CN1274010A - Characteristic nucleotide sequence and method for discriminating cordyceps - Google Patents
Characteristic nucleotide sequence and method for discriminating cordyceps Download PDFInfo
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Abstract
本发明属于利用分子生物学方法检测中药材的技术领域。本发明提供了冬虫夏草rDNA间隔区核苷酸序列及来源于该序列的核酸分子探针和包含该探针的冬虫夏草鉴定试剂盒,以及利用该序列、探针或试剂盒鉴别冬虫夏草的方法。利用本发明,可方便、快速、准确地鉴别冬虫夏草(包括商品药材、菌丝体和系列保健品等),还可判别样品的道地性,并且可以计算机化、仪器化,具有客观、准确、自动化等特点。The invention belongs to the technical field of detecting Chinese medicinal materials by means of molecular biology methods. The invention provides the nucleotide sequence of the rDNA spacer of Cordyceps sinensis, a nucleic acid molecular probe derived from the sequence, a Cordyceps identification kit containing the probe, and a method for identifying Cordyceps sinensis by using the sequence, the probe or the kit. The invention can conveniently, quickly and accurately identify Cordyceps sinensis (including commercial medicinal materials, mycelium and series of health care products, etc.), and can also determine the authenticity of samples, and can be computerized and instrumented, with objective, accurate and accurate identification. Features such as automation.
Description
The invention belongs to and utilize molecular biology method to detect the technical field of Chinese medicinal materials, specifically, relate to a kind of nucleotide sequence that is used to differentiate Cordyceps sinensis and nucleic acid molecular probe and differentiate the method for Cordyceps sinensis with it.
Cordyceps sinensis Cordyceps sinensis (Berk.) Sacc. is a kind of entomogenous fungi, for the traditional valuable ingredient of traditional Chinese medicine of China, has very high pharmaceutical use.People in pharmacy common to be the sophisticated sporophore of perfect stage of this fungi, form behind lepidopteran bat moth Hepialus spp. larva by fungi autoeciousness.Mainly be distributed in high altitude localitiess such as Sichuan, Yunnan, Qinghai, Gansu, Tibet.Because Cordyceps sinensis distribution height above sea level, vegetative period is long, and is unfavorable to existing worm grass resources protection, thereby source of goods shortage, and it is more to occur the puppet product at present, and common have Cordyceps militaris (L.) Link., a Cordyceps hawkesii Gary etc.The temperature of Cordyceps sinensis, sweet, the nourishing the lung and the kidney of flavor, beneficial vital essence, " Chinese pharmacopoeia is recorded only a kind of for Cordyceps sinensis; The pupa grass has anti-microbial effect, and is different fully with the Cordyceps sinensis effect.In addition, under the pressure of the depletion of the pressure and the natural resources of demand, there are much asexual fermentation myceliums replace its sexual sporophore stage to produce various " Chinese caterpillar fungus " patent medicine or healthcare products at present with Cordyceps sinensis or other Chinese caterpillar fungus.But people usually obtain the diverse anamorph kind of kind when cultivating Cordyceps fungus, Chinese Paecilomyces varioti is arranged; China pilose spore; the curved neck of China is mould etc., and the definite different scholar of Cordyceps sinensis anamorph kind is held different viewpoints, has caused the confusion of Cordyceps sinensis medicinal material or patent medicine.
Up to now, research and the patent about aspects such as Cordyceps sinensis artificial culture and industrialized preparing process has many reports in the world, but Shang Weijian is relevant for the technology and the Study on standards report of the differentiation of Cordyceps sinensis gene.The trend that goes back to nature along with people grows to even greater heights, and increasing people pay close attention to traditional Chinese materia medica.Chinese medicine will develop, just must be towards modernization and industrialization, and the core content of the modernization of Chinese medicine is the stdn of traditional Chinese medicine quality.In view of the present problem and the market requirement that exists of Cordyceps sinensis, adopt the novel method new technology quick and precisely to identify the Cordyceps sinensis true and false, instruct the suitability for industrialized production of exploitation Cordyceps fungus, to China's traditional Chinese medicine modernization, industrialization with move towards the world market and have great significance.
