CN102337347A - Characteristic nucleotide sequence for identifying ophiocordyceps crinalis, as well as probes and method thereof - Google Patents
Characteristic nucleotide sequence for identifying ophiocordyceps crinalis, as well as probes and method thereof Download PDFInfo
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Abstract
The invention discloses a characteristic nucleotide sequence for identifying ophiocordyceps crinalis, as well as probes and a method thereof. The characteristic nucleotide sequence for identifying the ophiocordyceps crinalis is derived from the transcription spacer in the rRNA (ribosomal ribose nucleic acid) of the ophiocordyceps crinalis, and has the sequence shown by SEQ ID No.1. The nucleic acid molecule probe I (shown by SEQ ID No. 2) and the nucleic acid molecule probe II (shown by SEQ ID No. 3) are designed based on the characteristic nucleotide sequence; an identification kit containing the nucleic acid molecule probes is disclosed; and the method for identifying the ophiocordyceps crinalis by the probes through polymerase chain reaction (PCR), nucleic acid hybridization or fluorescence quantitative PCR is also disclosed. The invention can be used for conveniently, accurately and rapidly identifying the ophiocordyceps crinalis and relevant products. By adopting the method of molecular biology, the artificial factors in identification are eliminated, so that the identification result is objective and accurate, the identification time is short, and the identification can be finished within half a day.
Description
Technical field
The invention belongs to the technical field of utilizing the molecular biology method detection Chinese medicinal materials true and false, be specifically related to be used to differentiate characteristic nucleotide sequence and the nucleic acid molecular probe and the careless method of discriminating hairworm of hairworm grass (Ophiocordyceps crinalis).
Background technology:
Hairworm grass (Ophiocordyceps crinalis) is a kind of entomogenous fungi that belongs to broad sense Cordyceps (Cordyceps s.l.), and this genus fungi also comprises entomophyte (Ophiocordyceps sinensis) and Cordyceps militaris (L.) Link. (Cordyceps militaris).Wild cordyceps is Chinese traditional Chinese medicine, has guarantor's lung kidney, mends marrow, reduces phlegm and end the effect of phthisical cough, can regulate immunologic function, the enhances human body resistibility.Because a large amount of excavating causes wild resource not enough, price significantly goes up opens at present.At present, still can't realize the artificial culture of Cordyceps sporophore, and its mycelium cultivation needs also low temperature to ferment for a long time, cost is too high.The hairworm grass is very approaching on sibship with entomophyte, has similar function with it, has the value that is developed as the Chinese caterpillar fungus product innovation.Therefore can rapid and precise differentiate that the method for the hairworm grass seed entity and the correlated product true and false is produced relevant enterprise with technology for the hairworm grass and related check mechanism will have great importance.
DNA is the biological material that stores genetic information, and each living species and even the individual characteristic nucleotide sequence that all has uniqueness can be used as this living species or individual other species or the individual sign of being different from.DNA is the element of cell biological, as the carrier of genetic information, has certain stability, can not receive the influence of phenotype and changes, and is to be used for differentiating species, individual reliable basis.Can find different plant species or individual characteristic nucleotide sequence through the analysis of information biology, utilize molecular biological means that this sequence is surveyed and can be differentiated species or individuality fast and accurately.The contriver has applied for and the patent of the Nucleotide characteristic sequence of differentiating tip Chinese caterpillar fungus and honeybee cephalont grass of being authorized two, the patent of not seeing the Nucleotide characteristic sequence relevant with the hairworm grass at present.
Summary of the invention:
First purpose of the present invention provides and derives from hairworm grass (Ophiocordyceps crinalis) rrna rRNA internal transcribed spacer district and 5.8S rRNA gene, is used to differentiate the characteristic nucleotide sequence of hairworm grass, and its sequence is shown in SEQ ID NO.1.
The characteristic nucleotide sequence that utilizes molecular biology method to differentiate the hairworm grass provided by the invention derives from hairworm grass rrna rRNA internal transcribed spacer district and 5.8S rRNA gene (rrna rRNA internal transcribed spacer district and 5.8S rRNA gene merge and be called for short the ITS district); Its sequence is shown in SEQ ID NO.1; Position and characteristic are seen Fig. 1, and this sequence comprises the sequence of ITS1,5.8S rDNA and the ITS2 of hairworm grass.This sequence can obtain through embodiment 1 described method, and the nucleotide sequence that also can on business-like dna synthesizer device, provide according to the contriver through the method for synthetic is synthetic.
