CN106399302A - Duplex fluorescent PCR method for efficiently detecting specific genes of transgenic soybean MON87701 strain and transgenic soybean MON87708 strain simultaneously - Google Patents
Duplex fluorescent PCR method for efficiently detecting specific genes of transgenic soybean MON87701 strain and transgenic soybean MON87708 strain simultaneously Download PDFInfo
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Abstract
The invention provides primer pairs and probe assemblies, and further provides applications of the primer pairs and the probe assemblies in detection or auxiliary detection of transgenic soybean MON87701 and transgenic soybean MON87708. The invention further provides a kit comprising the primer pairs and the probe assemblies, and applications of the kit, as well as a method for detecting the transgenic soybean MON87701 and the transgenic soybean MON87708 based on the primer pairs and the probe assemblies or the kit. The experiment proves that the primer pairs and the probe assemblies are good in specificity, high in sensitivity, and high in detection efficiency, a duplex fluorescent PCR method for detecting the transgenic soybean MON87701 and the transgenic soybean MON87708 established based on the primer pairs and the probe assemblies is high in flux, and is sensitive, accurate and rapid, and therefore, an effective method is provided for simultaneously detecting the transgenic soybean MON87701 and the transgenic soybean MON87708.
Description
Technical field
The present invention relates to field of biological detection, dual particularly to detection genetically engineered soybean MON87701 and MON87708
Fluorescence PCR method.
Background technology
It is within 2015 genetically modified crops commercialization 20 anniversary, up to 1.797 hundred million hectares of global genetically modified crops cultivated area,
Increase more than 100 times than the commercialization initial stage in 1996, it has also become using the most rapid crop technology in modern agriculture history.At present
The genetically modified crops of commercial growth mainly have Semen sojae atricolor, Semen Maydiss, Cotton Gossypii, Brassica campestris L etc., wherein grown worldwide area maximum for turning
Transgenic soybean, accounts for the half of the global genetically modified crops gross area.Although China does not also ratify any transgenic soybean lines
Carry out commercial growth, but China has become as use and the consumption big country of genetically engineered soybean, product is increasingly entering
The food production of China and consumption chain, the import total amount of genetically engineered soybean in 2015 has reached 80,000,000 tons.
The new varieties that genetically engineered soybean MON87701 and MON87708 Dou Shi Monsanto Chemicals research and develop in recent years, wherein
MON87701 is a kind of insect-resistant transgenic Semen sojae atricolor, is used as to process raw material by Chinese government's approval of import, and MON87708 is one
Plant herbicide-resistant genetically engineered soybean new varieties, also apply for import.Open for genetically engineered soybean MON87701 and MON87708
Exhibition efficiently accurately detection method research, be GMO bio-safety manage the technical support that relevant laws and regulations are smoothly implemented with
Ensure.
At present, adopt substance Fluorescence PCR assay the detection method being directed to genetically engineered soybean both at home and abroad, this technology is special more
The aspects such as property, sensitivity, repeatability have larger advantage, become one of the major technique in this field, but domestic for turning base
Detection because of Semen sojae atricolor MON87701 and MON87708 strain is rarely reported, and only has very small amount ring mediated isothermal nucleic acid amplification at present
(LAMP) detection method of technology is just in the patent application stage, and uses LAMP technology to detect the sensitivity of the primer of genetically engineered soybean
Property and specificity still not high it is impossible to meet actually detected needs.There is presently no and can detect genetically engineered soybean simultaneously
The method of the multiple fluorescence PCR technology of MON87701 and MON87708 strain-specific gene.
Content of the invention
The technical problem to be solved in the present invention be provide a kind of sensitive, accurate, efficient and can detection transgenic simultaneously
The double fluorescent PCR method of Semen sojae atricolor MON87701 and genetically engineered soybean MON87708, present invention also offers for the method
Specificity is good, sensitivity is high, detection efficiency is high and applied widely primer pair and probe combinations, and containing this primer pair and
The test kit of probe combinations and its application.
