CN103352075B - A kind of transgene component super sensitivity detection probe and primer - Google Patents
A kind of transgene component super sensitivity detection probe and primer Download PDFInfo
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Abstract
This application discloses feed transgene component method for detecting residue and reagent in a kind of animal product, this detection method comprises for target transgenosis design real-time fluorescence PCR primer and probe, then primer and probe is adopted to carry out real-time PCR detection to target transgenosis, wherein, have at least one lock nucleic acid in the sequence of probe, lock nucleic acid is selected from least one in A, C, G, T, U and mC.The detection method of the application and detection reagent, the short and small nucleotide fragments being particularly suitable for high degraded detects, thus the transgenosis residual component that can be applicable to low, the high degraded of content in the animal product such as animal meat product, milk preparation very well detects, improve the efficiency of feed transgene component residue detection in animal product, reduce undetected probability, for the feed transgene component residue detection in animal product provides a kind of new detection method and detection reagent, be particularly suitable for the departments such as Check and Examination of Port quarantine and use; There is good commercial value.
Description
Technical field
The application relates to the detection of transgene component, particularly relates to a kind of method for the residual feed detection of GMOs in animal product and primer and probe.
Background technology
Food safety involves huge numbers of families, and transgenic product exists potential security risk, is shown great attention to widely.China is every year from transgenic plant product such as a large amount of soybean of the national import such as the U.S., Canada, Argentina, beans Hectometer, soya-bean oil, rapeseed oil and feeds, and the transgene component in these plant prods can effectively be detected and supervise.But, there are a large amount of transgenic corns and soybean etc. to be used as animal-feed at home and abroad.Research finds, after poultry, domestic animal are fed transgenic plant feed, transgene component digestion is insufficient, is likely deposited in animal organ, particularly the internal organs of poultry; Its two, animal product in the course of processing owing to not rinsing well fully, likely by transgene component food pollution; What is more important has report to point out, feed transgene component, can enter animal blood and in the muscle tissue that appears at animal and milk preparation as foreign gene or DNA fragmentation through intestinal mucosa.At present, report in milk to contain or pollute have feed transgene component abroad more than once.These feeds turn based component and appear in animal product, certainly will affect the quality of product and the public to the acceptance of product; What is more important other countries likely it can be used as new international trade technology barriers.For Shenzhen, annual inspected meat products reaches more than 10,000 batches, only chicken's gizzard 300 batches nearby.Therefore carry out feed transgenosis residue detection in animal product and have its necessity.
According to current research, in animal product, feed transgene component residual content is low, and highly degrades, and gene or DNA fragmentation are very short, and usually all at below 100bp, detection difficulty is large.Conventional PCR is that the plant prod transgenic detection method of master is often helpless; Although real-time fluorescence PCR has higher detection sensitivity, but fluorescent probe used in current examination criteria is general longer, often at more than 30bp, in addition both sides primer, it detects foreign DNA target area generally at more than 100bp, therefore, foreign gene DNA is less than 100bp animality sample because of degraded can not be detected.Further, ready-made detection method can not contain all strains of genetically modified feed, easily undetected.Not yet set up animal product detection GMOs system so far, also never pay close attention to the detection of feed transgene component in animal product, do not set up its corresponding detection method and examination criteria.
Summary of the invention
The object of the application is to provide a kind of detection method residual for the residual feed transgene component of animal product newly, and the primer used in this detection method and probe, and based on the test kit of this probe and/or primer.
For achieving the above object, the application have employed following technical scheme:
This application discloses the detection method that in a kind of animal product, feed transgene component is residual, comprise for target transgenosis design real-time fluorescence PCR primer and probe, then described primer and probe is adopted to carry out real-time PCR detection to target transgenosis, have at least one lock nucleic acid in the sequence of its middle probe, this lock nucleic acid is selected from least one in A, C, G, T, U and mC.
In the application, target transgenosis comprises at least one in CaMV35S, NOS and FMV35S.
