Gene sequence and method for distinguishing cordyceps sinensis

Abstract

The present invention utilizes the singularity of the 18S rRNA gene sequence of the Cordyceps sinensis between the NS3/NS6 primer pair as the index for distinguishing the Cordyceps sinensis from other Cordyceps species.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention utilizes the singularity of the 18S rRNA gene sequence of the Cordyceps sinensis between the NS3/INS6 primer pair as the index for distinguishing the Cordyceps sinensis from other Cordyceps species.

2. Description of the Prior Art

In the literatures, the study of Cordyceps sinensis is only limited on species collection, description, and identification. For some Cordyceps sinensis with medical value, the research can only be restricted in the analysis of active ingredients metabolized therefrom for therapy purposes. However, due to the unclearness in the sexuality and the life cycle of the Cordyceps sinensis and the related species and due to the collection and storage difficulty, cultivating a stroma is still hard to achieve. Therefore, a clear picture in classification and a genuine relationship between the sex generation and the sexless generation can't be clearly understood so far. Such an unclearness in understanding genuine Cordyceps sinensis makes dangerous of wide-spreading usage upon so-called healthy Cordyceps sinensis' products in Chinese communities all over the world. It is quite possible that the manufacturers use fake Cordyceps sinensis to produce the products, or the customers have the so-called healthy products without active ingredients of the Cordyceps sinensis . Either of them detours a positive cycle in using the Cordyceps sinensis and makes less benefit from using the Cordyceps sinensis.

SUMMARY OF THE INVENTION

In view of lacking a standard process to identify real Cordyceps sinensis , the present invention introduces a new methodology of distinguishing Cordyceps sinensis by rRNA gene analysis. In a prior art, a 18S rRNA gene is successfully used to distinguish various fungi. Therefore, the gene analysis of the present invention also focuses on the 18S rRNA gene. Various specimens of candidate Cordyceps sinensis are collected at different locations and timings so that the characteristics of those candidate Cordyceps sinenis can be clearly observed. Further, specimens used in the present invention also include other Cordyceps, so-called Cordyceps sinensis reserved in some fungi centers, and candidate Cordyceps identified to be relatives of Cordyceps sinensis by a Gen Bank. By analyzing the data sorting from those candidate Cordyceps sinensis , the exclusive characteristics of a genuine Cordyceps sinensis can then be obtained.

According to the present invention, DNA extracts of all specimens are sent to PCR amplification of the 18S rRNA gene by well-known primer pairs as NS1(SEQ ID NO: 23)/NS2, NS3(SEQ ID NO: 24)/NS4(SEQ ID NO: 25), NS5/NS6(SEQ ID NO: 27) and NS7(SEQ ID NO: 28)/NS8(SEQ ID NO: 29). The gene sequences of the products from the PCR amplification are then determined by an automatic sequencing device. Then, comparisons between a genuine Cordyceps sinensis specimen and those candidate Cordyceps sinensis are carried out according to gene sequences. By the gene sequences, it is found that the 18S rRNA gene sequence of the genuine Cordyceps sinensis between primer pair NS3/NS6 is particularly different to those observed in relative Cordyceps. Based on the target sequence, a real Cordyceps sinensis can be easily determined. That is, the target sequence can be used as the flag to distinguish the Cordyceps sinensis . Further, after the 18S rRNA gene sequence between primer pair NS3/NS6 is determined as the flag to distinguish the Cordyceps sinensis , any fungus can be distinguished by amplifying its DNA extract by primer pairs NS3/NS4 and NS5/NS6 to determine the related PCR reaction of its 18S rRNA gene, by locating a gene sequence upon the PCR product with respect to the target gene sequence, and by comparing the gene sequence with the target gene sequence of the genuine Cordyceps sinensis . Following are operational embodiments upon above specimens. In the analysis, the 18S rRNA gene sequence between primer pairs NS1/NS2 and NS&/NS8 is not listed due to its vague role in the distinguishing process.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

The present invention will now be specified with reference to its preferred embodiments illustrated in the Tables and drawings, in which

FIG. 1 shows locations of primer pairs in the 18S rRNA genes;

FIG. 2 shows part of 18S rRNA gene sequences (SEQ ID NO: 3) in Table 5 between the primer pair NS3/NS4;

