CN102102126B

CN102102126B - Quick, simple and convenient detection method for transgenic cows with human lactoferrin gene

Info

Publication number: CN102102126B
Application number: CN 201010575492
Authority: CN
Inventors: 刘榜; 翟珊莉; 刘楚新
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2010-12-07
Filing date: 2010-12-07
Publication date: 2013-02-13
Anticipated expiration: 2030-12-07
Also published as: CN102102126A

Abstract

The invention belongs to the technical field of transgenic component detection and relates to a quick detection method for a transgenic component of transgenic cows with a human lactoferrin gene. The invention is associated with a loop-mediated isothermal amplification reaction. The method comprises the following steps: (1) designing a primer, namely designing a specific primer according to the sequence of the human lactoferrin gene, wherein the sequence of the primer is shown by sequences SEQID NO.1, SEQID NO.2, SEQID NO.3 and SEQID NO.4; (2) establishing a loop-mediated isothermal amplification reaction method, namely, performing isothermal amplification of the human lactoferrin gene by using FIP and BIP as internal primers and F3 and B3 as external primers to obtain a specifically amplified fragment; and (3) analyzing the product of the loop-mediated isothermal amplification, namely analyzing the amplification product by agarose gel electrophoresis and fluorescent dye dying, determining if there is a trapezoidal band according to the observation of a gel image or according to the result of the observation of the product of the loop-mediated isothermal amplification in sunlight by naked eye and determining if the product contains a transgenic component. The method is simple, convenient, quick, high in specificity, low in cost and suitable for basic use.

Description

A kind of fast and convenient detection method that is applicable to turn the human lactoferrin gene ox

Technical field

The invention belongs to the detection technique field of transgene component, be specifically related to a kind of fast and convenient detection method that turns the transgene component of human lactoferrin gene ox.

Background technology

The safety evaluation of genetically modified organism is genetically modified organism and products thereof marketization and commercial prerequisite, the detection of transgene component is an important content in safety evaluation, sets up that genetically modified organism is quick, easy, detection method is that safety evaluation and management lay the foundation accurately.

Human lactoferrin is a kind of native protein in the human breast milk, have mend iron, antibiotic, anticancer, improve the critical function such as immunity of organisms, it is the indispensable functional component of infant growth, professor Li Ning of China Agricultural University has successfully cultivated one and has write instructions and transfer the human lactoferrin transgenic dairy, and quantity has reached certain scale.This indicates that China's transgenic dairy rearing new variety and animal bioreactor technology have reached international most advanced level, in view of the maturation that turns human lactoferrin gene milk cow production technology with and the trend of product marketization, therefore set up a kind of method of quick, easy detection human lactoferrin, very important necessity and urgency is arranged.

Can carry out from nucleic acid and two levels of protein of foreign gene for the detection of transgene component at present, the detection of nucleic acid level is used more extensive.The detection of nucleic acid level, mainly for the nucleotide sequence of the foreign gene that changes over to and according to the homology design detection method of foreign gene and native gene, the main method that adopts is pcr amplification, namely according to promotor, goal gene, terminator and expression (restructuring) carrier information that changes foreign gene over to, the design Auele Specific Primer, carry out the polymerase chain reaction, having or not or how much determine whether transgenic product according to product.Can be different according to testing goal, the round pcr that detects genetically modified organism and products thereof is divided into two classes: qualitative PCR and quantitative PCR.Qualitative PCR mainly is after pcr amplification, and the result is positive or negative by the electrophoresis preliminary evaluation, and the PCR reaction has the characteristics of highly sensitive, high specific, high efficiency, is the main detection method in transgenic product primary dcreening operation stage.Qualitative PCR has also developed multiplex PCR, nest-type PRC, hot asymmetric interlaced PCR and electrochemiluminescence PCR etc. according to the research needs on conventional PCR basis.Quantitative PCR can quantize to detect to expression amount and the copy number of transgene in the test sample.In carrying out genetically modified organism and products thereof detection, select to carry out qualitative or detection by quantitative according to different needs and testing goal.

