CN101358963A - A method for rapid detection of malachite green in aquatic products using aptamers - Google Patents

Abstract

The present invention discloses a method which adopts the aptamer rapidly to detect the malachite green in water products. The sample to be detected, the malachite green aptamer and the report sequence with detection markers are mixed; after the report sequence and the malachite green are competitively combined with the malachite green aptamer, the markers on the report sequence is detected, and the content of the malachite green in the sample to be detected is calculated. The kit and the fast detecting test paper, which are prepared according to the method, can better satisfy the current urgent needs for detecting the malachite green residue in the water products, can be used in the detection of the customs and the food sanitation department, and can be easily promoted and applied in a large scale.

Description

A method for rapid detection of malachite green in aquatic products using aptamers

technical field

The invention relates to the field of food detection, in particular to a method for detecting malachite green in aquatic products.

Background technique

Malachite green (Malachite green, referred to as MG), belongs to triphenylmethane dyes, and its metabolite is colorless malachite green (Leucomalachite green, LMG). For a long time, because malachite green has good antifungal properties in aquaculture disease, freshness of aquatic products and low price, etc. Therefore, it has become the "fishing medicine" favored by many farmers to use, but these two substances have side effects such as high toxin, high residue, high teratogenicity and mutagenicity, not only It has brought huge obstacles to the export of aquatic products in our country, and more importantly, it has constituted a potential threat to people's health.

In view of the harmfulness of malachite green and its metabolites, many countries have listed it as a banned drug for aquaculture, and my country also listed it in the "List of Veterinary Drugs and Their Compounds Banned for Food Animals" in May 2002. However, Due to the weak awareness of aquaculture farmers' healthy breeding and the imperfect safety management mechanism of aquatic products, in 2005 alone, various aquatic products (such as: eel, eel, High residues of malachite green were detected in soft-shelled turtles, etc.).

At present, there are mainly three kinds of detection methods about malachite green and its metabolites, but these methods all have certain limitations: (1) high performance liquid chromatography, when the content is low, it is difficult to qualitatively utilize the retention time; 2) Gas chromatography method can only be used to qualitatively colorless malachite green; (3) Liquid chromatography-mass spectrometry (LC/MS) has a better effect but is expensive; therefore, malachite green and its metabolites The establishment of fast, stable and reliable residue detection methods in aquatic animals has become a technical problem to be solved urgently at home and abroad.

The core technology of immunological methods is to obtain malachite green-specific antibodies through experimental animals, but malachite green has strong immunotoxicity, and it is very difficult to prepare malachite green-specific antibodies, and the cycle of preparing an antibody is also very long. It takes a long time, generally more than 6 months. Therefore, obtaining specific antibodies through experimental animals has become a bottleneck problem in the study of rapid detection methods for malachite green.

Oligonucleotide aptamer technology is a new type of molecular imprinting technology developed in recent years, which is obtained by in vitro screening using SELEX technology. SELEX technology was established by Tuerk and Gold in the early 1990s, through multiple rounds of selection and amplification process (PCR or RT-PCR) to screen random oligonucleotide libraries for oligonucleotide adaptations that specifically recognize target substances It is an effective method to study the structure and function of nucleic acid. SELEX technology has developed rapidly since its inception. At present, due to its advantages of large library capacity, wide range of target molecules for screened aptamers, high affinity and strong specificity, it has been widely used in life science basic research, disease treatment, drug screening and clinical research. Diagnosis and other fields have been widely explored and applied.

Chinese invention patent application 200510060995.1 discloses a colorless malachite green indirect competition ELISA detection kit in aquatic products. The detection plate of the kit is a detachable 96-well ELISA plate coated with a colorless malachite green-albumin conjugate, and the anti-colorless malachite green antibody is immunoimmunized with a colorless malachite green-albumin conjugate The monoclonal antibody prepared from mice, the detection sample is firstly homogenized and then extracted with ethyl acetate and cyclohexane, adjusted the pH value and then diluted for detection. The judgment standard for sample detection is: the regression curve with the logarithmic value of the standard sample concentration as the abscissa and the inhibition rate as the ordinate. According to the inhibition rate of each sample, the concentration of the corresponding sample can be read from the curve. The detection sensitivity of this method is 0.0023 μg/ml, and the detection range is 0.0016-1 μg/ml.