FIG. 3 shows part of 18S rRNA gene sequences (SEQ ID NO: 4)in Table 5 between the primer pair NS5/NS6;

FIG. 4 shows part of 18S rRNA gene sequences (Lines 1 - 8 , SEQ ID NO: 5; Lines 9 , 10 , 13 , SEQ ID NO:6; Line 11 , SEQ ID NO:7; Line 12 , SEQ ID NO:8; Line 14 , SEQ ID NO:9; Line 15 , SEQ ID NO:10; Lines 16 - 18 , SEQ ID NO:11) of Cordyceps strains between the primer pair NS3/NS4;

FIG. 5 shows part of 18S rRNA gene sequences (Lines 1 - 5 , SEQ ID NO: 12; Line 6 , SEQ ID NO:13; Line 7 , SEQ ID NO:14; Line 8 , SEQ ID NO:15; Line 9 , SEQ ID NO:16; Line 10 , SEQ ID NO:17; Line 11 , SEQ ID NO:18, Line 12 , SEQ ID NO:19; Line 13 , SEQ ID NO:20; Line 14 , SEQ ID NO:21; Line 15 , SEQ ID NO:22) of Cordyceps strains between the primer pair NS5/NS6;

FIG. 6 is the phylogenetic relationship of Table 8;

FIG. 7 is the phylogenetic relationship of Table 9;

FIG. 8 is the phylogenetic relationship of Table 10; and

FIG. 9 shows the target 18S rRNA gene sequence (SEQ ID NOS; 1-2) of a genuine Cordyceps sinensis between the primer pair NS3/NS6.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention disclosed herein is directed to a method for distinguishing Cordyceps sinensis . In the following description, numerous details are set forth in order to provide a thorough understanding of the present invention. It will be appreciated by one skilled in the art that variations of these specific details are possible while still achieving the results of the present invention. In other instances, well-known components are not described in detail in order not to unnecessarily obscure the present invention.

Table 1 to Table 3 list specimens of Cordyceps sinensis , specimens of Cordyceps and strains of Cordyceps used in the present invention. In Table 1, each specimen of Cordyceps sinensis is further divided into a sclerotium part and a stroma part. Thereby, the difference between the sclerotium and the stroma in the same specimen can be identified. Firstly, each specimen is cultivated for extracting DNA. The DNA is then PCR amplified separately by various primer pairs listed in Table 4 for obtaining the 18S rRNA gene. Refer to FIG. 1 for correct action positions of respective primer pairs upon 18S rRNA gene. While PCR amplifying in accordance with the present invention, the reaction conditions are 2 minutes at 98° C. for the initial denaturing temperature, 45 seconds at 95° C. for the denaturing temperature, 45 seconds at 52° C. for the annealing denaturing temperature, 35 cycles under a 2-minute 72° C. extension temperature per cycle, and finally 10 minutes at 72° C. for the final denaturing temperature. The product after PCR amplification is then gone through a high pure PCR product purification kit for purification, gene ordered by an Applied Biosystems 373 DNA sequencer, and analyzed by a tag dye deoxy terminor cycle sequencing kit. The ordered gene sequence is then reported to the European Molecular Biology Laboratory for acquiring an accession number. The corresponding accession numbers for specimens listed in Tables 1, 2, and are shown in Tables 5, 6, and 7 respectively. Primer pairs NS3, NS4 & NS5, and NS6 are recorded separately in Tables 5, 6 and 7 respectively. In Table 2, the famous Saccharomyces cerevisiae is already known so that no new accession numbers is recorded in Table 6.

