WO2021098436A1 - Visualized non-cell in vitro color development system, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a visualized non-cell in vitro color development system, and a preparation method therefor and the use thereof. The preparation method comprises the following steps: (1) preparing engineering bacteria: knocking out beta-galactosidase genes in an Escherichia coli strain through a Red recombinant method to obtain the engineering bacteria; (2) preparing an engineering bacteria lysate: determining the growth curve of the engineering bacteria, and preparing the engineering bacteria lysate; and (3) compounding a color development system: compounding the color development system which comprises the engineering bacteria lysate, an energy supply system, an amino acid mixed solution and a substrate mixed solution which is necessary for the synthesis of other proteins, wherein the energy supply system contains phosphoenolpyruvate. The non-cell color development system can be applied to the quick detection of pathogenic microorganisms, and the reaction situation can be seen by the naked eye of a user.

Description

A visual acellular in vitro color-developing system and its preparation method and application Technical field

The invention relates to the field of genetic engineering and synthetic biology detection, in particular to the establishment and application of a visual micro-acellular in vitro color-developing system.

Background technique

The acellular expression system is an in vitro expression system based on cell lysates. It has been widely used in the synthesis of proteins that are difficult to express in the body, such as membrane proteins, toxic proteins, and in vitro genetic network research (Jessica G. Perez ,Jessica C.Stark, and Michael C.Jewett.Cell-Free Synthetic Biology: EngineeringBeyond the Cell[J]Cold Spring HarbPerspect Biol.2016; 1:8(12)). At present, two types of non-cellular expression systems, prokaryotic and eukaryotic have been developed. Prokaryotic expression systems, especially E. coli expression systems, have always been favored due to their high yield, good stability and low cost.

In the E. coli non-cellular expression system, E. coli lysate contains the most basic materials required for gene expression. The transcription of genes in this system is generally regulated by exogenously added T7 RNA polymerase. In addition, it is also necessary to add exogenous substrates required for protein synthesis such as amino acids, NTP and energy substances. The non-cellular expression system commonly used in laboratories involves the composition of multiple energy systems (Kim DM, Swartz JR. Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis[J]BiotechnolBioeng.2001;74(4):309- 16.), such as sodium pyruvate/CoA/NAD, PEP/PK, CP/CK, cAMP/CP/CK, each energy system composition has its own advantages and disadvantages, such as phosphoenolpyruvate system provides more energy Fast, high protein yield, but expensive; sodium pyruvate energy system is cheap, but protein yield is relatively low.

At present, there are various detection methods for pathogenic microorganisms (Mangal M, Bansal S, Sharma SK, et al. Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety[J] Crit Rev Food Sci Nutr. 2016; 56(9 ):1568-84.), such as common culture methods, RT-PCR, molecular beacon method, colloidal gold method, etc., but these detection methods require expensive instruments and equipment, extremely skilled, and require professional operators , Does not have the characteristics of simplicity, speed, and portability, so it is difficult to detect by these means in remote areas and on-site where there is a lack of professional and technical personnel. In summary, it is imminent to develop simple and fast detection methods that do not require expensive equipment.

Summary of the invention

In response to the above problems, we established a chromogenic expression system that relies on β-galactosidase to hydrolyze the substrate. First, knock out E. coli’s own β-galactosidase gene to eliminate background interference; then, optimize the non-cellular system The composition improves the expression efficiency of the non-cellular system. The main purpose of the present invention is to establish a simple, fast and visualized micro-acellular color development system.

The technical scheme of the present invention is as follows:

A method for preparing a visual acellular in vitro color-developing system includes the following steps:

(1) Preparation of engineering bacteria:

Using Red recombination method to knock out the β-galactosidase gene in E. coli strains to obtain engineered bacteria;

(2) Preparation of engineered bacteria lysate:

Determine the growth curve of the above-mentioned engineered bacteria, and prepare the engineered bacteria lysate;

(3) Preparation of color rendering system:

A color-developing system is prepared, including an engineered bacteria lysate, an energy supply system, an amino acid mixture and other substrate mixtures required for protein synthesis; the energy supply system contains phosphoenolpyruvate.

Preferably, the concentration of the energy supply system is 20-28 mM; the concentration of the amino acid mixture is 40-60 mM.

