CN102286621A - Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis - Google Patents
Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis Download PDFInfo
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Abstract
The invention discloses a characteristic nucleotide sequence, nucleic acid molecular probes and a method for identifying Cordyceps guangdongensis. The characteristic nucleotide sequence is shown as SEQ ID NO.1. The nucleic acid molecular probes comprise GDF of 5'-CTGTTGGCATCTTCTGAGTCTC-3' and GDR of 5'-GGTGCGAGGTTGTGCTACTA-3'. In the method, the nucleic acid molecular probes of GDF and GDR are used as polymerase chain reaction (PCR) primers, and a PCR method is used for identifying the Cordyceps guangdongensis; and steps are carried out according to a conventional PCR method. The GDF and the GDR are used as primers, the genome DNA of the Cordyceps guangdongensis is taken as a template, PCR is carried out according to the conventional PCR method, and the Cordyceps guangdongensis is amplified to a specific segment. Through experiments, only the Cordyceps guangdongensis can be amplified to the specific segment, while other Cordyceps samples such as Cordyceps sinensis, Cordyceps gunnii, Cordyceps militaris, supergene Cordyceps and Cordyceps brasiliensis are not amplified to any segment, so that the nucleic acid molecular probes of GDF and GDR have high specificity and can be used for quickly identifying truth and false of the Cordyceps guangdongensis (a fruit body and a mycelium) and related products.
Description
Technical field:
The invention belongs to and utilize molecular biology method to detect the technical field of the Chinese medicinal materials true and false, be specifically related to be used to identify Guangdong Cordyceps militaris (Cordyceps guangdongensis T.H.Li, Q.Y Lin ﹠amp; The method of characteristic nucleotide sequence B.Song) and nucleic acid molecular probe and evaluation Guangdong Cordyceps militaris.
Background technology:
Cordyceps sinensis (Chinese caterpillar fungus) is Chinese rare traditional Chinese medicine, has the effect of protecting the lung kidney, mending lean marrow, preventing phlegm from forming and stopping coughing, but often eats promoting digestion, adjusting immunologic function, strengthens resistance of human body, and the market requirement increases day by day.But along with people's excessive collection, make the wild Chinese caterpillar fungus resource rare day by day, be close in imminent dangerly that price significantly rises, form vicious cycle.Base in Mycelia of Cordyceps and Cordyceps militaris (L.) Link. (mycelium and sporophore) commercially produce the pressure of having alleviated Chinese caterpillar fungus market, have market potential preferably.Guangdong Cordyceps militaris (Cordyceps guangdongensis T.H.Li, Q.Y Lin ﹠amp; B.Song) be the peculiar Chinese caterpillar fungus new variety of newfound China, be preserved in Chinese typical culture collection center, address: China on May 6th, 2006, Wuhan, preserving number is CCTCCNO.M206051, and this Chinese caterpillar fungus safety non-toxic is edible, has anti-oxidant activity, antifatigue and the effect of lengthening the life preferably.Though the cordycepin of Guangdong Cordyceps militaris and adenosine content are lower but than Cordyceps sinensis height, its cordycepic acid content and Cordyceps sinensis are more approaching than Cordyceps militaris (L.) Link., have development and application values preferably.Therefore, differentiate that fast and accurately the method for the Guangdong Cordyceps militaris and the correlated product true and false thereof and technology are the important technologies that Guangdong Cordyceps militaris is commercially produced, the commercial production and the check of Guangdong Cordyceps militaris had great importance.
DNA is the carrier of biological heredity information, each living species and even the individual characteristic nucleotide sequence that all has uniqueness, and has certain stability, can not be subjected to condition effect such as environment or cultivation and change, be this living species or the individual important symbol that is different from other living species or individuality, thereby be to be used for identifying species or individual reliable basis.Utilize Protocols in Molecular Biology and means that species or individual characteristic sequence are analyzed and researched, thereby can survey then and can differentiate these species or individuality fast He exactly this characteristic nucleotide sequence exactly.At present, existing be used to differentiate that the Nucleotide characteristic sequence of Cordyceps sinensis, honeybee cephalont grass etc. patents, but the patent of the Shang Weijian Nucleotide characteristic sequence relevant with the Guangdong Cordyceps militaris evaluation.
Summary of the invention:
First purpose of the present invention provides a kind of molecular biology method that utilizes, and differentiates the characteristic nucleotide sequence of the Guangdong Cordyceps militaris and the correlated product true and false thereof from inheritance.
Of the present inventionly be used to differentiate that the characteristic nucleotide sequence of Guangdong Cordyceps militaris derives from rrna rDNA internal transcribed spacer district ITS1, the ITS2 of Guangdong Cordyceps militaris and the 5.8S rDNA gene between them, concrete sequence is shown in SEQ ID NO.1.