The purpose of this invention is to provide a kind of Cordyceps sinensis gene and screen standard and technology, it can differentiate the true and false of Cordyceps sinensis from the inheritance objective and accurately.Specifically comprise: 1, provide to can be used as the nucleotide sequence that is used to differentiate Cordyceps sinensis that gene is screened standard; 2, provide the Cordyceps specific nucleic acid molecular probe that derives from above-mentioned nucleotide sequence; 3, provide the test kit that is used to differentiate Cordyceps sinensis that comprises this nucleic acid molecular probe; 4, provide the method for utilizing above-mentioned nucleotide sequence, nucleic acid molecular probe or test kit to differentiate Cordyceps sinensis.
The present inventor finds in the research to Cordyceps sinensis and other Chinese caterpillar fungus and relevant fungi, the rDNA transcribed spacer (non-coding region) of Cordyceps sinensis has and other Chinese caterpillar fungus and the diverse nucleotide sequence of relevant fungi, can be used as the Cordyceps sinensis characteristic nucleotide sequence, be used to differentiate the true and false of Cordyceps sinensis.According to this characteristic nucleotide sequence, can design the specificity nucleic acid molecular probe of preparing Cordyceps sinensis rDNA transcribed spacer, set up the molecular biology method of fast, accurately differentiating Cordyceps sinensis.
Provided by the inventionly be used to differentiate that the nucleotide sequence of Cordyceps sinensis derives from the rDNA transcribed spacer of Cordyceps sinensis (being Cordyceps sinensis rrna rRNA internal gene transcribed spacers) sequence, the position of this sequence and constitutional features are shown in Fig. 1 and sequence table 1.
This sequence (SEQ ID No 1) can obtain by method as described in embodiment 1, perhaps forms and arrangement by the Nucleotide of known this sequence, is synthesized into according to a conventional method on business-like automatic dna synthesizer.
Cordyceps specific nucleic acid molecular probe of the present invention is to classify basic design as with above-mentioned Cordyceps sinensis rDNA transcribed spacer nucleotides sequence to come out.This nucleic acid molecular probe can be the complete sequence (being the sequence shown in the sequence table 1) that derives from this Cordyceps sinensis rDNA transcribed spacer nucleotide sequence, or one section oligonucleotide sequence that is no less than 18 nucleosides compositions in this sequence, or above-mentioned these sequences are modified again, change, and its Nucleotide variable quantity is no more than 5% sequence of total nucleotide amount.
The particularly preferred Cordyceps specific nucleic acid molecular probe of the present invention is two sequences shown in sequence table 2 and the sequence table 3 (following probe1 and the probe2 of abbreviating as respectively).
Above-mentioned Cordyceps specific nucleic acid molecular probe of the present invention, can be according in advance according to the good sequence of Cordyceps sinensis rDNA transcribed spacer sequences Design, the DNA synthetic method by routine is synthesized into (for example can use business-like automatic dna synthesizer to synthesize).These probes can be incorporated into row labels by radio isotope, enzyme, vitamin H, fluorescent agent or chemiluminescence element.
The Cordyceps specific nucleic acid molecular probe that the present invention is above-mentioned has high Cordyceps sinensis specificity, can react with the Cordyceps sinensis specificity, but not react with the DNA of other Chinese caterpillar fungus or relevant fungi.So, utilize this nucleic acid molecular probe, can differentiate Cordyceps sinensis (comprising the examination of Cordyceps sinensis polypide, mycelium, Chinese caterpillar fungus Chinese patent medicine and serial health protection product) fast and accurately by PCR method or making nucleic acid molecular hybridization method.
