CN104726580B - A kind of transgenic corns MON810 constant temperature probe method detection primer group, detection kit and detection method - Google Patents
A kind of transgenic corns MON810 constant temperature probe method detection primer group, detection kit and detection method Download PDFInfo
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- CN104726580B CN104726580B CN201510116604.7A CN201510116604A CN104726580B CN 104726580 B CN104726580 B CN 104726580B CN 201510116604 A CN201510116604 A CN 201510116604A CN 104726580 B CN104726580 B CN 104726580B
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Abstract
The invention discloses a kind of transgenic corns MON810 constant temperature probe method detection primer group, detection kit and detection method.Detection primer group includes outer primer, inner primer, ring primer and probe primer.To avoid false positive from expanding, the present invention introduces specific molecular probes in detection primer group, the probe can in real time with product amplification caused by target fragments specifically bind and send fluorescence signal, if amplified production is non-target gene, the probe is not combined with the product of amplification, unstressed configuration signal is sent, and testing result is still feminine gender.The detection method overcomes the shortcomings of Constant Temperature Detection technology, will further lead the popularization and application in the detection of Constant Temperature Detection technology.
Description
Technical field
The invention belongs to technical field of molecular biology, it is related to detection kit and its detection side of genetically modified plants kind
A kind of method, and in particular to constant temperature probe method detection primer group, detection kit and the detection method of transgenic corns MON810.
Background technology
Transgenic crop puts into commercial growth, and global genetically modified crops cultivated area is accumulative to already exceed 1,000,000,000 public affairs
Hectare.2010, the whole world 29 15,400,000 national peasant plantings totally 1.48 hundred million hectares of genetically modified crops.From 1996 extremely
2010, the cultivated area of global genetically modified crops added 87 times.Plant country's plantation of genetically modified crops ranking top ten
Area has exceeded 1,000,000 hectares first.At present, countries in the world have carried out the genetically modified crops of field test more than 5000 kinds,
The genetically modified crops of approval commercialization have kind more than 160, including corn and soybean, rape, tomato, potato, pimento, pawpaw, sweet tea
Dish, tobacco, rice etc..In terms of world wide, the popularization of transgenosis has had resulted in huge social and economic benefit.But turn
Gene corn there is also many problems, is concentrated mainly on the peace of GM food while huge social and economic benefit is brought
Full property and the security aspect to ecological environment, including the risk to humans and animals health, to ecological environment and the wind of agricultural
Danger, to the risk of nontarget organism.Recently cause the arguement of a GMO bio-safety in the world, support and oppose
Two groups fight for mastery relatively, mutually exclusive, distinguish and choose for ease of consumer, various countries propose to be identified GM food respectively
Management, and minimum content is made stipulations.Therefore fast and effectively detection method system is established, crop and food are made a distinction
It is particularly important.
The detection of genetically modified crops at present mainly detects whether sample contains exogenous proteins（Gene expression product）With
Whether foreign gene is contained（DNA）.Foreign protein can utilize the Enzyme-linked Immunosorbent Assay based on immunity principle in genetically modified crops
Method（ELISA）The methods of ELISA test strip, Western hybridization, is detected, but this method requires the anti-of high quality high stability
Body, otherwise because accuracy is inadequate, auxiliary detection means can only be used as, separately because detection target is albumen, the food egg through deep processing
It has been denatured in vain, therefore extraneous protein detection method has certain limitation for the food inspection of deep processing.Transgenosis is made
The nucleic acid detection method of thing mainly has PCR detection techniques, Constant Temperature Detection technology.But there are the following problems for PCR detection techniques, 1）
PCR method reaction system and operating process are more complicated, it is necessary to professional；2) PCR instrument is expensive needed for, detection time
It is long；3) electrophoresis common dyes EB is strong carcinogen, there is stronger toxicity, and is difficult to carry out Site Detection, so PCR detection techniques
It is not widely applied yet in basic unit.