In the application, the primer detecting target transgenosis CaMV35S is sequence shown in sequence and Seq ID No.2 shown in Seq ID No.1, and probe is sequence shown in Seq ID No.3; The primer detecting target transgenosis NOS is sequence shown in sequence and Seq ID No.5 shown in Seq ID No.4, and probe is sequence shown in Seq ID No.6; The primer detecting target transgenosis FMV35S is sequence shown in sequence and Seq ID No.8 shown in Seq ID No.7, and probe is sequence shown in Seq ID No.9.
Preferably, in the probe of sequence shown in Seq ID No.3, namely detect in the probe of target transgenosis CaMV35S, the base " G " of the base " A " of the 8th, the base " C " of the 11st and the 14th is lock nucleic acid; In the probe of sequence shown in Seq ID No.6, namely detect in the probe of target transgenosis NOS, the base " A " of the base " G " of the 6th and the 13rd is lock nucleic acid; In the probe of sequence shown in Seq ID No.9, namely detect in the probe of target transgenosis FMV35S, the base " T " of the base " A " of the 6th, the base " G " of the 10th and the 14th is lock nucleic acid.
The another side of the application discloses a kind of probe reagent for feed transgene component residue detection in animal product, in the sequence of this probe reagent, its 5 ' end and 3 ' end are marked with fluorescence radiation group and fluorescent quenching group respectively, the sequence length of probe reagent is 10 ~ 20bp, have at least one lock nucleic acid in the sequence of probe, this lock nucleic acid is selected from least one in A, C, G, T, U and mC.
The probe reagent of the application comprises the probe detecting transgenosis CaMV35S, the probe detecting transgenosis NOS and detects at least one in the probe of transgenosis FMV35S.Preferably, the probe detecting transgenosis CaMV35S has sequence shown in Seq ID No.3, and the probe detecting transgenosis NOS has sequence shown in Seq ID No.6, and the probe detecting transgenosis FMV35S has sequence shown in Seq ID No.9.Preferred, in the probe of the detection transgenosis CaMV35S of sequence shown in SeqID No.3, the base " G " of the base " A " of the 8th, the base " C " of the 11st and the 14th is lock nucleic acid; In the probe of the detection transgenosis NOS of sequence shown in Seq ID No.6, the base " A " of the base " G " of the 6th and the 13rd is lock nucleic acid; In the probe of the detection transgenosis FMV35S of sequence shown in Seq ID No.9, the base " T " of the base " A " of the 6th, the base " G " of the 10th and the 14th is lock nucleic acid.
The one side again of the application discloses and a kind ofly coordinates with the probe reagent of the application for the primer kit to feed transgene component residue detection in animal product, and this primer kit comprises at least one group in the primer sets of the primer sets detecting transgenosis CaMV35S, the primer sets detecting transgenosis NOS and detection transgenosis FMV35S; Wherein, the primer sets detecting transgenosis CaMV35S has sequence shown in sequence and Seq ID No.2 shown in Seq ID No.1, the primer sets detecting transgenosis NOS has sequence shown in sequence and Seq ID No.5 shown in Seq ID No.4, and the primer sets detecting transgenosis FMV35S has sequence shown in sequence and Seq ID No.8 shown in Seq ID No.7.
The application discloses a kind of test kit for feed transgene component residue detection in animal product more on the one hand, the probe reagent containing the application in this test kit, or containing the probe reagent of the application and the primer kit of the application.
Owing to adopting above technical scheme, the beneficial effect of the application is:
The detection method that in the animal product of the application, feed transgene component is residual and detection reagent, utilize the feature of lock nucleic acid, the specific probe of design containing lock nucleic acid, this probe is particularly suitable for the detection of the short and small nucleotide fragments of high degraded, thus can be good at being applicable to animal meat product, in the animal products such as milk preparation, content is low, the transgenosis residual component of high degraded detects, improve the efficiency of feed transgene component residue detection in animal product, reduce undetected probability, for the feed transgene component residue detection in animal product provides a kind of new detection method and detection reagent.The detection method of the application and probe, primer, compared with existing common detection methods, highly sensitive, ultrashort target sequence can be detected, be particularly suitable for the departments such as Check and Examination of Port quarantine and use; And there is good commercial application value.