The present invention is that template design obtains full length sequence or is no less than the oligonucleotide sequence that 16 Nucleotide are formed with the characteristic nucleotide sequence (shown in SEQ ID NO.1) of above-mentioned hairworm grass; And on the basis of above-mentioned full length sequence or oligonucleotide sequence through modifying, processing, changing and obtain nucleotide sequence, be also included within on the basis of above-mentioned full length sequence or oligonucleotide sequence at 5 ' end and add or change 10 Nucleotide below or change original nucleotides sequence number of columns and change the specificity nucleic acid molecule probe of the nucleotide sequence of 1 Nucleotide of less than as the evaluation of hairworm grass less than 6 Nucleotide of 10% and 3 ' end.Above-mentioned sequence can obtain according to the order that designs is synthetic through DNA synthesizers such as automatic dna synthesizers, and can carry out mark through modes such as enzyme, isotropic substance, vitamin H, chemiluminescence element, fluorescent agents.
Therefore second purpose of the present invention provides a kind of hairworm grass specificity nucleic acid molecule probe that is directed against the sequence shown in SEQ ID NO.1 in above-mentioned ITS district; It is characterized in that this hairworm grass specificity nucleic acid molecule probe comprises Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' (sequence is shown in SEQ ID NO.2) and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 ' (sequence is shown in SEQ ID NO.3).Further preferred, described hairworm grass specificity nucleic acid molecule probe also comprises Probe III:5 '-TCAGCGTCCG CAAGCAGAAATGAATCA-3 ' (sequence is shown in SEQ ID NO.4).This sequence can be used as the Taqman probe in order to identify the hairworm grass behind fluorescent mark.The particular location of Probe I, Probe II, Probe III is seen Fig. 2, and these sequence appearance are applicable to above-mentioned modification, processing and change, add operations such as restriction enzyme site, change partial nucleotide sequence and mark.
Above-mentioned hairworm grass specificity nucleic acid molecule probe provided by the invention: Probe I and Probe II have high specificity, can be with the narrow spectrum reaction of generation of hairworm grass react with Chinese caterpillar fungus or other fungies of other kinds.Utilize above-mentioned probe can detect hairworm grass and correlated product fast through the method for PCR or the method for nucleic acid hybridization.
The 3rd purpose of the present invention provides a kind of test kit that is used to differentiate the hairworm grass; It is characterized in that, comprise hairworm grass specificity nucleic acid molecule probe: Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 ' and PCR popular response reagent.
The described test kit that is used to differentiate the hairworm grass preferably also contains Cordyceps rrna rRNA internal transcribed spacer district universal primer CorF:5 '-CGTTGGTGAACCAGCGGAGGGAT-3 ' and CorR:5 '-TTGCCGCTTCACTCGCCGTTACT-3 '.With the right amplified production of this primer as positive control.
The described test kit that is used to differentiate the hairworm grass preferably also contains Probe III:5 '-TCAGCGTCCG CAAGCAGAAATGAATCA-3 '.Probe III can be used as the Taqman probe through behind the fluorescent mark, utilizes in the test sample that quantitative real time PCR Instrument can be real-time whether have the hairworm grass.
The 4th purpose of the present invention provides a kind of method that is used to differentiate the hairworm grass; It is characterized in that; Be with hairworm grass specificity nucleic acid molecule probe: Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 ' utilize the method for PCR or the method for nucleic acid hybridization or the method for quantitative fluorescent PCR to identify the hairworm grass as primer.
Described method with quantitative fluorescent PCR is identified the hairworm grass, preferably with behind Probe III:5 '-TCAGCGTCCG CAAGCAGAAATGAATCA-3 ' mark fluorescent, as the Taqman probe.
The method of described PCR is identified the hairworm grass, preferably with the amplified production of Cordyceps rrna rRNA internal transcribed spacer district universal primer CorF:5 '-CGTTGGTGAACCAGCGGAGGGAT-3 ' (shown in SEQ ID NO.5) and CorR:5 '-TTGCCGCTTCACTCGCCGTTACT-3 ' (shown in SEQ ID NO.6) as positive control.