Constant temperature gene amplification technology has the advantages of fast and convenient, operation is accurate, easily popularizes, be safe and reliable, establishes certainly
To be widely used, and develop in original basis using SYBR Green decoration methods, calcein method, nephelometry,
The Different Results reading partner method such as fluorescent dye determination, it is adapted to scene and quick detection, is widely applied in basic unit, but used
SYBR Green decoration methods, calcein method, nephelometry, fluorescent dye determination etc. there is inborn deficiency on Cleaning Principle,
All directly detected as target using nucleic acid amplification product or accessory substance, such as the non-specific amplification that happens is that in detection, detection
As a result still it is positive findings, i.e. the method for testing of the above has the problem of testing result is false positive（The problem of poor specificity）,
It is problem urgently to be resolved hurrily at present to develop a kind of detection GMOs kit for avoiding false positive.
To avoid false positive from expanding, the present invention introduces specific molecular probes in detection primer group, and the probe can be real-time
With product amplification caused by target fragments specifically bind and send fluorescence signal, if amplified production is non-target gene, the spy
Pin is not combined with the product of amplification, and unstressed configuration signal is sent, and testing result is still feminine gender.The detection method overcomes constant temperature to examine
The deficiency of survey technology, it will further lead the popularization and application in the detection of Constant Temperature Detection technology.
The content of the invention
It is an object of the present invention to provide the Constant Temperature Detection of transgenic corns MON810 a kind of to try primer sets.
It is another object of the present invention to provide a kind of transgenic corns MON810 Constant Temperature Detection kit.
It is another object of the present invention to provide a kind of transgenic corns MON810 Constant Temperature Detection method..
The technical solution adopted in the present invention is as follows：
Transgenic corns MON810 Constant Temperature Detection primer sets, including outer primer 1 and outer primer 2, inner primer 1 and inner primer
2nd, ring primer and probe primer, its nucleotide sequence difference are as follows：
Outer primer 1：5’-CATCGTTGAAGATGCCTCT-3’；
Outer primer 2：5’-GTACCGAAGACGGTAGATCT-3’；
Inner primer 1：5’-CGTCATCCCTTACGTCAGTGGAAGAAGACGTTCCAACCAC-3’；
Inner primer 2：5’-CCTTCGCAAGACCCTTCCTCGCTAGAGTCAGCTTGTCAG-3’；
Ring primer：5’-CATTTCATTTGGAGAGGACACG-3’；
Probe primer：FAM-ATCGACATCAATCCACTTGCTTTTCGAT-BHQ-1.
A kind of transgenic corns MON810 Constant Temperature Detection kit, it includes Constant Temperature Detection primer sets as described above.
Also include in the kit：Archaeal dna polymerase, LAMP reaction solutions, positive control and negative control.
The LAMP reaction solutions include 10mM dNTP, 150mM MgSO4The aqueous solution, the volume ratio of the two are 7~9：2~
4。
The positive control is transgenic corns MON810 DNA or the e. coli plasmid dna containing target gene, negative
Compare as the reaction mixture without target gene.
A kind of transgenic corns MON810 detection method, comprises the following steps：
（1）Extract measuring samples DNA.
（2）Constant temperature gene amplification reacts：Measuring samples are carried out using the Constant Temperature Detection primer sets described in claim 1 permanent
Detection pipe, is placed on the instrument that can provide constant temperature and fluoroscopic examination during amplification by temperature amplification, and every 30~60s gathers first order fluorescence
Signal；
25 μ l reaction systems of constant temperature gene amplification reaction system contain：1 0.2 μM of inner primer, 2 0.2 μM of inner primer, outside
1 1.6 μM of primer, 2 1.6 μM of outer primer, 0.8 μM of ring primer, 0.8 μM of probe primer, μ l, the DNA polymerizations of LAMP reaction solutions 12.5
The μ l of enzyme 8U, measuring samples DNA 2, with ultra-pure water polishing to 25 μ l；
Constant temperature gene amplification reaction program be：60~63 DEG C of 20~60min of reaction.