In carrying out the detection of foreign gene nucleic acid level, usually need to carry out two links of pcr amplification and electrophoresis detection, but common PCR, and higher nest-type PRC, the quantitative fluorescent PCR of sensitivity, all must rely on the precision instruments such as PCR instrument, testing cost is high, the testing staff there is higher technical requirements, can't carries out at the relatively poor laboratories of condition.

Summary of the invention

The object of the invention is to overcome the defective of prior art, a kind of fast and convenient detection method that is applicable to turn the human lactoferrin gene ox is provided, utilize ring mediated isothermal amplification method principle to set up the method for quick of foreign gene, accurately rapid detection goes out human lactoferrin gene in the transgenic cattle.The present invention does not need specific installation, and for the testing staff of basic unit provides a kind of fast and convenient method, method of the present invention also is that genetically modified organism and products thereof safety evaluation and management are offered reference simultaneously.

Realize that technical scheme of the present invention is as follows:

(1) design of primers and synthetic: according to gene order (Gene Bank accession:NC_000003.11) the design Auele Specific Primer of human lactoferrin, the right dna sequence dna of described primer is as follows:

FIP:5 '-CCACCTCCTTAGTGGAGTGAAGTGGTGCGAGGCCT-3 ' (corresponding sequence table SEQ ID NO:1);

BIP:5 '-TTCTCAGGGCTGTTCTTGGTAGCTAAGGAAAAGAGGA-3 ' (corresponding sequence table SEQ ID NO:2);

F3:5 '-AGATGGCAGATGGTAGGAGGT-3 ' (corresponding sequence table SEQ ID NO:3);

B3:5 ' TGCGACAAAAGGGCAGACA-3 ' (corresponding sequence table SEQ ID NO:4).

(2) loop-mediated isothermal amplification

Take FIP, BIP as inner primer, F3, B3 are that outer primer carries out constant-temperature amplification to human lactoferrin DNA.Reaction system is: 1M trimethyl-glycine, 400 μ M dNTPs, 2.5 μ l, 10 * Bst Buffer, Mg ²⁺2mM, 8 U Bst archaeal dna polymerases, 1 μ l template, each 0.2 μ M of outer primer F3 and B3, each 1.6 μ M of inner primer FIP and BIP mend ddH ₂O to 25 μ l places 200 μ l EP pipes with all the reagent mixings beyond above-mentioned the dezymotizing, 95 ℃ of 5min, and ice bath 1-2min adds 8U Bst archaeal dna polymerase immediately, in 61 ℃ of lower reaction 60min, then places 80 ℃ of lower 5min termination reactions;

(3) loop-mediated isothermal amplification product analysis

1) agarose gel electrophoresis: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer (40% glycerine, 0.2% tetrabromophenol sulfonphthalein, 59.8%0.25MTris-Hcl), mixing, again electrophoresis 30min in 2% sepharose, obtain gel imaging, observe whether the notch cuttype band is arranged, contain human lactoferrin gene (namely containing transgene component) if there is the notch cuttype band then to be judged to be, do not contain human lactoferrin gene if there is the notch cuttype band then to be judged to be.

2) fluorescent dyeing: SYBR Green I fluorescence dye dilution 100 times (by volume) afterwards as working fluid, is got the ring mediated isothermal amplification product of 5 μ l, add the working fluid of 0.5 μ lSYBR Green I again, the result detects by an unaided eye under daylight.If after adding SYBR Green I working fluid, amplified production becomes yellow-green colour, then be judged to be to contain human lactoferrin gene, if after adding SYBR Green I working fluid, amplified production is that brown color then is judged to be and does not contain human lactoferrin gene.

Two pairs of designed special primers of the present invention have successfully been set up the detection method quick, sensitive, special and simple and practical to human lactoferrin.Compare this invention with other normal PCR method following advantage arranged:

1. the present invention is economical and practical: reaction is to carry out under the condition of constant temperature, so do not need expensive PCR instrument, only needs the equipment that constant temperature can be provided, and for example water-bath gets final product.