Fluorescence quantitative PCR method: adopt foregoing hairworm narrow spectrum nucleic acid molecular probe Probe I of grass and Probe II as amplimer; As the Taqman probe, utilize whether there is the hairworm grass in the test sample that quantitative real time PCR Instrument can be real-time behind the Probe III mark fluorescent.Concrete steps and process are carried out according to conventional molecular biology method.
Making nucleic acid molecular hybridization method: adopt the narrow spectrum nucleic acid molecular probe of foregoing hairworm grass, hairworm grass and correlated product are identified through the mode (for example Southern hybridization or dot blot) of making nucleic acid molecular hybridization.Concrete steps and process are carried out according to the molecular biology method of routine.Wherein, hairworm grass and correlated product detect after can directly detecting the extraction that also can carry out DNA earlier and amplification again.
PCR method: adopt foregoing hairworm narrow spectrum nucleic acid molecular probe Probe I of grass and Probe II as one group of PCR primer; With Cordyceps ITS district universal primer CorF and CorR as previously mentioned as the PCR primer of positive controls; Utilize the PCR method amplification testing sample of above-mentioned two groups of primers respectively according to routine; Both are the hairworm grass at all can the increase sample of DNA fragment specific; The dna segment length of wherein utilizing Probe I and Probe II to obtain is 109bp, utilize dna segment length that CorF and CorR obtain for 530bp about.Only to access can not the increase sample of DNA fragment specific of PCR primer Probe I that the narrow spectrum nucleic acid molecular probe of specific fragment hairworm provided by the invention grass forms and Probe II then be not hairworm grass sample for Cordyceps ITS universal primer CorF and CorR.Wherein, PCR reaction can utilize the reagent in the test kit of the present invention or the hairworm grass sample total DNA extracted according to the molecular biology method of routine as template, also directly a spot of hairworm grass of picking sample adding PCR reaction system as template.
Hairworm of the present invention grass specificity nucleic acid molecule probe: Probe I and Probe II have high specificity, can be with the narrow spectrum reaction of generation of hairworm grass react with Chinese caterpillar fungus or other fungies of other kinds.Utilize above-mentioned probe can detect hairworm grass and correlated product fast through the method for PCR or the method for nucleic acid hybridization or quantitative fluorescent PCR.Whether the present invention is owing to have and adopt dna sequence dna as certification mark, therefore can detect unknown sample accurately and rapidly and be or contain the hairworm grass, and wherein sample can and include Chinese patent medicine preparation, the healthcare products of hairworm grass for sporophore, its grinding and processing product.When adopting PCR method to detect, required sample size is few, and method is simple, can accomplish detection in half a day.
Description of drawings:
Fig. 1 is a rrna rRNA gene structure synoptic diagram, and the scope that wherein is denoted as the ITS district is the dna sequence dna that the present invention relates to, i.e. rrna rRNA internal transcribed spacer district ITS1 and ITS2 and 5.8S rRNA gene;
Fig. 2 is the site plan that hairworm of the present invention grass characteristic nucleotide sequence complete sequence and being used to is identified two hairworms grass specificity nucleic acid molecule probe Probe I, Probe II and Probe III of hairworm grass; This figure shows the positive chain-ordering in ITS district; The left side is 5 ' end; The right side is 3 ' end, and wherein overstriking and underscore partly are respectively Probe I (77bp-94bp is positioned on the normal chain), ProbeII (163bp-185bp; Be positioned on the minus strand) and Probe III (103bp-129bp is positioned on the normal chain);
Fig. 3 is the PCR reaction result synoptic diagram among the embodiment 3, and wherein 1,2 is Cordyceps militaris (L.) Link.; 3,4 is the Dai Shi Chinese caterpillar fungus; 5,6 is entomophyte; 7,8 is honeybee cephalont grass; 9,10 is beauveria bassiana, and 11,12 is Lianzhou City Chinese caterpillar fungus; 13,14 is the hairworm grass, and M is a dna molecular amount standard;