In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:
First aspect present invention provides primer pair and probe combinations, and one of which primer pair comprises SEQ ID NO：1 He
SEQ ID NO：Nucleotide sequence shown in 2, the probe corresponding with this group primer pair comprises SEQ ID NO：Nucleoside shown in 3
Acid sequence；Another group of primer pair comprises SEQ ID NO：4 and SEQ ID NO：Nucleotide sequence shown in 5, organizes primer pair with this
Corresponding probe comprises SEQ ID NO：Nucleotide sequence shown in 6.
Second aspect present invention provides described primer pair and probe combinations in detection or auxiliary detection genetically engineered soybean
MON87701 and genetically engineered soybean MON87708, or preparation is detected or auxiliary detects that genetically engineered soybean MON87701 and transgenic are big
Application in the product of bean MON87708.
Third aspect present invention provides a kind of test kit, including described primer pair and probe combinations.
In a preference, also include fluorescent quantitative PCR reagent；
In a preference, described fluorescent quantitative PCR reagent includes dNTPs, Mg2+, PCR reaction buffer and Taq
At least one in archaeal dna polymerase；
In a preference, described dNTPs, Mg2+, PCR reaction buffer and Taq archaeal dna polymerase derive from Takara
The article No. of company is the reagent included in the test kit of RR390A；
In a preference, also include DNA extraction kit；
In a preference, the nucleic acid that described DNA extraction kit is included from the article No. reaching peace gene is DA-Z146 carries
Take test kit, Promega company article No. be A1120 Wizard Genomic DNA purification kit or
The article No. of TaKaRa company is the examination included in 9781 MiniBEST Plant Genomic DNA Extraction Kit
Agent；
In a preference, also include positive control and negative control.
Fourth aspect present invention provide described test kit in detection or auxiliary detection genetically engineered soybean MON87701 and
In genetically engineered soybean MON87708, or the product of preparation detection or auxiliary detection genetically engineered soybean MON87701 and MON87708
Application.
Fifth aspect present invention provides a kind of detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean
The double fluorescent PCR method of MON87708, including the step using described primer pair and probe combinations or described test kit.
In a preference, methods described comprises the steps：
1) double fluorescent PCR amplification
Carried out dual for template and described primer pair and probe combinations or described test kit with the DNA that biological sample contains
Fluorescent PCR expands；
2) judge whether biological sample is genetically engineered soybean MON87701 or turns base according to the result of double fluorescent PCR amplification
Because of Semen sojae atricolor MON87708, or whether biological sample is containing genetically engineered soybean MON87701's and/or genetically engineered soybean MON87708
Strain-specific gene composition；
In a preference, in step 1) it is additionally included in the step extracting DNA in biological sample before；
In a preference, the described DNA that extracts in biological sample is using the core reaching the article No. pacifying gene for DA-Z146
Sour extracts kit, Promega company article No. be A1120 Wizard Genomic DNA purification kit or
The article No. of TaKaRa company is that 9781 MiniBEST Plant Genomic DNA Extraction Kit is carried out.
In a preference, the reaction system of described double fluorescent PCR amplification is as follows：
Probes Master 2×conc 10μL
SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5
10 μm of ol/L, 0.4～0.6 μ L
SEQ ID NO：3 and SEQ ID NO：6 10 μm of ol/L, 0.05～0.3 μ L
DNA profiling 100～300ng/ μ L, 1～2 μ L
ddH2O complements to 20 μ L；
The article No. that described Probes Master 2 × conc derives from Takara company is the test kit of RR390A.
In a preference, the reaction system of described double fluorescent PCR amplification is as follows：Described SEQ ID NO：1、SEQ ID
NO：2、SEQ ID NO：4 and SEQ ID NO：5 consumption is 0.5 μ L, described SEQ ID NO：3 and SEQ ID NO：6 use
Amount is respectively 0.2 μ L and 0.1 μ L；
In a preference, described double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5～10s, 59 DEG C, 25
～40s, 40 circulations.
In a preference, described double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5s, 59 DEG C, 30s, 40
Individual circulation.
The biological sample of the present invention is genetically modified crops or the such as food of the processed goods containing transgene component etc..