It is the nucleic acid molecular probe that is used to differentiate Guangdong Cordyceps militaris of basic design that second purpose of the present invention provides a kind of characteristic nucleotide sequence (SEQ ID NO.1) with the discriminating Guangdong Cordyceps militaris and the correlated product true and false thereof, and this nucleic acid molecular probe sequence is as follows:
GDF：5`-CTGTTGGCATCTTCTGAGTCTC-3`；
GDR：5`-GGTGCGAGGTTGTGCTACTA-3`。
Above-mentioned nucleic acid molecular probe sequence GDF and GDR, their annealing temperature is close, all can not form primer dimer.This nucleic acid molecular probe has high specificity, only reacts with Guangdong Cordyceps militaris, and does not react with other Chinese caterpillar funguses or other fungies.Utilize this probe can differentiate Guangdong Cordyceps militaris (mycelium, sporophore) and correlated product thereof fast by pcr amplification.
The 3rd purpose of the present invention provides a kind of Guangdong Cordyceps militaris Rapid identification test kit, comprises above-mentioned nucleic acid molecular probe GDF and GDR, and conventional DNA extraction reagent and conventional PCR reaction reagent.
Described Guangdong Cordyceps militaris Rapid identification test kit preferably includes internal transcribed spacer district universal primer ITS1:5`-TCCGTAGGTGAACCTGCGG-3` and the ITS4:5`-TCCTCCGCTTATTGATATGC-3` of Cordyceps sinensis fungus rrna rDNA.This universal primer can be used as the positive control of identifying Guangdong Cordyceps militaris.
The 4th purpose of the present invention provides a kind of method of differentiating Guangdong Cordyceps militaris, is to adopt above-mentioned nucleic acid molecular probe GDF and GDR as the PCR primer, utilizes PCR method to identify Guangdong Cordyceps militaris, and concrete steps PCR method are routinely carried out.
Preferably, utilize ITS1 and ITS4, carry out conventional PCR as template, with as positive control with the genomic dna of Guangdong Cordyceps militaris as the PCR primer.
The present invention is based on the characteristic nucleotide sequence (SEQ ID NO.1) of differentiating the Guangdong Cordyceps militaris and the correlated product true and false thereof and designed specific nucleic acid molecular probe GDF and GDR, their annealing temperature is close, all can not form primer dimer, with this nucleic acid molecular probe GDF and GDR as primer, genomic dna with Guangdong Cordyceps militaris is a template, carries out PCR according to conventional PCR method, the dna fragmentation of acquisition, through checking order its concrete sequence shown in SEQ ID NO.2, its length is 325bp.And find through experiment, utilize nucleic acid molecular probe GDF of the present invention and GDR to increase, Guangdong Cordyceps militaris this specific fragment that can increase only, and other Chinese caterpillar fungus samples are as Cordyceps sinensis, Cordyceps gunnii (Berk.) Berk., Cordyceps militaris (L.) Link., hypergene Chinese caterpillar fungus, the Brazilian Chinese caterpillar fungus any fragment (as Fig. 2) that all do not increase, this shows that nucleic acid molecular probe GDF of the present invention and GDR have high specificity, can be used for differentiating fast the true and false of Guangdong Cordyceps militaris (sporophore and mycelium) and correlated product thereof.
The present invention adopts round pcr to detect, and material usage is few, and only 0.1g is just enough, and method is simple, and specificity is good, and the time spent is short, can finish in 1st.