Accompanying drawing explanation
Fig. 1: the comparing result figure being the detection method of real-time fluorescence PCR detection method conventional in the embodiment of the present application and the application, wherein A is conventional real-time PCR detection result, and B is the detected result of the detection method of the application;
Fig. 2: the comparing result figure being the detection method of real-time fluorescence PCR detection method conventional in another embodiment of the application and the application, wherein A is conventional real-time PCR detection result, and B is the detected result of the detection method of the application;
Fig. 3: the comparing result figure being the detection method of real-time fluorescence PCR detection method conventional in another embodiment of the application and the application, wherein A is conventional real-time PCR detection result, and B is the detected result of the detection method of the application.
Embodiment
In the animal product of the application, the detection method of feed transgene component and reagent design based on the feature of transgene component residual in animal product, as shown in study at present, because genetically modified crops are used by the increasing feed as animal, and, very likely appear in animal muscle tissue and milk preparation as the transgene component in the genetically modified crops of feed, make the pollution occurring transgene component in these animal products, affect food safety.Further, remain in transgene component in animal product and the transgene component in general crop and exist very large different, first is that content is low, and second is highly degrade; And mostly the detection method of existing detection transgene component is directly for the detection of the transgene component of crop or its direct products, the detection efficiency that the transgene component in the animal product of and highly degraded low for content remains is not high.The detection method of the application is based on real-time fluorescence PCR, the probe containing lock nucleic acid of special construction is adopted to detect the feed transgene component in animal product, whole detection target fragments can be shortened greatly, in an implementation of the application, the shortest fragment that effectively can detect fifties base sizes.It should be noted that, a key factor of the application is exactly the probe containing lock nucleic acid that have employed special construction, as long as be appreciated that the probe using the application in detection method, further, the corresponding primer of conventional design is adopted can to realize detecting in the upstream and downstream of probe binding site; Therefore, can have much with the primer of the Probe pairings of the application, be not specifically limited in this application.
The probe containing lock nucleic acid in the application, is also called LNA probe.LNA (Locked Nucleic Acid) is a kind of nucleic acid analog, and it and common nucleic acid molecular difference are have methylene bridge to form lock shape structure at the 2' Sauerstoffatom of its base carbocyclic ring and 4' carbon atom position, therefore also claim lock nucleic acid.LNA Nucleotide has A, and C, G, T, U, mC six kinds of bases, they all can introduce real-time fluorescence TaqMan probe.The TaqMan probe introducing LNA base had both been LNA-TaqMan probe, i.e. LNA probe.Often introduce a LNA base and can improve probe TM value 3-8 degree Celsius, so both can shorten the length of probe, greatly can increase again sensitivity and the stability of probe, thus make detection signal enhancing, signal to noise ratio increase.The application utilizes this characteristic just, devises applicable specific probe and primer.
In a kind of specific implementation of the application, from numerous primers, filter out the primer that can use with the Probe pairings of the application, although said above, be not specifically limited with the primer of Probe pairings; But, be appreciated that the primer of the application and the pairing of probe are used as a kind of preferred implementation, the advantage of the detection method of the application can greatly be played.Same, under the basic conception of the application, based on the probe of the application; can also design a lot of different from the primer of the application and with the primer of Probe pairings; its Detection results is perhaps suitable with the application, and perhaps comparatively the application is slightly poor, and this is still in the protection domain of the application.
It should be noted that; on the primer of the application and the basis of probe; primer or probe freeze-drying are made pulvis or directly make high density solution thus make test kit easy to use be hold facile; in such as this area; preserve for a long time to enable primer and be convenient to transport and often can be made into pulvis, add the TE buffered soln of sterile purified water or laboratory routine use in use.
Also it should be noted that, the transgenic strain containing foreign gene CaMV35S, FMV35S and NOS covers most transgenic strain in present stage feed, therefore, it is possible to just detect the transgene component of genetically modified feed is residual.
Also by reference to the accompanying drawings the application is described in further detail below by specific experiment example.Following experimental example is only further detailed the application, should not be construed as the restriction to the application.