It is obvious that the complete 18S rRNA gene sequences of a Cordyceps sinensis specimen are extremely long and only part of the sequences are helpful for identifying a genuine Cordyceps sinensis . After the analysis of the present invention, it is found that only the 18S rRNA gene sequences between the primer pairs NS3/NS4 and NS5/NS6 are valuable for the identification. FIG. 2 shows part of 18S rRNA gene sequences in Table 5 between the primer pair NS3/NS4. From columns 1 to 16 are listed specimens symbolized as Cs824af, Cs824b, W1023f, Cs7528A1, Cs7528A2, Cs7528Jf, Cs7528Jh, Cs7528Hf, Cs7528Hh, T1023f, S1023f, H1023f, K1023f, Cs1014df, Cs824ab, and Cs824ab respectively. FIG. 3 shows part of 18S rRNA gene sequences in Table 5 between the primer pair NS5/NS6. From columns 1 to 15 are listed specimens symbolized as Cs824af, Cs824ab, W1023f, Cs7528A1, Cs7528A2, Cs7528Jf, Cs7528Jh, Cs7528Hf, Cs7528Hh, T1023f, S1023f, H1023f, K1023f, Cs1014df, and Cs1014db respectively. As shown in FIG. 2 and FIG. 3 , all the Cordyceps sinensis specimens have the same 18S rRNA gene sequences between the primer pairs NS3/NS6. It is thus proved that all these wild Cordyceps sinensis specimens of Table 1 are originated from the same fungi species, even the collection source and timing are different. Also, it is proved that sclerotium and the stroma of the Cordyceps sinensis specimen keep the same 18S rRNA gene sequences, so that they are originated from the same fungi species.

According to the present invention, the nature of the Cordyceps sinensis and the 18S rRNA gene sequences between the primer pairs NS3/NS6 can be observed from FIG. 2 and FIG. 3 . However, it is doubtful that such evidence of gene sequences is sufficient to distinguish the genuine Cordyceps sinensis with other species. Further comparison upon the 18S rRNA gene sequences between the primer pairs NS3/NS6 among various specimens is still necessary. Aforesaid gene sequences are shown in FIG. 4 and FIG. 5 . FIG. 4 shows part of 18S rRNA gene sequences of Cordyceps strains between the primer pair NS3/NS4, and FIG. 5 shows part of 18S rRNA gene sequences of Cordyceps strains between the primer pair NS5/NS6. From columns 1 to 18 of FIG. 4 are listed specimens symbolized as C. Sinensis Cs824af, Cs7528A1, Cs7528Jf, Cs7528Hh, T1023f, H1023f, C. liangshanensis CC1014af, C. militaris Cm824a, LM1207f, P. ninchukispora CCRC 31900, C. memorabilis ATCC 36743, C. militaris ATCC 26848, C. ophioglossoides ATCC 36865, Cordyceps sp. ATCC 36337, C. sinensis CCRC 36421, C. capitata (from Gen Bank), and C. ophioglossoides (from Gen Bank) respectively. From FIG. 4 , difference in gene sequences between the genuine Cordyceps sinensis and other species can be located. Because only part of 18S rRNA gene sequences are shown in FIG. 4 , gene sequences of ATCC 36337 and CCRC 36421 are similar to those of a genuine Cordyceps sinensis . In column 8, gene sequences of CC1014af are identical to those of a genuine Cordyceps sinensis . Question arises now that if the 18S rRNA gene sequences between the primer pairs NS3/NS6 can be used as a flag to identify a genuine Cordyceps sinensis . However, according to the observation of the present invention upon the whole gene sequences, the interested part of gene sequences have 570 base pairs. Comparing these base pairs, the ATCC 36337 has 16 base pairs different to the respective pairs of the genuine Cordyceps sinensis , and the CC1014af has 4 base pairs different to the respective pairs of the genuine Cordyceps sinensis . Therefore, even though the gene sequences can be highly coherent between the genuine Cordyceps sinensis with some fungi species, difference among the 18S rRNA gene sequences between the primer pairs NS3/NS4 still exists therebetween and is sufficient to distinguish a genuine Cordyceps sinensis .

FIG. 5 shows part of 18S rRNA gene sequences of Cordyceps strains between the primer pair NS5/NS6. From columns 1 to 15 of FIG. 5 are listed specimens symbolized as C. Sinensis Cs7528A1, Cs7528Jf, Cs7528Hh, S1023f, H1023f, C. liangshanensis CC1014af, cc1014ab, C. militaris Cm824a, LM1207f, P. ninchukispora CCRC 31900, C. memorabilis ATCC 36743, C. militaris ATCC 26848, C. ophioglossoides ATCC 36865, Cordyceps sp. ATCC 36337, and C. sinensis CCRC 36421 respectively. From FIG. 5 , it can be observed that CC1014af and CC1014ab have most similar gene sequences with the genuine Cordyceps sinensis . However, among the 290 base pairs of the interested part of the 18S rRNA gene sequences between the primer pairs NS5/NS6, 14 base pairs of CC1014af and CC1014ab exist to be different to the respective base pairs of the genuine Cordyceps sinensis . So, difference between the genuine Cordyceps sinensis with some fungi species can still be told. From the gene sequences shown in FIG. 4 and FIG. 5 , a conclusion can be made that the 18S rRNA gene sequences between the primer pairs NS3/NS6 do present important features suitable for being used as a flag to distinguish a genuine Cordyceps sinensis among fungi species.