Beneficial effects of the present invention include：
(1) present invention is directed to a plurality of multi-primerses of two kinds of genetically engineered soybean designs, probe has carried out a large amount of screenings, comprehensive
Its specificity, sensitivity, pairing the influencing each other and different primers probe combinations and fluorescent PCR amplifing reagent of composite amplification
The suitability of box, finally filters out that specificity is good, sensitivity is high, the suitability is wide and can detect genetically engineered soybean MON87701 simultaneously
Primer pair and probe combinations with genetically engineered soybean MON87708.
(2) present invention be directed to establish on the basis of aforementioned primer pair and probe combinations genetically engineered soybean MON87701 and
The double fluorescent PCR detection method of genetically engineered soybean MON87708, can be simultaneously big to genetically engineered soybean MON87701 and transgenic
Bean MON87708 carries out qualitative and detection by quantitative, and specificity is good, flux is high, quick, improves detection efficiency, is that transgenic is big
While bean MON87701 and genetically engineered soybean MON87708, detection provides more effective way.
Brief description
Fig. 1 be in the embodiment of the present invention 5 using primer pair and probe combinations detect simultaneously genetically engineered soybean MON87701 and
The specificity verification result of the double fluorescent PCR method of genetically engineered soybean MON87708.The typical S curve of wherein A, B two is respectively
It is that double fluorescent PCR expansion is carried out for template with the genomic DNA of genetically engineered soybean MON87701 and genetically engineered soybean MON87708
Increase the fluorescence curve of the amplified production obtaining, remaining is with genetically engineered soybean GTS-40-3-2, genetically engineered soybean DAS81419, turns
Transgenic soybean DAS-44406-6, genetically engineered soybean DP305423, genetically engineered soybean A2704-12, genetically engineered soybean FG72, non-turn
The genomic DNA of transgenic soybean carries out the fluorescence curve of the amplified production that double fluorescent PCR amplification obtains for template.
Fig. 2 be in the embodiment of the present invention 6 using primer pair and probe combinations detect simultaneously genetically engineered soybean MON87701 and
The sensitivity the result of the double fluorescent PCR method of genetically engineered soybean MON87708.Wherein 1～4 respectively correspond to dilution factor according to
Secondary is 100To 10-3The genomic DNA of genetically engineered soybean MON87701 double fluorescent PCR reaction result, 5～8 correspond to respectively
Dilution factor is followed successively by 100To 10-3The genomic DNA of genetically engineered soybean MON87708 double fluorescent PCR reaction result, remaining
It is 10 for dilution factor-4The genomic DNA of genetically engineered soybean MON87701, dilution factor be 10-4Genetically engineered soybean MON87708
Genomic DNA and negative control double fluorescent PCR reaction result.
Specific embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiments and the drawings, the present invention will be described, it should be noted that these embodiments are only
It is illustrative, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, all according to normal
Rule experiment condition, such as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular
cloning:Alaboratory manual, 2001), or the condition according to manufacturer's description suggestion.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Embodiment 1
Present embodiments provide primer pair and probe combinations and its application.
The screening technique of two groups of primer pairs is：Product for genetically engineered soybean MON87701 and genetically engineered soybean MON87708
It is specific gene, designs different primers and probe combinations and carried out optimal screening, comprehensive its specificity, sensitivity, join
Influencing each other and different primers probe combinations and the suitability of fluorescent PCR amplification kit to composite amplification, finally screens
Go out that specificity is good, reproducible and sensitivity is high can detect that genetically engineered soybean MON87701, MON87708's is double as follows simultaneously
The primer pair of weight fluorescent PCR and probe combinations：
The nucleotide sequence of described primer pair and probe combinations is as follows：
MON87701-F：：5’-ATTTCCCGGACATGAAGCC-3’(SEQ ID NO：1)；
MON87701-R：5’-TGGTTAGTGAGTGGCAGTAATCG-3’(SEQ ID NO：2)；
The probe sequence of MON87701 is：5’-HEX-CAAGCTAATTCAAGAAAGTGAAGGCACG-BHQ1-3’(SEQ
ID NO：3)
MON87708-F：5’-GCTTATCCGATTTGAGCATTG-3’(SEQ ID NO：4)；
MON87708-R：5’-CGTTTCCCGCCTTCAGTTT-3’(SEQ ID NO：5)；
The probe sequence of MON87708 is：5’-FAM-ATGAGCCATTTAGTCTCACCTTCAAACA-BHQ1-3’(SEQ
ID NO：6)
Wherein HEX, FAM are fluorophor, and BHQ1 is quenching group.