Preferably, the E. coli strain is BL21(DE3); the engineered bacteria lysate is a BL21(DE3) lysate that knocks out the β-galactosidase gene, that is, BL21(DE3) ΔlacZ lysate.

Preferably, the amino acid mixture (containing 20 amino acids) is made of leucine, methionine, valine, lysine, phenylalanine, tryptophan, threonine, glycine, and arginine. Acid, histidine, alanine, aspartic acid, asparagine, isoleucine, proline, tyrosine, serine, cysteine, glutamic acid and glutamine are prepared by adding water The mixture of equimolar concentration.

Preferably, the substrate mixture required for the synthesis of other proteins is magnesium acetate, potassium glutamate, ammonium acetate, HEPES-KOH, tRNA, folic acid, dithiothreitol, putrescine, spermidine, oxalic acid, ATP, CTP, UTP, GTP, PEG8000, RNase inhibitor and chlorophenol red-β-D-galactopyranoside.

Preferably, trehalose or polyvinyl alcohol is added as a protective agent in the color development system, the concentration of trehalose is 0.2-0.6M, and the concentration of polyvinyl alcohol is 5%-10%.

The application of the visualized non-cellular in vitro chromogenic system in the detection of pathogenic microorganisms, specifically, a Toehold-Switch element plasmid for the detection target of pathogenic microorganisms is designed, and the 5'end of the plasmid contains a specific binding sequence with the detection target. The'end contains the β-galactosidase gene; the above plasmid and the sample to be tested are added to the non-cellular in vitro color development system, and the reaction is allowed to stand still. The color development is used to determine the presence or absence of the detection target in the system. When it is yellow, There is no corresponding detection target in the system. When it appears purple, the system has a corresponding detection target.

Preferably, the reaction temperature is 30±5°C, and the standing time is 20-60 min.

The establishment of a visual acellular color system includes the following steps:

(1) Construction of Escherichia coli BL21(DE3)ΔlacZ engineering bacteria

The Red recombination method was used to knock out the β-galactosidase gene in Escherichia coli BL21(DE3) to obtain BL21(DE3)ΔlacZ engineering bacteria.

(2) Preparation of engineering bacteria BL21(DE3)ΔlacZ lysate

Determine the growth curve of BL21(DE3)ΔlacZ engineering bacteria. According to the growth curve, IPTG induction is performed when the OD is 0.4-0.6, and the bacteria are collected when the OD is 3.5-4.0. Wash 3 times with S30 buffer, 2 times with high pressure disintegrator 20kpsi, 12,000g, 30min, take the supernatant, which is the lysate of engineering bacteria BL21(DE3)ΔlacZ.

(3) Optimization of the composition of the non-cellular color rendering system

① Optimization of the amount of T7 RNA polymerase added exogenously;

②Optimization of the composition of the energy system.

(4) Visual reaction expression of non-cellular color system

A 15μL system was prepared, containing the engineered bacteria BL21(DE3)ΔlacZ lysate, energy supply system, amino acid mixture and reaction buffer; the system was allowed to stand at 30°C for 20min to react for expression and color development.

(5) Optimization of the stability of the non-cellular color system

According to the storage temperature conditions, select the appropriate exogenous protective agent to improve the long-term stability of the non-cellular color-developing system.

(6) Application of acellular color system in the detection of pathogenic microorganisms

The Toehold-Switch element is a homologous RNA pair that regulates the target protein or biological activity by regulating the patent or translation process. It consists of two RNA chains called the switch and the trigger, called the Switch chain and the Trigger chain, respectively. Switch RNA contains the coding sequence of the regulated gene. The upstream of the coding sequence contains a strong ribosome binding site (RBS) and start codon (AUG) based hairpin processing module, and then the coding is added to A common 21 nt linking sequence of low molecular weight amino acids at the N-terminus of the target protein gene. The single-stranded leader sequence at the 5'end of the hairpin module provides the initial binding site of the Trigger RNA strand. The Trigger RNA contains an extended single-stranded region, which completes the process of branching and migration with the hairpin to expose the RBS and the initiation codon, thereby starting the translation and expression of the target protein gene.