2. of the present invention highly sensitive: as to adopt present method can detect the human lactoferrin goal gene of lower limit to 10 copy, than highly sensitive 100 times of common PCR.

3. high specificity of the present invention: the present invention adopts 4 primers, can identify 6 specific regions, has increased the specificity of being combined with goal gene.

4. recall rate of the present invention is high: 14 transgenic cattles that turn human lactoferrin are detected, all detect transgene component, recall rate is 100%.

5. the present invention detects simply: the colour-change of direct visual inspection reaction product after SYBR Green I dyeing qualitatively judges target sequence and whether increases.

Description of drawings

Sequence table SEQ ID NO:1-4 is that the primer of the amplification human lactoferrin gene specific fragment that designs of the present invention is to sequence.

Sequence table SEQ ID NO:5 is the specific fragment of the human lactoferrin gene that increases of the present invention.The fragment total length is 246bp.

Fig. 1: the specific fragment (sequence of this sequence and SEQ ID NO:5 is the same sequence) that is the human lactoferrin gene that increases of the present invention, the fragment total length is 246bp, the sequence underscore is the primer present position partly, and position shown in the square frame is the restriction enzyme site that restriction enzyme NcoI identifies in the sequence.

Fig. 2: pcr amplification human lactoferrin gene electrophorogram, among the figure: M:DL2000DNA marker; 1-5: template is the human gene group DNA; 6: template is cow genome group DNA; 7: water.

Fig. 3: LAMP amplification human lactoferrin gene electrophorogram is among the figure: M:DL2000 DNA marker; 1-5: template is the human gene group DNA; 6: template is cow genome group DNA; 7: water.

Fig. 4: LAMP amplification human lactoferrin gene visualization result, 1-5 among the figure: template is the human gene group DNA; 6: template is cow genome group DNA; 7: water.

Fig. 5: LAMP amplified production enzyme is cut the checking electrophorogram, among the figure: M:DL2000DNA marker; The 1:LAMP amplified production; 2: the LAMP amplified production after enzyme is cut; 3: water.

Fig. 6: pcr amplification human lactoferrin gene electrophorogram, among the figure: M:DL2000DNA marker; 1: template is the human gene group DNA; 2: water.

The test of Fig. 7: LAMP amplification human lactoferrin detection limit is among the figure: M:DL2000DNA marker; 1-7:pMD18T-LTF plasmid copy number successively 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ², 10,5 copies; 8: the genomic dna of ox; 9: water.

Fig. 8: LAMP amplification human lactoferrin detection limit test visualization result is among the figure: 1-7:pMD18T-LTF plasmid copy number successively 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ², 10,5 copies; 8: the genomic dna of ox.

Fig. 9: the test of pcr amplification human lactoferrin detection limit, among the figure: M:DL2000DNA marker; 1-7:pMD18T-LTF plasmid copy number successively 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ², 10,5 copies; 8: the genomic dna of ox.

Figure 10: LAMP detects the transgenic cattle that turns human lactoferrin, among the figure: M:DL2000DNA marker; 1-14: the transgenic cattle that turns human lactoferrin gene; 15: positive control, pMD18T-LTF plasmid; 16: negative control, cow genome group DNA; 17: water.

Figure 11: LAMP detects the transgenic cattle visualization result that turns human lactoferrin, among the figure: 1-14: the transgenic cattle that turns human lactoferrin gene; 15: positive control, pMD18T-LTF plasmid; 16: negative control, cow genome group DNA; 17: water.

Figure 12: PCR detects the transgenic cattle that turns human lactoferrin, among the figure: M:DL2000DNA marker; 1-14: the transgenic cattle that turns human lactoferrin gene; 15: positive control, pMD18T-LTF plasmid; 16: negative control, cow genome group DNA; 17: water.