（3）As a result judge：Judge yin and yang attribute result according to " S " type amplification curve is whether there is, have " S " type amplification curve for the positive
As a result, otherwise it is negative findings.
The beneficial effects of the present invention are：
The present invention is based on isothermal amplification technology, and specific probe primer is introduced in primer sets, can monitor reaction in real time
Process, and can effectively reduce the generation of false positive, the invention have high specific, high sensitivity, identification it is easy, rapidly and efficiently,
The beneficial effect such as easy to operate：
（1）High specific：The present invention designs according to transgenic corns MON810 foreign gene with endogenous gene junction
5 specific primers, using above-mentioned 5 primers, target sequence is expanded, there is very strong strain specificity, while in primer body
Specific probe primer is introduced in system, just has fluorescence signal to send when only specific probe primer is combined with amplification target fragment,
Instrument is just judged to the positive, effective further to improve specificity, avoids making because forming primer dimer and non-target gene magnification
Into false positive；
（2）Rapidly and efficiently：Whole amplification can only be completed with 20~60min, and amplification yield is up to 109~1010Individual copy；
（3）It is easy to operate：After the completion of amplification, without electrophoresis, uncap and add fluorescent dye, instrument can according to fluorescence probe with
Amplification target combines the fluorescence signal automatic interpretation testing result sent；
（4）High sensitivity：The lowest detection limit can reach 0.01%.
Brief description of the drawings
Fig. 1 is the sensitivity technique figure of embodiment 3；
Fig. 2 is the specific detection figure of embodiment 4；
Fig. 3 is the actual sample of embodiment 5 detection figure.
Embodiment
The present invention is further illustrated with reference to embodiments, but is not limited thereto.
The foundation of the transgenic corns MON810 of embodiment 1 Constant Temperature Detection kit
Transgenic corns MON810 Constant Temperature Detection kit, including following component：
（1）LAMP detection primer group, primer sequence are as follows：
Outer primer 1：5’-CATCGTTGAAGATGCCTCT-3’（SEQ ID NO.1）；
Outer primer 2：5’-GTACCGAAGACGGTAGATCT-3’（SEQ ID NO.2）；
Inner primer 1：5’-CGTCATCCCTTACGTCAGTGGAAGAAGACGTTCCAACCAC-3’（SEQ ID NO.3）；
Inner primer 2：5’-CCTTCGCAAGACCCTTCCTCGCTAGAGTCAGCTTGTCAG-3’（SEQ ID NO.4）；
Ring primer：5’-CATTTCATTTGGAGAGGACACG-3’（SEQ ID NO.5）；
Probe primer：FAM-ATCGACATCAATCCACTTGCTTTTCGAT-BHQ-1（SEQ ID NO.6）.
（2）Reaction solution：Containing 10mM dNTP, the 150mM MgSO4 aqueous solution, the volume ratio of the two is 7~9：2~4；
（3）Archaeal dna polymerase
（4）Control：Positive control is transgenic corns MON810 DNA or the e. coli plasmid dna containing target gene,
Negative control is the distilled water without target gene.
The transgenic corns MON810 of embodiment 2 Constant Temperature Detection method
Sample is detected using the transgenic corns MON810 of embodiment 1 Constant Temperature Detection kit, step is as follows：
（1）Extract measuring samples DNA.