The non-cellular chromogenic system of the present invention is used with Toehold-Switch element, the 5'end part of the element contains the specific binding sequence with the detection target, and the 3'end contains the β-galactosidase gene. If there is a detection target in the test sample, the element binds to the detection target and can successfully express β-galactosidase, then the system appears purple; on the contrary, if there is no corresponding detection target, the system appears yellow. Combining the chromogenic system with Toehold-Switch components for rapid detection of pathogenic microorganisms, such as Norovirus type GII.4, Norovirus type GII.17, human coronavirus, Zika virus and Staphylococcus aureus, etc. When the detection target appears in the system, the color of the system changes from light yellow to purple, and the naked eye can judge the presence or absence of the detection target and determine its specificity. The application of the non-cellular chromogenic system of the present invention is not limited to the above-mentioned several pathogenic microorganisms. As long as the corresponding Toehold-Switch element is designed, the pathogenic microorganisms can be detected.

The advantages of the present invention are: simple operation, short time-consuming, low cost, miniaturization (10-15 μL), and the reaction situation can be observed by naked eyes; the color-developing system can be added with a protective agent under corresponding storage conditions Extend the storage time. It provides convenience for its application; the color development system can realize the judgment of the reaction situation by observing the color change by naked eyes, and can be widely used in the detection of pathogenic microorganisms.

Description of the drawings

Figure 1 is a graph showing the growth curve of BL21(DE3)ΔlacZ engineering bacteria in Example 2.

Figure 2 is an optimized diagram of the components of the non-cellular color rendering system in Example 3; A is an optimized diagram of T7 RNA polymerase, and B is an optimized diagram of an energy composition system.

Figure 3 is an expression diagram of the pET28a-lacZ plasmid in Example 3 in a micro-acellular color-developing system.

Figure 4 shows the results of the non-cellular color-developing system in Example 4 under different protective agents and storage conditions; A is the results of the protective agent trehalose and polyvinyl alcohol under 4℃ storage conditions, and B is the protective agent trehalose And polyvinyl alcohol storage conditions at -20°C.

Figure 5 is a graph showing the detection results of the non-cellular color-developing system in Example 5 in GII.4-NoV, GII.17-NoV, HCoV and Zika; A is a graph showing the cross-detection results of 4 pathogenic microorganisms, and B is a graph of 4 pathogenic microorganisms. Cross detection color chart.

Fig. 6 is a graph showing the detection result of the non-cellular system in Example 5 in Staphylococcus aureus.

Detailed ways

In order to better understand the above-mentioned technical solution, the following detailed description will be given with the above-mentioned technical solution in conjunction with the drawings and specific implementations of the specification.

Example 1 Construction of Escherichia coli BL21(DE3) ΔlacZ engineering bacteria.

1. Design the homology arm sequence according to the knockout target gene sequence:

2. Using the pKD4 plasmid as a template, use the above homology arm primers to amplify the kana resistance gene. The specific steps are as follows:

(1) PCR reaction system (50μL):

(2) PCR amplification reaction program

(3) After the PCR reaction is completed, 1% (w/v) agarose gel electrophoresis is identified, and a gene fragment containing the linker sequence with a size of about 1.6 kb is obtained. The PCR product is purified and recovered to obtain a kana antibiotic gene. Homologous arm fragment, stored at -20℃ for later use

3. Using the principle and method of Red homologous recombination to knock out the lacZ gene of BL21(DE3) (purchased from Tiangen Biochemical Technology Co., Ltd.), the specific knockout method is as follows:

(1) The pKD46 plasmid is chemically transformed into the BL21(DE3) strain to be knocked out.

(2) The homology arm fragment containing the kana antibiotic gene recovered above was introduced into E. coli BL21(DE3)/pKD46 by electrotransformation. The electrotransformation parameters are as follows: electrotransformation equipment: eppendorf2510; electrotransformation voltage: 2.0KV , 4ms.

(3) A kanamycin-resistant LB plate with a final concentration of 50 μg/mL was added to screen positive clones and verified by colony PCR. The constructed mutant was named BL21(DE3)ΔlacZ.

Example 2 Preparation of E. coli BL21 (DE3) ΔlacZ lysate

1. Determination of the growth curve of Escherichia coli BL21(DE3)ΔlacZ engineering bacteria:

(1) Pick the single colony of BL21(DE3)ΔlacZ constructed above from the plate and inoculate it in 5mL LB medium, and cultivate overnight at 37°C in a shaker at 220 rpm.

