CN101392292B - Method for distinguishing between antler and horn of rein deer using real time pcr - Google Patents

Abstract

The present invention discloses a method for distinguishing reindeer antlers and antlers using real-time PCR, and more specifically, a purity test method for confirming the presence or absence of reindeer antlers that can be mixed into antler slices. By utilizing the present invention, the quality of sliced velvet antler, which is highly concerned by the society, can be ensured, and the distribution order of sliced velvet antler, which is a high-priced herbal medicine, can be established, thereby improving the reliability of people taking oriental medicine.

Description

Method for Distinguishing Reindeer Antlers and Antlers Using Real-time PCR

technical field

The present invention relates to a method for distinguishing reindeer antlers and antlers using PCR, and more particularly to a method for testing the purity of reindeer antlers that can be mixed into velvet slices. the

Background technique

Cervi Parvum Cornu is the most used animal crude drug in the Republic of Korea. It is recorded in the top-grade medicinal materials section of Shennong Herbal Classic. It has the effect of strengthening the body and is used as a representative whole-body tonic for various debilitating symptoms. And it can improve epilepsy, ulcer, anemia, rheumatism, stroke and other diseases, as well as insomnia, fatigue, headache, loss of appetite, convulsion and other symptoms. It has various effects such as promoting the recovery of sexual function, etc., and promoting the treatment of wounded patients. the

According to the results of biological activity, deer antler can promote the generation of hematopoietic stem cells, thereby increasing hematopoiesis and body vitality, improving glucose metabolism and promoting growth and development, and has the function of promoting the digestive system and strengthening the cardiovascular system. It has the effect of strengthening the heart and improving immunity. the

Medicinal velvet is defined as "deer velvet" in the Korean National Standard of Traditional Medicinal (Herbal and Botanical) Materials (Korean National Standard of Traditional Medicinal (Herbal and Botanical) Materials). , red deer (Cervus elaphus Linne), large deer (Cervus canadensis Erxleben, cervidae) male deer with dense hair and non-ossified or partially ossified antlers after drying". the

In addition, the types of antler used in Korea are roughly divided into sika deer and red deer antler, and Japanese deer (Cervusnippon Temminck), red deer (C.elaphus L.) and North American deer are used respectively. Dried young horns of the North American deer (C. canadensis Erxleben) called wapiti. the

In addition, velvet antler is divided according to the place of origin, Yuan velvet mainly from Russia, East red deer from Northeast China, West red deer from Xinjiang, and New Zealand, which is generally called red deer, and has a relatively large body and horns. Deer and other breeds obtained from large North American deer are distributed. the

Deer antler has been standardized by Korean medicine manufacturers and provided to Korean medical institutions, but the quality of velvet has never been questioned. This is because it is difficult to correctly distinguish deer antler that comes in a sliced state with the naked eye. the

The deer antlers mentioned in the Korean medical field include the velvet from Russia, the red deer from China, the sika deer bred in China, South Korea, Taiwan, North Korea and other places, the red deer from New Zealand, and the moose (Elk) from Canada. Antlers of the genus Rangifer used medicinally. the

However, in the case of reindeer in Alaska, both sexes have horns, and the origin species of the deer used for medicine is different from the animal family, and the ash content does not reach the standard content, so it cannot be used as velvet. Importation is prohibited, and the importation of Canadian moose is prohibited due to reasons such as mad cow disease infection. However, some drug dealers fraudulently sell illegally returned reindeer antlers as antlers. In the case of Russia, they formally registered with the Ministry of Food and Drug Safety to use reindeer as medicine, but the Korea Food and Drug Administration (KOREA FOOD & DRUG ADMINISTRATION) Notifications are content that cannot be identified. the

In the test item of "antler slice" purity in Korean National Standard of Traditional Medicinal (Herbal and Botanical) Materials, Korean Pharmacopoeia, there is a test for distinguishing antler horns from reindeer horns by genetic analysis method. the

