CN108441577B - Primer pair, kit and application thereof, and method for detecting whether alfalfa product contains soybeans - Google Patents
Primer pair, kit and application thereof, and method for detecting whether alfalfa product contains soybeans Download PDFInfo
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Abstract
The invention provides a primer pair, a kit and application thereof, and a method for detecting whether alfalfa products contain soybeans, and relates to the technical field of biology. The primer pair comprises a primer MD5-F and a primer MD5-R which have sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, and can simultaneously amplify DNA fragments with different fragment lengths of alfalfa and soybean, so that whether the sample to be detected contains alfalfa and/or soybean can be distinguished by one-time amplification. The primer pair is used for detecting whether the alfalfa product contains soybeans or not, has the advantages of strong sensitivity, high accuracy, strong repeatability, rapidness, convenience and the like, can be used for detecting the alfalfa product in large batches, and relieves the defect that the prior art lacks a method for detecting whether the alfalfa feed is doped with soybeans or not.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer pair, a kit and application thereof, and a method for detecting whether alfalfa products contain soybeans.
Background
Alfalfa (Medicago sativa L.) has the characteristics of high quality, high yield, high feeding value (crude protein content of 21%), good palatability, wide application and the like, and is known as the king of pasture in the world. After the pasture is granulated, the density is increased by more than 5 times compared with the original density, and the pasture has the advantages of volume reduction and convenient storage and transportation. The alfalfa grass particles are deeply favored by farmers due to the characteristics of small volume, convenience in feeding, high nutritional value and storage resistance, and are an important mode for the alfalfa to be used as a feed. More than 50% of cattle feed used in European and American countries is grass particles, the feeding of alfalfa particles can obviously improve the milk yield of cows and the quality of milk, and coarse feed granulation is also the development direction of feed industry in China. Alfalfa grass pellets are an important role in the feed trade.
Soybean [ Glycine max (Linn.) Merr.]Is one of the world important oil crops, and the planting area of Chinese soybean in 2008 is 955 ten thousand hm2The addition of the Chinese medicinal preparation is 9.1% compared with 2007, and the innovation history is high. Although the application range of the soybean is wide, the utilization value of the straw of the soybean is not high. Because the soybean straws are hard in texture and high in lignin content, the soybean straws are only used as coarse fodder for cattle and sheep when forage grass is in short supply and the forage grass is expensive in spring, if the soybean straws are crushed and added into alfalfa grass particles, the quality of the grass particles is reduced, the soybean straws directly used as the fodder have low nutritive value, and the rumen effective degradation rate of dry matters is only 17.98%, so that the rights and interests of consumers are hurt.
When the alfalfa is made into grass powder to be used for manufacturing grass particles, whether the soybean straws are mixed in the grass powder or not can not be distinguished by naked eyes, if a user purchases a large number of alfalfa particles containing the soybean straws, the rights and interests of consumers are greatly damaged, a large amount of manpower, material resources and financial resources are consumed for detecting the quality of the alfalfa particles by using a conventional chemical analysis method, and a unified and effective detection method does not exist at home and abroad at present. The accurate and rapid detection of whether the alfalfa grass particles contain other crop straws is a big problem in the quality inspection of the alfalfa grass particles. Therefore, a method for detecting whether the alfalfa grass granulated feed is doped with the soybean straws is needed in the current market.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a primer pair for detecting alfalfa and soybean, which alleviates the technical problem of insufficient detection technology in the prior art, the primer pair can amplify DNA fragments of alfalfa and soybean, and whether a sample to be detected contains alfalfa and/or soybean can be detected simultaneously because the lengths of the amplified product fragments of alfalfa and soybean are different.
The second purpose of the invention is to provide a kit containing the primer pair, and the kit has wide application in matching with other reagents commonly used in molecular biology experiments.