Embodiment
One, reagent and instrument
1, main agents
Taq archaeal dna polymerase, dNTP are all by commercially available purchase.The component of 2*PCR damping fluid comprises: 100mmol/L Tris-HCl(pH8.3), 500mmol/L KCl, 15mmol/L MgCl
2; The component of CTAB extracting solution comprises: 2%CTAB, 100mmol/L Tris-HCl(pH8.0), 1.4mol/L NaCl, 20mmol/LEDTA(pH8.0); The component of CTAB precipitated liquid comprises: CTAB5g/L, 0.04mol/L NaCl; The concentration of NaCl solution is 1.2mol/L.This example reagent used is analytical pure or biochemical reagents, and experimental water meets the specification of one-level water in GB/T6682.All reagent is all with the containers polluted without DNA enzymatic.
2, key instrument
Real-time fluorescence PCR instrument, whizzer: maximum centrifugal force >=16000g, micropipet: 10 μ L, 100 μ L, 200 μ L, 1000 μ L, refrigerator: 2 DEG C-8 DEG C ,-20 DEG C, autoclave, nucleic acid-protein analyser or ultraviolet spectrophotometer.
Two, test sample
1. for examination material
Transgenosis standard substance: transgene rape RT73(or GT73), transgene cotton MON15985, transgenic corns Bt176, CBH351 and T25, genetically engineered soybean A5547-127.
2. test sample
Import chicken leg and chicken wings, all purchased from supermarket.
Three, sample preparation
1, DNA extraction
Animal uses a large amount of genetically modified feed in feeding process.These transgenic plant feeds, what have is insufficient because digesting, and can be deposited in digestive organ, particularly poultry and fish; What have is attached to animal product surface because thoroughly not removing in the course of processing; Only a few transgene component DNA short-movie section can be passed enteron aisle capillary wall and enter the blood of animal, emulsion and muscle tissue etc. in addition.Therefore, for different animal products, its different position should be got as detection sample.The DNA extraction step of this example is as follows:
(1) get fresh or freezing animal tissues block 0.1g, shred as far as possible.Be placed in glass homogenizer, the cell lysis buffer solution homogenate adding 1mL, to loseing tissue block, proceeds in 1.5mL centrifuge tube, adds Proteinase K (500ug/ml) 20 μ L, mixing.Water-bath 30min in 65 DEG C of thermostat water baths, also can proceed to 37 DEG C of water-bath 12 ~ 24h, and interrupted oscillation centrifuge tube for several times.In desk centrifuge with the centrifugal 5min of 12000rpm, get supernatant liquor and enter in another centrifuge tube.
(2) add 2 times of volume isopropanol, after reversing mixing, can filament be seen, choose (or centrifugation DNA) with 100uL suction nozzle, airing, again dissolve (can carry out PCR reaction etc., what needs were further purified follows these steps to carry out) with 200uL TE.
(3) phenol of equivalent is added: chloroform: primary isoamyl alcohol vibration mixing, centrifugal 12000rpm, 5min.
(4) get upper solution to manage to another, add isopyknic chloroform: primary isoamyl alcohol, vibration mixing, centrifugal 12000rpm, 5min.
(5) get upper solution to manage to another, add the 7.5mol/L acetic acid ammonia of 1/2 volume, add the dehydrated alcohol of 2 times of volumes, precipitation at room temperature 2min after mixing, centrifugal 12000rpm, 10min.
(6) carefully outwell supernatant liquor, centrifuge tube is inverted on thieving paper, the residual droplets investing tube wall is removed.
(7) 1mL70% washing with alcohol throw out 1 time is used, centrifugal 12000rpm, 5min.
(8) carefully outwell supernatant liquor, centrifuge tube is inverted on thieving paper, remove the residual droplets investing tube wall, drying at room temperature.
(9) add 200uL TE dissolution precipitation thing again, be then placed in 4 DEG C or-20 DEG C save backup.
Or be added in the animal tissues of 0.1g porphyrize with 1mL CTAB extracting solution, add Proteinase K (500ug/ml) 20 μ L, mixing.Water-bath 30min in 65 DEG C of thermostat water baths, also can proceed to 37 DEG C of water-bath 12 ~ 24h, and interrupted oscillation centrifuge tube for several times.Extract according to the CTAB method specified in GB/T19495.3 afterwards.