Embodiment

Embodiment 1: human lactoferrin gene purpose fragment is increased

1, the extraction and purification of DNA: gather human blood sample and ox blood sample according to ordinary method; adopt the phenol-chloroform extraction process of report (with reference to the Pehanorm Brooker; Huang Peitang translates. molecular cloning experiment guide [M]. and Science Press. Beijing: 2005 editions methods) distinguish extracting people and cow genome group DNA, obtain the genomic dna of people or ox.

2. conventional pcr amplification

Pcr amplification method (Pehanorm Brooker with routine, Huang Peitang translates. molecular cloning experiment guide [M]. and Science Press. Beijing: 2005) namely be numbered bovine lactoferrin gene purpose fragment (its nucleotide sequence is seen sequence table SEQ ID NO:5 or shown in Figure 1, and the sequence total length is 246bp) among the primer amplification human gene group DNA of F3 (sequence table SEQ ID NO:3), B3 (sequence table SEQ ID NO:4) with two outer primers.Simultaneously with cow genome group DNA as negative control.

1) reaction system: upstream primer F30.2 μ M, downstream primer B30.2 μ M, 1 μ l PCR buffer, 1.5mM Mgcl ₂, 75 μ M dNTPs, the 0.5UDNA polysaccharase, human gene group DNA 1 μ L is with distilled water polishing to 10 μ L.

2) response procedures: 95 ℃, 5min; 34 circulations: 95 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 20s; 72 ℃, 5min.

3) result judges: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer (prescription: 40% (v/v) glycerine, 60% (v/v) 0.25MTris-Hcl, 0.2% (w/v) tetrabromophenol sulfonphthalein), mixing is then in 2% sepharose behind the electrophoresis 30min, by the gel imaging system imaging, visual length is the amplified fragments of 246bp, and the result as shown in Figure 2.

3, LAMP amplification

Nucleotides sequence with primers F IP (sequence table SEQ ID NO:1), primer BIP (sequence table SEQ ID NO:2) is classified inner primer as, with primers F 3 (sequence table SEQ ID NO:3); , the primer shown in the primer B3 (sequence table SEQ ID NO:4) is that outer primer carries out constant-temperature amplification to the human gene group DNA, simultaneously with cow genome group DNA as negative control.

1) reaction system: reagent is composed as follows: 1M trimethyl-glycine, 400 μ M dNTPs, 2.5 μ l, 10 * Bst Buffer, Mg ²⁺2mM, 8U Bst archaeal dna polymerase, 1 μ l template adds each 0.2 μ M of outer primer F3, B3, and each 1.6 μ M of inner primer FIP, BIP mend distilled water to cumulative volume 25 μ l.

2) response procedures: all the reagent mixings beyond dezymotizing in the above-mentioned reaction system are placed 200 μ l EP pipes, in 95 ℃ of reaction 5min, ice bath 1-2min adds 8U Bst archaeal dna polymerase more immediately, in 61 ℃ of lower reaction 60min, then put 80 ℃ of lower 5min termination reactions.

3) interpretation of result and judgement

1. agarose gel electrophoresis: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer (prescription: 40% (v/v) glycerine, 60% (V/V) 0.25MTris-Hcl, 0.2% (w/v) tetrabromophenol sulfonphthalein), mixing, again electrophoresis 30min in 2% sepharose, imaging under gel systems, whether have notch cuttype band occur, contain human lactoferrin gene if there is the notch cuttype band then to be judged to be if observing, do not contain human lactoferrin gene if there is the notch cuttype band then to judge.

2. fluorescent dyeing: as working fluid, get the ring mediated isothermal amplification product of 5 μ l after 100 times of the SYBR Green I fluorescence dye dilutions, add the working fluid of 0.5 μ lSYBR Green I again, the result detects by an unaided eye under daylight.If after adding SYBR Green I working fluid, amplified production becomes yellow-green colour, judge then and contain human lactoferrin gene that if after adding SYBR Green I working fluid, amplified production is that brown color then is judged to be and does not contain human lactoferrin gene.