Above-mentioned primer pair and probe are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
Genetically engineered soybean MON87701 and genetically engineered soybean MON87708 can be simultaneous for using above-mentioned primer pair and probe
Carry out double fluorescent PCR amplification.
In application, above-mentioned primer pair and probe combinations can be applicable to detect or assist detection genetically engineered soybean MON87701
On genetically engineered soybean MON87708；Can also be used to preparation detection or auxiliary detection genetically engineered soybean MON87701 and transgenic
The product of Semen sojae atricolor MON87708.
Embodiment 2
Present embodiments provide a kind of test kit and its application, described test kit includes：
(1)SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5 and SEQ
ID NO：6 (from embodiments 1)；
(2)dNTPs、Mg2+, PCR reaction buffer, at least one in Taq archaeal dna polymerase.
Above-mentioned dNTPs, Mg2+, PCR reaction buffer, Taq archaeal dna polymerase be preferably derived from the article No. of TaKaRa company and be
The test kit of RR060A.
Further preferably include DNA extraction kit, described DNA extraction kit preferably includes from the goods reaching peace gene
Number for the nucleic acid extraction kit of DA-Z146, Promega company article No. be A1120 Wizard Genomic DNA
The article No. of purification kit or TaKaRa company is 9781 MiniBEST Plant Genomic DNA
Reagent included in Extraction Kit.
Further preferably include positive control and negative control.
It is demonstrated experimentally that the article No. of above-mentioned DNA extraction kit, TaKaRa company is the test kit of RR060A, and and SEQ
ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5 and SEQ ID NO：6 combine makes
With effect is more superior, is in particular in that high specificity, reproducible and sensitivity are high.
In application, mentioned reagent box can be applicable to detect or assist detection genetically engineered soybean MON87701 and transgenic big
On bean MON87708；Can also be used to preparation detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean
The product of MON87708.
Embodiment 3
Present embodiments provide a kind of detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean
According to the result of double fluorescent PCR amplification, the method for MON87708, for example, judge whether biological sample is genetically engineered soybean
MON87701 or genetically engineered soybean MON87708, or whether biological sample contain genetically engineered soybean MON87701 and/or transgenic
The strain-specific gene composition of Semen sojae atricolor MON87708；The method use primer pair and probe combinations or the enforcement of embodiment 1
The test kit of example 2.
Said method comprises the steps：
Step one：Double fluorescent PCR expands
With the DNA of biological sample as template, double fluorescent P CR amplification is carried out using primer pair and probe, arranges simultaneously simultaneously
If deionized water is negative control.
Double fluorescent pcr amplification reaction system is as shown in table 1.Wherein, Probes Master 2 × conc (includes
DNTP, Mg2+, Taq enzyme etc.) (test kit being RR390A from the article No. of Takara company) 10 μ L, the embodiment of 10 μm of ol/L
1 primer SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5 each 0.4～0.6 μ L, 10 μm of ol/L
Embodiment 1 probe SEQ ID NO：3 and SEQ ID NO：6 each 0.05～0.3 μ L, DNA profiling 1～2 μ L (concentration 100～
300ng/ μ L), ultra-pure water is mended to 20 μ L.
Preferably, above-mentioned primer SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5 interpolation
Amount is 0.5 μ L, probe SEQ ID NO：3 and SEQ ID NO：6 addition is respectively 0.2 μ L and 0.1 μ L.
Double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5～10s, 59 DEG C, 25～40s, 40 circulations.
