CN102952857B

CN102952857B - Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat

Info

Publication number: CN102952857B
Application number: CN201110248300.8A
Authority: CN
Inventors: 章桂明; 向才玉; 凌杏园; 潘广; 程颖慧; 康林; 李鹤遥
Original assignee: Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Current assignee: Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Priority date: 2011-08-26
Filing date: 2011-08-26
Publication date: 2015-03-25
Anticipated expiration: 2031-08-26
Also published as: CN102952857A

Abstract

The invention discloses a primer and a method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat. The primer is high in specificity and can be used for PCR amplification and DHPLC analysis. The method for detection for the endogenous genes of the wheat is simple in operation, good in extensibility and high in sensitivity. PCR amplification products are analyzed by means of DHPLC, the segment size resolution of the PCR amplification products can reach multiple basic groups, and the resolution ratio is high. The primer and the method have the advantages that the method is a simple, convenient, effective and reliable method for detection for the endogenous genes of the wheat, and the primer and the method are particularly suitable for port inspection and quarantine departments and the like.

Description

Wheat endogenous gene PCR-DHPLC detects primer and detection method

Technical field

The present invention relates to a kind of gene test primer and method, the PCR-DHPLC particularly relating to a kind of wheat endogenous gene detects primer and detection method.

Background technology

At present the detection methods such as Standard PCR, real-time fluorescence PCR, PCR-gene chip are mainly adopted to the detection method of wheat cdna.Traditional Standard PCR detection method has certain limitation in platform extension, and along with the increase of target to be checked, need to re-start optimization to the consumption often overlapping primer in system and ratio, and take into account the factors such as amplification efficiency, workload is larger; On the other hand, the method for the gel electrophoresis analysis amplified production usually adopted, distinguish efficiency not high, detected result is undesirable.Although multiple real time fluorescence PCR more multiple Standard PCR detection method in detection sensitivity etc. has superiority, but due to the restriction that current instrument self and fluorescence dye are developed, non-interfering 4 fluorescence channels only can be provided simultaneously, also limit this technology in the expansion of detection flux.PCR-gene chip detection method, complex operation step, and be not suitable for large-scale high throughput testing.Denaturing high performance liquid chromatography (Denaturing High-performance LiquidChromatography, DHPLC) be a kind of method for nucleic acid analysis of simple, quick, non-gel, have that extendability is strong, good resolution, sensitivity advantages of higher.The method is analytic sample under 50 DEG C of conditions, and the wash-out at sample peak only determines elution order by the quantity of base pair, and the acetonitrile concentration having served as post improves, and nucleic acid fragment can according to molecular weight order from small to large by wash-out out.To be compared with marker by the elution peak that obtains and determine molecular size range, determine whether containing target detect gene.At present, the PCR of wheat endogenous gene is not still had in conjunction with the detection technique (PCR-DHPLC) of DHPLC.

Summary of the invention

The object of this invention is to provide the primer that a kind of PCR-DHPLC for wheat endogenous gene detects.

Another object of the present invention is to provide that a kind of scalability based on above-mentioned primer is good, the PCR-DHPLC detection method of sensitivity and the high wheat endogenous gene of resolving power.

For achieving the above object, present invention employs following technical scheme:

The invention discloses the primer that the PCR-DHPLC for wheat endogenous gene detects, the upstream primer of described primer contains sequence shown in Seq ID No.1, and downstream primer contains sequence shown in Seq ID No.2;

Seq ID No.1：5’-CGACAACACCATCGCTATCC-3’

Seq ID No.2：5’-TGAACACGACGACTTTTCTTCTT-3’。

It is pointed out that above-mentioned Seq ID No.1 and Seq ID No.2 is the distinguished sequence with the detection target sequence complementary pairing of wheat endogenous gene, there is very strong specific recognition.

Further, 5 ' end of described upstream primer also comprises sequence shown in Seq ID No.3, and 5 ' end of described downstream primer also comprises sequence shown in Seq ID No.4;

Seq ID No.3：5’-CGTGGCCTCGCGATCTGACT-3’

Seq ID No.4：5’-CTCAGCGGCGGAGCTACAGA-3’。

It is to be noted, above-mentioned Seq ID No.3 and Seq ID No.4 holds the one section sequence irrelevant with detecting target sequence of adding at 5 ' of upstream primer and downstream primer respectively, this sequence can be the sequence of other species far with detecting target sequence homology, also can be the sequence of one section of stochastic generation.This sequence may be used for the size of pcr amplification product, so that the further analysis of pcr amplification product.