3. the result judges: above-mentioned analytical procedure 1. in; become at gel under the ultraviolet lamp of phase system; template is the human gene group DNA; when namely containing people's bovine lactoferrin gene; can observe the notch cuttype band, and template does not namely contain people's bovine lactoferrin gene when being cow genome group DNA; then do not observe the notch cuttype band, its result as shown in Figure 3.Above-mentioned analytical procedure 2. in, when the genomic dna that template is behaved, when namely containing people's bovine lactoferrin gene, amplified production becomes yellow-green colour, when template was cow genome group DNA, amplified production was brown color, its result is as shown in Figure 4.

4) LAMP amplified production specificity check: the LAMP amplified production is carried out enzyme cut checking.

The LAMP amplified production is not to present a band in electrophoresis detection, but presents the notch cuttype band that is comprised of multi-ribbon, and this is that the open-loop products different in size that links together causes because this method forms with the purpose fragment in amplification procedure.

Bovine lactoferrin gene purpose fragment (its nucleotide sequence is seen sequence table SEQ ID NO:5 or shown in Figure 1) size is 246bp among the present invention, the restriction enzyme site that exists restriction enzyme A luI to identify at the 140bp place, therefore can use NcoI that the LAMP amplified production is carried out enzyme cuts, whether the notch cuttype band that checking LAMP amplification obtains is comprised of bovine lactoferrin gene purpose fragment, enzyme is cut if the notch cuttype band can be identified by NcoI, then enzyme is cut rear electrophoresis and is detected the notch cuttype band and do not exist, then be judged to be the notch cuttype band and formed by bovine lactoferrin gene purpose fragment, that is the LAMP resulting product that increases is bovine lactoferrin gene purpose fragment.

1. endonuclease reaction system: get 3 μ lLAMP amplified productions, add 0.8 μ l restriction enzyme NcoI (available from Fermentas company) and 1 μ lbuffer, distilled water is mended to 10 μ l.

2. response procedures: enzyme was cut 6 hours in 37 ℃ of constant incubators.

3. the result judges: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer (prescription: 40% (v/v) glycerine, 60% (v/v) 0.25MTris-Hcl, 0.2% (w/v) tetrabromophenol sulfonphthalein), mixing, electrophoresis 30min in 2% sepharose again, imaging under gel systems, observe enzyme and cut later LAMP amplified production and no longer include the notch cuttype band, the result as shown in Figure 5.The LAMP resulting product that increases is bovine lactoferrin gene purpose fragment among presentation of results the present invention.

Embodiment 2: the test of human lactoferrin LAMP detection method detection limit

Make template with the plasmid pMD18T-LTF that contains human lactoferrin gene, checking detects the detection limit of human lactoferrin LAMP method, and compares with the detection limit of PCR method.Concrete steps are as follows:

1, the structure of plasmid pMD18T-LTF

1) the recovery purifying of human lactoferrin gene purpose fragment PCR products

Be bovine lactoferrin gene purpose fragment (its nucleotide sequence is seen sequence table SEQ ID NO:5 or shown in Figure 8, and the sequence total length is 246bp) among primers F 3 (sequence table SEQ ID NO:3), primer B3 (sequence table SEQ ID NO:4) the amplification human gene group DNA with PCR method with two outer primers.Reaction system: upstream primer F30.2 μ M, downstream primer B30.2 μ M, 1 μ l PCR buffer, 1.5mM Mgcl ₂, 75 μ M dNTPs, the 0.5UDNA polysaccharase, human gene group DNA 1 μ L is with distilled water polishing to 10 μ L.Response procedures: 95 ℃, 5min; 34 circulations: 95 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 20s; 72 ℃, 5min.

Get 50 μ L PCR product electrophoresis 30min in 2% sepharose, gel becomes imaging on the phase system.The result can see the fragment of 246bp size, and the result is (its nucleotide sequence is seen sequence table SEQ ID NO:5 or shown in Figure 1) as shown in Figure 6.The goal gene of amplification is downcut from sepharose, use sepharose to reclaim test kit (available from the white Bioisystech Co., Ltd in Yuanping City, Beijing) and reclaim DNA.

