WO2021026693A1 - 鉴定发酵虫草菌粉的特征性核苷酸、探针、试剂盒及方法 - Google Patents

鉴定发酵虫草菌粉的特征性核苷酸、探针、试剂盒及方法 Download PDF

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WO2021026693A1
WO2021026693A1 PCT/CN2019/100046 CN2019100046W WO2021026693A1 WO 2021026693 A1 WO2021026693 A1 WO 2021026693A1 CN 2019100046 W CN2019100046 W CN 2019100046W WO 2021026693 A1 WO2021026693 A1 WO 2021026693A1
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cordyceps
cordyceps powder
fermented cordyceps
nucleic acid
identifying
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PCT/CN2019/100046
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French (fr)
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赵祥伟
葛芹玉
章汝恺
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宁芯(南京)生物医学技术研究院有限公司
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Priority to PCT/CN2019/100046 priority Critical patent/WO2021026693A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

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  • the invention relates to characteristic nucleotides, probes, kits and methods for identifying fermented cordyceps powder and belongs to the technical field of biomedicine.
  • Cordyceps is a complex formed by fungi parasitizing in insects. It belongs to the kingdom of Fungi, Fungi, Ascomycotina, Sclerotomycetes, Hypocrea, and Claviceps, and currently nearly 400 species are reported worldwide. As a traditional Chinese herbal medicine, many of them have a long history of medicinal use, such as Cordyceps sinensis, Cordyceps milliarus, Cordyceps gunnii, Cordyceps liangshanensis, etc. Cordyceps is an important class of entomogenous fungi, and its medicinal research has attracted wide attention from the world. At present, many literatures report that Cordyceps can produce a variety of active substances.
  • cordycepin has attracted worldwide attention due to its wide biological activity.
  • Cordycepin has pharmacological effects such as inhibiting the growth of microorganisms, anti-tumor, regulating immunity, and anti-inflammatory.
  • New drugs with cordycepin as the main ingredient have been clinically tried for the treatment of leukemia. Therefore, Cordyceps has good development and application value.
  • cordyceps In recent years, with the increase in demand for cordyceps, natural cordyceps will soon fail to meet market demand, and prices have also formed a market supply and demand situation that is rising day by day. At this time, people began to seek artificial methods of cultivating Cordyceps, and even used fakes to make huge profits. Even some cheap Cordyceps species are closely related to expensive Cordyceps and similar in appearance and shape. They are common confusing products of Cordyceps and are difficult to identify by routine identification. The detection method of cordyceps powder promulgated by the Ministry of Health of the People's Republic of China is complicated and time-consuming, and it is impossible to detect the authenticity of cordyceps powder anytime and anywhere.
  • DNA is the carrier of genetic information. Different species of Cordyceps fungi have their characteristic nucleotide sequences, and they are stable and will not be affected by external factors. It is important to distinguish this individual from other individuals. Sign. Therefore, the characteristic nucleotide sequence used to identify Cordyceps fungus is very reliable. Based on this, the present invention uses the PCR method to amplify the characteristic nucleotide sequences of multiple species of Cordyceps fungi as primers, and then conducts qualitative detection through gel electrophoresis samples. It can provide the most direct detection for Cordyceps fungi. , The most valuable and accurate information.
  • the purpose of the present invention is to provide characteristic nucleotides, probes, kits and methods for identifying fermented cordyceps powder in response to the above-mentioned defects in the prior art, so as to identify the authenticity of related fermented cordyceps powder based on genetic nature.
  • the nucleic acid hybridization created by adopting this technical solution can quickly, conveniently and accurately improve the efficiency of authenticating.
  • the first object of the present invention is to provide a characteristic nucleotide that uses molecular biology means to identify the authenticity of cordycepin and its related products from the genetic nature.
  • the present invention is used to identify the characteristic nucleotide sequence of Cordyceps fermenting bacteria powder (GenBank number: AB067721.1) ribosomal rDNA internal transcription spacer ITS1, ITS4 and related conservative region genes (such as SEQ ID NO: 2, 615 bp), its sequence is shown in SEQ ID NO: 1, and its length is 247 bp.