Description of drawings:
Fig. 1 utilizes ITS1 and ITS4 according to the PCR method of routine different Chinese caterpillar fungus samples to be carried out the electrophorogram of the product of PCR as primer, wherein 1, Cordyceps sinensis, and 2, Cordyceps gunnii (Berk.) Berk., 3, Cordyceps militaris (L.) Link., 4, Guangdong Cordyceps militaris, 5, the hypergene Chinese caterpillar fungus, 6, Brazilian Chinese caterpillar fungus, M is the dna molecular amount standard of 2Kb;
Fig. 2 utilizes nucleic acid molecular probe GDF of the present invention and GDR according to the PCR method of routine different Chinese caterpillar fungus samples to be carried out the electrophorogram of the product of PCR as primer, wherein 1, Cordyceps sinensis, 2, Cordyceps gunnii (Berk.) Berk., 3, Cordyceps militaris (L.) Link., 4, Guangdong Cordyceps militaris, 5, hypergene Chinese caterpillar fungus, 6, Brazilian Chinese caterpillar fungus, M is the dna molecular amount standard of 2Kb.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
The acquisition of the extraction of Guangdong Cordyceps militaris genomic dna and ITS rDNA gene
Get 0.1g Guangdong Cordyceps militaris sample and place aseptic mortar, liquid nitrogen is poured into and ground fast, add 2% (w/v) CTAB extraction buffer, 600 μ l, wherein contain the SDS of 2% (quality volume fraction), continue to grind after several seconds and pour in the 1.5ml Eppendorf tube 65 ℃ of water bath heat preservation 1h into.After being cooled to room temperature, contain to 1.5ml and to add isopyknic saturated phenol in the Eppendorf tube of sample: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1) solution, put upside down mixing 2min, the centrifugal 15min of 12000rpm, with 200 μ l pipettors supernatant liquor is changed in the new centrifuge tube, abandon precipitation.Add isopyknic with it chloroform in the supernatant liquor: primary isoamyl alcohol (volume ratio 24: 1) solution, put upside down mixing 2min, the centrifugal 15min of 12000rpm, the upper strata (water) that to contain DNA with the micropipet of 200 μ l moves in the new pipe carefully, if at water and organic phase intersection adularescent throw out, then extracting organic phase again is 1-2 time, merges water.The micropipet of final supernatant liquor with 200 μ l carefully changed in the new centrifuge tube.The sodium acetate of the 3mol/L pH5.2 of 1/10 volume of adding supernatant liquor flicks the centrifuge tube wall with finger and makes it mixing several times in the past supernatant liquor (dna solution).Adding and the isopyknic Virahol of dna solution (saline solns) (4 ℃ of precoolings) be mixing gently, leaves standstill 2h in-4 ℃.The centrifugal 15min of 12000rpm abandons supernatant liquor, is twice of 75% aqueous ethanolic solution washing throw out with volume fraction.Naturally dry under the room temperature.It is resuspended to add the aseptic ultrapure water of 20 μ l after the seasoning, and (the genome purification kit of bio tech ltd is contained in east, and its article No. is: N1091) the resuspended DNA of purifying, and the genomic dna of acquisition Guangdong Cordyceps militaris to select the DNA purification kit for use.
Get fungi ITS rDNA universal primer ITS1:5`-TCCGTAGGTGAACCTGCGG-3` and ITS4:5`-TCCTCCGCTTATTGATATGC-3`, primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.(the HS-taq Mix test kit of bio tech ltd is contained in east, and its article No. is: P2081), carry out the PCR reaction, reaction system is 25 μ L cumulative volumes, ddH to utilize the HS-taqMix test kit
2O 12.8 μ L, 10 times of damping fluid (Mg
2+) 8 μ L, dNTP (2.5mmol/L) 2 μ L, ITS1 (10 μ mol/L) 0.5 μ L, ITS4 (10 μ mol/L) 0.5 μ L, HotStart Taq (5u/ μ L) 0.2 μ L, template DNA 1 μ L.Response procedures is 94 ℃ of pre-sex change of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, 35 circulations, 72 ℃ of 8min.The PCR product send Beijing China big gene sequencing, obtains ITS rDNA gene order, and its sequence is shown in SEQ ID NO.1.
Extract Cordyceps sinensis according to ordinary method, Cordyceps gunnii (Berk.) Berk., Cordyceps militaris (L.) Link., the genomic dna of hypergene Chinese caterpillar fungus and Brazilian Chinese caterpillar fungus, with it in contrast, with fungi ITS rDNA universal primer ITS1:5`-TCCGTAGGTGAACCTGCGG-3` and ITS4:5`-TCCTCCGCTTATTGATATGC-3` as primer, carry out PCR according to the method described above, the PCR product carries out electrophoresis, and its electrophorogram as shown in Figure 1, as seen from Figure 1, utilize primer I TS1 and the ITS4 can be, Cordyceps gunnii (Berk.) Berk., Cordyceps militaris (L.) Link. from Cordyceps sinensis, Guangdong Cordyceps militaris amplifies sequence in the genomic dna of hypergene Chinese caterpillar fungus and Brazilian Chinese caterpillar fungus.
The pcr amplification of Guangdong Cordyceps militaris Auele Specific Primer
According to Guangdong Cordyceps militaris ITS rDNA gene order (SEQ ID NO.1), utilize primer-design software primer6 design to obtain the pair of sequences that 18～24 Nucleotide are formed, i.e. GDF and GDR, its sequence is as follows,
GDF：5`-CTGTTGGCATCTTCTGAGTCTC-3`；
GDR：5`-GGTGCGAGGTTGTGCTACTA-3`。
Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.