2) the purpose fragment PCR products is connected with the pMD18-T carrier: will reclaim product and be connected with pMD18-T carrier (available from Wuhan strong wind bio tech ltd), linked system is: 0.5 μ L pMD-18T carrier, 3.5 μ L solution I, 3 μ L reclaim product, and 4 ℃ of connections behind the mentioned reagent mixing are spent the night.

3) connect the conversion of product, evaluation and the extraction of pMD18T-LTF plasmid: get 5 μ L connection product and add in the 100 μ L bacillus coli DH 5 alpha competent cells, ice bath 30min, thermal shock 90s in 42 ℃ of recirculated waters, ice bath 1-2min makes the cell cooling fast, add 500 μ L LB liquid nutrient mediums, cultivate 45min on 37 ℃ of shaking tables, at the centrifugal 5min of 4000rpm, discard 500 μ L supernatant liquors, with remaining liquid piping and druming evenly, draw 100 μ L and coat on the LB agar plate that contains penbritin (100 μ g/ml), cultivate 12h for 37 ℃.With through (121 ℃ of autoclavings, high pressure steam sterilization 30min) the LB liquid nutrient medium that bacterium colony places 800 μ L penbritins (100 μ g/ml) is got in 10 μ L pipettor choicests, 4h is cultivated in 37 ℃ of shaking table concussions, then select positive bacteria liquid by bacterium liquid PCR, then serve the sea and give birth to the order-checking of worker's biotechnology company limited, sequencing result is after comparison is confirmed, with the little extraction reagent kit of plasmid (available from TIANGEN company, the method of introducing according to the specification sheets of test kit operates) extract plasmid pMD18T-LTF, and measure plasmid concentration.

4) calculate plasmid copy number

Calculate plasmid copy number, its formula is:

(6.23 * 10 ²³Copy/mol) * (concentration g/ml) ÷ [base number * (660 dalton/base)]=copy number/ml

Plasmid pMD18T-LTF copy number is as calculated: 10.0 * 10 ¹³Copy number/ml plasmid

With distilled water plasmid pMD18T-LTF is carried out doubling dilution, making its final concentration is 25 * 10 ⁷Copy/μ l, 25 * 10 ⁶Copy/μ l, 25 * 10 ⁵Copy/μ l, 25 * 10 ⁴Copy/μ l, 25 * 10 ³Copy/μ l, 25 * 10 ²Copy/μ l, 25 * 10 ¹Copy/μ l.

2, the LAMP method detects the detection limit test:

Utilizing the known plasmid pMD18T-LTF that contains human lactoferrin gene testing goal fragment (246bp) copy number that calculates in the present embodiment is template, adopts LAMP method and PCR method to detect the detection limit test.

1) the LAMP method plasmid pMD18T-LTF of different copy numbers that increases

Take primers F IP (sequence table SEQ ID NO:1), primer BIP (sequence table SEQ ID NO:2) as inner primer, take primers F 3 (sequence table SEQ ID NO:3), B3 sequence table SEQ ID NO:4) the plasmid pMD18T-LTF that step 1 in the present embodiment obtained as outer primer carries out the LAMP amplification, makes to contain plasmid copy number in the system and be followed successively by: 10 ⁶Individual, 10 ⁵Individual, 10 ⁴Individual, 10 ³Individual, 10 ²Individual, 10,5.

1. reaction system: reagent is as follows: 1M trimethyl-glycine, 400 μ M dNTPs, 2.5 μ l, 10 * Bst Buffer, Mg ²⁺2mM, 8U Bst archaeal dna polymerase, 1 μ l template adds each 0.2 μ M of outer primer F3, B3, and each 1.6 μ M of inner primer FIP, BIP mend distilled water to cumulative volume 25 μ l.

2. response procedures: all the reagent mixings beyond above-mentioned reaction system dezymotized place 200 μ l EP pipes, 95 ℃ of reaction 5min, ice bath 1-2min adds 8U Bst archaeal dna polymerase more immediately, in 61 ℃ of lower reaction 60min, then put 80 ℃ of lower 5min termination reactions.