The purity test is a test to analyze the concomitant substances in the crude drug, and the purity test of deer antler is a test to prevent the mixing of reindeer antlers. However, the test method of this test is cumbersome and the test steps are complicated, so there is a possibility of contamination during the test. the

In addition, it is impossible to distinguish a small amount of reindeer horns mixed in velvet slices due to the usual test method, so the development of a new test method that conforms to the purity test method needs to be proposed. the

Contents of the invention

In order to solve the above disadvantages, the present invention aims to provide a primer set or primers for real-time PCR capable of distinguishing deer antlers from reindeer antlers. the

In addition, the present invention aims to provide a highly reliable antler purity test method capable of ensuring the quality of sliced antler. the

Furthermore, the purpose of the present invention is to provide a detection kit that is easy to use and can quickly and accurately determine the purity of deer antler in a short period of time. the

The present invention is realized in the following manner. The present invention provides a primer set for detecting deer, which includes a pair of deer-specific primers having the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2. the

Furthermore, the present invention provides a real-time PCR detection set for deer detection, which includes a deer-specific primer pair having the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2, and a primer pair having the base sequence of SEQ ID NO: 3 Deer-specific probes. the

Furthermore, the present invention includes a method for distinguishing deer-derived substances, including the steps of: performing real-time PCR on DNA extracted from a specimen using the above-mentioned real-time PCR detection group for deer detection; and confirming that the above-mentioned real-time PCR results to determine whether the specimen is stage of material from deer. the

The above real-time PCR stage can use the real-time PCR detection group for reindeer detection at the same time. A reindeer-specific probe having the base sequence of SEQ ID NO:6. the

Moreover, the present invention further provides a kit for antler purity test including the above-mentioned real-time PCR detection set for deer detection, the above-mentioned kit further includes a real-time PCR detection set for reindeer detection, and the real-time PCR detection set for reindeer detection is The set includes a primer pair specific to reindeer having the base sequences of SEQ ID NO: 4 and SEQ ID NO: 5, and a probe specific to reindeer having the base sequence of SEQ ID NO: 6. the

Description of drawings

Fig. 1 is a schematic diagram showing the manufacturing process of the real-time PCR detection group for deer detection of the present invention;

Fig. 2 is a comparative arrangement table showing the base sequences in the mitochondrial D-loop region of deer and reindeer;

3a-c are schematic diagrams showing the results of real-time PCR reactions using the deer and/or reindeer detection real-time PCR detection group of the present invention, a represents the real-time PCR results using reindeer DNA as a template, and b represents using deer DNA as a template c represents the real-time PCR result using a mixture of reindeer and deer DNA. the

Fig. 4 shows the results of analyzing PCR products by real-time PCR after PCR reaction using the real-time PCR detection group for detection of deer and/or reindeer of the present invention. the

Detailed ways

Hereinafter, the present invention will be described in detail. the

In order to provide a new purity test method that can confirm whether the reindeer horns are mixed in the state where the antler slices and a small amount of reindeer horns are mixed in a crushed shape, the inventors of the present invention used PCR, which is the latest genetic analysis method The reindeer of method and the distinguishing method of deer, complete the present invention on this basis. the

In the present invention, the DNA of the specimen is analyzed by real-time PCR, and the presence or absence of the deer specimen and/or the reindeer specimen in the specimen is preferably evaluated as the presence or absence of the reindeer specimen in the deer specimen. the

The above-mentioned specimens can be cells, tissues, organs or body parts of all animals, preferably horns of any animal, more preferably deer and/or reindeer horns, such as antlers, antlers, and antlers. The above-mentioned specimens are most preferably deer antlers, deer antlers, deer antlers that are commonly used or will be used as medicinal materials. the

The above-mentioned deer is preferably all kinds of deer whose antlers can be used as medicinal materials, such as sika deer (Cervus nippon Temminck), red deer (Cervus elaphus Linne) and large deer (Cervus canadensis Erxleben, Cervidae). The above-mentioned reindeer may be any kind of reindeer in the world, especially the reindeer (Rangifertarandus) which is misused as a medicinal herb.