The third objective of the present invention is to provide an application of the primer pair or the kit in detecting alfalfa and/or soybean, the application is very wide, and the primer pair or the kit can be applied to detecting whether a sample to be detected contains alfalfa and/or soybean, and the primer pair and the kit comprising the primer pair are suitable for detecting various samples to be detected, and even if the sample to be detected contains less alfalfa and/or soybean, the primer pair and the kit comprising the primer pair can detect alfalfa and/or soybean.
The fourth purpose of the invention is to provide a method for detecting whether alfalfa products contain soybeans, and to alleviate the technical problem that the prior art lacks a method for detecting whether alfalfa products are doped with soybeans.
In order to solve the technical problems, the invention adopts the following technical scheme:
a primer pair for detecting alfalfa and soybean comprises a primer MD5-F and a primer MD 5-R;
the primer MD5-F has a sequence shown as SEQ ID NO.1, and the primer MD5-R has a sequence shown as SEQ ID NO. 2.
Further, the primer MD5-F and/or the primer MD5-R are modified;
further, the modification comprises adding a label at the 5 'end and/or 3' end of the primer;
further, the label includes a fluorescent label, a biotin label or a digoxigenin label.
The invention also provides a kit containing the primer pair.
Further, the kit also comprises Mg2+dNTP, DNA polymerase and PCR Buffer.
The invention also provides an application of the primer pair or the kit in detecting alfalfa and/or soybean.
The invention also provides a method for detecting whether the alfalfa product contains soybeans, which comprises the following steps: carrying out PCR amplification on DNA of the alfalfa product, and detecting whether the amplified product contains DNA fragments of soybean or not, wherein if the product contains the DNA fragments of the soybean, the alfalfa product contains the soybean;
wherein the primer used in the PCR amplification comprises the primer pair.
Further, the alfalfa product is alfalfa grass pellet feed.
Further, the PCR amplification system is a 20. mu.l system, which comprises 2 XPCR Master 10. mu.l, 1. mu.l DNA template with the concentration of 50 ng/. mu.l, 1. mu.l primer MD 5-F1. mu.l with the concentration of 10nM, 1. mu.l primer MD 5-R1. mu.l primer with the concentration of 10nM and ddH2O 7μl；
Wherein the 2 XPCR Master comprises 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq and 2 XPCR Buffer.
Further, the PCR amplification conditions are a) pre-denaturation at 94 ℃ for 4 minutes; b) denaturation at 94 ℃ for 30 seconds; c) annealing at 55-65 deg.C for 30 s; d) extension at 72 ℃ for 30 seconds; the steps b) to d) are circulated for 25 to 40 times; e) extension at 72 ℃ for 7 min;
wherein steps b) to d) are preferably cycled 30 to 38 times; more preferably from 32 to 36 cycles;
the annealing temperature is preferably 55-62 ℃; more preferably from 57 to 59 ℃.
Further, detecting the amplification product by using a gel electrophoresis method; wherein the gel is 6-10% of PAGE gel; the electrophoresis conditions are as follows: the voltage is 180-220V, and the electrophoresis time is 90-120 min.
Compared with the prior art, the invention has the following beneficial effects:
the primer pair for detecting the alfalfa and the soybean can simultaneously amplify DNA fragments of the alfalfa and the soybean, and the amplification products of the alfalfa and the soybean are different in length, so that whether the sample to be detected contains the alfalfa and/or the soybean can be distinguished by amplifying the sample to be detected once, and false positive or false negative caused by the difference of amplification efficiency or errors of manual operation during separate amplification is avoided.
According to the kit containing the primer pair, the primer pair is prepared into the kit in advance, so that the experimental efficiency can be improved, and the kit has wide application in cooperation with other common reagents in molecular biology experiments.
The primer pair or the kit provided by the invention has very wide application in detecting alfalfa and/or soybean. The primer pair can detect whether the sample to be detected contains alfalfa and/or soybean. And because the primer pair has high sensitivity, the primer pair and the kit containing the primer pair are suitable for detecting various samples to be detected, and even if the samples to be detected contain less alfalfa and/or soybean, the primer pair and the kit containing the primer pair can also detect the alfalfa and/or the soybean. In actual production, the alfalfa is widely applied and is rich in nutrition, the soybean straw is rich in yield but not suitable for the feed, and the problem that the alfalfa feed is not fully mixed with the soybean straw exists, so that the primer pair or the kit is very valuable when being applied to detection of the alfalfa and/or the soybean.