In addition, because the characteristic of rDNA transcribed spacer hypermutation, the Cordyceps sinensis in the different places of production lists at its rDNA transcribed spacer nucleotides sequence and also shows difference, the difference that is the nucleotide sequence of rDNA transcribed spacer can reflect that the distance of the sibship between the different Cordyceps funguss (is that sibship is near more, this difference is more little, genuineness is also high more, and vice versa).Therefore, Cordyceps sinensis rDNA transcribed spacer sequence provided by the present invention and specificity nucleic acid molecular probe also can be used for the detection of Cordyceps sinensis genuineness.
The present invention also provides a kind of Cordyceps sinensis identification kit of being made up of Cordyceps specific nucleic acid molecular probe and relevant PCR primer and reagent, can be used for differentiating quickly and easily the true and false of Cordyceps sinensis.This test kit specifically comprises: a, two foregoing Cordyceps specific nucleic acid molecular probes (isotopic labeling or fluorescent mark); B, three pairs of PCR primers: with above-mentioned two nucleic acid molecular probes is a pair of primer, and above-mentioned two nucleic acid probes are paired into two pairs of primers in addition with another fungi universal PC R primer respectively; C, Cordyceps sinensis micro-example DNA prepare reagent: comprise and extract damping fluid, glass powder, sodium iodide and washing lotion.
Two nucleic acid molecular probes in the mentioned reagent box are preferably probe1 and probe2; With these two nucleic acid molecular probe paired fungi universal PC R primers can be LH2 (5 ' TAGGTGAACCTGCGGAAGGATCA 3 ') and D1a (5 ' TTCCCTGTTCACTCGCCGTTACT3 '); The extraction damping fluid composition that sample DNA prepares in the reagent is: 0.5～3%SDS, 5～10mM EDTA (pH7.5～8.5), 10mM Tris-HCl (pH7.5～8.5), 10mM NaCl, the washing lotion composition is: 84mM Tris-HCl, 40 μ M EDTA, 60% ethanol, 80mM KAc.
Utilize the mentioned reagent box, can differentiate Cordyceps sinensis quickly and easily according to the PCR method of routine.
Cordyceps sinensis discrimination method provided by the present invention can adopt making nucleic acid molecular hybridization method or PCR method, and division is as follows:
Making nucleic acid molecular hybridization method: adopt foregoing nucleic acid molecular probe of the present invention, with general making nucleic acid molecular hybridization mode (for example Southern hybridization or dot blot), to the Cordyceps sinensis evaluation of testing.Concrete steps and process (comprising the pre-treatment of the mark and the testing sample of probe), molecular biology method routinely carries out.
This method can directly detect Cordyceps fungus, Chinese patent medicine goods or the healthcare products sample of the dried medicinal material of Cordyceps sinensis commodity, industrial fermentation, also can earlier the Cordyceps sinensis DNA in the sample be extracted amplification, is checked again.
PCR method: with two foregoing nucleic acid molecular probes of the present invention is one group of PCR primer, perhaps matches with another fungi universal PC R primer respectively with these two nucleic acid molecular probes, carries out the PCR reaction.Concrete steps, the process operating process of PCR method are routinely carried out.
PCR primer in the above-mentioned PCR method can be by following selection setting:
1. with fungi 18S rRNA coding region 3 ' terminal conserved regions a fungi universal PC R primer LH2 is set, matches as another primer with rDNA transcribed spacer Cordyceps sinensis specialized oligonucleotides probe probe 2 then, the about 550bp of pcr amplification product is long.
2. with rDNA transcribed spacer Cordyceps sinensis specific molecular probe probe1 as a primer, in 25S rRNA coding region 5 ' terminal conserved regions a fungi universal primer D1a is set in addition then, the about 550bp of probe1/D1a pairing amplified production grows.
3. carry out PCR with Cordyceps sinensis specialized oligonucleotides probe probe1 and probe2 as primer and detect the about 400bp of PCR product.