Fig. 4 is the PCR reaction result synoptic diagram among the embodiment 4, and wherein 1,2 is Cordyceps militaris (L.) Link.; 3,4 is the Dai Shi Chinese caterpillar fungus; 5,6 is entomophyte; 7,8 is honeybee cephalont grass; 9,10 is beauveria bassiana, and 11,12 is Lianzhou City Chinese caterpillar fungus; 13,14 is the hairworm grass, and M is a dna molecular amount standard.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
The preparation of hairworm grass rRNA internal transcribed spacer district and 5.8S rRNA gene (sequence in ITS district)
Get hairworm grass sample 0.1-0.5g, pulverize in the liquid nitrogen, (SDS 1.5% to add 300 μ l urea extracting solutions; Urea 7mol/L, Tris-HCl pH8.00.05mol/L, NaCl 0.5mol/L); Move in the miniature centrifuge tube of 1.5ml, in liquid nitrogen, cool off, move on to 65 ℃ of thawings rapidly.Behind the multigelation 3～5 times, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), concussion, the centrifugal 10min of 13000r/min; Supernatant moves into and adds the equal-volume chloroform in the new pipe: primary isoamyl alcohol (24: 1), and concussion, the centrifugal 10min of 13000r/min, supernatant move in the new pipe and add micro-Rnase A; 37 ℃ of insulation 30min add 3 times of volume absolute ethyl alcohols, 3M NaAc (pH5.2) ,-20 ℃ place at least 30min after; The centrifugal 15min of 13000rpm reclaims the DNA deposition, with 70% ethanol and absolute ethanol washing, drains or air-dry; Every pipe adds 50ul TE, and (pH8.0) Hui Rong obtains total DNA for 100mmol/L Tris-Cl, 10mmol/L EDTA.
Get one of 0.2ml PCR pipe, add 20ul PCR reaction solution and (comprise 10xPCR damping fluid 2ul, primer CorF, each 40ng of CorR, 2mmol/L dNTP 1ul; 1 unit of Taq enzyme adds ultrapure water to 20ul), add 0.5ul hairworm grass total DNA extraction liquid, close tight pipe lid; Place on the PCR appearance by following program and carry out the PCR reaction: 94 ℃ of preparatory sex change 3min, 93 ℃ of sex change 1min then, 55 ℃ of renaturation 1min; 72 ℃ are extended 2min, totally 30 circulations, and last 72 ℃ are extended 10min.The purifying that utilizes business-like purification kit to carry out product after reaction is accomplished also directly checks order on the dna sequencing appearance.Obtain nucleotide sequence and remove the sequence that resulting sequence after 18S rRNA gene and the 28S rRNA gene order is hairworm grass rRNA internal transcribed spacer district and 5.8S rRNA gene, its sequence is shown in SEQ ID NO.1.
Embodiment 2:
Hairworm grass specific molecular probe (primer): Probe I, the preparation of Probe II and ProbeIII
The ITS region sequence of hairworm grass and the homologous sequence of other Cordyceps are compared, obtain to be suitable for differentiating hairworm grass oligonucleotide fragment composition most and to put in order, i.e. Probe I; Probe II and Probe III; As shown in Figure 2, Probe I position is 77bp-94bp, is positioned on the normal chain; Probe II position is 163bp-185bp, is positioned on the minus strand; Probe III position is 103bp-129bp, is positioned on the normal chain.According to Probe I; The Nucleotide of Probe II and Probe III is arranged and formed can be through the synthetic Probe I (shown in SEQ ID NO.2) of the method for chemosynthesis on business-like dna synthesizer; Probe II (shown in SEQ ID NO.3) and Probe III (shown in SEQ ID NO.4) promptly can be used as specificity PCR primer.On this basis, can utilize conventional molecular biology method to utilize enzyme, isotropic substance, vitamin H, chemiluminescence element, fluorescent agent etc. that Probe I and Probe II are carried out mark again, promptly can be used as hybridization and use probe.The mark that Probe III is carried out fluorescence and quenching group according to the Taqman probe method promptly can utilize it to carry out quantitative fluorescent PCR as the Taqman probe.
Embodiment 3:
Identify testing of hairworm grass over against shining
This test is in order to confirm that DNA samples met PCR requires to obtain the specific PCR product.