（2）Constant temperature gene amplification reacts：Measuring samples are carried out using the Constant Temperature Detection primer sets described in claim 1 permanent
Detection pipe, is placed on the instrument that can provide constant temperature and fluoroscopic examination during amplification by temperature amplification, such as ABI7500 instruments, every 30~
60s gathers first order fluorescence signal；
25 μ l reaction systems of constant temperature gene amplification reaction system contain：1 0.2 μM of inner primer, 2 0.2 μM of inner primer, outside
1 1.6 μM of primer, 2 1.6 μM of outer primer, 0.8 μM of ring primer, 0.8 μM of probe primer, μ l, the DNA polymerizations of LAMP reaction solutions 12.5
The μ l of enzyme 8U, measuring samples DNA 2, with ultra-pure water polishing to 25 μ l；
Constant temperature gene amplification reaction program be：60~63 DEG C of 20~60min of reaction.
（3）As a result judge：Judge yin and yang attribute result according to " S " type amplification curve is whether there is, have " S " type amplification curve for the positive
As a result, otherwise it is negative findings.
The sensitivity test of embodiment 3
Using the kit in embodiment 1 and the method for embodiment 2, the known sample of various concentrations is detected, had
Body process is as follows：
（1）Measuring samples DNA extraction：Using the sample DNA of CTAB methods extraction concentration known, transgenic corns MON810
Content it is as follows：1%th, 0.1%, 0. 05%, 0.01%, 0.005%, 0.001% sample；
（2）Constant temperature gene amplification reacts：Reaction system is prepared in 200 μ l PCR pipes：Primer liquid 1 μ l, the μ of reaction solution 12.5
L, archaeal dna polymerase 1 μ l, the μ l of DNA 2 to be checked, with sterile deionized water polishing to 25 μ l；With the Escherichia coli matter containing target gene
Grain DNA is used as negative control as positive control by the use of distilled water；Centrifuged after the PCR pipe prepared is mixed, reaction tube is placed
On ABI7500 instruments, 1min is set to gather 1 signal, 63 DEG C of reaction 45min；
（3）As a result judge：After reaction terminates, according to " S " type amplification curve judged result.As a result display density be 1%,
0.1%th, 0. 05%, 0.01% sample has obvious " S " type amplification curve, shows that kit sensitivity can reach 0.01%, such as schemes
Shown in 1.
The specific test of embodiment 4
Specificity verification is carried out with the kit of embodiment 1 and the method for embodiment 2, respectively the transgenosis to isolating and purifying
Corn MON810, BT176, BT11, EVENT98140,59122, MON810, MON88017, MON89034, MON863, GA21,
Genetically engineered soybean MON89788, transgenic paddy rice BT63, transgenic potato EH92-527-1, transgene rape T45, transgenic cotton
Flower GHB614, non-transgenic corn, rice, wheat, rape, cotton are identified.
Qualification result is shown：Transgenic corns BT176, BT11, EVENT98140,59122, MON810, MON88017,
MON89034, MON863, GA21, genetically engineered soybean MON89788, transgenic paddy rice BT63, transgenic potato EH92-527-1,
Transgene rape T45, transgene cotton GHB614, non-transgenic corn, rice, wheat, rape, cotton reaction tube are without " S "
Type amplification curve, transgenic corns MON810 reaction tube have " S " type amplification curve, the results showed that the kit has good
Specificity, as shown in Figure 2.
The actual sample of embodiment 5 detects
Utilize the kit of embodiment 1 and the method for embodiment 2 the MON810 corn sample positive to known 2 and 2
The corn sample of known negative is detected, and as a result MON810 corn samples positive known to display have obvious " S " type amplification
Curve, it is known that negative corn sample is without " S " type amplification curve, as shown in Figure 3.
Above example shows that detection method of the invention can effectively reduce the generation of false positive, has high specific, height
Sensitivity, identification be easy, rapidly and efficiently, the beneficial effect such as easy to operate.