In the present invention, preferably, described upstream primer has sequence shown in Seq ID No.5, and downstream primer has sequence shown in Seq ID No.6;

Seq ID No.5：5’-CGTGGCCTCGCGATCTGACTCGACAACACCATCGCTATCC-3’

Seq ID No.6：5’-CTCAGCGGCGGAGCTACAGATGAACACGACGACTTTTCTTCTT-3’。

It should be noted that, described Seq ID No.5 and Seq ID No.6 sequence add regulating and controlling sequence at its 5 ' end respectively by above-mentioned Seq ID No.1 and Seq ID No.2 sequence to form, illustrate that although above-mentioned regulating and controlling sequence shown in Seq IDNo.3 and Seq ID No.4 can be the sequence of stochastic generation, but, be not that arbitrary sequence can be added, specific in the present invention, the unified of the physico-chemical property considering sequence shown in regulating and controlling sequence and Seq ID No.1 and Seq IDNo.2 is needed to coordinate, and need to consider that shown in the SeqID No.3 after adding regulating and controlling sequence and Seq ID No.4, sequence is as the overall performance detecting primer.

The invention also discloses a kind of PCR-DHPLC detection method for wheat endogenous gene, described detection method comprises the above-mentioned primer of employing, is that template carries out pcr amplification, carries out dhplc analysis to pcr amplification product with Wheat DNA.

Further, it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker is compared.

Preferred above-mentioned detection method comprises the steps:

(A) adopt above-mentioned primer, take Wheat DNA as template, carry out pcr amplification;

(B) be that sample carries out DHPLC analysis with the pcr amplification product of step (A), be that reference standard carries out DHPLC analysis with marker simultaneously;

(C) the DHPLC analytical results of sample in step (B) and the DHPLC analytical results of marker are compared, determine the molecular size range of sample, thus judge whether the target sequence containing detecting.

Owing to adopting above technical scheme, beneficial effect of the present invention is:

Wheat endogenous gene test primer of the present invention has stronger specificity, can be used in follow-up pcr amplification and DHPLC analysis.The present invention is directed to the problem that traditional electrophoretic analysis pcr amplification result is undesirable, PCR and DHPLC combined, provide a kind of easy and simple to handle, scalability good, sensitivity and the high wheat endogenous gene tester of resolving power.Utilize DHPLC to analyze pcr amplification product, several base can be reached to the PCR primer fragment differentiation rate of different size, even can distinguish the fragment of 1 base difference in size.Primer of the present invention and detection method are that wheat endogenous gene test provides a kind of simple, convenient, effective, reliable detection method, are particularly suitable for the departments such as Check and Examination of Port quarantine and use.

Accompanying drawing explanation

Figure is the partial results that in the embodiment of the present invention, DHPLC analyzes; In figure, top-down curve is labeled as M, 1-10 respectively, M be marker, 1 for wheat samples 1,2 for wheat samples 2,3 for wheat samples 3,4 is non-transgenic rape, 5 is potato strain EH92-527-1,6 is non-transgenic paddy rice, 7 is non-transgenic cotton, 8 is non-transgenic corn, 9 is pawpaw, 10 contrasts for water.

Embodiment

The present invention discloses the primer that the PCR-DHPLC for wheat endogenous gene detects, and this primer designs for the distinguished sequence of wheat endogenous gene acc1.Further, also the upstream and downstream of primer with the addition of respectively one section with detect target sequence and have nothing to do or regulating and controlling sequence that homology is far, for realizing the regulation and control of the clip size to amplified production.It is to be noted, described regulating and controlling sequence add just in order to obtain more excellent Detection results, therefore, the present invention is preferred, be the primer adding regulating and controlling sequence for the PCR of wheat endogenous gene in conjunction with the primer of DHPLC technology for detection, there is sequence respectively shown in Seq ID No.5 to Seq ID No.6.

The invention also discloses a kind of PCR-DHPLC detection method for wheat endogenous gene, described detection method comprises the described primer of employing, is that template carries out pcr amplification, carries out dhplc analysis to pcr amplification product with Wheat DNA.Further, it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker are compared, judge the size of pcr amplification product according to comparison result, thus judge whether the target sequence containing detecting.In the present invention, the DHPLC elution peak of described primer extension product is 337bp.

Also by reference to the accompanying drawings the present invention is described in further detail below by specific experiment example.Following experimental example is only further detailed the present invention, should not be construed as limitation of the present invention.