2, DNA concentration determination
Nucleic acid-protein analyser or ultraviolet spectrophotometer is used to detect light absorption value A260 and A280 at 260nm and 280nm place respectively.The concentration of DNA is according to formulae discovery: c=A × N × 50/1000; Wherein, c represents DNA concentration, and unit is the every microlitre of microgram (μ g/ μ L), and A represents the light absorption value at 260nm place; N represents nucleic acid extension rate.
When the A260/A280 ratio of this example is between 1.7 ~ 1.9, be suitable for pcr amplification.
3, diluted sample
Extract DNA with for examination material transgenosis standard substance, DNA sample is mixed with 6 kinds of DNA concentration altogether: 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L and 1pg/ μ L.
Four, primer and probe design
Respectively for gene order design specific detection primer and the probe of online transgene component CaMV35S, NOS and FMV35S of announcing, and, by primer Synesis Company synthetic primer and probe, wherein, design some lock nucleic acid according to testing requirement in probe, the lock nucleic acid particular case in primer, probe and probe is as table 1.Meanwhile, this example has also been synthesized, the probe as shown in table 1 not containing lock nucleic acid, and the probe namely shown in table 1 conventionally synthesizes, wherein containing lock nucleic acid, with as a comparison.
Table 1 detection of GMOs primer and probe
Note: wherein " just " represents forward primer, and negation represents reverse primer, " probe " the i.e. probe of real-time PCR detection, the capitalization in each probe sequence represents that this base is for lock nucleic acid.
Five, detecting step
1, Fluorescence PCR system
Real-time fluorescence PCR reaction system, in table 2, should make sample DNA solution fall into reaction solution completely, not adhere on tube wall, should cover tightly pipe lid as early as possible after application of sample during application of sample.
Table 2 real-time fluorescence PCR reaction system
Note: 1, in table, DNA profiling is the template amount of raw material, and the visual processing stage of converted products suitably increases template amount; Also can as the case may be or different reaction cumulative volumes suitably adjust; 2, the commercial kit of real-time fluorescence PCR reaction mixture available equivalents is as AB company
universal PCR Master Mix replaces.
2, Fluorescence PCR parameter
Real-time fluorescence PCR reaction conditions slightly changes with instrument difference, is generally: 50 DEG C of 2min; 95 DEG C of 10min; 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
3, real-time fluorescent PCR amplification
(1) sample is arranged
Each sample arranges two parallel reaction systems.Positive control, negative control and blank should be set up respectively in testing process.Make positive control with genetically engineered soybean, corn and rape standard substance or known positive material, make negative control with non-transgenic genetically engineered soybean, corn and rape, replace template DNA to make blank with isopyknic ddH2O.
More than detect, the primer adopting this example to design respectively and the probe containing lock nucleic acid, and the primer of this example with do not carry out simultaneous test containing the probe locking nucleic acid.
(2) specific detection
The primer adopting this example to design respectively and probe, carry out specific detection to non-transgenic material, negative sample and positive respectively.
(3) relative sensitivity detects
Genetically engineered soybean, cotton and rape sample are joined in corresponding non-transgenic material the sample made containing 0.1% and 0.01% transgene component, extract DNA, carry out sensitivity technique.
(4) animal sample detects
With the import chicken leg bought in supermarket, chicken wings for detected sample, extract its DNA, the primer adopting this example to design respectively and probe detect it.
Six, detected result
1, containing lock nucleic acid with not containing the detected result contrast of the probe of lock nucleic acid
The probe sequence of this example design, when synthesizing, has synthesized respectively containing lock nucleic acid with not containing the probe of lock nucleic acid.Then these two groups of probes are adopted to detect different samples respectively.Concrete, (1) adopts respectively containing lock nucleic acid with not containing the probe of lock nucleic acid, and detect the CaMV35S gene in cotton transgenic strain MON15985 genomic dna, the system of detection is all identical with condition; (2) adopt respectively containing lock nucleic acid with not containing the probe of lock nucleic acid, detect the FMV35S gene in rape transgenic strain RT73 genomic dna, the system of detection is all identical with condition; (3) adopt respectively containing lock nucleic acid with not containing the probe of lock nucleic acid, detect the NOS gene in cotton transgenic strain MON15985 genomic dna, the system of detection is all identical with condition.
