CN101358963A - Method for rapidly detecting malachite green in aquatic products by Aptamer - Google Patents
Method for rapidly detecting malachite green in aquatic products by Aptamer Download PDFInfo
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- CN101358963A CN101358963A CNA2007100703810A CN200710070381A CN101358963A CN 101358963 A CN101358963 A CN 101358963A CN A2007100703810 A CNA2007100703810 A CN A2007100703810A CN 200710070381 A CN200710070381 A CN 200710070381A CN 101358963 A CN101358963 A CN 101358963A
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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
Technical field
The present invention relates to the food inspection field, relate in particular to the detection method of malachite green in a kind of aquatic products.
Background technology
Malachite green (Malachite green, be called for short MG), belong to triphenylmethane dye, its metabolin is leucomalachite green (Leucomalachite green, LMG), for a long time, because malachite green has good antimycotic in aquaculture, preservation of fishery and advantage such as cheap, therefore, " the fishing medicine " that become many raisers' favors uses, but these two kinds of high toxin that material all has, high residue and high teratogenesis, spinoffs such as mutagenesis, brought huge obstacle not only for the outlet of China's aquatic products, and what is more important has constituted potential threat for people's health.
Harmfulness in view of malachite green and metabolin thereof, many countries all classify it as the aquaculture forbidden drugs, China also classifies it as in " veterinary drug and the compound inventory thereof of food animal forbidding " in May, 2002, yet, because the consciousness of aquaculture family healthy aquaculture is thin, and the reasons such as imperfection of aquatic products security management mechanism, only 2005, just detect the high residue of malachite green at home in the multiple aquatic products (as: eel, soft-shelled turtle etc.) in many places (especially Fujian, Jiangxi and Zhejiang).
At present, mainly contain three kinds about the detection method of malachite green and metabolin thereof, but these methods all exist certain limitation: (1) high performance liquid chromatography, when content was low, it was qualitative to utilize retention time to be difficult to; (2) gas chromatography mass spectrometry method, can only be used for to leucomalachite green carry out qualitative; (3) LC-MS method (LC/MS), effect is better, but costs an arm and a leg; Therefore, the foundation quick in aquatic livestock, reliable and stable method for detecting residue of malachite green and metabolin thereof becomes the technical barrier that needs to be resolved hurrily both at home and abroad.
The core technology of immunological method be exactly by experiment animal obtain the malachite green specific antibody, but malachite green has very strong immunotoxicity, preparation malachite green specific antibody is the very work of difficulty, and it is also long to prepare a kind of antibody cycle, generally needs more than 6 months.Therefore animal obtains the bottleneck problem that specific antibody becomes the research malachite green fast detecting method by experiment.
The oligonucleotide aptamer technology is a kind of novel molecular imprinting of development in recent years, is to adopt SELEX technology in-vitro screening to obtain.The SELEX technology is set up in the early 1990s by Tuerk and Gold etc., selection and amplification procedure (PCR or RT-PCR) by many wheels screens the oligonucleotide aptamer of specific recognition target material from the random oligonucleotide library combinatorial chemistry technique is a kind of effective ways of studying nucleic acid structure and function.The SELEX technology has obtained fast development since founding, have because of it that storage capacity is big, the target molecule scope of the adaptive son of screening is wide, affinity is high at present and advantage such as high specificity has obtained exploring widely and using in fields such as life science fundamental research, disease treatment, drug screening and clinical diagnosises.
Leucomalachite green indirect competitive ELISA kit for detecting in a kind of aquatic products is disclosed in the Chinese invention patent application 200510060995.1.The check-out console of kit is for wrapping by the removable 96 hole ELISA Plate of leucomalachite green and albumin conjugate, the monoclonal antibody of anti-leucomalachite green antibody for utilizing leucomalachite green and albuminous conjugate immune mouse to prepare, test sample is regulated the dilution of pH value back and is used for detecting earlier through extracting with ethyl acetate and cyclohexane after the even matter.The criterion of pattern detection is: the logarithm value with standard specimen concentration is a horizontal ordinate, and inhibiting rate is the regression curve of ordinate.Just can read the concentration of corresponding sample from curve according to the inhibiting rate of each sample.The sensitivity that this method detects is 0.0023 μ g/ml, and sensing range is 0.0016-1 μ g/ml.