(2) Transfer the seed solution to 2 bottles of 300 mL of 2×YTPG medium at a ratio of 1:100, and continue culturing on a shaker at 37°C at 220 rpm.

(3) Sampling and measuring OD600 after 1 hour, and then sampling once every half an hour to detect OD600, repeat 3 times and record.

(4) When the OD600 is between 0.4 and 0.6, add 1 mM isopropyl thiogalactoside (IPTG) to a bottle of culture medium for induction, take samples every half an hour together with the control group to determine OD600, repeat and record 3 times.

(5) Plot the growth curve of the BL21(DE3)ΔlacZ engineering bacteria based on the data, as shown in Figure 1.

2. Preparation of E. coli BL21(DE3)ΔlacZ lysate:

(1) Pick the single colony of BL21(DE3)ΔlacZ constructed above from the plate and inoculate it in 5mL LB medium, and cultivate overnight at 37°C in a shaker at 220 rpm.

(2) Transfer the seed solution to 300 mL of 2×YTPG medium at a ratio of 1:100, and continue culturing in a shaker at 37°C at 220 rpm.

(3) According to the growth curve determined in 1, when cultured for about 2 hours and the bacterial concentration OD600 is 0.4-0.6, add 1mM IPTG for induction and continue the culture. When the OD600 value of the bacterial concentration reaches 3.5 to 4, the bacterial cells are collected.

(4) Resuspend the bacteria in pre-chilled S30Buffer (10mM Tris (pH8.2), 14mM magnesium acetate, 60mM potassium glutamate, 2mM dithiothreitol), mix thoroughly with a shaker, and centrifuge at 4°C at 8000g 7min, pour the supernatant. Repeat this step 3 times.

(5) Add 1mL of pre-cooled S30Buffer per 1g of bacteria and mix well.

(6) Crush the cells twice with a high-pressure cell disruptor at 20 kpsi. Centrifuge the lysate at 12,000g for 20min at 4°C, and take the supernatant.

(7) Centrifuge again at 12,000g for 20 minutes at 4°C, and take the supernatant, which is the lysate. Aliquot the supernatant into 0.5mL EP tubes, 50μL per tube, quick-frozen in liquid nitrogen, and store in the refrigerator at -80℃.

Example 3 Establishment and verification of a cell-free coloring system

1. pET28a-lacZ vector construction:

The E. coli β-galactosidase (lacZ) gene was amplified by PCR, and then connected to the expression vector pET28a (preserved in the laboratory) to construct a recombinant expression plasmid that can successfully express β-galactosidase. The specific construction method is as follows:

(1) Design primers for amplification of lacZ gene:

(2) PCR reaction system (50μL):

(3) PCR amplification reaction program:

After the PCR reaction was completed, 1% (w/v) agarose gel electrophoresis was used for identification, and the gel was cut to recover the target fragment of about 3100bp.

(4) The recovered product and pET28a plasmid were subjected to BamHI/Hind III double digestion at the same time, and the digestion system was as follows (50μL):

Place it in a constant temperature PCR instrument at 37°C to react for 3 hours. After the enzyme digestion reaction is completed, it is purified and recovered.

(5) Connect the recovered product obtained in step (4) with T4 ligase, and the specific operation is as follows: connect the reaction system (20 μL):

Placed in a 16°C constant temperature PCR machine to react for 12 hours.

(6) After the ligation reaction is completed, transform the ligation product into Escherichia coli DH5α, and then apply the bacterial solution to the LB solid plate containing kanapenicillin (final concentration of 50μg/mL), and place it in a 37°C incubator overnight. . The conversion method uses a chemical conversion method.

(7) Sequencing and identification of the single clones grown in the plate. The plasmid pET28a-lacZ which correctly carries the lacZ gene was screened out.

2. Establishment of non-cellular color rendering system:

(1) Optimization of system components

The BL21(DE3)ΔlacZ engineering bacteria itself can produce T7RNA polymerase after being induced by IPTG. Since T7RNA polymerase is indispensable in the color-developing system, it is guessed whether the addition of T7RNA polymerase can improve the rapid and large-scale expression of the color-developing system. , To achieve a better color rendering effect. It was experimentally verified that when T7 RNA polymerase (purchased from NEB, #M0251S) was additionally added, the expression level of the reaction system decreased instead, and the result is shown in Figure 2A. Therefore, the color-developing system does not require additional T7 RNA polymerase.