However, it is very difficult to obtain high-efficiency monoclonal antibodies in this technology, which also increases the cost.

Contents of the invention

The invention provides a rapid detection method for malachite green in aquatic products with convenient operation and high accuracy.

A method for quickly detecting malachite green in aquatic products using aptamers, mixing the sample to be tested with the malachite green aptamer and a reporter sequence with a detection marker, the reporter sequence is mixed with the malachite green in the sample to be tested After competitively binding to the malachite green aptamer, detect (detect with an enzyme-relative chromogenic substrate) the marker on the reporter sequence, and calculate the content of malachite green in the sample to be tested.

The detection marker is horseradish peroxidase, chemiluminescent group, chemical fluorescent group or colloidal gold and the like.

Preparation of the reporter sequence: According to the obtained malachite green adapter sequence, design and synthesize a reporter sequence complementary to the adapter sequence part (5' end palindromic complementary sequence), and mark the detection marker on the reporter sequence .

The malachite green aptamer is prepared through the following steps:

(1) Construct a random ssDNA library with a sequence length of about 80 bases. The two ends of the ssDNA sequence have fixed sequences with restriction endonuclease sites, and the middle is 35 random bases. According to the two ends of the ssDNA sequence Fixed sequence design of PCR primers;

(2) using asymmetric PCR method or biotin-streptavidin magnetic bead method to amplify the ssDNA library obtained in step (1);

(3) SELEX screening was carried out from the amplified ssDNA library by using malachite green-OVA conjugate coated microporous plate medium method or high-efficiency capillary electrophoresis method to obtain ssDNA specifically combined with malachite green, that is, malachite green gamete.

The asymmetric PCR method described in step (2) promptly introduces primers of different concentrations in the PCR amplification cycle (generally adopting the primer concentration of 50:1-100:1 ratio), and in the initial 10-15 cycles, the main product It is double-stranded DNA (dsDNA), but after the low-concentration primers are exhausted, a high-concentration primer-mediated PCR reaction can produce a large amount of ssDNA.

The biotin-streptavidin magnetic bead method described in step (2) is in the PCR system, the 5' end of the downstream primer is designed and synthesized to be labeled with biotin, and the ssDNA library is used as a template for PCR amplification to obtain The dsDNA product with biotin at the 3' end was separated and purified and combined with streptavidin magnetic beads (the dsDNA binds to the streptavidin on the magnetic beads through the biotin at the 3' end), and then with a certain concentration (0.15mol /L) NaOH denatures the dsDNA and dissolves it. A strand with biotin binds to streptavidin and stays on the magnetic beads, while a strand without biotin is dissociated. After separation and purification, it is used for the next step. One round of screening, repeating the above operation cycle enrichment (generally about 10 times) until the binding rate of malachite green and aptamer reaches 90%.

The screening method in which the microporous plate described in step (3) is a medium is to use a 96-well ELISA plate of malachite green and albumin conjugate (MG-OVA), and set albumin (OVA) to coat the hole and the blank at the same time. The control wells were all blocked with 3% BSA. The ssDNA library amplified and purified by PCR was first screened against the blank control wells to remove the ssDNA bound to BSA, and then screened with the OVA-coated wells to remove the ssDNA bound to OVA, and then transferred The malachite green-OVA conjugate is bound to the coated hole, and the ssDNA specifically combined with malachite green is obtained after elution, separation and purification.

The high-efficiency capillary electrophoresis method described in step (3) is to incubate a certain amount of PCR-amplified and purified ssDNA library with malachite green, and then separate the unbound sequence from the malachite green-bound complex by high-efficiency capillary electrophoresis, and collect the bound Sequence, repeat the above operation cycle enrichment-separation and finally get the ssDNA specifically combined with malachite green (ie malachite green aptamer).

According to the rapid detection method of the present invention, relevant reagents can be packaged or fixed to make a kit or rapid detection test paper.