But the acquisition of monoclonal antibody efficiently is very difficult in this technology, has also increased cost.
Summary of the invention
The invention provides a kind of easy to operate, the method for quick of malachite green in the high aquatic products of accuracy.
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, detect the label on (relative chromogenic substrate detects with enzyme) report sequence, calculate malachite green content in the testing sample.
Described certification mark thing is horseradish peroxidase, chemiluminescent groups, chemiluminescence group or collaurum etc.
The preparation of described report sequence: according to the adaptive subsequence of malachite green that obtains, design is synthesized and the complementary report sequence of adaptive subsequence part (5 ' end palindrome complementary series), and the certification mark substance markers is being reported on the sequence.
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.
Asymmetric PCR method described in the step (2) is promptly introduced variable concentrations in the pcr amplification circulation primer (generally adopts 50: 1-100: the primer concentration of 1 ratio), primary product is double-stranded DNA (dsDNA) in initial 10-15 circulation, but after the low concentration primer was depleted, the PCR reaction of high density primer mediation can produce a large amount of ssDNA.
Biotin-streptavidin paramagnetic particle method described in the step (2) is promptly in the PCR system, 5 ' the end that designs and synthesizes downstream primer is marked with biotin, with the ssDNA library is template, carry out pcr amplification and can obtain the dsDNA product that 3 ' end has biotin, through combine (dsDNA combines with streptavidin on the magnetic bead by the biotin of 3 ' end) after the separation and purification with streptavidin magnesphere, use the NaOH of finite concentration (0.15mol/L) that the dsDNA sex change is unwind then, a chain that has biotin combines with streptavidin to be stayed on the magnetic bead, and do not dissociated out with a chain of biotin, after separation and purification, be used for the next round screening, repeat aforesaid operations circulation enrichment (general about 10 times) and reach 90% until the combination rate of malachite green and adaptive son.
Microwell plate described in the step (3) is that the screening technique of medium is promptly used malachite green and albumin conjugate (MG-OVA) 96 hole ELISA Plate, establish albumin (OVA) bag simultaneously by hole and blank hole, all with 3% BSA sealing, pcr amplification is removed the ssDNA that combines with BSA with anti-sieve the in blank hole earlier with the ssDNA library of purifying, removed the ssDNA that combines with OVA by the hole primary dcreening operation with the OVA bag again, be transferred to malachite green-OVA conjugate bag then by the hole combination, obtain ssDNA with the malachite green specific bond through wash-out, isolation and purification.
High performance capillary electrophoresis method described in the step (3) is about to the ssDNA library of a certain amount of pcr amplification, purifying and malachite green hatch after, to not by high performance capillary electrophoresis, binding sequence separates in conjunction with compound with malachite green, collect binding sequence, repetition aforesaid operations circulation enrichment-separation obtains the ssDNA (being the adaptive son of malachite green) with the malachite green specific bond at last.
According to method for quick of the present invention, can be with related reagent encapsulation or fixing back generate a reagent box or quick detection test paper.
The present invention also provides the kit that is used for detecting the aquatic products malachite green, comprises the test pieces base (as 96 hole ELISA Plate or nitrocellulose membrane etc.) that is coated with the adaptive son of malachite green at least, also comprises binding buffer liquid, and bag is cushioned liquid.
The adaptive son bag of malachite green by on the test pieces base time, can be connected the adaptive subsequence 3 ' end of catenation sequence (as 3 '-ACTCATCTGTGA-5 ') 5 ' end and malachite green and synthesize the adaptive son-catenation sequence of malachite green; The fixed sequence program (as 3 '-ATGAGTAGACACT-5 ') with the catenation sequence complementation is synthesized in design, with the immobilization group (as Avidin being fixed on the surface of NC film, the Biotin-ssDNA that singly connects that will have a biotin by Avidin fixes again) be marked at 3 ' end of fixed sequence program, by immobilization group and catenation sequence with the adaptive son bag of malachite green quilt on the test pieces base.