By providing the aforesaid methodology of the present invention, a genuine Cordyceps sinensis can be told by judging the 18S rRNA gene sequences between the primer pairs NS3/NS6. Following will verify the species of the Cordrceps sinensis by analyzing the phylogenetic relationship, for furier ascertaining the application of the present invention. The analysis will focus on the relationship study of Cordyceps listed in Tables 1-3 and those in the Gen bank. As listed in Table 8, the 18S rRNA gene sequences of Cordyceps sinensis between the primer pairs NS3/NS6 are ordered according to the Gen bank by an Applied Biosystems 373 DNA sequencer. Then, the S. cerevisiae is applied as the out group to calculate the phylogenetic relationship by PAUP4.0 (Phylogenetic Analysis Using Parsimony 4.0) and the phylogenetic relationship is established by Tree View 3.0 (Diving of Environmental and Evolutionary Biology IBLS). Observed from FIG. 6 , it is verified that all Hypocreales including the Cordyceps sinensis are unique grouped. In addition, the fungi listed in Tables 9 and 10 are also ordered and analyzed to have the phylogenetic relationships shown in FIG. 7 and FIG. 8 , respectively. In FIG. 7 , an H. luta is used as the out group to analyze the 18S rRNA gene sequences between the primer pairs NS3/NS4. On the other hand, in FIG. 8 , an A. terveus is used as the out group to analyze the 18S rRNA gene sequences between the primer pairs NS5/NS6. As shown in FIG. 7 and FIG. 8 , all the specimens of the present invention provided upon different sources and timings belong to the same group, so that a unique species is verified. Though the C. liangshanensis has the closer relationship with the genuine Cordyceps sinensis , yet small difference in between can still be told. By providing the results shown from FIG. 6 to FIG. 8 , it is proved that the specimens of the present invention are all genuine Cordyceps sinensis , even from different sources.

According to the aforesaid description, it is further verified that the 18S rRNA gene sequences between the primer pairs NS3/NS6 shown in FIG. 9 , in accordance with the present invention can be used as a flag to determine a genuine Cordyceps sinsis.

While the present invention has been particularly shown and described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be without departing from the spirit and scope of the present invention.

TABLE 1
The specimens of Cordyceps sinensis used in this invention.
	Specimen	Tissue used to
Species	No.	prepare DNA	Source	Location	Date
Cordyceps	Cs1014df	Stroma		Obtained from Nantong Tonghui Edible	Oct. 1994
sinensis				Fungi Trading Center of Jiangsu, China
C. sinensis	Cs1014db	Sclerotium		Obtained from Nantong Tonghui Edible	Oct. 1994
				Fungi Trading Center of Jiangsu, China
C. sinensis	Cs824af	Sclerotium	Sze Chuan, China	Purchased at Chinese drug store in Taipei	Oct. 1995
C. sinensis	C5824ab	Sclerotium	Sze Chuan, China	Purchased at Chinese drug store in Taipei	Oct. 1995
C. sinensis	Cs824b	Sclerotium	Sze Chuan, China	Purchased at Chinese drug store in Taipei	Oct. 1995
C. sinensis	Cs7528A1	Sclerotium	Tibet, China	Collected in Tibet, China	May 1997
C. sinensis	Cs7528A2	Stroma	Tibet, China	Collected in Tibet, China	May 1997
C. sinensis	Cs7528Jf	Stroma		Purchased at Chinese drug store in Taipei	May 1997
C. sinensis	Cs7528Jh	Sclerotium		Purchased at Chinese drug store in Taipei	May 1997
		(head)
C. sinensis	Cs7528Hf	Stroma		Purchased at Chinese drug store in Taipei	May 1997
C. sinensis	Cs7528Hh	Sclerotium		Purchased at Chinese drug store in Beijing	May 1997
		(head)
C. sinensis	W1023f	Stroma	Sze Chuan, China	Purchased at Chinese drug store in Taipei	Sep. 1996
C. sinensis	S1023f	Stroma	Tibet, China	Purchased at Chinese drug store in Taipei	Oct. 1998
C. sinensis	T1023f	Stroma	Qinghai, China	Purchased at Chinese drug store in Taipei	Oct. 1998
C. sinensis	H1023f	Stroma	Qinghai, China	Purchased at Chinese drug store in Qinghai,	Oct. 1998
				China
C. sinensis	K1023f	Stroma		Purchased at Chinese drug store in Taipei	Oct. 1998