  • the second object of the present invention is to provide a nucleic acid molecular probe designed on the basis of identifying characteristic nucleotides of fermented cordyceps powder. Its sequence is as follows:
  • nucleic acid molecular probe sequences F and R designed based on the characteristic nucleotide sequence have close annealing temperatures and no primer dimer formation between each other. They have extremely high specificity. This sequence can be used as a primer to ferment Cordyceps The bacterial powder DNA is efficiently amplified and identified.
  • the third object of the present invention is to provide a kit for efficiently identifying fermented cordyceps powder.
  • the kit includes the aforementioned nucleic acid molecular probe, DNA extraction reagent and PCR reaction reagent.
  • kit also includes universal primers for the inner transcribed spacer of rDNA:
  • ITS1 5’-TCCGTAGGTGAACCTGCGG-3’;
  • ITS4 5'-TCCTCCGCTTATTGATATGC-3'; this universal primer sequence can be used as a sample control for identifying fermented cordyceps powder.
  • the fourth object of the present invention is to provide a method for identifying fermented cordyceps powder, which uses the above-mentioned nucleotide sequence molecular probe as a primer and uses PCR for identification.
  • the specific nucleotide molecular probes F and R designed in the present invention have similar annealing temperatures and cannot form primer dimers.
  • the molecular probe is used as a primer, and fermented cordyceps powder DNA is used as a template. Amplify to obtain a DNA fragment, after sequencing, its specific sequence is shown in SEQ ID NO: 1, and the length is 247bp.
  • SEQ ID NO: 1 the specific sequence is shown in SEQ ID NO: 1
  • the length is 247bp.
  • Experiments have verified that by using the molecular probe designed in the present invention for PCR amplification, only related products containing fermented cordyceps powder can obtain specific nucleic acid fragments, while other non-cordyceps products do not obtain any amplified fragments.
  • the invented nucleic acid molecular probes F and R have extremely high specificity.
  • the present invention adopts PCR technology for detection, the amount of materials is small, 0.1g is enough, the method is simple, the specificity is good, the time is short, and it can be completed in half a day.
  • Figure 1 shows the electrophoresis map
  • the lanes 1 to 9 are Cordyceps militaris, Cordyceps taii, Cordyceps spp., Beauveria bassiana, Cordyceps gunis, Cordyceps braziliani, Cordyceps sinensis (Inner Mongolia Dajinjiu Pharmaceutical Co., Ltd.), Epiphytic Cordyceps, Cordyceps sinensis (Nanjing Huanpeng Medical Technology Co., Ltd.).
  • Example 1 Extraction of DNA from fermented cordyceps powder and acquisition of characteristic sequences.
  • Fermented Cordyceps powder (this product is the Cordyceps fungus isolated from Cordyceps sinensis (Berk.) Sacc of the ergot family Cordyceps sinensis (Berk.) Sacc-Paecilomyces hepiali Chen et Dai (Cs-4). Nanjing Huanpeng Medical Technology Limited The company) was washed 3 times with sterile deionized water, and centrifuged at 5000r/min each time, and the supernatant was poured out. Then, the washed mycelium was dried overnight in a 45°C incubator, and put in a mortar, and then liquid nitrogen was poured into the mortar and quickly ground to produce bacterial powder.
  • ITS1 and ITS4 Take Cordyceps universal primers ITS1 and ITS4.
  • the primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd., using takara's Premix Ex Taq TM Hot Start Version (RR030Q), and carrying out the PCR reaction according to the instructions.
  • the total reaction volume was 25 microliters.
  • Water 17.3 microliters, 10-fold buffer contains 2.5 microliters of magnesium chloride, dNTP mix (2.5mmol/L each) 2 microliters, ITS1 primer (10uM) 0.5 microliters, ITS4 primer (10uM) 0.5 microliters, HotstartTaq enzyme ( 5U/uL) 0.2 microliters, template DNA 2 microliters.