Therefore, in the present invention, the specific nucleotide sequences of deer and/or reindeer were analyzed using databases such as GeneBank, and based on the site suitable for real-time PCR detection, each deer and/or reindeer Preparation and review of reindeer-specific primer pairs and probes. The deer and/or reindeer-specific primer pairs and probes of the present invention are described in Table 1 below. the

【Table 1】

The present invention provides a method for distinguishing deer and/or reindeer using the primer pair and probe specific to the deer and/or reindeer of the present invention. the

The above method is to amplify by PCR a reindeer-specific nucleotide sequence that is likely to be mixed into deer, and at the same time react with a probe that can react with the amplified product and is labeled as a detectable device, thereby distinguishing deer by real-time PCR. and reindeer. the

The reindeer detection sets of serial numbers 4 to 6 and the deer detection sets of serial numbers 1 to 3 can be used alone or in combination. In this case, the probe is marked as a detectable device, and especially when two types are used at the same time, it is marked as a device capable of different detection. the

The above-mentioned detectable device refers to a compound, biomolecule or biomolecule mimetic, etc., which are linked to, bound to, or adhered to a probe to confirm the density, concentration, amount, etc. in a usual manner. the

Examples thereof include commonly used fluorescent labeling factors, luminescent substances, luminescent substances, isotopes, and the like, but are not limited thereto. As another example, a fluorescent labeling factor is most preferable. the

Various fluorescent labeling factors are commercially available and thus can be easily purchased. Exemplary fluorescent labeling factors include FAM (carboxyfluorescein), VIC, TAMRA (6-carboxytetramethylrhodamine), JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein), ROX (6- carboxy-X-rhodamine), NED, HEX (2′, 4′, 5′, 7′-tetrachloro-6-carboxy-4, 7-dichlorofluorescein), TET (2′, 7′-dichloro-6-carboxy- 4,7-dichlorofluorescein), SYBR Green, Cy3, Texas Red, Cy5 _TM , etc., but not limited thereto. Depending on the type of fluorescent labeling factor and the different excitation and emission wavelengths, the method of use is also different. Considering this problem, the fluorescent labeling factor used together with the same PCR reaction should be judged whether it can be detected separately and used selectively. Use different colors. The details and selection of the above-mentioned fluorescent labeling factors will be obvious to those of ordinary skill in the technical field to which the present invention belongs.

As an example, the fluorescent labeling factor is labeled with the primers or probes included in the deer and/or reindeer detection set of the present invention by a usual method. Labeling methods include interchelating method, TaqMan _TM probe method and molecular channel (Molecualr beacon) method, etc.

The interchelating method refers to a method in which a reagent that expresses fluorescence (inter-chelator: SYBR Green I, EtBr, etc.) that binds to double-stranded DNA is added to the PCR reaction to detect the fluorescence that accompanies amplification. The double-stranded DNA synthesized by the PCR reaction binds to emit fluorescence, and the intensity of the fluorescence is detected to measure the amount of the amplified product produced.

In the TaqMan _TM probe method, a TaqMan _TM probe (TaqManTM probe) modified at the 5' end with a fluorescent labeling factor (FAM, etc.) and at the 3' end with a quencher (quencher) substance (TAMRA, etc.) is added to PCR In the method in the reaction solution, the TaqManTM probe specifically hybridizes with the template DNA in the annealing stage, and the occurrence of fluorescence is inhibited due to the restriction of the quenching substance on the probe. In the elongation stage, TaqManTM The 5'→3'exonuclease (exonuclease) activity of DNA polymerase (Polymerase) only decomposes the TaqMan _TM probe hybridized with the template, so that the fluorescent pigment is freed from the probe, and the inhibition caused by quenching is relieved , thereby emitting fluorescence.