The method for detecting whether the alfalfa product contains the soybeans can effectively detect whether the alfalfa product contains the soybean straws or not at a level of 1%. The method has the advantages of strong sensitivity, high accuracy, strong repeatability, rapidness, convenience and the like, can be used for detecting mass alfalfa products, and provides a feasible basis for ensuring the purity detection of the alfalfa product grass granulated feed. The method can be used for rapidly, efficiently and accurately detecting whether the alfalfa product is mixed with the soybeans or not, is time-saving and labor-saving, is simple and convenient to operate, and can be well finished in a short time under laboratory conditions. The method can make alfalfa product better serve for animal husbandry production, and lay a solid foundation for guaranteeing forage grass safety.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an electrophoretogram of PCR amplification products provided in example 4 of the present invention;
FIG. 2 is an electrophoretogram of PCR amplification products provided in example 4 of the present invention;
FIG. 3 is an electrophoretogram of PCR amplification products provided in example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a primer pair for detecting alfalfa and soybean, which comprises a primer MD5-F and a primer MD 5-R; wherein the primer MD5-F has a sequence shown as SEQ ID NO.1, and the primer MD5-R has a sequence shown as SEQ ID NO. 2.
The primer MD5-F and the primer MD5-R can simultaneously amplify DNA fragments of alfalfa and soybean, and the lengths of amplification products of the alfalfa and the soybean are different, so that whether the sample to be detected contains the alfalfa and/or the soybean can be distinguished by amplifying the sample to be detected once, and false positive or false negative caused by the difference of amplification efficiency or errors of manual operation during separate amplification is avoided.
It can be understood that, when the sample to be tested only contains alfalfa, the primer pair can be used for amplifying the DNA fragment of the alfalfa; when the sample to be detected only contains soybeans, the primer pair can be used for amplifying the DNA fragment of the soybeans; when the sample to be detected contains the alfalfa and the soybean at the same time, the primer pair can amplify DNA fragments of the alfalfa and the soybean at the same time, amplification products of the alfalfa and the soybean are different, and the two products amplified by the primer pair can be distinguished after amplification, so that the sample to be detected can be known to contain the alfalfa and the soybean at the same time from the amplification products of the primer pair.
In an amplification product of the primer pair, a DNA fragment of the alfalfa amplified by the primer MD5-F and the primer MD5-R has a sequence shown as SEQ ID NO.3, the length of the sequence fragment is 194bp, and a complementary sequence of the sequence fragment is shown as SEQ ID NO. 4; the DNA fragment of soybean amplified by the primer MD5-F and the primer MD5-R has a sequence shown as SEQ ID NO.5, the length of the sequence fragment is 203bp, and the complementary sequence of the sequence fragment is shown as SEQ ID NO. 6.
In some alternative embodiments, primer MD5-F and/or primer MD5-R are modified; the modification may be, for example, but not limited to, phosphorylation, biotin labeling, digoxigenin labeling, fluorophore labeling, thio-modification, deoxyuracil modification, or deoxyhypoxanthine modification. It is to be understood that the present invention is not limited to the modification of the primer as long as it complies with the conventional experimental principles of molecular biology experiments. In some preferred embodiments, the primers are modified using fluorescent labels, and the primers modified with fluorescent labels can be used for fluorescent quantitative PCR.