Adopt this PCR method can detect discriminating Cordyceps sinensis quickly and accurately, and amount of samples is few.
The present invention has following beneficial effect:
1. non-coding region (transcribed spacer) is a hypervariable region among the Chinese caterpillar fungus rDNA, the rDNA spacer structure difference of different Chinese caterpillar fungus monoids is very big, and this difference can also reflect the distance of sibship between the different Cordyceps, and (be that sibship is near more, difference is more little, genuineness is high more, and vice versa).According to Cordyceps sinensis rDNA transcribed spacer sequence provided by the present invention, (standard is: the genuineness of Nucleotide variation range 0.00～5%) differentiating sample according to the variation size of Nucleotide in the test sample sequence, and computerizable, instrumentation, have characteristics such as objective, accurate, automatization.
2. the Cordyceps sinensis nucleic acid molecular probe that is provided is the Cordyceps sinensis specificity sequence in (the Cordyceps mycelium stage that comprises natural cordyceps polypide stage and artificial culture), therefore under the prerequisite of sample the unknown, can by whether having identical therewith nucleotide sequence, the true and false of test sample quickly and easily in the test sample.
3. because the application of PCR method, make this method oversimplify more, usually need only small amount of sample, just can reach requirement as several thecaspores of picking or a small amount of sclerotium tissue or mycelium, and do not need to extract in a large number DNA, in addition, also can detect Cordyceps sinensis herbal medicine patent medicine (pulvis or tablet) and Chinese caterpillar fungus health product.
Fig. 1 is a rrna rRNA gene structure synoptic diagram, and ITS1 wherein and ITS2 are polynucleotide sequence (rDNA transcribed spacer sequence) involved in the present invention;
Fig. 2 is Cordyceps sinensis rDNA transcribed spacer polynucleotide sequence (rRNA internal gene transcribed spacers sequence) structural representation, and wherein (142bp～163bp and 506bp～485bp) are respectively Cordyceps sinensis specialized oligonucleotides molecular probe probe1 of the present invention and probe2 with the black matrix part that goes up line;
Fig. 3 is the PCR result schematic diagram of embodiment 4, and wherein 1 is blank, and 2 is the 2kb molecular weight gradient, and 3 is the natural cordyceps polypide, and 4 is artificial culture Cordyceps fungus (China pilose spore), and 5 is Cordyceps militaris (L.) Link., and 6 is Cordyceps gunnii (Berk.) Berk., and 7 is Chinese Paecilomyces varioti;
Fig. 4 is the pcr amplification result schematic diagram of embodiment 5, and wherein 1 is blank, and 2 is the 2kb molecular weight gradient, and 3 is the natural cordyceps polypide, and 4 is artificial culture Cordyceps fungus (China pilose spore), and 5 is Cordyceps militaris (L.) Link., and 6 is Cordyceps gunnii (Berk.) Berk., and 7 is Chinese Paecilomyces varioti.
