target is increased, and the color of the B tube is deepened from orange red to deep red or even purple red; the lowest concentration distinguishable by the naked eye was 0.1nM, and above 500nM, the purple color of the B tube was deeper and significant sedimentation occurred.
6. The method of claim 5, wherein: the reaction conditions in step (1) are: reacting for 1-2 hours at 30-37 ℃; the reaction conditions in step (2) are: reacting for 1-2 hours at 30-37 ℃; the reaction conditions in step (3) are: the reaction was carried out at 30℃for 1 hour.
7. The method of detecting according to claim 6, wherein: the reaction conditions in step (1) are: reacting for 2 hours at 32 ℃; the reaction conditions in step (2) are: reacting for 2 hours at 32 ℃; the reaction conditions in step (3) are: the reaction was carried out at 30℃for 1 hour.
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