The invention also provides a kit containing the primer pair. The primer pair is prepared into a kit in advance, and the kit canThe kit can be widely used by matching with other common reagents in molecular biology experiments. For example, the kit optionally includes Mg2+PCR reagents such as dNTPs and DNA polymerases can be used as a PCR amplification kit, and when a fluorescent dye is further included, the PCR reagent can be used as a fluorescent quantitative PCR kit. In some preferred embodiments, the kit further comprises 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq enzyme and 2 XPCR Buffer. It can be understood that the kit can be used for detecting whether the sample to be detected independently contains alfalfa or soybean, and can also be used for detecting whether the sample to be detected contains alfalfa and soybean simultaneously; when the kit also comprises primers for detecting other species, the kit can also be used for simultaneously detecting samples of multiple species.
The invention also provides an application of the primer pair or the kit in detecting alfalfa and/or soybean. Because the primer pair has high sensitivity, the primer pair and the kit containing the primer pair are suitable for detecting various samples to be detected, and even if the samples to be detected contain less alfalfa and/or soybean, the primer pair and the kit containing the primer pair can also detect the alfalfa and/or the soybean. The primer pair can detect whether the sample to be detected contains the alfalfa and/or the soybean, and more importantly, whether the sample to be detected containing the alfalfa is mixed with the soybean or not.
The invention also provides a method for detecting whether the alfalfa product contains soybeans, which comprises the following steps: carrying out PCR amplification on DNA of the alfalfa product, and detecting whether the amplified product contains DNA fragments of soybean or not, wherein if the product contains the DNA fragments of the soybean, the alfalfa product contains the soybean; wherein the primers used for PCR amplification comprise the primer pair, namely a primer MD5-F and a primer MD 5-R.
It is understood that the primers used in the PCR amplification may comprise other primer pairs in addition to the primer MD5-F and the primer MD5-R, so as to achieve the purpose of simultaneously amplifying other target fragments. For example, when it is required to detect whether alfalfa is doped with other crops, corresponding primers may be added to perform composite PCR, and the present invention does not limit the addition of other primer pairs, and it can be understood that the addition of another primer pair should not affect the amplification efficiency of the primer pair of the present invention. It is understood that the PCR may be, for example, but not limited to, general PCR, fluorescence quantitative PCR, or high resolution solubility curve (HRM), and the invention is not limited to the type of PCR, as long as the primer pair provided by the invention can be used to perform a chain polymerase reaction on DNA of a sample to be tested; the invention can also further improve the PCR system and the used reagent to enhance the reaction efficiency of PCR, but the improvement is based on the primer pair provided by the invention when in amplification.
The method for detecting whether the alfalfa product contains the soybeans can effectively detect whether the alfalfa product contains the soybean straws or not at a level of 1%. The method has the characteristics of strong sensitivity, high accuracy, strong repeatability, rapidness, convenience and the like, can be used for detecting mass alfalfa products, and provides a feasible basis for ensuring the purity detection of the alfalfa products. The method can quickly, efficiently and accurately detect whether the alfalfa product is mixed with soybeans or not. The method is time-saving and labor-saving, is simple and convenient to operate, and can be well completed in a short time under laboratory conditions. The alfalfa product is better served for the production of animal husbandry, and a solid foundation is laid for guaranteeing the safety of forage grass.
In some alternative embodiments, the alfalfa product is a alfalfa grass pellet feed. After the pasture is granulated, the density is increased by more than 5 times compared with the original density, and the pasture has the advantages of volume reduction and convenient storage and transportation. The alfalfa grass particles are deeply favored by farmers due to the characteristics of small volume, convenience in feeding, high nutritional value and storage resistance, and are an important mode for the alfalfa to be used as a feed. The mode that soybean straws are added into alfalfa grass particles as the feed is a sub-full main mode, but when alfalfa is ground into grass powder to be used for making the grass particles, whether the soybean straws are mixed in the alfalfa grass particles or not cannot be distinguished by naked eyes. The method provided by the invention is used for identifying whether the alfalfa grass granulated feed contains soybean straws or not by detecting whether the alfalfa grass granulated feed contains soybean DNA or not, avoids visual observation and chemical detection, has the advantages of high speed, high flux, capability of completing detection only by few samples and higher sensitivity and accuracy.