The present invention adopts a kind of adaptive son (aptamer) technology that does not rely on animal used as test, phyletic evolution technology (systematic evolution of ligands by exponentialenrichment by exponential enrichment part, SELEX) in-vitro screening is to the adaptive son of high-affinity specific recognition malachite green, and the antibody that substitutes in traditional immunological method is set up a kind of fast detecting malachite green method.
Detection method advantage of the present invention is
1. the screening cycle lacked for 3 weeks;
2. the high specificity that combines with malachite green is highly sensitive;
3. the synthesis cycle of adaptive son is short, is beneficial to large-scale production;
4. detectable stability is strong, and easy renaturation after the sex change is beneficial to transportation, stores.
According to the kit and the quick detection test paper of the inventive method development, can better satisfy at present pressing for that the aquatic products malachite green vestigial detects, be used for customs and detect, the detection of food hygiene department is easy to apply on a large scale.
Description of drawings
Fig. 1 is adaptive son preparation of malachite green and malachite green detection method schematic flow sheet;
Fig. 2 is a fixedly synoptic diagram of the adaptive son of malachite green;
Fig. 3 is that the adaptive son competition of malachite green detects suitable figure;
Embodiment
The preparation of the adaptive son of malachite green
(1) makes up a sequence length and be about 80 (scopes: 75～90) library of ssDNA at random of individual base, ssDNA sequence two ends have the fixed sequence program of restriction endonuclease sites, described ssDNA sequence is: (5 ' ATAGAGCTCATGGAGTCTCCCTCGG-(N35)-TTTGGATCCGGCAGTGGTGGGCGGGC3 '), centre are 35 sequences bases (N35) at random, according to the fixed sequence program design PCR primer at ssDNA sequence two ends;
Upstream primer: 5 '-ATAGAGCTCATGGAGTCTCC-3 ',
Downstream primer: 5 '-GCCCGCCCACCACTGCCGG-3 '
(2) adopt the asymmetric PCR method to be increased in the ssDNA library that step (1) obtains.
The operating process of asymmetric PCR method is:
The primer of introducing variable concentrations in the pcr amplification circulation (generally adopts 50: 1-100: the primer concentration of 1 ratio), primary product is double-stranded DNA (dsDNA) in initial 10-15 circulation, but after the low concentration primer was depleted, the PCR reaction of high density primer mediation can produce a large amount of ssDNA.PCR reaction system: dsDNA template 2 μ l, 10 * PCR damping fluid, 10 μ l, MgCl
26 μ l, dNTPs100 μ mol/L, Tag enzyme 2 μ l, upstream primer 60pmol, downstream primer 1pmol adds deionized water to 100 μ l.The PCR reaction conditions: 94 ℃ of sex change 5min, 94 ℃ of sex change 30s of 40 circulations then, 65 ℃ of annealing 1min, 72 ℃ are extended 2min, last 72 ℃ of 10min.
Also can adopt the biotin-streptavidin paramagnetic particle method to be increased in the ssDNA library that step (1) obtains.
The operating process of biotin-streptavidin paramagnetic particle method is:
In the PCR system, 5 ' the end that designs and synthesizes downstream primer is marked with biotin, with the ssDNA library is template, carry out pcr amplification and can obtain the dsDNA product that 3 ' end has biotin, through combine (dsDNA combines with streptavidin on the magnetic bead by the biotin of 3 ' end) after the separation and purification with streptavidin magnesphere, use the NaOH of finite concentration (0.15mol/L) that the dsDNA sex change is unwind then, a chain that has biotin combines with streptavidin to be stayed on the magnetic bead, and do not dissociated out with a chain of biotin, ssDNA is dissolved in the 20ul TE damping fluid, measure absorbance (A) value, can be used for the next round screening through separation and purification, repeat aforesaid operations circulation enrichment (general about 10 times) and reach 90% until the combination rate of malachite green and adaptive son.