  • the thermal reaction parameters are 94°C for 5min pre-denaturation, 98°C for 15 seconds, renaturation at 55°C for 20 seconds, 72°C extension for 45 seconds, 40 cycles, 72°C, and further extension for 8 minutes.
  • the PCR product was routinely sequenced in Nanjing GenScript Biotechnology Co., Ltd. to obtain the gene sequence of rDNA in the ITS region. The sequence is shown in SEQ ID NO: 2.
  • a general sequence specific to Cordyceps fermenting bacteria is obtained, as shown in SEQ ID NO: 2.
  • the primer design software primer5 and the online primer design software primer3 are used to design the amplification primer probes.
  • the obtained probe sequences are combined with the respective annealing temperature and product length for manual fine-tuning, and finally a set of theoretically specific and non-specific Dimer primer combination.
  • the upstream and downstream primers are:
  • the length of the specific sequence defined between the primer combinations is 247bp, as shown in SEQ ID NO:1.
  • the sequence of the primer combination can be synthesized by chemical synthesis on a commercial DNA synthesizer, and can be used as a nucleotide molecular probe (primer) for the identification of fermented cordyceps powder. On this basis, ordinary PCR or real-time fluorescent quantitative PCR can be used for identification.
  • Example 3 Specific identification method of fermented cordyceps powder
  • the various samples used in this experiment were obtained from Nanjing Huanpeng Medical Technology Co., Ltd. and Inner Mongolia Dajinjiu Pharmaceutical Co., Ltd.
  • the total DNA was extracted according to Example 1.
  • samples Cordyceps militaris, Cordyceps dai , Cordyceps sinensis, Beauveria bassiana, Cordyceps muscari, Cordyceps brazilianis, Cordyceps epiphyte, and Cordyceps sinensis obtained from the above two companies
  • the system is 20 ⁇ L PCR reaction solution (including 10 ⁇ PCR buffer 2 ⁇ L, primer F (10 ⁇ M), primer R (10 ⁇ M) 0.5 ⁇ L each, 2mM dNTP mix (2.5mM each) 2 ⁇ L, Taq DNA polymerase 1 unit, plus super (Pure water value 19 ⁇ L), add 1 ⁇ L of each sample DNA extraction solution, close the tube cap, place it on the PCR machine and perform
  • Figure 1 shows the results of the experiment carried out according to the above method, showing that except for two kinds of Cordyceps (raw material of fermented cordyceps fungus powder), the other cordyceps have obtained correct specific bands. This indicates that the PCR primers can be used with PCR amplification methods to specifically detect fermented Cordyceps powder.
  • the gel electrophoresis map uses 1-9 respectively.
  • Lanes 1 to 9 are Cordyceps militaris, Cordyceps taii, Cordyceps sinensis, Beauveria bassiana, Cordyceps gunis, Cordyceps braziliani, Cordyceps sinensis (Inner Mongolia Dajin Jiuyao) Industry Co., Ltd.), Epigenetic Cordyceps, Cordyceps sinensis (Nanjing Huanpeng Medical Technology Co., Ltd.).