In some alternative embodiments, the PCR amplification system is a 20. mu.l system comprising 2 XPCR Master 10. mu.l, 1. mu.l DNA template concentration of 50 ng/. mu.l, primer MD 5-F1. mu.l concentration of 10nM, primer MD 5-R1. mu.l concentration of 10nM and ddH2O7 mu l; wherein the 2 XPCR Master comprises 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq and 2 XPCR Buffer. In the PCR amplification reaction for detection, 20 mul system can meet the requirement of the detection dosage of the amplified product. It will be appreciated that the PCR system described above can be adapted and optimised to the actual experimental and testing conditions.
In some alternative embodiments, the PCR amplification conditions are a) pre-denaturation at 94 ℃ for 4 minutes; b) denaturation at 94 ℃ for 30 seconds; c) annealing at 55-65 deg.C for 30 s; d) extension at 72 ℃ for 30 seconds; e) extension at 72 ℃ for 7 min; wherein steps b) -d) are cycled 25-40 times. Alternatively, the annealing temperature may be, for example, but not limited to, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ or 65 ℃, preferably 57 to 59 ℃, more preferably 58 ℃; alternatively, the number of cycles of steps b) -d) may be, for example but not limited to, 25, 28, 30, 32, 35, 38 or 40, preferably 32-36. The PCR amplification conditions can be adjusted and optimized according to the change of the PCR system.
In some alternative embodiments, for example, but not limited to, detecting the PCR amplification product by using polyacrylamide gel electrophoresis, agarose gel electrophoresis, capillary gel electrophoresis, or the like, directly sequencing the PCR product, or optionally detecting the PCR amplification product without using additional technical means when using a method such as fluorescence quantitative PCR or HRM, or the like, which can display the product during PCR amplification, and thus it is understood that the invention is not limited to the detection method of the PCR product. In a preferred embodiment, the method for detecting the amplification product by polyacrylamide gel electrophoresis is low in cost and high in speed, and does not need to modify a primer additionally; PAGE gels with 6% -10% gels are preferably used, and may be, for example and without limitation, PAGE gels with 6%, 7%, 8%, 9% or 10% concentration, preferably PAGE gels with 8% concentration; performing electrophoresis for 90-120min under the conditions of voltage 180-220V; alternatively, the voltage condition may be, for example, but not limited to, 180V, 185V, 190V, 200V, 210V or 220V, preferably 200V; optionally, the electrophoresis time may be, but is not limited to, 90min, 100min, 105min, 110min or 120min, for example; preferably 100 min; the electrophoretic band can be clearer by adjusting and optimizing the conditions of electrophoresis.
The following examples are provided to further illustrate the advantageous effects of the present invention.
Example 1 primer set for detecting alfalfa and soybean
The embodiment provides a primer pair for detecting alfalfa and soybean, which comprises a primer MD5-F and a primer MD5-R, wherein the primer MD5-F has a sequence shown as SEQ ID NO.1, and the primer MD5-R has a sequence shown as SEQ ID NO. 2.
MD5-F(5′-3′)：TCCGTACCAACTCAAGATATGC
MD5-R(5′-3′)：TAAGCTCCAATTGCATCATAGG
Example 2A kit for detecting alfalfa and soybean
The embodiment provides a kit for detecting alfalfa and soybeans, which comprises the following components: primer MD5-F and primer MD5-R, 2 XPCR Master and ddH2O；
Wherein the 2 XPCR Master contains 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq and 2 XPCR Buffer.