The molecular channel (Molecular beacon) method refers to a molecular channel probe that forms a hairpin-type secondary structure by modifying both ends with fluorescent labeling factors (FAM, TAMRA, etc.) and quencher substances (DABCYL, etc.) (MolecularBeacon probe) method added to the PCR reaction. The molecular channel probe has a hairpin structure in the free state, and is close to the fluorescent labeling substance and the quencher substance to inhibit the occurrence of fluorescence. During hybridization, the distance between the fluorescent labeling factor and the quencher substance increases to release the inhibition caused by quencher, so that the fluorochrome on the probe shows fluorescence. However, unhybridized molecular channel probes do not show fluorescence due to maintaining the hairpin structure. the

This method can be applied to the real-time PCR method for detecting deer and/or reindeer of the present invention. The above methods are obvious to those of ordinary skill in the art, so specific methods can be selected according to reaction efficiency, time, and type of fluorescent labeling factor. In the present invention, the TaqMan _™ probe method can be used as an example.

In the method for distinguishing deer and/or reindeer according to the present invention, the real-time PCR reaction conditions can be used in a normal method, and as an example, the following conditions can be adopted, that is, the following conditions are used for UDC activity at 50°C for 2 minutes, and at 95°C. After initial denaturation (initial denaturation) for 10 minutes, denaturation (15 seconds at 94° C.) and annealing (60 seconds at 60° C.) were performed. the

In addition, the deer and/or reindeer detection group of the present invention can be implemented using a common real-time PCR method and device. the

The above-mentioned real-time PCR method is a method for detecting and quantifying the fluorescence performed in real time according to each cycle of PCR based on the principle of DNA polymerase and fluorescence resonance energy transfer (FRET). the

With this method, specific amplification products can be confirmed by distinguishing specific amplification products from non-specific amplification products, and analysis results can be easily obtained through automation (Aspects). the

The real-time PCR instrument that can be used in the present invention has Real-time PCR instrument 7900, 7500, 7300 of ABI company, Mx3000p of Stratagene company, and Chromo4 instrument of BioRad company etc., but not limited thereto. At the end of PCR, the laser of this real-time PCR instrument detects the fluorescent labeling factor of the probe labeled in the amplified PCR product, forming the peak as shown in Fig. 3a-c. Thus, the software provided in the instrument can be operated under the condition of no electrophoresis process, thereby automatically analyzing the results. the

Through the deer and/or reindeer detection group of the present invention, and the method for distinguishing deer and/or reindeer of the present invention using this, the accuracy of the usual antler purity test method can be improved and the required time and cost can be significantly reduced, and can be quickly Confirm the test results. Through these methods, low-concentration DNA fragments and PCR products less than 100 bp that are difficult to distinguish by electrophoresis can be confirmed by laser sensitization, and there is no contamination caused by PCR products, from the analysis results to the end of detection All the testing process is run by an automatic system, thereby reducing the probability of the tester's error and detection error, etc., the data collection and analysis are easy, and the required detection time and manpower can be minimized. the

Furthermore, the present invention provides a kit for antler purity test including a real-time PCR detection set for deer detection, the above-mentioned kit also includes a real-time PCR detection set for reindeer detection, and the real-time PCR detection set for reindeer detection may include A primer pair specific to reindeer having the base sequence of SEQ ID NO: 4 and SEQ ID NO: 5, and a probe specific to reindeer having the base sequence of SEQ ID NO: 6. the

In the kit of the present invention, the above-mentioned primer pairs and probes can be packaged in a reaction container, a strip, or a microplate, and packaged by a method well known to those of ordinary skill in the art . the

And, the kit of the present invention further includes one or more items selected from the group consisting of Taq polymerase (Taqpolymerase), a reaction buffer including MgCl ₂ , dNTPs, and a stabilizer (stabilizer), and may further include the Other reagents known in the art.