TABLE 2
The Cordyceps spp. specimens and Saccharomyces cerevisiae used in this invention.
		Tissue used to
Species	Specimen No.	prepare DNA	Source	Location	Date
Cordyceps militaris	Cm824a	stroma	China	purchased at Chinese drug store in	May 1997
				Taipei
C. militaris	Cm1014c	stroma	China	purchased at Chinese drug store in	May 1997
				Taipei
C. militaris	LM1207f	stroma	China	obtained from Sericultural Science	May 1997
				Research Institute of Jilin, China
C. liangshanensis	CC1014af	stroma	China
C. liangshanensis	CC1014ab	sclerotium	China
Saccharomyces	Y824a	cells	this laboratory	purchased in Taipei	1991
cerevisiae

TABLE 3
The Cordyceps strains and reference strains used
in this invention.
Species	Collection No.	Source
Cordyceps memorabilis	ATCC 36743	American Type Culture
		Collection, U.S.A.
C. militaris	ATCC 26848	American Type Culture
		Collection, U.S.A.
C. ophioglossoides	ATCC 36865	American Type Culture
		Collection, U.S.A.
Cordyceps sp.	ATCC 36337	American Type Culture
		Collection, U.S.A.
C. sinensis	CCRC 36421	Culture Collection &
		Research Center,
		Hsinchu, Taiwan.
Phytocordyceps	CCRC 31900	Culture Collection &
ninchukispora		Research Center,
(reference species)		Hsinchu, Taiwan.

TABLE 4
List of primers used in this invention.
Primer
designation	Primer sequences (5′→3′)	Position
N S 1	GTAGTCATATGCTTGTCTC	18S rRNA gene 1-19
N S 3	GCAAGTCTGGTGCCAGCAGCC	18S rRNA gene 553-573
N S 4	CTTCCGTCAATTCCTTTAAG	18S rRNA gene 1131-1150
N S 5	AACTTAAAGGAATTGACGGAAG	18S rRNA gene 1131-1148
N S 6	GCATCACAGACCTGTTATTGCCTC	18S rRNA gene 1413-1435
N S 7	GAGGCAATAACAGGTCTGTGATGC	18S rRNA gene 1413-1436
N S 8	TCCGCAGGTTCACCTACGGA	18S rRNA gene 1790-1810

TABLE 5
The 18S rRNA gene sequence accession numbers
of Cordyceps sinensis specimens.
			NS3,4	NS5,6
	Species	Specimen No.	Acc. No.	Acc. No.
Cordyceps sinensis	Cs1014df	◯	◯
C. sinensis	Cs1014db	◯	◯
C. sinensis	Cs824af	AJ238505	◯
C. sinensis	Cs824ab	◯	◯
C. sinensis	Cs824b	AJ238506	◯
C. sinensis	Cs7528A1	AJ009676	AJ007566
C. sinensis	Cs7528A2	AJ009677	AJ007567
C. sinensis	Cs7528Jf	AJ009678	AJ007568
C. sinensis	Cs7528Th	AJ009679	AJ007569
C. sinensis	Cs7528Hf	AJ238504	◯
C. sinensis	Cs7S28Hh	AJ238689	◯
C. sinensis	W1023f	AJ238690	◯
C. sinensis	T1023f	AJ238691	◯
C. sinensis	S1023f	AJ238691	◯
C. sinensis	H1023f	AJ238693	◯
C. sinensis	K1023f	AJ238692	◯
◯: Pending

TABLE 6
The 18S rRNA gene sequence accession numbers
of Cordyceps spp. specimens.
		Specimen	NS3,4	NS5,6
	Species	No.	Acc. No.	Acc. No.
	Cordyceps militaris	Cm824a	AJ009682	AJ007571
	C. militaris	Cm1014C	AJ009683	AJ242435
	C. militaris	LM1207f	AJ009681	AJ242436
	C. liangshanensis	CC1014af	AJ238503	AJ239070
	C. liangshanensis	CC1014ab	AJ238503	AJ239071