Partial detection is in Table 3-table 5, and part AFLP system is shown in Fig. 1-Fig. 3; Wherein, the comparison and detection result figure of Fig. 1 to be the comparison and detection result figure of CaMV35S gene, Fig. 2 be FMV35S gene, Fig. 3 is the comparison and detection result figure of NOS gene.Result shows, and the Ct value containing the probe of lock nucleic acid can shift to an earlier date at most more than 6 circulations, and namely its detection sensitivity at least can improve nearly a hundred times.
The comparison of table 3CaMV35S two kinds of Taqman probe Ct values
The comparison of table 4FMV35S two kinds of Taqman probe Ct values
The comparison of table 5NOS terminator two kinds of Taqman probe Ct values
2, specific detection
Adopt the primer of this example and the probe containing lock nucleic acid, test sample is detected, result shows, the primer of detection CaMV35S gene and probe specificly can only amplify the sample containing CaMV35S gene, and to other negative samples and water contrast, all do not increase, namely there is no Ct value, collection of illustrative plates does not have peak value yet; The primer of detection FMV35S gene and probe specificly can only amplify the sample containing FMV35S gene, and to other negative samples and water contrast, all do not increase, namely do not have Ct value, collection of illustrative plates does not have peak value yet; The primer of detection NOS gene and probe specificly can only amplify the sample containing NOS gene, and to other negative samples and water contrast, all do not increase, namely do not have Ct value, collection of illustrative plates does not have peak value yet.
Visible, the detection method of this example and detect primer and have good specificity containing the probe of lock nucleic acid, can go out target sequence by specific amplified, and not have non-specific amplification containing the sample of target sequence to other.
3, relative sensitivity detects
With 6 kinds of real-time PCR detection containing LNA probe accordingly containing 0.1% and 0.01% transgene component sample, all there is specific amplification in result, shows that the detection sensitivity of its detection method reaches 0.1%(W/W).
4, animal sample detects
The animals products bought from supermarket is all tested with target transgene component.Join in the animals products of purchase by genetically engineered soybean, cotton and rape sample according to 0.01% weight ratio, then extract DNA and detect, result shows, and all detects and adds composition accordingly.
The real-time fluorescence detection probes of this example, introduces lock nucleic acid, improves combination stability and the bonding strength of probe and target in its sequence, while shortening probe sequence, has ensured the binding ability of probe, thus can detect shorter target sequence.In this example, through a large amount of optimal screening, finally obtain the probe containing lock nucleic acid that specificity is good, highly sensitive, simultaneously, through optimizing, also obtain the primer coordinated with probe, finally establish be suitable for highly degraded, content is low, detection method that feed transgene component in animal product that fragment is short and small is residual.Be appreciated that primer and the probe of this example, be equally also suitable for common detection of GMOs, be not restricted to the detection that the feed transgene component in animal product remains.The probe of this example and primer, due to the Detection results that it is special, have good commercial value, can be made into pulvis or highly concentrated solution is sold.
Above content is the further description done the application in conjunction with concrete embodiment, can not assert that the concrete enforcement of the application is confined to these explanations.For the application person of an ordinary skill in the technical field, under the prerequisite not departing from the application's design, some simple deduction or replace can also be made, all should be considered as the protection domain belonging to the application.