Through the kit of the present invention, it is easy to confirm whether the specimen, especially the deer antler medicinal materials used or/and approved for medicinal purposes, is mixed with the reindeer specimen, for example, the detection minimum concentration of deer antler and reindeer gene in the real-time PCR method It is 100pg/uL. When converted by the mixing ratio of reindeer and velvet, it can be detected when only 15% reindeer is mixed in the velvet sample, but this value is only an estimated value. In the present invention, it can be accurately judged according to the DNA assurance degree of the sample. Broken reindeer specimens mixed or not. the

As described above, the method of distinguishing reindeer and deer by real-time PCR of the present invention can provide a purity test method for confirming the presence or absence of reindeer horns that may be mixed into velvet slices, and can secure velvet slices that are highly concerned by society. The quality of velvet antler, which is a high-priced Korean medicinal material, has been established, and the distribution order of velvet slices, which is a high-priced Korean medicinal material, has been established, thereby improving the trust that people can safely take Korean medicinal materials. the

1. DNA extraction method DNA extraction method using silica gel column (PowerPrepTMDNA Extraction from Food and Feed kit)

*Instruments and reagents

①Chloroform

② TE buffer is prepared with distilled water to make the final state of TE buffer reach 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0), and then sterilized at 121°C for 15 minutes. the

③Vortex

④ Spectrometer (Spetrometer)

⑤Protease K (protease)-dissolved in sterilized distilled water at 10 mg/ml for use. the

⑥Inverter (Inverter, mixer)

⑦Small glass tube (cuvette)

⑧Micropipette: 10μl-20μl, 100μl-200μl, 1,000μl

*Method of implementation

1) Use a pulverizer to pulverize the specimen evenly. the

2) Transfer 100mg-200mg of crushed specimen to a 1.5ml tube. the

3) Add 400 μl of Lysis Buffer, 40 μl of Lysis Buffer II and 5 μl of 20 mg/ml proteinase K (protease), and mix with a Vortex. the

4) React at 65°C for 30 minutes. the

5) Add 500 μl of chloroform and mix with a vortexer, mixing vigorously for 15 seconds. the

6) Centrifuge at 12,000 rpm for 10 minutes, and transfer 350 μl of the supernatant to a new 1.5 ml tube. the

7) To bind DNA to the binding column, add 1050 μl of DNA binding buffer to sample No. 6 and mix (the vortexer is not used at this time). the

- At this point perform a slight center separation to collect the solution stuck to the lid. the

8) Add 600 μl of No. 7 solution to the DNA binding column, and separate at 8,000 rpm for 2 minutes. After the center of the circle is separated, remove the supernatant. the

9) Repeat stage 8. the

10) Add 600 μl of washing buffer I to the DNA binding column, and separate at 8,000 rpm for 2 minutes. At this point, the solution passing through the column was removed. the

11) Repeat stage 10. the

Option: You can go directly to stage 12. However, if it is necessary to completely remove the washing buffer according to the DNA extraction process, after removing the solution in the filter tube, perform center separation at 12,000 rpm for 2 minutes to remove the washing buffer. the

12) Put 150 μl of lysis buffer into a completely dry DNA binding column, and react at room temperature or 65°C for 10 minutes. the

13) Put 150 μl of lysis buffer into the DNA binding column, and react at room temperature or 65° C. for 10 minutes. the

14) Perform centroid separation at 8,000 rpm for 3 minutes to recover DNA. the

*DNA purity confirmation and preservation

A certain DNA sample is diluted 10 times, and the concentration of the certain amount of DNA sample can be measured after being diluted 10 times, and then the ultraviolet absorption spectrum (Spectrum) is measured at 260nm and 280nm, and each absorbance (absorbency) is recorded. The absorbance 1 at O.D.260 is the DNA concentration of 50 ng/μl, and the DNA concentration is calculated based on this. the

For example, when the absorbance at 260nm is 0.21,

DNA concentration = absorbance * dilution unit (10 times here) * O.D.