Double fluorescent pcr amplification reaction condition can be more preferably：95℃3min；95 DEG C, 5s, 59 DEG C, 30s, 40
Circulation.
Step 3：The fluoroscopic examination of double fluorescent pcr amplification product
The 59 DEG C of stages circulated at each terminate after collect FAM and HEX fluorescence signal.
The standard that the result of described double fluorescent PCR amplification judges is as shown in table 1.
Table 1
In table 1 "+" representing Ct value≤35 for the positive, it is feminine gender that "-" represents Ct value ＞ 35.
If biological sample does not also extract DNA, can also include before said method step (1) double fluorescent PCR amplification
Using be the nucleic acid extraction kit of DA-Z146 from the article No. reaching peace gene, the article No. of Promega company be A1120's
The article No. of Wizard Genomic DNA purifi cation kit or TaKaRa company is 9781 MiniBEST Plant
The step that Genomic DNA Extraction Kit is extracted to the DNA in biological sample according to kit specification.
Embodiment 4
Detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean that the present embodiment is set up to embodiment 3
The method of MON87708 has carried out validation verification, and material to be tested used is as follows：Genetically engineered soybean MON87701 powder, turn base
Because of Semen sojae atricolor MON87708 powder, (AOCS, biotechnology is spreaded far and wide in Shenzhen to be purchased from American Oil Chemists'Society
Corporate agent).
Step one：The extraction of genomic DNA
Take genetically engineered soybean MON87701 powder and each 10mg of genetically engineered soybean MON87708 powder in 1.5mL centrifuge tube
Interior, using RNA isolation kit (MiniBEST Plant Genomic DNA Extraction Kit, TaKaRa：9781) extract base
Because organizing DNA, the constant volume of final DNA is 50 μ L.
Step 2：Double fluorescent PCR expands
With the genomic DNA mixed liquor of genetically engineered soybean MON87701 and genetically engineered soybean MON87708 extraction as template,
Carry out double fluorescent PCR amplification, arrange simultaneously and set deionized water as negative control.
Double fluorescent pcr amplification reaction system：Probes Master 2 × conc (includes dNTP, Mg2+, Taq enzyme
Deng) (test kit being RR390A from the article No. of Takara company) 10 μ L, the primer SEQ ID of the embodiment 1 of 10 μm of ol/L
NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5 each 0.5 μ L, the probe SEQ of the embodiment 1 of 10 μm of ol/L
ID NO：3 and SEQ ID NO：6 are respectively 0.2 μ L and 0.1 μ L, DNA profiling 1 μ L (concentration 100～300ng/ μ L), and ultra-pure water is mended
To 20 μ L.
Double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5s, 59 DEG C, 30s, 40 circulations.
Step 3：The fluoroscopic examination of double fluorescent pcr amplification product
The 59 DEG C of stages circulated at each terminate after collect FAM and HEX fluorescence signal.
Interpretation of result：
From result, under FAM passage, Ct value is 24.51, shows to contain genetically engineered soybean MON87708 product in this sample
It is specific gene, under HXE passage, Ct value is 24.13, show special containing genetically engineered soybean MON87701 strain in this sample
Property gene.The detection or auxiliary that the primer pair based on embodiment 1 of the present invention and the test kit of probe and embodiment 2 are set up is described
The method helping genetically engineered soybean MON87701 and genetically engineered soybean MON87708 can be with effective detection genetically engineered soybean MON87701
With genetically engineered soybean MON87708.
Embodiment 5
The present embodiment has carried out specificity verification to the primer pair of embodiment 1 and probe combinations, and material to be tested used is such as
Under：Genetically engineered soybean MON87701 (AOCS, American Oil Chemists'Society U.S. oiling scholar association), turn
Transgenic soybean MON87708 (AOCS, American Oil Chemists'Society U.S. oiling scholar association), transgenic are big
Bean GTS-40-3-2 (EU criteria material and measurement Research institute, IRMM), genetically engineered soybean DAS81419 (are stored in China's inspection
Quarantine research institute), genetically engineered soybean DAS-44406-6 (EU criteria material and measurement Research institute, IRMM), transgenic big
Bean DP305423 (EU criteria material and measurement Research institute, IRMM), genetically engineered soybean A2704-12 (AOCS, American
Oil Chemists'Society U.S. oiling scholar learn), genetically engineered soybean FG72 (AOCS, American Oil
Chemists'Society U.S. oiling scholar learn), (the local market of farm produce, is identified as non-transgenic to Non-transgenic soybean
Semen sojae atricolor).