(3) utilize malachite green-OVA conjugate bag to be carried out SELEX screening by the microwell plate medium 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.
Malachite green-OVA conjugate bag by the operating process of microwell plate medium method is:
By on elisa plate, 4 ℃ are spent the night with MG-OVA albumen bag, and coating buffer is 0.05mol/LNaHCO
3, the damping fluid of pH9.6 is established blank simultaneously.C albumen bag is all sealed 2h with 3%BSA37 ℃ by hole and blank hole.SsDNA library and a certain amount of tRNA are earlier at binding buffer liquid SHCMK liquid (20mmol/L Hepes pH 7.35,120mmol/L NaCl, 5mmol/LKCl, 1mmol/L CaCl at random
2, 1mmol/L MgCl
2) in combine 40min for 37 ℃ with blank hole through 3%BSA sealing.Wash (SHAMK liquid+0.05% polysorbas20) 6 times with the buffering washing lotion), the unconjugated ssDNA of flush away, add elution buffer (20mmol/L Tris-HCl again, the 4mol/L guanidinium isothiocyanate, 1mmol/L DDT, pH8.3) in 80 ℃ of effect 10min, the following and protein bound ssDNA of C of wash-out, through phenol-chloroform extracting, precipitation with alcohol, ssDNA is dissolved in the 20 μ l TE damping fluids.SsDNA is become the dsDNA of an end band biotin with the primer of mark biotin through pcr amplification, separate ssDNA through the biotin-streptavidin magnetic bead, as the ssDNA library of next round screening.Repetition aforesaid operations circulation enrichment-separation obtains the ssDNA with the malachite green specific bond at last.
Also can utilize the high performance capillary electrophoresis method to carry out SELEX screening from the ssDNA library after the amplification, the ssDNA that obtains with the malachite green specific bond is the adaptive son of malachite green.
The operating process of high performance capillary electrophoresis method is:
After the ssDNA library of a certain amount of pcr amplification, purifying and malachite green hatched, by high performance capillary electrophoresis not binding sequence separate in conjunction with compound with malachite green, collect binding sequence and can be used for the next round screening.
The related reagent that the adaptive son of malachite green uses when carrying out SELEX screening and obtaining all has openly in the article " foundation of random single-stranded DNA banks SELEX screening oligonucleotide aptamer method " (" biological chemistry and biophysics progress " 2,003 30 volumes (1) 151～155) of Zhan Linsheng.
Screening and order-checking through above step have obtained following high specific bond sequence (being the adaptive son of malachite green):
5’-GTACAAAAAAGTTGGCCTTTAAGACAATCGTTGTGAA-3’
5’-CTGAAAGAAACTAAAATCTCATGGCGACGACGTCGAC-3’
5’-ATTAGAAAATGGCAGAACAGAAACAAATGGAAGTGGT-3’
Detect the kit of malachite green in the aquatic products
The adaptive son bag of malachite green by on the test pieces base time, can be connected catenation sequence 5 ' end (3 '-ACTCATCTGTGA-5 ') and synthesize the adaptive son-catenation sequence of malachite green with the adaptive subsequence 3 ' end of malachite green; The synthetic fixed sequence program (3 '-ATGAGTAGACACT-5 ') with the catenation sequence complementation of design, immobilization group (as biotin) is marked at 3 ' of fixed sequence program holds, can the adaptive son of malachite green be wrapped quilt on the test pieces base by the catenation sequence of immobilization sequence and complementation like this.