The present invention will be further described by the following examples:
The preparation of embodiment 1 Cordyceps sinensis rDNA transcribed spacer polynucleotide sequence
Get Cordyceps sinensis sample 0.1-0.5g in mortar, add liquid nitrogen and be ground to Powderedly, divide the 1.5ml that packs into to extract (1%SDS in the damping fluid, 10mM EDTA pH 8.0,10mM Tris-HCl pH 7.6) in the little vial, shake all, 65 ℃ are incubated 0.5-2 hour, centrifugal (8000rpm/min), get supernatant, change new vial over to, add 3M NaAc and equal-volume isopropanol precipitating 15 minutes, centrifugal (10000rpm/min, 10min), the supernatant that inclines, precipitation is dried, add 50-100 μ l TE (Tris-HCl, 10mM, EDTA, 1mM, pH 7.8) dissolving, obtain the total DNA of Cordyceps sinensis.Get 1 little vial of 0.5ml, add 100 microlitre PCR reaction solutions (100 microlitre PCR reaction solution compositions: 10 * TaqDNA polymerase buffer, 10 microlitres; 2mM dNTP solution 10 microlitres; Primer is LH2 0.5 microgram; Primer is D1a 0.5 microgram; TaqDNA polysaccharase 2.5 units add sterilized water and are settled to 100 microlitres).Add the total DNA1 microlitre of Cordyceps sinensis that obtains then, add 50 microlitre mineral oil, be put on the PCR instrument, carry out the PCR reaction by follow procedure: 94 ℃ of 5min, 94 ℃, 55 ℃ and 72 ℃ of each 1min, 30 circulations are 72 ℃ of 5min then.After reaction is finished, remove mineral oil, (QIAGEN, Germany) purified pcr product obtain required nucleotide sequence with the PCR purification kit.PCR product behind the purifying is measured on business-like automatic dna sequencer, obtains to form and arrangement as the Nucleotide of sequence table 1.
The preparation of embodiment 2probe1 and probe2
On the basis that obtains Cordyceps sinensis rDNA transcribed spacer nucleotide sequence, institute's calling sequence and other Chinese caterpillar fungus or fungi homologous sequence are arranged comparison, and the Nucleotide that draws probe1 and probe2 (142bp-163bp as shown in Figure 2 and 506bp-485bp) is formed and arranged is to be used for the best oligonucleotide fragment that Cordyceps sinensis is differentiated.Form and arrangement according to the Nucleotide of probe1 and probe2, on business-like automatic dna synthesizer, carry out chemosynthesis, can obtain the nucleotide sequence shown in sequence table 2 and sequence table 3.
The preparation of embodiment 3 Cordyceps sinensis micro-example DNA
With a small amount of thecaspore of toothpick picking Cordyceps sinensis polypide or a small amount of sclerotium tissue, or a small amount of asexual mycelium → put into contains an amount of extraction damping fluid (0.5-3%SDS, 5-10mM EDTA (pH7.5-8.5), 10mM Tris-HCl (pH7.5-8.5), 10mM NaCl) in the little vial of 0.5 microlitre → concussion → adding 1ul glass powder and 60 microlitre 6M NaI → centrifugal → abandon supernatant → adding 100ul washing lotion (84mMTris-HCl, 40uMEDTA, 60% ethanol, 80mMKAc), centrifugal → as to abandon supernatant → precipitation airing, standby.
The discriminating of embodiment 4 Cordyceps sinensis (conventional PCR method)
(1) sample pretreatment (sample preparation to be detected can adopt following method I or method II)
Method I. sample thief 0.1～0.5 restrains in mortar, adding liquid nitrogen is ground to Powdered, dividing packs into contains 1.5 milliliters of extraction damping fluid (1%SDS, 10mM EDTA pH8.0,10mM Tris-Hcl pH7.6) in the little vial, shake all, 65 ℃ are incubated 0.5～2 hour, centrifugal (8000 rev/mins), get supernatant, move into pipe newly, add 3M NaAc and equal-volume isopropanol precipitating 15 minutes, centrifugal (10000 rev/mins, 10 minutes), the supernatant that inclines adds 50～100 microlitre TE (Tris-HCl 10mM behind the precipitation airing, EDTA 1mM, pH 7.8) dissolving.
Method II. with toothpick picking small amount of sample (for example: a small amount of spore of Cordyceps sinensis polypide or a small amount of sclerotium tissue, or a small amount of asexual mycelium), put into and contain 20 microlitres and extract damping fluid (the damping fluid composition is with method I), shake all, [comprise glass powder with the DPS purified reagent, 6M NaI, washing lotion (84mMTris-HCl, 40uMEDTA, 60% ethanol, 80mMKAc)] behind the purifying, airing, standby.