<110>Suzhou Hua Mai bio tech ltd
<120>A kind of transgenic corns MON810 constant temperature probe method detection primer group, detection kit and detection side
Method
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
catcgttgaa gatgcctct 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gtaccgaaga cggtagatct 20
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<400> 3
cgtcatccct tacgtcagtg gaagaagacg ttccaaccac 40
<210> 4
<211> 39
<212> DNA
<213>Artificial sequence
<400> 4
ccttcgcaag acccttcctc gctagagtca gcttgtcag 39
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
catttcattt ggagaggaca cg 22
<210> 6
<211> 28
<212> DNA
<213>Artificial sequence
<400> 6
atcgacatca atccacttgc ttttcgat 28
Claims (8)
1. transgenic corns MON810 Constant Temperature Detection primer sets, including outer primer 1 and outer primer 2, inner primer 1 and inner primer 2,
Ring primer and probe primer, its nucleotide sequence difference are as follows：
Outer primer 1：5’-CATCGTTGAAGATGCCTCT-3’；
Outer primer 2：5’-GTACCGAAGACGGTAGATCT-3’；
Inner primer 1：5’-CGTCATCCCTTACGTCAGTGGAAGAAGACGTTCCAACCAC-3’；
Inner primer 2：5’-CCTTCGCAAGACCCTTCCTCGCTAGAGTCAGCTTGTCAG-3’；
Ring primer：5’-CATTTCATTTGGAGAGGACACG-3’；
Probe primer：FAM-ATCGACATCAATCCACTTGCTTTTCGAT-BHQ-1.
2. a kind of transgenic corns MON810 Constant Temperature Detection kit, it is characterised in that the kit includes claim 1
Described Constant Temperature Detection primer sets.
3. transgenic corns MON810 according to claim 2 Constant Temperature Detection kit, it is characterised in that the reagent
Also include in box：Archaeal dna polymerase, LAMP reaction solutions, positive control and negative control.
4. transgenic corns MON810 according to claim 3 Constant Temperature Detection kit, it is characterised in that the LAMP
Reaction solution includes 10mM dNTP, 150mM MgSO4The aqueous solution, the volume ratio of the two are 7~9：2~4.
5. transgenic corns MON810 according to claim 3 Constant Temperature Detection kit, it is characterised in that the positive
The DNA for transgenic corns MON810 or the e. coli plasmid dna containing target gene are compareed, negative control is without purpose base
The reaction mixture of cause.
6. a kind of transgenic corns MON810 detection method, comprise the following steps and feature：
（1）Extract measuring samples DNA；
（2）Constant temperature gene amplification reacts：Constant temperature expansion is carried out to measuring samples using the Constant Temperature Detection primer sets described in claim 1
Increase, detection pipe is placed on the instrument that can provide constant temperature and fluoroscopic examination during amplification, every 30~60s collections first order fluorescence letter
Number；
（3）As a result judge：Judge yin and yang attribute result according to " S " type amplification curve is whether there is, there is " S " type amplification curve to be tied to be positive
Fruit, on the contrary it is negative findings.
7. transgenic corns MON810 according to claim 6 detection method, it is characterised in that the constant temperature gene expands
The 25 μ l reaction systems for increasing reaction contain：1 0.2 μM of inner primer, 2 0.2 μM of inner primer, 1 1.6 μM of outer primer, outer primer 2
1.6 μM, 0.8 μM of ring primer, 0.8 μM of probe primer, the μ l of LAMP reaction solutions 12.5 μ l, archaeal dna polymerase 8U, measuring samples DNA 2
, with ultra-pure water polishing to 25 μ l.
8. transgenic corns MON810 according to claim 6 detection method, it is characterised in that the constant temperature gene expands
Increasing the program reacted is：60~63 DEG C of 20~60min of reaction.
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| CN111020053A (en) * | 2019-12-24 | 2020-04-17 | 广州迪澳生物科技有限公司 | Transgenic CAMV35S constant-temperature fluorescence detection primer group capable of avoiding false negative and kit thereof |
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| CN101812527B (en) * | 2010-04-20 | 2014-09-17 | 北京农业职业学院 | Method for quickly detecting six kinds of genetically modified corns |
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