When the absorbance at 260 is 1, the concentration is 50ng/μl=0.21*10*50ng/μl=105ng/μl. the

And, when the ratio of O.D.260/O.D.280 is 1.6-2.0, it means that the state of DNA purification is very good. However, when the ratio is less than 1.0, it is best to re-purify the DNA and use it. the

The required amount of the obtained DNA was diluted with sterilized distilled water, and the remaining DNA was frozen and stored below -20°C. Extracted DNA is not all diluted and stored. The remaining diluted DNA after use should not be stored and discarded. the

In addition, when the stock solution concentration of the extracted DNA does not reach the predetermined concentration required for the PCR reaction, it can be concentrated or the DNA can be directly used in the PCR reaction when the concentration is less than 20% of the predetermined concentration. In addition, when the absorbance in DNA concentration measurement is less than 0.02, 10 μl of DNA is directly used for PCR reaction. the

*DNA concentration and purification method

The concentration and purity of the extracted DNA sample are not suitable for PCR reaction. the

-Concentration method-

Based on the volume of 100μl of the initial DNA sample

①Add 1/10 times (10μl) of 3M sodium acetate (pH5.2) (or 1/5 times when using 10M ammonium acetate) the DNA volume, and then add 2.5 times (250μl) 99% or more of EtOH while mixing. the

②React the above-mentioned mixed specimen at -20°C for more than 20 minutes. the

③Separate the center of the sample ② at 12,000rpm at 4°C for 15 minutes. the

④ Carefully remove the supernatant to obtain a precipitate (the precipitate may not be visible). the

⑤ Put 800μl of 70%-80% EtOH solution and shake gently. the

⑥ Perform center separation at 12,000rpm for 10 minutes, carefully remove the supernatant from the sediment (the sediment may not be visible). the

⑦Dry the precipitate and dissolve it in sterilized distilled water or 50μl of TE buffer. At this time, complete drying is not performed. the

*When the DNA sample is a small amount, sterilized distilled water or TE buffer can be added to increase the initial volume, and then concentrated. the

Example 1: Production of primers and probes for deer and reindeer detection

The mitochondrial (mitochondria) D-loop region of reindeer and deer was amplified, and the base sequence of the amplified PCR product was analyzed by an automatic base sequence analyzer (ABI310 Genetic Analyzer, PE, USA). The results were compared with the nucleotide sequences registered in the gene bank. the

Reindeer was compared and analyzed centering on ral (AY970667), and deer was comparing and analyzing each nucleotide sequence of the D-loop centering on ce6 (AF016957), and the specific sequence was analyzed, and the following standards were produced. Primers specific for deer. the

① The length of each primer is set to 18-30 nucleotides (Nucleotide). the

② The optimum annealing temperature is set at around 60°C. the

③The ratio of GC:AT can be set as 50:50, and the highest ratio of GC should not exceed 65%, and the lowest ratio should not be lower than 40%. the

④ It is set so that no secondary structure occurs inside. the

⑤ In order to prevent non-specific reactions in the PCR reaction, specific primers were produced. the

As a result, the positions that were analyzed as specific positions were confirmed based on ral and ce6 ( FIG. 1 ). Based on the results, the nucleotide sequences of each primer and probe were prepared and listed in Table 2. the

【Table 2】

The specificity of each primer and probe sequence in Table 2 above was confirmed. The base sequence specificity of the primers was confirmed through gene bank databases (NCBI, Blast, EMBL, GenBank), and as a result, none of the base sequences of animals, plants, and microorganisms was 100% identical. the

Example 2: The difference between deer and reindeer

Antlers from deer (Cervus elaphus) and reindeer were prepared separately, and DNA was purified from the same specimens as above. the

For each extracted DNA sample, the reaction composition shown in Table 3 below was prepared, and then PCR was carried out under the PCR reaction conditions shown in Table 4. the

【table 3】

PCR reaction composition

【Table 4】

PCR reaction conditions

And, a reindeer- and deer-specific distinction was made by two methods in parallel:

1. A method for distinguishing amplification curves using real-time PCR. the

2. Distinguish method of using post read function after performing real-time PCR. the

There is no difference between the two methods in terms of accuracy, but method No. 1 is easier to analyze a small number of samples, and method No. 2 is easier to analyze a large number of samples. the