Example 3 method for detecting soybeans in alfalfa grass granulated feed
The embodiment provides a method for detecting soybeans in alfalfa grass granulated feed, which comprises the following steps:
A) collecting alfalfa grass particles, and extracting the genomic DNA of the grass particles for later use;
B) performing PCR amplification on the genomic DNA extracted in the step A) by using a primer MD5-F and a primer MD 5-R;
the PCR amplification system is a 20-mu-l system which comprises 2 XPCR Master10 mu l, 1 mu l DNA template with the concentration of 50 ng/. mu.l, 1 mu l primer MD 5-F1 mu l primer with the concentration of 10nM, 1 mu l primer MD5-R with the concentration of 10nM and ddH2O7 mu l; wherein the 2 XPCR Master comprises 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq and 2 XPCR Buffer;
PCR amplification procedure: a) pre-denaturation at 94 ℃ for 4 min; b) denaturation at 94 ℃ for 30 seconds; c) annealing at 58 ℃ for 30 seconds; d) extension at 72 ℃ for 30 seconds; the steps b) to d) are cycled for 35 times; e) extension at 72 ℃ for 7 min;
C) and (3) detecting a PCR product: detection was performed using 8% PAGE gel, 100ml system, 53.0ml H2O, 26.3ml of polypropylene, 20ml of 5 XTBE, 700. mu.l of 35% ammonium persulfate, 70. mu.l of TEMED, solidifying for 50min, performing electrophoresis at 200V for 1h40min in a vertical plate electrophoresis apparatus, then dyeing with nucleic acid dye, and performing photography on a gel imager;
if the amplified product contains the soybean DNA fragment, the alfalfa grass granulated feed to be detected is doped with soybean.
Example 4 validation of the primers MD5-F and MD5-R
Extracting 10 alfalfa varieties, wherein genome DNA of 5 individuals of each variety is randomly extracted, and the concentration of the genome DNA is diluted to 50 ng/mu l and used as a DNA template in PCR (polymerase chain reaction) as shown in a table 1;
10 soybean varieties were extracted, and as shown in Table 2, 3 individuals of genomic DNA were randomly extracted from each variety, and the concentration was diluted to 50 ng/. mu.l; used as a DNA template in PCR;
TABLE 1 information description of alfalfa cultivars reference material
TABLE 2 information description of the materials tested for the soybean variety
Performing PCR amplification on the extracted alfalfa genome DNA and soybean genome DNA by using a primer MD5-F and a primer MD 5-R;
the PCR amplification system is a 20. mu.l system, which comprises 10. mu.l of 2 XPCR Master (Shanghai products code: SK2082), 1. mu.l of DNA template with the concentration of 50 ng/. mu.l, 1. mu.l of primer MD 5-F1. mu.l with the concentration of 10nM, 1. mu.l of primer MD 5-R1. mu.l with the concentration of 10nM and ddH2O 7μl；
The PCR amplification procedure was as follows: a) pre-denaturation at 94 ℃ for 4 min; b) denaturation at 94 ℃ for 30 seconds; c) annealing at 58 ℃ for 30 seconds; d) extension at 72 ℃ for 30 seconds; the steps b) to d) are cycled for 35 times; e) extension at 72 ℃ for 7 min;
and (3) detecting a PCR product: detection was performed using 8% PAGE gel, 100ml system, 53.0ml H20, 26.3ml of polypropylene, 20ml of 5 XTBE, 700. mu.l of 35% ammonium persulfate, 70. mu.l of TEMED, 50min of coagulation, electrophoresis at 200V for 1h40min in a riser electrophoresis apparatus, followed by staining with a nucleic acid dye and photographing on a gel imager, wherein each lane represents the sample shown in tables 1 and 2, and lane M represents Marker;
wherein, fig. 1 and fig. 2 show that electrophoresis bands using 50 single plant genome DNAs of alfalfa of 10 varieties as templates are clear and bright, electrophoresis bands using 30 genome DNAs of soybeans of 10 varieties as templates are also clear and bright, and are basically consistent with predicted bands, and the difference between the electrophoresis bands is 9bp, so that the soybeans and the alfalfa can be obviously distinguished.
Example 5: verifying the amplification products of the primer MD5-F and the primer MD5-R
To further verify the effectiveness of primer MD5-F and primer MD5-R, DNA fragments of the amplified products of alfalfa and soybean were sequenced. The results show that the amplification products of alfalfa and soybean DNA both contain primer MD5-F and primer MD 5-R.