TABLE 7
The 18S rRNA gene sequence accession numbers
of Cordyceps spp. strains.
	Collection	NS3,4	NS5,6
Species	No.	Acc. No.	Acc. No.
Cordyceps memorabilis	ATCC 36743	AJ238501	AJ242432
C. militaris	ATCC 26848	AJ238500	AJ242430
C. ophioglossoides	ATCC 36865	AJ238498	AJ242431
Cordyceps sp.	ATCC 36337	AJ242429	AJ242433
Phytocordyceps	CCRC 31900	AJ238499	AJ242434
ninchukispora
C. sinensis	CCRC 36421	AJ238502	AJ239072
C. sinensis	CsRSbH1	AJ009680	AJ067570
C. sinensis	RS2	AJ133815	AJ242427
C. sinensis	RS3	AJ238685	◯
C. sinensis	RS4-2	AJ238686	◯
C. sinensis	RS5-2	AJ238687	AJ242427
C. sinensis	RS6-3	AJ238688	AJ242428
C. sinensis	MCs1014b	AJ238496	AJ242437
C. sinensis	MCs119	AJ238497	◯
◯: Pending

TABLE 8
The species and sequences accession numbers used
in FIG. 6.
	Accession
Species	Number	Order	Source
Cordyceps sinensis	AJ009676	Hypocreales	inventor
Cs7528A1
C. sinensis	AJ238502	Hypocreales	inventor
CCRC 36421
C. ophioglossoides	AJ238498	Hypocreales	inventor
ATCC 36865
C. memorabilis	AJ238501	Hypocreales	inventor
ATCC 36743
C. militaris	AJ009683	Hypocreales	inventor
ATCC 26848
C. liangshanensis	AJ238503	Hypocreales	inventor
CC1014af
P. ninchukispora	AF238499	Hypocreales	inventor
CCRC 31900
Saccharomyces	J01353	Saccharomycetales	GenBank
cerevisiae
Gaeumannomyces	AF050488	Diapothales	GenBank
graminis
Hypoxylon atroroseum	U32411	Xylariales	GenBank
Daldinia concentrica	U47828	Xylariales	GenBank
Sclerotinia sclerotiorum	X69850	Leotiales	GenBank
Neurospora crassa	NCRRNAS	Sordariales	GenBank
Aspergillus terreus	AB008409	Eurotiales	GenBank
Claviceps purpurea	U44040	Hypocreales	GenBank
Claviceps paspali	U32401	Hypocreales	GenBank
Hypomyces polyporius	U32410	Hypocreales	GenBank
Sphaerostilbella	U32415	Hypocreales	GenBank
aureonitens
Neocosmospora	U32414	Hypocreales	GenBank
vasinfecta
Melanospora zamiae	U78356	Melanosporales	GenBank
Hypocrea lutea	U00IFOC	Hypocreales	GenBank
Cordyceps capitata	U44041	Hypocreales	GenBank
Cordyceps	U46881	Hypocreales	GenBank
ophioglossoides
Xylaria carpophila	Z49785	Xylariales	GenBank
Xylaria hypoxhlon	U20378	Xylariales	GenBank

TABLE 9
The species and sequences accession numbers used
in FIG. 7.
	Accession
Species	Number	Source
Cordyceps sinensis Cs7528A1	AJ009676	inventor
C. sinensis Cs7528Jf	AJ009678	inventor
C. sinensis Cs7528Hf	AJ238504	inventor
C. sinensis Cs824b	AJ238506	inventor
C. sinensis W1023f	AJ238690	inventor
C. sinensis K1023f	AJ238692	inventor
C. sinensis sisH1023f	AJ238693	inventor
C. sinensis CCRC 36421	AJ238502	inventor
C. ophioglossoides ATCC 36865	AJ238498	inventor
C. memorabilis ATCC 36743	AJ238501	inventor
Cordyceps sp. ATCC 36337	AJ242429	inventor
C. militaris ATCC 26848	AJ009683	inventor
C. militaris Cm824a	AJ009682	inventor
C. militaris LM1207f	AJ009681	inventor
C.liangshanensis CC1014af	AJ238503	inventor
P. ninchukispora CCRC 31900	AJ238499	inventor
Hypocrea lutea	U00IFOC	GenBank
0 capitata	U44041	GenBank
C. ophioglossoides	U46881	GenBank