Composition in (2) the 100 microlitre PCR mixed solutions
Cordyceps sinensis specific molecular probe Probe1 0.5 microgram
Cordyceps sinensis specific molecular probe Probe2 0.5 microgram
10 * Taq dna polymerase buffer liquid, 10 microlitres
2mMdNTP solution 10 microlitres
TaqDNA polysaccharase 2.5 units
Be 100 microlitres more than with the sterilized water constant volume
(3) PCR operation
Get 7 0.5 milliliter of little vials, add 20 microlitre PCR mixed solutions respectively, add sample DNA then respectively, one of them pipe only adds PCR mixed solution 20 microlitres (contrast).Add 50 microlitre mineral oil in each pipe, be put on the PCR instrument, carry out PCR reaction by follow procedure: 94 ℃ 5 minutes, 94 ℃, 55 ℃ and 72 ℃ each 1 minute, and circulate continuously 30 times, then 72 ℃ 5 minutes.After reaction was finished, every pipe added 5 microlitre load sample liquid (30%Fcol, 0.25% bromjophenol blue).Get 4 microlitre point samples in 2% sepharose, electrophoresis result as shown in Figure 3.
The result of Fig. 3 clearly illustrates: adopt primer Probe1/Probe2 to carry out PCR and detect, the all positive reaction of Cordyceps fungus (China pilose spore) of sexual polypide stage of natural cordyceps and artificial culture, and Cordyceps gunnii (Berk.) Berk., the negative reaction of anamorph bacterial strain that other Chinese caterpillar fungus such as Cordyceps militaris (L.) Link. is relevant has illustrated the specificity of this combination of primers.
Present method has following advantage:
(1) easy to be quick.Conventional DNA prepares needs 1 to a few hours, and this method only need get final product (except the proliferation time) in 20～30 minutes as if employing " method II " when sample pretreatment.
(2) amount of samples is few, only needs small amount of sample just can finish entire operation, does not influence the integrity of sample.
(3) accurate, highly sensitive, probe1 and probe2 are Cordyceps specific nucleic acid molecular probe, if pseudo-product, negative reaction.
(4) do not use toxic reagent.
The discriminating of embodiment 5 Cordyceps sinensis (with reference to the primer method)
(1) two kind of fungi rDNA transcribed spacer PCR universal primer
LH2：5’TAGGTGAACCTGCGGAAGGATCA?3’、
D1a:5 ' TTCCCTGTTCACTCGCCGTTACT3 ' is all synthetic with automatic dna synthesizer, all primers or probe be dissolved in TE (Tris-HCl 10mM, EDTA 1mM, pH7.8) solution, concentration is 0.5 microgram/microlitre.
(2) sample pretreatment is with embodiment 1.
The preparation of (3) 100 microlitre PCR mixed solutions
Cordyceps sinensis specific molecular probe Probe1 0.5 microgram
Cordyceps sinensis specific molecular probe Probe2 0.5 microgram
Fungi rDNA transcribed spacer PCR universal primer LH2 0.5 microgram
Fungi rDNA transcribed spacer PCR universal primer D1a 0.5 microgram
10 * Taq dna polymerase buffer liquid, 10 microlitres
2mMdNTP solution 10 microlitres
Taq archaeal dna polymerase 2.5 units
Be 100 microlitres more than with the sterilized water constant volume
(4) the PCR operation is with embodiment 1, and the result as shown in Figure 4.
Can clearly be seen that from the result of Fig. 4: adopt and detect with reference to the primer PCR method, the Cordyceps fungus (China pilose spore) of sexual polypide stage of natural cordyceps and artificial culture has 3 pcr amplification bands, and the relevant anamorph bacterial strain of other Chinese caterpillar funguses such as Cordyceps gunnii (Berk.) Berk., Cordyceps militaris (L.) Link., Chinese Paecilomyces varioti only has 1 amplified band, thereby clear, differentiate Cordyceps sinensis and relevant Cordyceps accurately and rapidly.