The present invention also provides a test kit for detecting malachite green in aquatic products, comprising at least a test piece coated with malachite green aptamers (such as 96-well microplate or nitrocellulose membrane, etc.), buffer, coating buffer.

When the malachite green aptamer is coated on the test substrate, the 5' end of the linking sequence (such as 3'-ACTCATCTGTGA-5') can be connected with the 3' end of the malachite green aptamer sequence to synthesize the malachite green aptamer. Gamete-linker sequence; design and synthesize a fixed sequence complementary to the linker sequence (such as 3'-ATGAGTAGACACT-5'), fix the immobilized group (such as avidin on the surface of the NC membrane, and then pass the avidin The single-chain Biotin-ssDNA immobilized with biotin) is marked at the 3' end of the immobilized sequence, and the malachite green aptamer is coated on the test substrate through the immobilized group and the connecting sequence.

The present invention adopts an aptamer technology that does not rely on experimental animals, and through the systematic evolution of ligands by exponential enrichment (SELEX) in vitro screening to identify malachite with high affinity The green aptamer replaces the antibody in the traditional immunological method to establish a rapid detection method for malachite green.

The detection method of the present invention has the advantages of

1. The screening period is as short as 3 weeks;

2. Strong specificity and high sensitivity for binding to malachite green;

3. The synthesis cycle of aptamers is short, which is conducive to large-scale production;

4. The detection reagent has strong stability and is easy to refold after denaturation, which is convenient for transportation and storage.

The kit and rapid detection test paper developed according to the method of the present invention can better meet the current urgent need for malachite green residual detection in aquatic products, be used for customs detection and food hygiene department detection, and be easy to popularize and apply on a large scale.

Description of drawings

Fig. 1 is the schematic flow chart of malachite green aptamer preparation and malachite green detection method;

Figure 2 is a schematic diagram of malachite green aptamer immobilization;

Figure 3 is a suitable map for malachite green aptamer competition detection;

Detailed ways

Preparation of malachite green aptamer

(1) Construct a random ssDNA library with a sequence length of about 80 (range: 75 to 90) bases, with fixed sequences with restriction endonuclease sites at both ends of the ssDNA sequence, and the ssDNA sequence is: ( 5'ATAGAGCTCATGGAGTCTCCCTCGG-(N35)-TTTGGATCCGGCAGTGGTGGGCGGGC3'), with 35 random bases (N35) in the middle, and PCR primers were designed according to the fixed sequences at both ends of the ssDNA sequence;

Upstream primer: 5'-ATAGAGCTCATGGAGTCTCC-3',

Downstream primer: 5'-GCCCGCCCACCACTGCCGG-3'

(2) The asymmetric PCR method is used to amplify the ssDNA library obtained in step (1).

The operation process of asymmetric PCR method is as follows:

Introduce different concentrations of primers in the PCR amplification cycle (generally using a primer concentration in the ratio of 50:1-100:1), the main product is double-stranded DNA (dsDNA) in the first 10-15 cycles, but low-concentration primers are eliminated. After depletion, PCR reactions mediated by high concentrations of primers can generate large amounts of ssDNA. PCR reaction system: dsDNA template 2 μl, 10×PCR buffer 10 μl, MgCl ₂ 6 μl, dNTPs 100 μmol/L, Tag enzyme 2 μl, upstream primer 60 pmol, downstream primer 1 pmol, add deionized water to 100 μl. PCR reaction conditions: denaturation at 94°C for 5min, followed by 40 cycles of denaturation at 94°C for 30s, annealing at 65°C for 1min, extension at 72°C for 2min, and finally 10min at 72°C.

The ssDNA library obtained in step (1) can also be amplified by the biotin-streptavidin magnetic bead method.

The operation process of the biotin-streptavidin magnetic bead method is as follows:

In the PCR system, the 5' end of the downstream primer is designed and synthesized to be labeled with biotin, and the ssDNA library is used as a template to perform PCR amplification to obtain a dsDNA product with biotin at the 3' end, which is separated and purified to bind to the chain affinity Binding to prime magnetic beads (dsDNA binds to streptavidin on the magnetic beads through biotin at the 3' end), and then denatures and melts the dsDNA with a certain concentration (0.15mol/L) of NaOH, and one strand with biotin is combined with Streptavidin binds and stays on the magnetic beads, and a strand without biotin is dissociated. The ssDNA is dissolved in 20ul TE buffer, and the absorbance (A) value is measured. After separation and purification, it can be used for the next round of screening , repeat the above operation cycle enrichment (generally about 10 times) until the binding rate of malachite green and aptamer reaches 90%.