In the non-cellular system, there are many system components that can provide energy. We chose the cheap pyruvate (SP) energy system and the phosphoenolpyruvate (PEP) energy system that quickly provides ATP. After comparing experiments, it is found that the effect of PEP is better than that of SP. As shown in Figure 2B, the reason may be that PEP can quickly supply energy, so that the system can quickly achieve the color rendering effect.

(2) The establishment and visual expression of system components

The dosage of each component in the system is shown in Table 1:

Reagent Use concentration Storage temperature Mixture A To -20℃ RNase inhibitor 0.27U/μL -20℃ UTP 0.86mM -20℃ ATP 0.86mM -20℃ GTP 0.86mM -20℃ CTP 0.86mM -20℃ PEG8000 2% -20℃ PEP 20mM -20℃ 20 kinds of amino acid mixture 40～60mM -20℃ Engineering bacteria lysate 25% -80℃ CPRG 0.6mg/mL -20℃

The composition of mixture A is shown in Table 2:

Reagent Use concentration Storage temperature Magnesium acetate 12mM -20℃ Potassium Glutamate 90mM -20℃ Ammonium acetate 80mM -20℃

HEPES-KOH 57mM -20℃ DTT 2mM -20℃ Putrescine 1mM -20℃ Spermidine 1.5mM -20℃ oxalic acid 4mM -20℃ Folinic acid 0.034mg/mL -20℃ tRNA 0.171mg/mL -20℃

Prepare a 15μL reaction system according to Table 1 and Table 2, add plasmid pET28a-lacZ (13.3ng/μL), stand at 30°C for 20 minutes, observe the color change, and determine the OD570 value. The result is shown in Figure 3: static at 30°C After standing for 20 minutes, the color of the reaction system changed from light yellow to purple.

Example 4 Exploration of the stability of the non-cellular color-developing system

The non-cellular color-developing system contains a variety of enzyme components. After being placed at 4°C or -20°C for 1 week, the activity decreases significantly. As a new type of biomolecular protective agent, trehalose has been extensively studied and is often used as a protein stabilizer; there are also reports in the literature that polyvinyl alcohol is often used as an additive to maintain the activity of enzymes in the reaction system. In order to improve the stability of the color-developing system, trehalose (purchased from Beijing Probosin Biotechnology Co., Ltd., CAS#99-20-7) and polyvinyl alcohol (PVA) (purchased from Sigma-Aldrich, 360627) influence on the stability of the color-developing system. The experimental results are shown in Figure 4. As the storage time increases, the activity of the color-developing system decreases; under the storage condition of 4℃, the stabilization effect of adding trehalose to the system is better than that of polyvinyl alcohol; but at -20℃ Under storage conditions, the stabilizing effect of adding polyvinyl alcohol to the system is better than that of trehalose. Therefore, according to different storage conditions, different protective agents can be selected to extend the storage time of the system.

Example 5 Application of the non-cellular chromogenic system in the detection of pathogenic microorganisms

This embodiment is applied to the Toehold-Switch switch for RNA detection. The detection target is Norovirus type GII.4. The Toehold-Switch element is designed for this target, combined with the non-cellular color-developing system, and the presence of the detection target is judged by the change of the system color. Happening.

The Toehold-Switch element for Norovirus GII.4, Norovirus GII.17, and human coronavirus, as well as the Toehold-Switch element for Zika virus reported in the literature, etc., obtained by laboratory screening, were applied to the above non-cellular in vitro display. The color system determines the presence or absence of the detection target in the system by color development. Prepare a cell-free coloring system according to Tables 1 and 2. The plasmid is the Toehold-Switch element plasmid for each detection target. After interacting with Trigger plasmids of different targets at 30°C for 1 hour, observe the color change of the system and determine the OD570. The results are as follows Shown in Figure 5. The results show that when there is no GII.4 Norovirus target Trigger plasmid in the system, the system color does not change, showing light yellow, and the OD value is low; when there is GII.4 Norovirus target Trigger plasmid in the system, the system The color changes from light yellow to purple, and the OD570 value increases.