Sequence table 11. sequence signatures: length: 615bp, type: nucleic acid, chain number: two strands, geometry: linearity; 2. molecule type: rDNA 3. sources: Cordyceps sinensis 4. sequence descriptions: SEQ ID No.1
GTAGGTGAAC?CTGCGGAAGG?ATCCTTATCG?AGTCACCACT?CCCAAACCCC?CTGCGAACAC
CACAGCAGTT?GCCTCGGCGG?GACCGCCCCG?GCGCCCCAGG?GCCCGGACCA?GGGCGCCCGC
CGGAGGACCC?CCAGACCCTC?CTGTCGCAGT?GGCATCTCTC?AGTCAAGAAG?CAAGCAAAGG
AATCAAAACT?TTCAACAACG?GATCTCTTGG?TTCTGGCATC?GATGAAGAAC?GCAGCGAAAT
GCGATAAGTA?ATGTGAATTG?CAGAATTCAG?TGAACCATCG?AATCTTTGAA?CGCACATTGC
GCCCGCCAGC?ACTCTGGCGG?GCATGCCTGT?CCGAGCGTCA?TCTCAACCCT?CGAGCCCCCC
GCCTCGCGGC?GGCGGGGCCC?GGCCTTGGGG?GTCACGGCCC?CGCGCCGCCC?CCTAAACGCA
GTGGCAACCC?CGCCGCGGCT?CCCCTGCGCA?GTAGCTCGCT?GAGAACCTCG?CACCGGGAGC
GCGGAGGCGG?TCACGCCGTG?AAACCACCAC?ACCCTCCAGT?TGACCTCGGA?TCAGGTAGGG
ATACCCGCTG?AACTTAAGCA?TATCAATAAG?CGGAGGAAAA?GAAACCAACA?GGGATTGCCC
CAGTAACGGC?GAGTG
Sequence table 21. sequence signatures: length: 22bp, type: nucleic acid, chain number: strand, geometry: linearity; 2. molecule type: DNA3. originates: Cordyceps sinensis 4. sequence descriptions: SEQ ID No.2
TGTCGCAGTG?GCATCTCTCA?GT
Sequence table 31. sequence signatures: length: 22bp, type: nucleic acid, chain number: strand, geometry: linearity; 2. molecule type: DNA3. originates: Cordyceps sinensis 4. sequence descriptions: SEQ ID No.3
TGGTTTCACG?GCGTGACCGC?CT
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| CN110408721A (en) * | 2019-08-09 | 2019-11-05 | 宁芯(南京)生物医学技术研究院有限公司 | Identify characteristic nucleotide, probe, kit and the method for fermentation cordyceps |
| WO2021026693A1 (en) * | 2019-08-09 | 2021-02-18 | 宁芯(南京)生物医学技术研究院有限公司 | Characteristic nucleotide, probe, kit and method for identifying fermented cordyceps powder |
| CN110484650A (en) * | 2019-09-25 | 2019-11-22 | 成都图径生物科技有限公司 | The primer and probe of fluorescence quantitative PCR detection Hirsutella sinensis |
| CN113462685A (en) * | 2021-07-21 | 2021-10-01 | 翌圣生物科技(上海)股份有限公司 | Probe composition for preventing reverse transcription of fungus conserved region and application thereof |
| CN113462685B (en) * | 2021-07-21 | 2023-08-22 | 翌圣生物科技(上海)股份有限公司 | Probe composition for preventing reverse transcription of fungus conserved region and application thereof |
| CN113832247A (en) * | 2021-09-29 | 2021-12-24 | 中国食品药品检定研究院 | Primer, probe and identification method of hirsutella sinensis |
| NL2033157A (en) * | 2021-09-29 | 2023-04-04 | Nat Inst Food & Drug Control | Primer pair, probe and identification method of hirsutella sinensis |
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