(3) SELEX screening was carried out from the amplified ssDNA library by using the malachite green-OVA conjugate coated microwell plate medium method to obtain ssDNA that specifically binds to malachite green, that is, the malachite green aptamer.

The operation process of malachite green-OVA conjugate coated microporous plate medium method is as follows:

The MG-OVA protein was coated on the enzyme-linked plate, overnight at 4°C, the coating solution was 0.05mol/L NaHCO ₃ , buffer solution of pH 9.6, and a blank control was set at the same time. Protein C coated wells and blank control wells were blocked with 3% BSA at 37°C for 2h. The random ssDNA library and a certain amount of tRNA were mixed with 3% BSA in the binding buffer SHCMK solution (20mmol/L Hepes pH 7.35, 120mmol/L NaCl, 5mmol/L KCl, 1mmol/L CaCl ₂ , 1mmol/L MgCl ₂ ) The blocked blank control wells were combined at 37°C for 40min. Wash with buffer washing solution (SHAMK solution + 0.05% Tween 20) 6 times), wash off unbound ssDNA, and then add elution buffer (20mmol/L Tris-HCl, 4mol/L guanidine isothiocyanate, 1mmol /L DDT, pH 8.3) at 80°C for 10 min, the ssDNA bound to protein C was eluted, extracted with phenol-chloroform, precipitated with ethanol, and dissolved in 20 μl TE buffer. The ssDNA was amplified by PCR with biotin-labeled primers into dsDNA with biotin at one end, the ssDNA was separated by biotin-streptavidin magnetic beads, and used as the ssDNA library for the next round of screening. Repeat the above operation cycle enrichment-separation to obtain ssDNA specifically combined with malachite green.

High-efficiency capillary electrophoresis can also be used to perform SELEX screening from the amplified ssDNA library to obtain ssDNA that specifically binds to malachite green, that is, the malachite green aptamer.

The operation process of high performance capillary electrophoresis method is as follows:

After incubating a certain amount of PCR-amplified and purified ssDNA library with malachite green, the unbound sequence was separated from the malachite green-bound complex by high-performance capillary electrophoresis, and the bound sequence was collected for the next round of screening.

Reagents used for malachite green aptamers obtained by SELEX screening are in Zhan Linsheng's article "Establishment of SELEX Screening Oligonucleotide Aptamer Method for Random Single-Stranded DNA Libraries" ("Progress in Biochemistry and Biophysics" 2003 Volume 30 (1) 151 to 155) are disclosed.

After the screening and sequencing of the above steps, the following highly specific binding sequences (ie malachite green aptamers) were obtained:

5'-GTACAAAAAAGTTGGCCTTTAAGACAATCGTTGTGAA-3'

5'-CTGAAAGAAACTAAAATCTCATGGCGACGACGTCGAC-3'

5'-ATTAGAAAATGGCAGAACAGAAACAAATGGAAGTGGT-3'

Kit for detecting malachite green in aquatic products

When the malachite green aptamer is coated on the test substrate, the 5' end of the linking sequence (3'-ACTCATCTGTGA-5') can be connected to the 3' end of the malachite green aptamer sequence to synthesize the malachite green aptamer Sub-connection sequence; design and synthesize a fixed sequence (3'-ATGAGTAGACACT-5') complementary to the connection sequence, and label the immobilization group (such as biotin) at the 3' end of the fixed sequence, so that the immobilized sequence and Complementary linker sequences coat the malachite green aptamer on the test substrate.