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Abstract

一种鉴定发酵虫草菌粉的特征性核苷酸、核酸分子探针、试剂盒及方法。该特征性核苷酸序列如SEQ ID NO：1所示。该核酸分子探针基于发酵虫草菌粉的核糖体rDNA内转录间隔区ITS1、ITS4及相关的保守区域的基因获取的特征性核苷酸序列设计。该核酸分子探针序列F和R的退火温度接近，相互之间没有引物二聚体形成，具有极高的特异性，结合PCR技术进行检测，方法简单，特异性好，用时短，半天即可完成，可以对发酵虫草菌粉DNA进行高效的扩增鉴定。

Description

鉴定发酵虫草菌粉的特征性核苷酸、探针、试剂盒及方法 技术领域
本发明涉及鉴定发酵虫草菌粉的特征性核苷酸、探针、试剂盒及方法，属于生物医药技术领域。
背景技术
虫草是真菌寄生在昆虫体内之后形成的复合体，它隶属于真菌界、真菌门、子囊菌亚门、核菌纲、肉座菌目、麦角菌科，目前在全世界报道近400种。作为传统的中草药，其中很多菌种有着悠久的药用历史，如冬虫夏草(Cordyceps sinensis)、蛹虫草(Cordyceps milliarus)、古尼虫草(Cordyceps gunnii)、凉山虫草(Cordyceps liangshanensis)等。虫草属是一类重要的虫生真菌，药用研究受到世人广泛的关注。目前很多文献报道虫草可产多种活性物质，其中虫草素由于其广泛的生物活性，已引起世界范围内的普遍关注。虫草素具有抑制微生物生长、抗肿瘤、调节免疫、抗炎等药理作用，以虫草素为主要成分的新药已在临床上试用于白血病的治疗。因此，虫草属菌具有较好的开发应用价值。
近年来，随着对虫草需求量的增加，天然虫草很快就不能满足市场的需求，价格也形成了一日一涨的市场供求状况。这时人们开始寻求人工培养虫草的方法，甚至以假乱真，牟取暴利。甚至有部分廉价的虫草种类与昂贵的冬虫夏草亲缘关系近、外观形状类似，是常见的冬虫夏草混淆品，常规鉴定较难鉴别。中华人民共和国卫生部颁布的虫草菌粉的检测方法步骤比较复杂，耗时长，不能够随时随地对虫草菌粉的真伪进行检测。其他虫草菌检验的相关专利仅针对某个地区的某个品种进行检验，不能用与其他物种的检验。虽然市场上有相关虫草菌试剂盒检测，但是外界其他因素可能会对测定结果有影响，使得灵敏度下降，从而难以判断虫草菌及相关制品的真伪。
DNA是遗传信息的载体，来自不同种类的虫草属真菌都有其特征性核苷酸序列，并且具有一定的稳定性，不会受到外界因素的影响而改变，是该个体区别与其他个体的重要标志。因此，特征性的核苷酸序列用来鉴定虫草属真菌是非常可靠的。基于此，本发明通过PCR的方法将多个种类的虫草属真菌的特征性核苷酸序列作为引物进行扩增，随后通过凝胶电泳样本进行定性检测，它能为检测虫草属真菌提供最直接、最有价值及最为准确的信息。
发明内容
本发明目的在于针对以上现有技术的缺陷，提供鉴定发酵虫草菌粉的特征性核苷酸、探针、试剂盒及方法，从遗传本质上鉴别相关发酵虫草菌粉的真伪。采用该技术方案创建的核酸杂交 能够快速、方便、准确的提高鉴别真伪的效率。
本发明的第一目的是提供一种利用分子生物学手段，从遗传本质上来鉴别虫草菌素及其相关制品真伪的特征性核苷酸。
本发明用于鉴定发酵虫草菌的特征性核苷酸序列来源于发酵虫草菌粉(GenBank号:AB067721.1)的核糖体rDNA内转录间隔区ITS1、ITS4及相关的保守区域的基因(如SEQ ID NO：2所示，615bp)，其序列如SEQ ID NO：1所示，长度为247bp。
本发明的第二目的是提供一种以鉴别发酵虫草菌粉的特征性核苷酸为基础设计的核酸分子探针，其序列如下所示：
F:5’-GATGAAGAACGCAGCGAAAT-3’；
R:5’-GGTTCTCAGCGAGCTACTGC-3’。
基于特征性核苷酸序列设计的上述核酸分子探针序列F和R，其退火温度接近，相互之间没有引物二聚体形成，具有极高的特异性，可以利用该序列作为引物对发酵虫草菌粉DNA进行高效的扩增鉴定。