Embodiment 1DNA extracts

CTAB method is adopted to extract sample DNA, specific as follows:

A) take sample 5g, in mortar, add the powder that liquid nitrogen grinding to sample is about 0.5mm size;

B) take the sample that 300mg grinds, proceed to rapidly in 2mL centrifuge tube, add the CTAB extracting solution 700 μ L of 65 DEG C of preheatings, mixing, puts into 65 DEG C of water-bath water-bath 30min;

C) 5 μ L RNase (10mg/mL) are added, 37 DEG C of water-bath 30min;

D) add the saturated phenol of equal-volume Tris, fully mix, the centrifugal 15min of 12000r/min;

E) get supernatant, add the mixing of isopyknic chloroform/primary isoamyl alcohol (24: 1), the centrifugal 15min of 12000r/min;

F) get supernatant, add the mixing of isopyknic chloroform/primary isoamyl alcohol (24: 1), the centrifugal 15min of 12000r/min;

G) add the Virahol of equal-volume precooling, jiggle, be placed in-20 DEG C of refrigerators and leave standstill 30min, the centrifugal 15min of 12000r/min;

H) abandon supernatant, add 70% ethanol 500 μ L, the centrifugal 3min of 12000r/min, removes supernatant, repeats 2 times;

I) obtain DNA precipitation, carry out drying, add 50 μ L ~ 100 μ L TE or aseptic deionized waters with freeze drier, after fully dissolving, the purity of measurement DNA and concentration are placed in-20 DEG C of refrigerators preserves.

Embodiment 2DNA concentration determination

Concentration and purity testing are carried out to the sample DNA extracted; Adopt ultraviolet spectrophotometer to measure 260nm and 280nm place absorption value, calculate purity and the concentration of nucleic acid respectively, calculation formula is as follows:

DNA purity=OD260/OD280

DNA concentration=50 × OD260mg/mL

The purity ratio of DNA is between 1.7 ~ 1.9, and concentration is greater than 10ng/ μ L.

Embodiment 3PCR increases

The binding site of specific detection primer is designed according to wheat endogenous gene acc1, and add regulating and controlling sequence at 5 ' end of specific detection primer binding site, the detection primer (table 1) of synthesis containing regulating and controlling sequence, adopt the up/down trip primer adding regulating and controlling sequence to carry out pcr amplification, sample arranges and comprises: DNA, the DNA of non-transgenic corn of the DNA of the DNA of 3 wheat samples and the DNA of non-transgenic rape, potato strain EH92-527-1, the DNA of non-transgenic paddy rice, non-transgenic cotton, the DNA of pawpaw and water blank.

Table 1 wheat endogenous gene acc1 detects primer binding site and primer

PCR reaction system cumulative volume is 50 μ L, each composition is respectively: multi-PRC reaction mixed solution Multiplex PCR Mix (TaKaRa) 25 μ L, 10 μm of ol/L primer each 1 μ L, DNA 2 μ L, 5U/ μ L Taq enzyme 0.25 μ L, supplying with sterilizing distilled water is 50 μ L.

PCR reaction conditions: 94 DEG C of sex change 1min; Then 35 circulations are entered, 94 DEG C of 30s, 57 DEG C of 1min, 72 DEG C of 1min; After loop ends, 72 DEG C extend 10min.

Embodiment 4DHPLC detects

PCR primer is placed on the automatic sampling platform of DHPLC; Open DHPLC control software design Navigator, in detection method, select Multiplex, be i.e. multiple clips analysis, set upper limit of detection is 600bp simultaneously, and lower limit is 70bp; Run two blank, i.e. empty needles, balance chromatographic column; Run a marker, for reference to contrast, detect the nucleic acid fragment size of sample; Analyze sample to be tested successively, partial results is shown in Fig. 1.

Result judges: observe the elution peak that DHPLC obtains, by the DNA fragmentation size with marker comparison and WAVE 4500 automatic analysis determination elution peak position, judge accordingly.

To the sample containing Wheat components, its analytical results shows, and this sample contains the elution peak of 146bp; Other transgenosis or non-transgenic sample and water contrast all do not have elution peak.

Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.

Claims

1., for the primer that wheat endogenous gene PCR-DHPLC detects, it is characterized in that: the upstream primer of described primer is sequence shown in Seq ID No.5, and downstream primer is sequence shown in Seq ID No.6;

Seq ID No.5：5’-CGTGGCCTCGCGATCTGACTCGACAACACCATCGCTATCC-3’

Seq ID No.6：5’-CTCAGCGGCGGAGCTACAGATGAACACGACGACTTTTCTTCTT-3’。

2. the PCR-DHPLC detection method for wheat endogenous gene, it is characterized in that: described detection method comprises employing primer according to claim 1, be that template carries out pcr amplification with Wheat DNA, dhplc analysis is carried out to pcr amplification product.

3. detection method according to claim 2, it is characterized in that: it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker is compared.