Detection of Malachite Green in Aquatic Products

50pmol malachite green aptamer labeled with [γ-32P]ATP at the 5' end and 1μmol/L malachite green (standard sample) reacted in the binding buffer at 37°C for 40min, then suction filtered on the nitrocellulose membrane, and used 5ml After rinsing with binding buffer, dry the membrane, remove it and place it in a scintillation cup, add 3ml of PPO-POPOP-xylene solution, measure its radioactivity with a Beckman LS liquid scintillation instrument, and calculate the malachite green aptamer and malachite green The percent binding was 90%. The dissociation constant is 50pmol/L.

The aquatic product (test sample) was first homogenized and then extracted with ethyl acetate and cyclohexane. After adjusting the pH value, it was diluted and then combined with 50 pmol 5' end-labeled [γ-32P]ATP malachite green aptamer After acting in the buffer solution at 37°C for 40 minutes, filter it on a nitrocellulose membrane, wash it with 5ml of binding buffer, dry the membrane, remove it and place it in a scintillation cup, add 3ml of PPO-POPOP-xylene solution, and use Beckman LS The radioactivity was measured by a liquid scintillation instrument, and multiplied by a factor of 90% (measured by the combination of the malachite green aptamer and the malachite green standard sample in the previous step) to obtain the malachite green content of the sample species. Figure 2 and Figure 3 illustrate the principle of malachite green aptamer competition detection. There are a total of four sequences in Figure 2. The upper 3' end is an aptamer sequence that has specific binding ability to malachite green screened by SELEX technology, and its two ends have palindromic complementary structures; the upper 5' end It is the connection sequence designed and synthesized; the lower 3' end is an immobilized sequence complementary to the connection sequence, the bases of this part of the sequence have been biotinylated and can be fixed on a 96-well plate or nitrocellulose membrane; the sequence at the lower 5' end is the reporter sequence, which is enzyme-linked and responsible for the color development of the reporter. In the absence of malachite green, the 5' end of this part of the sequence is complementary to the palindromic complementary sequence at the 5' end of the aptamer. In the state shown in Figure 3, there is malachite green in the environment, and the conformation of the aptamer is changed due to the combination of malachite green, and the palindromic complementary sequences at the 3' and 5' ends are self-associated and compete with the reporter sequence , the reporter sequence drops off. After the steps of elution and color development, the content of malachite green in the test object can be seen by comparing with the control.

Claims (4)

1, a kind of method of utilizing malachite green in the adaptive sub-fast detecting aquatic products, adaptive son of testing sample and malachite green and the report sequence that has the certification mark thing are mixed, malachite green in report sequence and the testing sample can be competitively with after the adaptive son of malachite green combines, label on the examining report sequence calculates malachite green content in the testing sample;

Described certification mark thing is horseradish peroxidase, chemiluminescent groups, chemiluminescence group or collaurum etc.;

The adaptive subsequence part of described report sequence and malachite green is complementary.

2, the method for claim 1 is characterized in that: the adaptive son of described malachite green makes as follows:

(1) make up the library of ssDNA at random that a sequence length is about 80 bases, fixed sequence program, centre that ssDNA sequence two ends have restriction endonuclease sites are 35 sequences bases at random, according to the fixed sequence program design PCR primer at ssDNA sequence two ends;

(2) adopt asymmetric PCR method or biotin-streptavidin paramagnetic particle method to be increased in the ssDNA library that step (1) obtains;

(3) utilize malachite green-OVA conjugate bag to be carried out the SELEX screening by microwell plate medium method or high performance capillary electrophoresis method from the ssDNA library after the amplification, the ssDNA that obtains with the malachite green specific bond is the adaptive son of malachite green.

3, a kind of kit that detects malachite green in the aquatic products comprises the test pieces base, it is characterized in that: be coated with the adaptive son of malachite green on the described test pieces base.

4, kit as claimed in claim 3 is characterized in that: the adaptive son bag of malachite green by on the test pieces base time, is connected the adaptive subsequence 3 ' end of catenation sequence 5 ' end and malachite green and synthesizes the adaptive son-catenation sequence of malachite green; Design fixed sequence program synthetic and the catenation sequence complementation, the immobilization group is marked at 3 ' of fixed sequence program holds, with combining of catenation sequence the adaptive son of malachite green is wrapped by on the test pieces base by fixed sequence program.

Images