In the same way, this embodiment combines the Toehold-Switch switch, the detection target is Staphylococcus aureus, the Toehold-Switch element is designed for this target, combined with the non-cellular color-developing system, and the existence of the detection target is judged by the color change of the system.

The Toehold-Switch elements for Staphylococcus aureus, Listeria monocytogenes, Salmonella and Vibrio parahaemolyticus, etc. obtained by laboratory screening are applied to the above-mentioned non-cellular in vitro color development system, and the detection in the system is determined by color development. The presence or absence of goals. Prepare the non-cellular color system according to Table 1 and 2. The plasmid is the Toehold-Switch element plasmid for each detection target. After interacting with the Trigger plasmid of different targets at 30℃ for 0.7h, observe the color change of the system and measure the OD570. The result As shown in Figure 6. The results show that when there is no Staphylococcus aureus target Trigger plasmid in the system, the color of the system does not change, showing light yellow, and the OD value is low; when there is a Staphylococcus aureus target Trigger in the system, the system color changes from light yellow It turns purple and the OD570 value increases.

In summary, we have established a visual micro-acellular in vitro color-developing system, which can be applied to the detection of pathogenic microorganisms. When the detection target appears in the system, the color of the system changes from light yellow to purple, which can be done by the naked eye. Determine the presence or absence of detection targets.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

A method for preparing a visual acellular in vitro color-developing system, which is characterized in that it comprises the following steps: (1) Preparation of engineering bacteria: Using Red recombination method to knock out the β-galactosidase gene in E. coli strains to obtain engineered bacteria; (2) Preparation of engineered bacteria lysate: Determine the growth curve of the above-mentioned engineered bacteria, and prepare the engineered bacteria lysate; (3) Preparation of color rendering system: A color-developing system is prepared, including an engineered bacteria lysate, an energy supply system, an amino acid mixture and other substrate mixtures required for protein synthesis; the energy supply system contains phosphoenolpyruvate. The preparation method according to claim 1, wherein the concentration of the energy supply system is 20-28 mM; the concentration of the amino acid mixture is 40-60 mM. The preparation method according to claim 1, wherein the E. coli strain is BL21(DE3); the engineered bacteria lysate is BL21(DE3) lysate with β-galactosidase gene knocked out, namely BL21(DE3)ΔlacZ lysate, the concentration is 20-30%. The preparation method according to claim 1, wherein the amino acid mixture is made of leucine, methionine, valine, lysine, phenylalanine, tryptophan, and threonine. , Glycine, arginine, histidine, alanine, aspartic acid, asparagine, isoleucine, proline, tyrosine, serine, cysteine, glutamic acid and glutamine A mixture of equimolar concentration prepared by adding amide to water. The preparation method according to any one of claims 1 to 4, wherein the substrate mixture required for the synthesis of other proteins is magnesium acetate, potassium glutamate, ammonium acetate, HEPES-KOH, tRNA, folic acid , Dithiothreitol, putrescine, spermidine, oxalic acid, ATP, CTP, UTP, GTP, PEG8000, RNase inhibitor and chlorophenol red-β-D-galactopyranoside. The preparation method according to claim 5, characterized in that trehalose or polyvinyl alcohol is added as a protective agent in the color-developing system. The preparation method according to claim 6, wherein the concentration of the trehalose is 0.2-0.6M, and the concentration of the polyvinyl alcohol is 5%-10%. A visualized non-cellular in vitro color development system prepared by the method of any one of claims 1-7. The application of the visualized non-cellular in vitro chromogenic system in the detection of pathogenic microorganisms according to claim 8, characterized in that the Toehold-Switch element plasmid for the detection target of pathogenic microorganisms is designed, and the 5'end of the plasmid contains the specificity of the detection target. The binding sequence contains the β-galactosidase gene at the 3'end. The above plasmid and the sample to be tested are added to the non-cellular in vitro color-developing system, and the reaction is allowed to stand still. The color development is used to determine the presence or absence of the detection target in the system. When it is yellow, there is no corresponding detection target in the system, and when it is purple, there is a corresponding detection target in the system. The application according to claim 9, characterized in that the reaction temperature is 30±5°C, and the standing time is 20-60 min.

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