2) the PCR method plasmid pMD18T-LTF of different copy numbers that increases

Simultaneously carry out pcr amplification with two outer primer F3 (sequence table SEQ ID NO:3), B3 (sequence table SEQ ID NO:4), make to contain plasmid copy number in the system and be followed successively by: 10 ⁶Individual, 10 ⁵Individual, 10 ⁴Individual, 10 ³Individual, 10 ²Individual, 10,5.

1. reaction system: upstream primer F30.2 μ M, downstream primer B30.2 μ M, 1 μ l PCR buffer, 1.5mM Mgcl ₂, 75 μ M dNTPs, the 0.5UDNA polysaccharase, human gene group DNA 1 μ L is with distilled water polishing to 10 μ L.

2. response procedures: 95 ℃, 5min; 34 circulations: 95 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 20s; 72 ℃, 5min.

1) interpretation of result:

Through obtaining the result behind the agarose gel electrophoresis: when the pMD18T-LTF plasmid is 10 copies, the LAMP method can amplify the notch cuttype band, and when the pMD18T-LTF plasmid is 5 copies, the LAMP method fails to amplify the notch cuttype band, therefore judge to be limited to 10 copies under the amplification of LAMP to the pMD18T-LTF plasmid, the result as shown in Figure 7.In the LAMP amplified production, get 5 μ l and add 0.5 μ lSYBR Green I working fluid, the result that under daylight, detects by an unaided eye, the result: plasmid copy number is followed successively by: 10 ⁶Individual, 10 ⁵Individual, 10 ⁴Individual, 10 ³Individual, 10 ²Individual, in the time of 10, amplified production becomes yellow-green colour, and plasmid copy number is 5 o'clock, and amplified production then is brown color.The result as shown in Figure 8.

Behind agarose gel electrophoresis, obtain the result: when the pMD18T-LTF plasmid is 10 ³During individual copy, PCR method can amplify specific band, when the pMD18T-LTF plasmid is 10 ²During individual copy, PCR method fails to amplify specific band, therefore judges under the amplification of PCR method to the pMD18T-LTF plasmid to be limited to 10 ³Individual copy, the result as shown in Figure 9.

Therefore, in the present embodiment, the LAMP method is highly sensitive, adopts present method can detect the human lactoferrin goal gene of lower limit to 10 copy, than highly sensitive 100 times of common PCR.

Embodiment 3: the transgenic cattle that turns human lactoferrin is carried out LAMP detect

14 transgenic cattles that turn human lactoferrin are carried out the LAMP detection, use simultaneously the method for PCR in contrast.The genomic dna of used transgenic cattle and people's genome DNA sample in the test all is diluted to 20ng/ μ l.

1) LAMP amplification procedure: take FIP (sequence table SEQ ID NO:1), BIP (sequence table SEQ ID NO:2) as inner primer, F3 (sequence table SEQ ID NO:3), B3 (sequence table SEQ ID NO:4) carry out constant-temperature amplification for outer primer to human lactoferrin DNA, reaction system is: the 1M trimethyl-glycine, 400 μ M dNTPs, 2.5 μ l 10 * BstBuffer, Mg ²⁺2mM, 8U BstNDA polysaccharase large fragment, 1 μ l template, each 0.2 μ M of outer primer, each 1.6 μ M of inner primer mend ddH ₂O to 25 μ l places 200 μ l EP pipes with all the reagent mixings beyond above-mentioned the dezymotizing, 95 ℃ of reaction 5min, and ice bath 1-2min adds 8U Bst DNA polymerase large fragment immediately, 59 ℃ of reaction 60min, termination reaction behind 80 ℃ of reaction 5min;

2) pcr amplification process: with purpose fragment (246bp) among two outer primer F3, B3 amplification human lactoferrin gene group DNA, reaction system is: upstream primer F30.2 μ M, downstream primer B30.2 μ M, 1 μ l PCR buffer, 1.5mM Mgcl ₂, 75 μ M dNTPs, the 0.5U archaeal dna polymerase, human gene group DNA 1 μ L is with distilled water polishing to 10 μ L.The mentioned reagent mixing is placed in the PCR instrument, and reaction parameter is set to: 95 ℃, and 5min; 34 circulations: 95 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 20s; 72 ℃, 5min.