TABLE 10
The species and sequences accession numbers used
in FIG. 8.
	Accession
Species	Number	Order	Source
Cordyceps sinensis	AJ007566	Hypocreales	inventor
Cs7528A1
C. sinensis Cs7528Jf	AJ007568	Hypocreales	inventor
C. sinensis W1023f	◯	Hypocreales	inventor
C. sinensis K1023f	◯	Hypocreales	inventor
C. sinensis S1023f	◯	Hypocreales	inventor
C. sinensis	AJ239072	Hypocreales	inventor
CCRC 36421
C. ophioglossoides	AJ242431	Hypocreales	inventor
ATCC 36865
C. memorabilis	AJ242432	Hypocreales	inventor
ATCC 36743
Cordyceps sp.	AJ242433	Hypocreales	inventor
ATCC 36337
C. militaris	AJ242430	Hypocreales	inventor
ATCC 26848
C. militaris Cm824a	AJ007571	Hypocreales	inventor
C. militaris LM1207f	AJ242436	Hypocreales	inventor
C. liangshanensis	AJ239070	Hypocreales	inventor
CC1014af
C. liangshanensis	AJ239071	Hypocreales	inventor
CC1014ab
P. ninchukispora	AJ242434	Hypocreales	inventor
CCRC 31900
Hypocrea lutea	U00IFOC	Hypocreales	GenBank
Saccharomyces	J01353	Saccharo-	GenBank
cerevisiae		mycetales
Sclerotinia	X69850	Leotiales	GenBank
sclerotiorum
Neurnspora crassa	NCRRNAS	Sordariales	GenBank
Aspergillus terreus	AB008409	Eurotiales	GenBank
◯: Pending