50 ng/. mu.L of alfalfa genome DNA is taken as a template, and a primer MD5-F and a primer MD5-R are adopted for PCR amplification. The PCR system and method were the same as in example 4. Detecting the PCR product in 2% agarose, recovering DNA segment of 190bp length, connecting pGEM-T vector to obtain recombinant plasmid pGEM-T-MD 5-alfalfa, and transforming the plasmid into colibacillus DH5 alpha competent cell. Coating the Escherichia coli containing pGEM-T-MD 5-alfalfa plasmid on LB culture medium containing Amp, IPTG and X-Gal, culturing overnight at 37 ℃, selecting 3 positive clone bacterial plaques, and culturing overnight in LB liquid culture medium containing Amp at 37 ℃ with shaking. After the detection of the bacterial liquid, the bacterial liquid conforms to the size of the expected band, the bacterial liquid is sent to Shanghai biological engineering Co., Ltd for sequencing, and 3 positive cloned alfalfa fragments are tested. The result shows that the primer MD5-F and the primer MD5-R can well amplify the gene fragment of 194bp of alfalfa. Wherein the amplified fragment of the alfalfa has a sequence shown as SEQ ID NO.3, the length of the sequence fragment is 194bp, and the complementary sequence of the sequence fragment is shown as SEQ ID NO. 4.
PCR amplification was performed using 50 ng/. mu.L soybean genomic DNA as a template and primer MD5-F and primer MD 5-R. The PCR system and method were the same as in example 4. Detecting the PCR product in 2% agarose, recovering DNA fragment with length about 200bp, connecting with pGEM-T vector to obtain recombinant plasmid pGEM-T-MD 5-soybean, and transforming the plasmid into Escherichia coli DH5 alpha competent cell. Spreading Escherichia coli containing pGEM-T-MD 5-soybean plasmid on LB culture medium containing Amp, IPTG and X-Gal, culturing at 37 deg.C overnight, picking 3 positive clone bacterial plaques, and culturing in LB liquid culture medium containing Amp at 37 deg.C overnight with shaking. After the detection of the bacterial liquid, the size of the bacterial liquid is consistent with the size of an expected strip, the bacterial liquid is sent to Shanghai biological engineering company Limited for sequencing, and 3 positive cloned soybean fragments are detected. The result shows that the primer MD5-F and the primer MD5-R can well amplify the gene fragment of soybean 203 bp. Wherein the amplified soybean fragment has a sequence shown as SEQ ID NO.5, the length of the sequence fragment is 203bp, and the complementary sequence is shown as SEQ ID NO. 6.
Example 6 testing of alfalfa and Soybean genome in different ratios
The DNA of the soybean and the alfalfa are mixed according to the mass ratio of 100:0, 99:1, 75:25, 50:50, 25:75, 1:99 and 0:100 respectively, then PCR and electrophoresis are carried out according to the detection method provided by the embodiment 4, and the primer MD5-F and the primer MD5-R can clearly distinguish 1% of soybean genes contained in the alfalfa, so that the quality of the alfalfa particles can be detected accurately. Results are shown in FIG. 3, in which lanes 1-7 represent: the DNA of soybean and alfalfa were mixed at 100:0, 99:1, 75:25, 50:50, 25:75, 1:99 and 0:100 ratios, and lane M indicated Marker.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Lanzhou university
Primer pair, kit and application thereof, and method for detecting whether alfalfa product contains soybeans
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
tccgtaccaa ctcaagatat gc 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
taagctccaa ttgcatcata gg 22
<210> 3
<211> 194
<212> DNA
<213> alfalfa (Medicago sativa L)
<400> 3
tccgtaccaa ctcaagatat gcttataggc ctttatgtat taactagcgg aaatcgtcga 60
ggtatttgtg caaacaggta taatccgttt aattgcagaa attctaaaaa tgaaaaaatt 120
agcaataata actctaagta tatgaaaaaa aaagaaccct ttttttgcaa ttcctatgat 180
gcaattggag ctta 194
<210> 4
<211> 194
<212> DNA
<213> alfalfa (Medicago sativa L.)