3) interpretation of result:

1. agarose gel electrophoresis: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer (40% (v/v) glycerine, 60% (v/v) 0.25MTris-Hcl, 0.2% (w/v) tetrabromophenol sulfonphthalein), mixing, again electrophoresis 30min in 2% sepharose, obtain gel imaging, observe whether the notch cuttype band is arranged, if there is the notch cuttype band then to contain human lactoferrin gene, if there is not the notch cuttype band then not contain human lactoferrin gene.

2. fluorescent dyeing: with after 100 times of the SYBR Green I fluorescence dye dilutions as working fluid, get the ring mediated isothermal amplification product of 5 μ l, add again the working fluid of 0.5 μ lSYBR Green I, the result detects by an unaided eye under daylight.If after adding SYBR Green I working fluid, amplified production becomes yellow-green colour, then be judged to be to contain human lactoferrin gene, if after adding SYBR Green I working fluid, amplified production is that brown color then is judged to be and does not contain human lactoferrin gene.

3. the result judges: the LAMP detection method of present embodiment and PCR method have in contrast all detected the bovine lactoferrin gene composition of positive transgenic cattle, and the result is respectively such as Figure 10, Figure 11, shown in Figure 12.

Claims

One kind turn the human lactoferrin gene ox fast, convenient detection method, its step comprises:

(1) design of primers and synthetic: according to the gene order design Auele Specific Primer of human lactoferrin, the dna sequence dna of described primer is as follows:

FIP：5’-CCACCTCCTTAGTGGAGTGAAGTGGTGCGAGGCCT-3’；

BIP：5’-TTCTCAGGGCTGTTCTTGGTAGCTAAGGAAAAGAGGA-3’；

F3：5’-AGATGGCAGATGGTAGGAGGT-3’；

B3：5’-TGCGACAAAAGGGCAGACA-3’。

(2) loop-mediated isothermal amplification:

Take FIP, BIP as inner primer, F3, B3 are that outer primer carries out constant-temperature amplification to human lactoferrin gene, and its reaction system is: 1M trimethyl-glycine, 400 μ M dNTPs, 2.5 μ l, 10 * Bst Buffer, Mg ²⁺2mM, 8U Bst DNA polymerase, 1 μ l template, each 0.2 μ M of outer primer F3 and B3, each 1.6 μ M of inner primer FIP and BIP, mend distilled water to 25 μ l, all the reagent mixings beyond above-mentioned the dezymotizing are placed 200 μ l EP pipes, in 95 ℃ of reaction 5min, ice bath 1-2min immediately after the reaction, add 8U Bst DNA polymerase, in 61 ℃ of reaction 60min, termination reaction behind 80 ℃ of lower reaction 5min;

(3) loop-mediated isothermal amplification product analysis:

1) agarose gel electrophoresis: get 2.5 μ l amplified productions, add 1 μ l sample-loading buffer, this damping fluid comprises concentration 40% glycerine, 0.2% tetrabromophenol sulfonphthalein, 59.8%0.25M Tris-Hcl, mixing, electrophoresis 30min in 2% sepharose obtains the gel imaging again, whether observe has the notch cuttype band, contain human lactoferrin gene if there is the notch cuttype band then to be judged to be, namely contain transgene component, do not contain human lactoferrin gene if there is the notch cuttype band then to be judged to be;

2) with after 100 times of the SYBR Green I fluorescence dye dilutions as working fluid, get the ring mediated isothermal amplification product of 5 μ l, add again the working fluid of 0.5 μ lSYBR Green I, the result detects by an unaided eye under daylight.