Therefore, PCR reactions were performed using the above two methods. The PCR reaction was performed with the composition and conditions in Table 3 and Table 4, however, for the simultaneous detection of reindeer and deer, the PCR reaction was performed without adding distilled water using 2 μl of each template DNA (reindeer and deer). the

The result is shown in Fig. 3 and Fig. 4 . the

Figure 3a-c shows the results of real-time PCR reactions, a shows the PCR results using reindeer DNA as a template, b shows the PCR results using deer DNA as a template, and c shows the PCR results using a mixture of reindeer and deer DNA result. In Fig. 3a-c, the respective amplification curves for reindeer and deer can be confirmed, and two curves corresponding to reindeer and deer can be confirmed when they are used in combination. the

FIG. 4 shows the results of analyzing the PCR products by real-time PCR after the PCR reaction was carried out by general PCR equipment without using real-time PCR. As shown in Fig. 3a–c, reindeer and deer were detected separately and simultaneously. From the results in Fig. 3a-c and Fig. 4, it can be seen that both methods mentioned above can be used as analytical methods.

<110> Korea Food and Drug Administration

<120> Distinguishing method of reindeer horns and antlers using real-time PCR

<130>DP070182

<160>6

<170>KopatentIn1.71

<210>1

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>1

gtcctactaa ttacacagca 20

<210>2

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>2

tgtactatta tacataatat gtcc 24

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> probe

<400>3

catgtataac agtacatgag 20

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>4

cgtacatata tggtcctgta c 21

<210>5

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>5

gtactatgta ctgtaaataa tgtc 24

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> probe

<400>6

ccccatgctt ataagcaagt 20

Claims (6)

1. one kind is used for difference and detects group from the PCR in real time of the material of deer and reinder, comprising to the special primer of deer to the special probe of deer and to the special primer of reinder to the probe special to reinder, wherein the right base sequence of the special primer of deer is shown in SEQ ID NO:1 and SEQ ID NO:2; Base sequence to the special probe of deer shows in SEQ ID NO:3; The base sequence right to the special primer of reinder shows in SEQ ID NO:4 and SEQ ID NO:5; Base sequence to the special probe of reinder shows that in SEQ ID NO:6 wherein said material is the part of health.

2. PCR in real time according to claim 1 detects group, and wherein, 3 ' terminal or 5 ' end of above-mentioned probe is the fluorescent mark factor institute mark of selecting with the group that consists of from FAM, VIC, TAMRA, JOE, ROX, NED, HEX and TET.

3. a difference is from the method for the material of deer and reinder, comprises the steps: take the DNA that extracts from sample as object, utilizes to be used for difference and to detect the stage that group is carried out PCR in real time from the PCR in real time of the material of deer and reinder; And by confirming that above-mentioned PCR in real time result judges that whether sample is from the material of deer and/or whether sneaks into stage from the material of reinder, wherein said for difference from the PCR in real time of the material of deer and reinder detect group comprise to the special primer of deer to the special probe of deer and to the special primer of reinder to the probe special to reinder, wherein, 3 ' terminal or 5 ' end of above-mentioned probe, with from FAM, VIC, TAMRA, JOE, ROX, NED, the fluorescent mark factor institute mark of selecting in the group that HEX and TET consist of wherein shows in SEQ ID NO:1 and SEQ ID NO:2 the right base sequence of the special primer of deer; Base sequence to the special probe of deer shows in SEQ ID NO:3; The base sequence right to the special primer of reinder shows in SEQ ID NO:4 and SEQ ID NO:5; Base sequence to the special probe of reinder shows that in SEQ ID NO:6 wherein said material is the part of health.

4. difference according to claim 3 is from the method for the material of deer and reinder, and above-mentioned substance is organic cell, tissue, organ.

5. difference according to claim 3 is from the method for the material of deer and reinder, and above-mentioned substance is the angle.

6. the purity test test kit of a pilose antler comprises that the difference that is used for of claim 1 or claim 2 detects group from the PCR in real time of the material of deer and reinder.

Images