Step one：The extraction of genomic DNA
Extract genetically engineered soybean MON87701 respectively, turn using the extracting method of the genomic DNA of the step one of embodiment 4
Transgenic soybean MON87708, genetically engineered soybean GTS-40-3-2, genetically engineered soybean DAS81419, genetically engineered soybean DAS-44406-
6th, genetically engineered soybean DP305423, the genome of genetically engineered soybean A2704-12, genetically engineered soybean FG72 and Non-transgenic soybean
DNA.
Step 2：Double fluorescent PCR expands
Respectively with the genomic DNA of each genetically engineered soybean of step one as template, carry out double fluorescent PCR amplification, dual
The method of fluorescent PCR amplification is with the step 2 of embodiment 4.
Step 3：The fluoroscopic examination of double fluorescent pcr amplification product
The 59 DEG C of stages circulated at each terminate after collect FAM and HEX fluorescence signal.
Interpretation of result：
Result is as shown in figure 1, the typical S curve of A, B two is respectively with genetically engineered soybean MON87701 and genetically engineered soybean
The genomic DNA of MON87708 carries out, for template, the fluorescence curve of amplified production that double fluorescent PCR amplification obtains, remaining be with
Genetically engineered soybean GTS-40-3-2, genetically engineered soybean DAS81419, genetically engineered soybean DAS-44406-6, genetically engineered soybean
DP305423, genetically engineered soybean A2704-12, genetically engineered soybean FG72, the genomic DNA of Non-transgenic soybean are carried out for template
The fluorescence curve of the amplified production that double fluorescent PCR amplification obtains.I.e. genetically engineered soybean MON87701 and genetically engineered soybean
MON87708 has amplification can smoothly detect, and remaining genetically engineered soybean and Non-transgenic soybean all no expand, and thus proves the present invention
The primer pair of embodiment 1 and probe have high specific；Further, the present invention is based on primer pair and probe and test kit is set up
Detection or auxiliary genetically engineered soybean MON87701 and genetically engineered soybean MON87708 method can be accurately big to transgenic
Bean MON87701 and genetically engineered soybean MON87708 is detected.
Embodiment 6
The present embodiment has carried out sensitivity checking to the primer pair of embodiment 1 and probe combinations, and material to be tested used is such as
Under：Genetically engineered soybean MON87701, genetically engineered soybean MON87708, are purchased from American Oil Chemists Society
(AOCS, biotech company agency is spreaded far and wide in Shenzhen).
Step one：The extraction of genomic DNA
The step one that the extracting method of genomic DNA is shown in embodiment 4.The genomic DNA that will extract after test nucleic acid concentration
Mix, then deionized water carries out gradient dilution, respectively obtaining dilution factor is respectively 100、10-1、10-2、10-3、10-4Turn
(corresponding concentration is respectively 260ng/ μ L, 26ng/ μ L, 2.6ng/ μ L, 0.26ng/ μ L and .0.026ng/ to transgenic soybean MON87701
μ L) and genetically engineered soybean MON87708 (corresponding concentration be respectively 240ng/ μ L, 24ng/ μ L, 2.4ng/ μ L, 0.24ng/ μ L and
0.024ng/ μ L) the mixed genomic DNA, that is, five groups：
Group 1：The genomic DNA of genetically engineered soybean MON87701 of 260ng/ μ L and the genetically engineered soybean of 240ng/ μ L
The mixed liquor of the genomic DNA of MON87708；
Group 2：The genomic DNA of genetically engineered soybean MON87701 of 26ng/ μ L and the genetically engineered soybean of 24ng/ μ L
The mixed liquor of the genomic DNA of MON87708；
Group 3：The genomic DNA of genetically engineered soybean MON87701 of 2.6ng/ μ L and the genetically engineered soybean of 2.4ng/ μ L
The mixed liquor of the genomic DNA of MON87708；
Group 4：The genomic DNA of genetically engineered soybean MON87701 of 0.26ng/ μ L and the genetically engineered soybean of 0.24ng/ μ L
The mixed liquor of the genomic DNA of MON87708；
Group 5：The genomic DNA of genetically engineered soybean MON87701 of 0.026ng/ μ L and the genetically engineered soybean of 0.024ng/ μ L
The mixed liquor of the genomic DNA of MON87708.