29 1 579 DNA Cordyceps sinensis 1tctggtgcca gcagccgcgg taattccagc tccaatagcg tatattaaag ttgttgtggt 60taaaaagctc gtagttgaac cttgggcctg gctggccggt ccgcctcacc gcgtgtactg 120gtccggccgg gcctttccct ctgtggaacc ccatgccctt cactgggcgt ggcggggaaa 180caggactttt actttgaaaa aattagagtg ctccaggcag gcctatgctc gaatacatta 240gcatggaata atgaaatagg acgcgcggtt ctattttgtt ggtttctagg accgccgtaa 300tgattaatag ggacagtcgg gggcatcagt attcaatggt cagaggtgaa attcttggat 360ccattgaaga ctaactactg cgaaagcatt tgtcaaggat gttttcatta atcaggaacg 420aaagttaggg gatcgaagac gatcagatac cgtcgtagtc ttaaccataa actatgccga 480ctagggatcg gacgatgtta ttttttgact cgttcggcac cttacgagaa atcaaagtgc 540ttgggctcca gggggagtat ggtcgcaagg ctgaaactt 579 2 370 DNA Cordyceps sinensis 2aataacaggt ctgtgatgcc cttagatgtt ctgggccgca cgcgcgctac actgacggag 60ccagcgagtc ctcccttggc cggaaggccc gggtaatctt gttaaacttc gtcgtgctgg 120ggatagagca ttgcaattat tgctcttcaa cgaggaatcc ctagtaagcg caagtcatca 180gcttgcgttg actacgtccc tgccctttgt acacaccgcc cgtcgctact accgattgaa 240tggctcagtg aggcgtccgg actggcccag gggggtggga aaccgccccc cagggccggg 300aagctctcca aactcggtca tttagaggaa gtaaaagtcg taacaaggtc tccgtaggtg 360aacctgcgga 370 3 58 DNA Cordyceps sinensis misc_feature (1)..(58) Fig. 2 lines 1-16. 3atacattagc atggaataat gaaataggac gcgcggttct attttgttgg tttctagg 58 4 57 DNA Cordyceps sinensis misc_feature (1)..(57) Fig. 3, lines 1-15. 4gcccgtactg ctccggcagt gcgccggctt cttagaggga ctatcggctc aagccga 57 5 58 DNA Cordyceps sinensis misc_feature (1)..(58) Fig. 4, lines 1-8. 5tacattagca tggaataatg aaataggacg cgcggttcta ttttgttggt ttctagga 58 6 58 DNA Cordyceps militaris misc_feature (1)..(58) Fig. 4, lines 9-10, 13. 6tacattagca tggaataata aaataggacg cgtggttcta ttttgttggt ttctagga 58 7 58 DNA Cordyceps memorabilis 7tacattagca tggaataatg aaataggacg cgtggttcta ttttgttggt ttctagga 58 8 58 DNA Cordyceps militaris 8tacattagca tggaataata aaataggacg tgtggttcta ttttgttggt ttctagga 58 9 58 DNA Cordyceps sp. 9tacattagca tggaataatg aaataggacg tgcggttcta ttttgttggt ttctagga 58 10 58 DNA Cordyceps sinensis 10tacattagca tggaataata aaataggacg cgcggttcta ttttgttggt ttctagga 58 11 58 DNA Cordyceps capitata misc_feature (1)..(58) Fig. 4, lines 16-18. 11tacattagca tggaataatg aaataggacg tgcggttcta ttttgttggt ttctagga 58 12 52 DNA Cordyceps sinensis misc_feature (1)..(52) Fig. 5, lines 1-5. 12atagcccgta ctgctccggc agtgcgccgg cttcttagag ggactatcgg ct 52 13 52 DNA Cordyceps liangshanensis 13atagcccgcc ctgctccggc ggcgcgccgg ttttttagag ggactatcgg tt 52 14 52 DNA Cordyceps liangshanensis 14ttacccggcc ctgctccggc ggcccgccgg tttttaagag ggactttggg tt 52 15 52 DNA Cordyceps militaris 15atagcctgta ttgctttggc agtacgctgg cttcttaaag ggactatcgg ct 52 16 52 DNA Cordyceps militaris 16atagcctgta ttgctttggc agtacgctgg cttcttagag ggactatcgg ct 52 17 52 DNA Phytocordyceps ninchukispora 17atagcccgta ttgctttggc agtacgccgg cttcttagag ggactatcgg ct 52 18 52 DNA Cordyceps memorabilis 18ctagcccgta ttgctttggc agtacgctgg cttcttagag ggactatcgg ct 52 19 52 DNA Cordyceps militaris 19atagcctgta ttgctttggc agtacgctgg cttcttagag ggactatcgg ct 52 20 52 DNA Cordyceps ophioglossoides 20atagcccgta ttgctttggc agtacgctgg cttcttagag ggactatcgg ct 52 21 52 DNA Cordyceps sp. 21atagtcagta ttgctatggc agtacgcggg cttcttagag ggactatcgg ct 52 22 52 DNA Cordyceps sinensis 22atagcccgta ttgctttggc agtacgctgg cttcttagag ggactatcgg ct 52 23 19 DNA primer NS1 23gtagtcatat gcttgtctc 19 24 21 DNA primer NS3 24gcaagtctgg tgccagcagc c 21 25 20 DNA primer NS4 25cttccgtcaa ttcctttaag 20 26 22 DNA primer NS5 26aacttaaagg aattgacgga ag 22 27 24 DNA primer NS6 27gcatcacaga cctgttattg cctc 24 28 24 DNA primer NS7 28gaggcaataa caggtctgtg atgc 24 29 20 DNA primer NS8 29tccgcaggtt cacctacgga 20

Claims

1. Gene sequences for distinguishing Cordyceps sinensis, by judging a PCR-amplified 18S rRNA gene between primer pairs NS3/NS6, comprising:the DNA sequence (SEQ ID NO: 1) between the primer pairs NS3/NS4; and the DNA sequence (SEQ ID NO: 2) between the primer pairs NS5/NS6.

2. A method for distinguishing Cordyceps sinensis by:obtaining an 18S rRNA gene by PCR; amplifying an extracted DNA from a specimen through primer pairs NS3/NS4 and NS5/NS6; applying a DNA sequencer to order gene sequences of PCR amplified products; and comparing the gene sequences of the PCR amplified products with the gene sequences of claims to determine whether the specimen is a genuine Cordyceps sinensis.

3. The method of claim 2, wherein the DNA sequencer is an Applied Biosystems 373 DNA sequencer.