The detection of malachite green in the aquatic products
The adaptive son of malachite green of 50pmol 5 ' end mark [γ-32P] ATP and 1 μ mol/L malachite green (standard specimen) in binding buffer liquid 37 ℃ act on 40min after, suction filtration is on nitrocellulose membrane, after the flushing of 5ml binding buffer liquid, film is dried, take off as in the scintillating disc, add PPO-POPOP-dimethylbenzene liquid 3ml, measure its radioactivity with Beckman LS liquid glimmer instrument, combine percent with malachite green be 90% to the adaptive son of malachite green as calculated.Dissociation constant is at 50pmol/L.
Aquatic products (test sample) are extracted with ethyl acetate and cyclohexane after sparing matter earlier, the adaptive son of malachite green of regulating dilution back, pH value back and 50pmol 5 ' end mark [γ-32P] ATP in binding buffer liquid 37 ℃ act on 40min after, suction filtration is on nitrocellulose membrane, after the flushing of 5ml binding buffer liquid, film is dried, take off as in the scintillating disc, add PPO-POPOP-dimethylbenzene liquid 3ml, measure its radioactivity with Beckman LS liquid glimmer instrument, multiply by the malachite green content that coefficient 90% (recording with combining of malachite green standard specimen by the adaptive son of malachite green of last step) promptly obtains this sample kind.Fig. 2 and Fig. 3 have illustrated the adaptive son of malachite green to compete the principle that detects.Always have four sections sequences among Fig. 2, the part of top 3 ' end is the adaptive subsequence that has specific binding capacity through the same malachite green that the SELEX technology screening is crossed, and its two ends are according to palindrome complementary structure is arranged; Top 5 ' end is the synthetic catenation sequence of design; Bottom 3 ' end is the immobilization sequence with the catenation sequence complementation, and the base of this part sequence can be fixed on 96 orifice plates or the nitrocellulose membrane by biotinylation; The sequence of bottom 5 ' end is the report sequence, and this sequence is that the enzyme connection is modified, and is responsible for the report colour developing.Do not having under the state of malachite green, 5 ' end of this part sequence is to hold the complementary combination of palindrome complementary series with 5 ' of adaptive son.Under state shown in Figure 3, malachite green has been arranged in the environment, adaptive son is because of combining malachite green, and conformation changes for it, and 3 ' and 5 ' holds palindrome complementary series from combining, and forms competition with the report sequence, reports that sequence comes off thereupon.Through wash-out, the step of colour developing, by with the contrast of contrast just as can be seen, the content of malachite green in the determinand.
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.
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| CN101949846A (en) * | 2010-08-11 | 2011-01-19 | 昆明理工大学 | Leucomalachite green detection method |
| CN102181430A (en) * | 2011-02-28 | 2011-09-14 | 新乡医学院 | Method for preparing target deoxyribonucleic acid (DNA) fragment with cohesive terminuses of restriction enzyme at both ends |
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| CN101949846B (en) * | 2010-08-11 | 2013-08-28 | 昆明理工大学 | Leucomalachite green detection method |
| CN102645430B (en) * | 2011-02-17 | 2014-10-15 | 中国人民解放军第三军医大学第一附属医院 | Method and biosensor for detecting target microbe |
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| KR101545228B1 (en) * | 2013-06-28 | 2015-08-19 | 호서대학교 산학협력단 | Nucleic acid aptamer specifically binding to malachite green |
| CN105018461A (en) * | 2014-04-29 | 2015-11-04 | 中国科学技术大学 | Method for rapid screening of nucleic acid aptamer |
| CN105018461B (en) * | 2014-04-29 | 2018-05-01 | 中国科学技术大学 | A kind of rapid screening method of aptamer |
| TWI655426B (en) * | 2018-03-31 | 2019-04-01 | 國立交通大學 | Structure and manufacturing method of fast screening wafer, and detection method of malachite green concentration using the fast screening wafer |
| CN108715847A (en) * | 2018-05-31 | 2018-10-30 | 江南大学 | A kind of fast detection method and its application enhancing malachite green fluorescence based on DNA |
| CN109507126A (en) * | 2018-12-07 | 2019-03-22 | 集美大学 | A kind of Determination of Malachite Green in Aquatic Products |
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