Utilize east to contain the HS-taq Mix test kit of bio tech ltd, its article No. is P2081, carries out the PCR reaction, is template with the genomic dna of Guangdong Cordyceps militaris, and reaction system is 25 μ L cumulative volume: ddH
2O 12.8 μ L, 10 times of damping fluid (Mg
2+) 8 μ L, dNTP (2.5mmol/L) 2 μ L, GDF (10 μ mol/L) 0.5 μ L, GDR (10 μ mol/L) 0.5 μ L, HotStart Taq (5u/ μ L) 0.2 μ L, template DNA 1 μ L.Response procedures is 94 ℃ of pre-sex change of 5min, 94 ℃ of 30s, 60 ℃ of 20s, 72 ℃ of 60s, 35 circulations, 72 ℃ of 8min.The PCR product is through order-checking, and its sequence is shown in SEQ ID NO.2.
With Cordyceps sinensis, Cordyceps gunnii (Berk.) Berk., Cordyceps militaris (L.) Link., the genomic dna of hypergene Chinese caterpillar fungus and Brazilian Chinese caterpillar fungus is as template, with GDF and GDR as primer, carry out PCR in contrast according to the method described above, the PCR product carries out electrophoresis, its result as shown in Figure 2, as seen from Figure 2, Guangdong Cordyceps militaris this specific fragment that can increase only, any fragment and other Chinese caterpillar fungus samples do not increase, this shows that nucleic acid molecular probe of the present invention has high specificity, and specificity is good, therefore can be used for differentiating fast Guangdong Cordyceps militaris (sporophore and mycelium) and correlated product thereof.
Claims (6)
1. one kind is used to differentiate Guangdong Cordyceps militaris (Cordyceps guangdongensis T.H.Li, Q.Y Lin; B.Song) characteristic nucleotide sequence is characterized in that, this characteristic nucleotide sequence is shown in SEQ ID NO.1.
2. a nucleic acid molecular probe that is used to differentiate Guangdong Cordyceps militaris is characterized in that, this nucleic acid molecular probe is:
GDF：5`-CTGTTGGCATCTTCTGAGTCTC-3`；
GDR：5`-GGTGCGAGGTTGTGCTACTA-3`。
3. a Guangdong Cordyceps militaris Rapid identification test kit is characterized in that, comprises claim 2 described nucleic acid molecular probe sequence GDF and GDR, and conventional DNA extraction reagent and conventional PCR reaction reagent.
4. Guangdong Cordyceps militaris Rapid identification test kit according to claim 3, it is characterized in that, also comprise internal transcribed spacer district universal primer ITS1:5`-TCCGTAGGTGAACCTGCGG-3` and the ITS4:5`-TCCTCCGCTTATTGATATGC-3` of Cordyceps sinensis fungus rrna rDNA.
5. a method of differentiating Guangdong Cordyceps militaris is characterized in that, is to adopt described nucleic acid molecular probe GDF of claim 2 and GDR as the PCR primer, utilizes PCR method to identify Guangdong Cordyceps militaris, and concrete steps PCR method are routinely carried out.
6. the method for discriminating Guangdong Cordyceps militaris according to claim 5, it is characterized in that, also utilize ITS1:5`-TCCGTAGGTGAACCTGCGG-3` and ITS4:5`-TCCTCCGCTTATTGATATGC-3` as the PCR primer, carry out conventional PCR with the genomic dna of Guangdong Cordyceps militaris as template, with as positive control.
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| CN103233062A (en) * | 2012-12-03 | 2013-08-07 | 中国食品发酵工业研究院 | Duplex PCR authentication method of cordyceps sinensis original powder |
| CN104342495A (en) * | 2014-11-06 | 2015-02-11 | 广东省微生物研究所 | Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus |
| CN105087805A (en) * | 2015-09-09 | 2015-11-25 | 广东省微生物研究所 | Characteristic nucleotide sequence, nucleic acid molecule primers and method for quantitative determination of cordyceps guangdongensis |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103232995A (en) * | 2012-12-03 | 2013-08-07 | 中国食品发酵工业研究院 | Cordyceps sinensis original powder high-purity genome DNA extraction method |
| CN103233062A (en) * | 2012-12-03 | 2013-08-07 | 中国食品发酵工业研究院 | Duplex PCR authentication method of cordyceps sinensis original powder |
| CN104342495A (en) * | 2014-11-06 | 2015-02-11 | 广东省微生物研究所 | Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus |
| CN105087805A (en) * | 2015-09-09 | 2015-11-25 | 广东省微生物研究所 | Characteristic nucleotide sequence, nucleic acid molecule primers and method for quantitative determination of cordyceps guangdongensis |
| CN105087805B (en) * | 2015-09-09 | 2018-06-22 | 广东省微生物研究所 | A kind of characteristic nucleotide sequence, nucleic acid molecule primers and method for quantitatively detecting Guangdong Cordyceps militaris |
| CN109536635A (en) * | 2019-01-28 | 2019-03-29 | 中国医学科学院药用植物研究所 | A kind of cordyceps sinensis rapid identification method based on Taqman probe and Portable fluorescence quantitative PCR apparatus |
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