<400> 4
aggcatggtt gagttctata cgaatatccg gaaatacata attgatcgcc tttagcagct 60
ccataaacac gtttgtccat attaggcaaa ttaacgtctt taagattttt acttttttaa 120
tcgttattat tgagattcat atactttttt tttcttggga aaaaaacgtt aaggatacta 180
cgttaacctc gaat 194
<210> 5
<211> 203
<212> DNA
<213> Soybean (Glycine max (Linn.) Merr.)
<400> 5
tccgtaccaa ctcaagatat gcttattgga ctttacatat taacgagcgg gaatcgtcga 60
ggtatttatt caaataggta taatccacgg aattgcggaa attttagaaa tcttaaaaat 120
gaaagaattc gagataataa ctataagtat acgaaaaaaa aagaaccctt tttttgtaat 180
tcctatgatg caattggagc tta 203
<210> 6
<211> 203
<212> DNA
<213> Soybean (Glycine max (Linn.) Merr.)
<400> 6
aggcatggtt gagttctata cgaataacct gaaatgtata attgctcgcc cttagcagct 60
ccataaataa gtttatccat attaggtgcc ttaacgcctt taaaatcttt agaattttta 120
ctttcttaag ctctattatt gatattcata tgcttttttt ttcttgggaa aaaaacatta 180
aggatactac gttaacctcg aat 203
Claims (9)
1. A primer pair for detecting alfalfa and soybean is characterized by comprising a primer MD5-F and a primer MD 5-R;
the nucleotide sequence of the primer MD5-F is SEQ ID NO.1, and the nucleotide sequence of the primer MD5-R is SEQ ID NO. 2.
2. The primer pair according to claim 1, wherein primer MD5-F and/or primer MD5-R is modified;
the modification comprises adding a label at the 5 'end and/or 3' end of the primer;
the label comprises a fluorescent label, a biotin label or a digoxigenin label.
3. A kit comprising the primer pair of claim 1 or 2.
4. The kit of claim 3, further comprising Mg2+dNTP, DNA polymerase and PCR Buffer.
5. Use of the primer pair of claim 1 or 2 or the kit of claim 3 or 4 for detecting alfalfa and soybean.
6. A method for detecting whether alfalfa products contain soybeans or not is characterized by comprising the following steps:
carrying out PCR amplification on DNA of the alfalfa product, and detecting whether the amplified product contains DNA fragments of soybean or not, wherein if the product contains the DNA fragments of the soybean, the alfalfa product contains the soybean;
wherein, the primers used in the PCR amplification are the primer pair of claim 1 or 2;
the alfalfa product is alfalfa grass pellet feed;
the length of the amplified DNA fragment of the alfalfa is 194bp, and the length of the amplified DNA fragment of the soybean is 203 bp.
7. The method according to claim 6, characterized in that the PCR amplification system is a 20 μ l system, which comprises 2 XPCR Master10 μ l, 1 μ l DNA template with a concentration of 50ng/μ l, primers MD 5-F1 μ l with a concentration of 10nM, primers MD 5-R1 μ l and ddH with a concentration of 10nM2O 7µl；
Wherein the 2 XPCR Master comprises 3mM MgCl20.2mM dNTP, 0.1U/. mu.l Taq and 2 XPCR Buffer.
8. The method of claim 7, wherein the PCR amplification conditions are a) pre-denaturation at 94 ℃ for 4 minutes; b) denaturation at 94 ℃ for 30 seconds; c) annealing at 58 ℃ for 30 seconds; d) extension at 72 ℃ for 30 seconds; the steps b) to d) are cycled for 35 times; e) extension at 72 ℃ for 7 minutes.
9. The method of claim 6, wherein the amplification product is detected using a gel electrophoresis method;
wherein the gel is 8% PAGE gel; the electrophoresis conditions are as follows: voltage 200V, electrophoresis time 1h40 min.
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