Method 1: total DNA extraction carries out with reference to embodiment 1, and different samples (Cordyceps militaris (L.) Link., Dai Shi Chinese caterpillar fungus, entomophyte, honeybee cephalont grass, beauveria bassiana, Lianzhou City Chinese caterpillar fungus, hairworm grass) is extracted DNA respectively, carries out pcr amplification again.The PCR reaction system of each sample is: 20ul PCR reaction solution (comprises 10xPCR damping fluid 2ul, primer CorF, each 40ng of CorR, 2mmol/L dNTP 1ul, 1 unit of Taq enzyme; Add ultrapure water to 20ul), the total DNA extraction liquid of each sample of adding 0.5ul closes tight pipe lid; Place on the PCR appearance by following program and carry out the PCR reaction: 94 ℃ of preparatory sex change 3min, 93 ℃ of sex change 1min then, 55 ℃ of renaturation 1min; 72 ℃ are extended 2min, totally 30 circulations, and last 72 ℃ are extended 10min.The product of amplification carries out electrophoresis on 2% the sepharose that contains the DNA dyestuff, under suitable ultraviolet source, check and take pictures.
Method 2: with each an amount of sample of the direct picking of toothpick (pulverizing the correlated product of sporophore, pulverizing) (Cordyceps militaris (L.) Link., Dai Shi Chinese caterpillar fungus, entomophyte, honeybee cephalont grass; Beauveria bassiana, Lianzhou City Chinese caterpillar fungus, hairworm grass) plant stirring at the 0.2ml PCR pipe of dress 20ul ultrapure water, and rare respectively to 0.1 times and 0.01 times; The sample suspension liquid adding of getting above-mentioned three concentration of 2ul contains 20ul PCR reaction solution and (comprises 10xPCR damping fluid 2ul, primer CorF, each 40ng of CorR, 2mmol/L dNTP 1ul; 1 unit of Taq enzyme adds ultrapure water to 20ul) 0.2ml PCR pipe in, fully mixing closes tight pipe lid; All the PCR pipe places and carries out following PCR reaction on the PCR appearance: 94 ℃ of preparatory sex change 3min, 93 ℃ of sex change 1min then, 60 ℃ of renaturation 1min; 72 ℃ are extended 2min, totally 30 circulations, and last 72 ℃ are extended 10min.The product of amplification carries out electrophoresis on 2% the sepharose that contains the DNA tinting material, under suitable ultraviolet source, check and take pictures.
The experimental result of Fig. 3 for being undertaken by method 1 shows that (1,2 is Cordyceps militaris (L.) Link. to seven kinds of Chinese caterpillar funguses; 3,4 is the Dai Shi Chinese caterpillar fungus; 5,6 is entomophyte; 7,8 is honeybee cephalont grass; 9,10 is beauveria bassiana, and 11,12 is Lianzhou City Chinese caterpillar fungus; 13,14 are the hairworm grass) all can increase normally obtains ITS district DNA specific fragment, and this result shows that total DNA of five kinds of Chinese caterpillar funguses extractions to be measured meets the requirement of pcr amplification, can be as the testing sample of specificity detection.
Embodiment 4:
Hairworm grass specificity is differentiated
In order to narrow spectrum amplification hairworm grass from the sample that meets the PCR requirement, thereby differentiate the hairworm grass.
Method 1: use to meet the sample that the PCR reaction requires among the embodiment 3, as template, carry out pcr amplification with reference to method among the embodiment 1 with total DNA of these samples; Wherein primer replaces with Probe I and Probe II, and reaction conditions is 94 ℃ of preparatory sex change 3min, 93 ℃ of sex change 1min then; 60 ℃ of renaturation 1min; 72 ℃ are extended 2min, totally 30 circulations, and last 72 ℃ are extended 10min.The product of amplification carries out electrophoresis on 2% the sepharose that contains the DNA dyestuff, under the light source of suitable ultraviolet, check and take pictures.
Method 2: use to meet the method 2 said PCR of carrying out reactions among sample suspension liquid that the PCR reaction requires such as the embodiment 3 among the embodiment 3; Wherein primer replaces with Probe I and Probe II, and reaction conditions is 94 ℃ of preparatory sex change 3min, 93 ℃ of sex change 1min then; 60 ℃ of renaturation 1min; 72 ℃ are extended 2min, totally 30 circulations, and last 72 ℃ are extended 10min.The product of amplification carries out electrophoresis on 2% the sepharose that contains the DNA dyestuff, under suitable ultraviolet source, check and take pictures.