Step 2：Double fluorescent PCR expands
Genomic DNA and genetically engineered soybean using the genetically engineered soybean MON87701 of the variable concentrations of step one
The mixed liquor (organizing 1- group 5) of the genomic DNA of MON87708, as template, carries out double fluorescent PCR amplification, double fluorescent
The method of PCR amplification is with the step 2 of embodiment 4.Each dilution factor makees 3 repetitions, and sets deionized water as negative control.
Step 3：The fluoroscopic examination of double fluorescent pcr amplification product
The 59 DEG C of stages circulated at each terminate after collect FAM and HEX fluorescence signal.
Interpretation of result：
Result as shown in Fig. 2 wherein 1～4 respectively correspond to dilution factor be followed successively by 100To 10-3Genetically engineered soybean
The double fluorescent PCR reaction result of the genomic DNA of MON87701,5～8 correspond to dilution factor respectively is followed successively by 100To 10-3Turn
The double fluorescent PCR reaction result of the genomic DNA of transgenic soybean MON87708, remaining is 10 for dilution factor-4Transgenic big
Bean MON87701 and the mixed genomic DNA of MON87708 and the double fluorescent PCR reaction result of negative control.It can thus be appreciated that its
Detection lower bound is up to 10-4, you can detect the genomic DNA of genetically engineered soybean MON87701 and genetically engineered soybean MON87708 simultaneously
The strain-specific gene of middle 0.26ng and 0.24ng, is equivalent to the corresponding intended transgenic composition of detectable level about 0.1%.
Although, above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. primer pair and probe combinations are it is characterised in that one of which primer pair comprises SEQ ID NO：1 and SEQ ID NO：2
Shown nucleotide sequence, the probe corresponding with this group primer pair comprises SEQ ID NO：Nucleotide sequence shown in 3；Another
Group primer pair comprises SEQ ID NO：4 and SEQ ID NO：Nucleotide sequence shown in 5, organizes the corresponding probe of primer pair with this
Comprise SEQ ID NO：Nucleotide sequence shown in 6.
2. primer pair according to claim 1 and probe combinations in detection or auxiliary detection genetically engineered soybean MON87701 and
Genetically engineered soybean MON87708, or preparation detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean MON87708
Product in application.
3. a kind of test kit is it is characterised in that include the primer pair described in claim 1 and probe combinations.
4. test kit according to claim 3 is it is characterised in that also include fluorescent quantitative PCR reagent；
Optional, described fluorescent quantitative PCR reagent includes dNTPs, Mg2+, PCR reaction buffer and Taq archaeal dna polymerase
In at least one；
Optional, described dNTPs, Mg2+, PCR reaction buffer and Taq archaeal dna polymerase derive from the article No. of Takara company
Reagent included in test kit for RR390A；
Optional, also include DNA extraction kit；
Optional, described DNA extraction kit include from the article No. reaching peace gene be DA-Z146 nucleic acid extraction kit,
The article No. of Promega company is the Wizard Genomic DNA purification kit of A1120 or the goods of TaKaRa company
Number for 9781 MiniBEST Plant Genomic DNA Extraction Kit included in reagent；
Optional, also include positive control and negative control.
5. the test kit described in claim 3 or 4 is in detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean
Application in MON87708, or the product of preparation detection or auxiliary detection genetically engineered soybean MON87701 and MON87708.