本发明的第三目的是提供一种高效鉴定发酵虫草菌粉的试剂盒，所述试剂盒包括上述核酸分子探针以及DNA提取试剂和PCR反应试剂。
进一步的，所述试剂盒还包括rDNA的内转录间隔区通用引物：
ITS1:5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4:5’-TCCTCCGCTTATTGATATGC-3’；该通用引物序列可以作为鉴定发酵虫草菌粉的样本对照。
本发明的第四目的是提供发酵虫草菌粉的鉴定方法，采用上述核苷酸序列分子探针作为引物，利用PCR进行鉴定。
本发明设计的特异性的核苷酸分子探针F和R，其退火温度相近，不能形成引物二聚体，以该分子探针为引物，以发酵虫草菌粉DNA为模板，采用PCR方法进行扩增，获得DNA片段，经测序，其具体序列如SEQ ID NO：1所示，长度为247bp。经实验验证，利用本发明设计的分子探针进行PCR扩增，只有含有发酵虫草菌粉的相关制品能够获得特异性的核酸片段，而其他非虫草制品没有获得任何的扩增片段，这表明本发明的核酸分子探针F和R具有极高的专一性。可以用于快速的鉴定虫草及相关制品的真伪。此外，本发明采用PCR技术进行检测，材料用量少，0.1g足够，方法简单，特异性好，用时短，半天即可完成。
附图说明
图1为电泳图谱；其中泳道1到9分别为蛹虫草、戴氏虫草、蜂头虫草、球孢白僵菌、古尼虫草、巴西虫草、冬虫夏草(内蒙古大金九药业有限责任公司)、表生虫草、冬虫夏草(南京环朋医疗技术有限公司)。
具体实施方式
以下实施例是对本发明的进一步说明，而不是本发明的限制。
实施例1：发酵虫草菌粉DNA的提取和特征性序列的获取。
(1)样本的前处理
将发酵虫草菌粉(本品为从麦角菌科冬虫夏草Cordyceps sinensis(Berk.)Sacc中分离所得的虫草菌-蝙蝠蛾拟青霉Paecilomyces hepiali Chen et Dai(Cs-4)。南京环朋医疗技术有限公司)经无菌去离子水洗涤3次，每次均经5000r/min离心后倾去上清夜。然后将洗涤后的菌丝置于45℃温箱过夜干燥，放入研钵中，之后将液氮倒入研钵中并快速研磨，制成菌粉。
(2)核酸的提取
1)取0.1g菌粉至2mL离心管中，并加入缓冲液(100mmol/L Tris—HCl(pH＝8.0)，10mmol/L EDTA(pH＝8.0)，0.5％SDS，75ug/mL蛋白酶K)1mL，混匀后50℃水浴2h(间隔10～15min晃动2～3次)；
2)冰浴结束后，12000r/min离心15min，并取上清夜；
3)在上清夜中加入等体积酚：氯仿：异戊醇(25：24：1)和30％体积的无水乙醇对杂质多糖进行沉淀，充分混匀，放置片刻。然后用12000r/min离心15min，并用移液枪将上清夜转入新的离心管中，弃沉淀；
4)在离心管中加入等体积的氯仿：异戊醇(24：1)抽提，漩涡混匀后12000r/min离心15min；
5)取上清液(即DNA溶液)至新的离心管中，加入2/3体积预冷异丙醇，混匀，室温放置30min。12000r/min离心10min，弃上清。用70％乙醇洗涤沉淀2次，室温下自然晾干后加适量TE缓冲液溶解，保存于-20℃备用。
取虫草菌通用引物ITS1和ITS4，引物由南京金斯瑞生物科技有限公司合成，利用takara的Premix Ex Taq TM Hot Start Version(RR030Q)，按照说明书进行PCR反应，反应总体积25微升，双蒸水17.3微升，10倍的缓冲液含有氯化镁2.5微升，dNTP mix(2.5mmol/L each)2微升，ITS1引物(10uM)0.5微升，ITS4引物(10uM)0.5微升，HotstartTaq酶(5U/uL)0.2微升，模板DNA2微升。热反应参数为94℃5min预变性，98℃15秒变性，55℃20秒复性，72℃延伸45秒，40个循环，72℃，进一步延伸8分种。PCR产物于南京金斯瑞生物科技有限 公司进行常规测序，获得ITS区rDNA的基因序列，序列如SEQ ID NO：2所示。