Claims (4)
1. the probe reagent for detection of GMOs, in the sequence of described probe reagent, its 5 ' end and 3 ' end are marked with fluorescence radiation group and fluorescent quenching group respectively, it is characterized in that: the sequence length of described probe reagent is 10 ~ 20bp, have at least one lock nucleic acid in the sequence of described probe, described lock nucleic acid is selected from least one in A, C, G, T, U and mC;
Described probe reagent comprises the probe detecting transgenosis CaMV35S, the probe detecting transgenosis NOS and detects the probe of transgenosis FMV35S;
The probe of described detection transgenosis CaMV35S is sequence shown in Seq ID No.3, and the probe of described detection transgenosis NOS is sequence shown in Seq ID No.6, and the probe of described detection transgenosis FMV35S is sequence shown in Seq ID No.9;
In the probe of the detection transgenosis CaMV35S of sequence shown in Seq ID No.3, the base " G " of the base " A " of the 8th, the base " C " of the 11st and the 14th is lock nucleic acid;
In the probe of the detection transgenosis NOS of sequence shown in Seq ID No.6, the base " A " of the base " G " of the 6th and the 13rd is lock nucleic acid;
In the probe of the detection transgenosis FMV35S of sequence shown in Seq ID No.9, the base " T " of the base " A " of the 6th, the base " G " of the 10th and the 14th is lock nucleic acid.
2. coordinate the primer kit being used for detection of GMOs with probe reagent according to claim 1, it is characterized in that: described primer kit comprises at least one group in the primer sets of the primer sets detecting transgenosis CaMV35S, the primer sets detecting transgenosis NOS and detection transgenosis FMV35S;
The primer sets of described detection transgenosis CaMV35S is sequence shown in sequence and Seq ID No.2 shown in Seq ID No.1, the primer sets of described detection transgenosis NOS is sequence shown in sequence and Seq ID No.5 shown in Seq ID No.4, and the primer sets of described detection transgenosis FMV35S is sequence shown in sequence and Seq ID No.8 shown in Seq ID No.7.
3. the detection method of a transgene component, comprise for target transgenosis design real-time fluorescence PCR primer and probe, then described primer and probe is adopted to carry out real-time PCR detection to target transgenosis, it is characterized in that: have at least one lock nucleic acid in the sequence of described probe, described lock nucleic acid is selected from least one in A, C, G, T, U and mC;
Described target transgenosis comprises CaMV35S, NOS and FMV35S;
The primer detecting target transgenosis CaMV35S is sequence shown in sequence and Seq ID No.2 shown in Seq ID No.1, and probe is sequence shown in Seq ID No.3;
The primer detecting target transgenosis NOS is sequence shown in sequence and Seq ID No.5 shown in Seq ID No.4, and probe is sequence shown in Seq ID No.6;
The primer detecting target transgenosis FMV35S is sequence shown in sequence and Seq ID No.8 shown in Seq ID No.7, and probe is sequence shown in Seq ID No.9;
In the probe of sequence shown in Seq ID No.3, the base " G " of the base " A " of the 8th, the base " C " of the 11st and the 14th is lock nucleic acid;
In the probe of sequence shown in Seq ID No.6, the base " A " of the base " G " of the 6th and the 13rd is lock nucleic acid;
In the probe of sequence shown in Seq ID No.9, the base " T " of the base " A " of the 6th, the base " G " of the 10th and the 14th is lock nucleic acid.
4. for a test kit for detection of GMOs, it is characterized in that: containing probe reagent according to claim 1 and primer kit according to claim 2 in described test kit.
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| CN1385541A (en) * | 2002-05-17 | 2002-12-18 | 中山大学 | Method for detecting transgenic plant and products thereof |
| CN101962690A (en) * | 2010-11-01 | 2011-02-02 | 中华人民共和国北京出入境检验检疫局 | Nucleotide sequences and kit for testing H1 and H3 subtypes of influenza viruses |
| CN102344966A (en) * | 2011-10-21 | 2012-02-08 | 浙江省疾病预防控制中心 | Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof |
-
2013
- 2013-06-13 CN CN201310233912.9A patent/CN103352075B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1385541A (en) * | 2002-05-17 | 2002-12-18 | 中山大学 | Method for detecting transgenic plant and products thereof |
| CN101962690A (en) * | 2010-11-01 | 2011-02-02 | 中华人民共和国北京出入境检验检疫局 | Nucleotide sequences and kit for testing H1 and H3 subtypes of influenza viruses |
| CN102344966A (en) * | 2011-10-21 | 2012-02-08 | 浙江省疾病预防控制中心 | Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms;Gaspparic MB et al.;《BMC Biotechnology》;20080306;第8卷(第26期);摘要，表2 * |
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