The experimental result of Fig. 4 for being undertaken by method 1 shows that (1,2 is Cordyceps militaris (L.) Link. to seven kinds of Chinese caterpillar funguses; 3,4 is the Dai Shi Chinese caterpillar fungus; 5,6 is entomophyte; 7,8 is honeybee cephalont grass; 9,10 is beauveria bassiana, and 11,12 is Lianzhou City Chinese caterpillar fungus; 13; 14 is the hairworm grass); Wherein have only the hairworm grass to be obtained the DNA fragment specific about 109bp by narrow spectrum amplification, and other Chinese caterpillar funguses all do not have DNA fragment specific and increased, this result shows; Hairworm grass specific molecular probe (primer) Probe I and Probe II can identify the hairworm grass accurately from multiple Chinese caterpillar fungus, got rid of the possibility of false negative (not meeting the negative findings that the pcr amplification condition causes) simultaneously according to embodiment 3.
Claims (9)
1. a characteristic nucleotide sequence that is used to differentiate hairworm grass (Ophiocordyceps crinalis) is characterized in that its sequence is shown in SEQ ID NO.1.
2. one kind is used to differentiate the careless specificity nucleic acid molecule of the careless hairworm of hairworm probe; It is characterized in that this hairworm grass specificity nucleic acid molecule probe comprises Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 '.
3. hairworm grass specificity nucleic acid molecule probe according to claim 2 is characterized in that, described hairworm grass specificity nucleic acid molecule probe also comprises Probe III:5 '-TCAGCGTCCG CAAGCAGAAATGAATCA-3 '.
4. test kit that is used to differentiate the hairworm grass; It is characterized in that, comprise the described hairworm grass of claim 2 specificity nucleic acid molecule probe: Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 ' and PCR popular response reagent.
5. test kit according to claim 4; It is characterized in that this test kit also contains Cordyceps rrna rRNA internal transcribed spacer district universal primer CorF:5 '-CGTTGGTGAACCAGCGGAGGGAT-3 ' and CorR:5 '-TTGCCGCTTCACTCGCCGTTACT-3 '.
6. test kit according to claim 4 is characterized in that this test kit also contains Probe III:5 '-TCAGCGTCCGCAAGCAGAAATGAATCA-3 '.
7. method that is used to differentiate the hairworm grass; It is characterized in that; Be with the described hairworm grass of claim 2 specificity nucleic acid molecule probe: Probe I:5 '-GACCCAGACCCCGCTGTC-3 ' and Probe II:5 '-CATTTCGCTGCGTTCTTCATCG-3 ' utilize the method for PCR or the method for nucleic acid hybridization or the method for quantitative fluorescent PCR to identify the hairworm grass as primer.
8. the method that is used to differentiate the hairworm grass according to claim 7; It is characterized in that; Described method with quantitative fluorescent PCR is identified the hairworm grass, is to carry out quantitative fluorescent PCR again as the Taqman probe behind Probe III:5 '-TCAGCGTCCG CAAGCAGAAATGAATCA-3 ' mark fluorescent group.
9. the method that is used to differentiate the hairworm grass according to claim 7; It is characterized in that; Described method with PCR is identified the hairworm grass, also with the amplified production of Cordyceps rrna rRNA internal transcribed spacer district universal primer CorF:5 '-CGTTGGTGAACCAGCGGAGGGAT-3 ' and CorR:5 '-TTGCCGCTTCACTCGCCGTTACT-3 ' as positive control.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN106636449A (en) * | 2017-03-06 | 2017-05-10 | 山西省农业科学院食用菌研究所 | Molecular marker, primer and probe for identifying lentaria patouillardii edible mushrooms |
| CN107012240A (en) * | 2017-05-08 | 2017-08-04 | 山东大学 | A kind of method of Rapid identification epothilone gene cluster positive strain |
| 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 |
| CN113151538A (en) * | 2021-03-09 | 2021-07-23 | 贵州大学 | Candidate DNA bar code, primer and method for identifying cordyceps fungus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106636449A (en) * | 2017-03-06 | 2017-05-10 | 山西省农业科学院食用菌研究所 | Molecular marker, primer and probe for identifying lentaria patouillardii edible mushrooms |
| CN107012240A (en) * | 2017-05-08 | 2017-08-04 | 山东大学 | A kind of method of Rapid identification epothilone gene cluster positive strain |
| 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 |
| CN113151538A (en) * | 2021-03-09 | 2021-07-23 | 贵州大学 | Candidate DNA bar code, primer and method for identifying cordyceps fungus |
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| CN102337347B (en) | 2013-05-22 |
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