实施例2：核酸分子探针的制备
将发酵虫草菌的ITS区序列与其他虫草菌的同源序列进行比较，获得发酵虫草菌特异的通用序列，即如SEQ ID NO：2所示。利用引物设计软件primer 5以及在线引物设计软件primer3进行扩增引物探针的设计，获得的探针序列结合各自的退火温度与产物长度等进行人工微调，最后确定一组理论上特异性强、无二聚体的引物组合。上下游引物分别为：
F:5’-GATGAAGAACGCAGCGAAAT-3’；
R:5’-GGTTCTCAGCGAGCTACTGC-3’。
该引物组合间界定的特异性序列长度为247bp，如SEQ ID NO：1所示。根据该引物组合的序列可以在商用DNA合成仪上通过化学合成的办法合成，及可以作为发酵虫草菌粉的鉴定的核苷酸分子探针(引物)。在此基础上可以利用PCR方法进行普通PCR或实时荧光定量PCR进行鉴定。
实施例3：发酵虫草菌粉的专一性鉴定方法
本实验所用的各种不同的样本均从南京环朋医疗技术有限公司和内蒙古大金九药业有限责任公司获取，总DNA的提取参照实施例1，对不同的样本(蛹虫草、戴氏虫草、蜂头虫草、球孢白僵菌、古尼虫草、巴西虫草、表生虫草、和从上述两家公司获取的冬虫夏草各一份)分别提取DNA，再进行PCR扩增，每个样本的反应体系为20μL PCR反应液(包括10×PCR缓冲液2μL，引物F(10μM)，引物R(10μM)各0.5μL，2mM dNTP mix(2.5mM each)2μL，Taq DNA聚合酶1个单位，加超纯水值19μL)，加入每个样本DNA提取液1μL，闭紧管盖，置于PCR仪上按照以下程序进行PCR反应：95℃预变性2min，然后94℃变性30秒，58℃复性30秒，72℃延伸40秒，共35循环，最后72℃延伸5min。扩增产物在2％的含有DNA染料的琼脂糖凝胶上进行电泳，在紫外光源下查看及拍照。
图1为按上述方法进行的实验结果，显示除了两种冬虫夏草(发酵虫草菌粉原料)，其余虫草均为获得正确的特异性条带。表明该PCR引物可以和PCR扩增方法可以用于专一性检测发酵虫草菌粉。凝胶电泳图谱上用到1-9分别为其中泳道1到9分别为蛹虫草、戴氏虫草、蜂头虫草、球孢白僵菌、古尼虫草、巴西虫草、冬虫夏草(内蒙古大金九药业有限责任公司)、表生虫草、冬虫夏草(南京环朋医疗技术有限公司)。

Claims (6)

  1. 一种鉴定发酵虫草菌粉的特征性核苷酸，其特征在于，该核苷酸来源于发酵虫草菌粉的核糖体rDNA内转录间隔区ITS1、ITS4及相关保守区域的基因，其序列如SEQ ID NO：1所示。
  2. 一种鉴定发酵虫草菌粉的核酸分子探针，其特征在于，该核酸分子探针序列为：
    F:5’-GATGAAGAACGCAGCGAAAT-3’；
    R:5’-GGTTCTCAGCGAGCTACTGC-3’。
  3. 一种鉴定发酵虫草菌粉的试剂盒，其特征在于，包括权利要求1所述的分子探针序列F和R，以及DNA提取试剂和PCR反应试剂。
  4. 根据权利要求3所述的试剂盒，其特征在于，还包括虫草属真菌核糖体rDNA的内转录间隔区通用引物：
    ITS1:5’-GGTCATTTAGAGGAAGTAA-3’；ITS4:5’-TCCTCCGCTTATTGATATGC-3’。
  5. 一种鉴定发酵虫草菌粉的方法，其特征在于，采用权利要求1所述的核酸分子探针F和R作为PCR引物，利用PCR方法检测发酵虫草菌粉。
  6. 根据权利要求5所述的鉴定发酵虫草菌粉的方法，其特征在于，还利用ITS1和ITS4作为PCR引物，以虫草菌粉提取的DNA作为模板进行常规的PCR，以作为阳性对照。
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