6. the double fluorescent PCR side of a kind of detection or auxiliary detection genetically engineered soybean MON87701 and genetically engineered soybean MON87708
Method requires the primer pair described in 1 and any one of probe combinations or claim 3-4 institute it is characterised in that including usage right
The step stating test kit.
7. method according to claim 6 is it is characterised in that comprise the steps：
1) double fluorescent PCR amplification
The DNA being contained with biological sample is in template and the primer pair and probe combinations or claim 3-4 described in claim 1
Test kit described in any one carries out double fluorescent PCR amplification；
2) judge whether biological sample is genetically engineered soybean MON87701 or transgenic is big according to the result of double fluorescent PCR amplification
Bean MON87708, or the biological sample whether strain containing genetically engineered soybean MON87701 and/or genetically engineered soybean MON87708
Specific gene composition.
8. method according to claim 7 is it is characterised in that in step 1) before be additionally included in biological sample and extract
The step of DNA；
Optional, the described DNA that extracts in biological sample is using the nucleic acid extracting reagent reaching the article No. pacifying gene for DA-Z146
Box, the article No. of Promega company are Wizard Genomic DNA purification kit or the TaKaRa company of A1120
Article No. be that 9781 MiniBEST Plant Genomic DNA Extraction Kit is carried out.
9. method according to claim 8 it is characterised in that：The reaction system of described double fluorescent PCR amplification is as follows：
Probes Master 2×conc 10μL
SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5
10 μm of ol/L, 0.4～0.6 μ L
SEQ ID NO：3 and SEQ ID NO：6 10 μm of ol/L, 0.05～0.3 μ L
DNA profiling 100～300ng/ μ L, 1～2 μ L
ddH2O complements to 20 μ L；
The article No. that described Probes Master 2 × conc derives from Takara company is the test kit of RR390A.
10. method according to claim 9 it is characterised in that：The reaction system of described double fluorescent PCR amplification is as follows：
Described SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4 and SEQ ID NO：5 consumption is 0.5 μ L, described SEQ
ID NO：3 and SEQ ID NO：6 consumption is respectively 0.2 μ L and 0.1 μ L；
Optional, described double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5～10s, 59 DEG C, 25～40s, 40 follow
Ring.
Optional, described double fluorescent pcr amplification reaction condition：95℃3min；95 DEG C, 5s, 59 DEG C, 30s, 40 circulations.
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| CN201610486875.6A CN106399302A (en) | 2016-06-24 | 2016-06-24 | Duplex fluorescent PCR method for efficiently detecting specific genes of transgenic soybean MON87701 strain and transgenic soybean MON87708 strain simultaneously |
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| CN201610486875.6A CN106399302A (en) | 2016-06-24 | 2016-06-24 | Duplex fluorescent PCR method for efficiently detecting specific genes of transgenic soybean MON87701 strain and transgenic soybean MON87708 strain simultaneously |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108866224A (en) * | 2018-05-25 | 2018-11-23 | 苏州市食品检验检测中心 | 13 kinds of genetically engineered soybeans detection multiple PCR reagent kit and detection methods |
| CN111218520A (en) * | 2019-10-26 | 2020-06-02 | 大连海关技术中心 | Transgenic soybean GTS-40-3-2 strain EFIRM detection probe and application thereof |
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| CN102596984A (en) * | 2009-09-17 | 2012-07-18 | 孟山都技术公司 | Soybean transgenic event MON 87708 and methods of use thereof |
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| CN101861392A (en) * | 2007-11-15 | 2010-10-13 | 孟山都技术公司 | Soybean plant and seed corresponding to transgenic event MoN87701 and methods for detection thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108866224A (en) * | 2018-05-25 | 2018-11-23 | 苏州市食品检验检测中心 | 13 kinds of genetically engineered soybeans detection multiple PCR reagent kit and detection methods |
| CN111218520A (en) * | 2019-10-26 | 2020-06-02 | 大连海关技术中心 | Transgenic soybean GTS-40-3-2 strain EFIRM detection probe and application thereof |
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