Method for identifying cordyceps sinenis

Abstract

The present invention is drawn to a method for identifying Cordyceps sinensis, by amplifying a specimen's 18S rRNA polymorphism by PCR using primer pair NS3 and NS6; digesting the PCR product with restriction enzyme Cfo I; and identifying a genuine Cordyceps sinensis specimen by determining the presence of a PCR product digestible with the restrictions enzyme Cfo I and a DNA fragment in the polymorphism of the specimen belonging to a specific DNA fragment in the polymorphism of Cordyceps sinensis.

Description

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The invention relates to a method for distinguishing Cordyceps sinensis , and more particularly to a laser level apparatus that is easy to use and has multipurpose uses.

(2) Description of the Prior Art

Cordyceps sinensis ( C. sinensis ) is a complex having a sclerotium and a stroma and is grown from a bug-parasitized fungus that parasitizes in a worm of hepialus armoricanus oberthur. According to the Alexopoulos classification of 1996 , C. sinensis belongs to the Cordyceps Fr of the Clavicipiyaceae of the Clavicipitales of Ascomycetes. Currently, there are about 400 species found on the planet, which belong to the Cordyceps Fr genus. Among those species, few possess medical ingredients; such as C. sinensis, C. sobolifera, C. militaris, C. hawkesii , and so on. Therein, C. sinensis is the most popular one. After pharmacological study, C. sinensis was found to have active ingredients for curing tumors, kidney inflammation and aging, and for improving immunizability. Therefore, C. sinensis has become one of the important topics with studies of medical fumgi.

Due to the important medical potential of C. sinensis , healthy foods containing C. sinensis ingredients are popular products for health-care and age-care. However, while C. sinensis is widely accepted, questions arise to what C. sinensis really is. What are actual ingredients of a C. sinensis ? What characteristics does a genuine C. sinensis have? How to identify a real C. sinensis ? So far, no assured answer for any of the preceding questions is provided. Until now, people studying C. sinensis or the related Cordyceps can only work on the species collection, description, and identification. Regarding the medical potential of C. sinensis , “existing some metabolic products with some active ingredients for disease-prevention” is the only conclusion that can be provided. Of course, in some literatures, some efforts have been made to further understand the aspect of the Cordyceps, particularly C. sinensis , in view of the nature variety of the sexless Cordyceps. However, due to the collection difficulty, identification and reservation of C. sinensis , and also due to the difficulty in artificially cultivating the stroma, a clear picture in classification and a genuine relationship between the sex generation and the sexless generation has not been found so far. Hence, the confusion in standardizedly distinguishing C. sinensis is still there.

SUMMARY OF THE INVENTION

Since current classification methodologies by configuration, by physiology, or by biochemistry cannot provide a satisfactory and accurate classification of C. sinensis ; a real C. sinensis can't be certainly identified. Therefore, a new method in classifying the Cordyceps is urgently needed. Accordingly, the present invention teaches a method for distinguishing Cordyceps sinensis . In view of gene ordering, the difference between C. sinensis and other Cordyceps can be determined. Further, the method of the present invention can be applied to distinguish so-called C. sinensis or fungi in the market or in the fungi centers. By providing the method of the present invention, specific nature characteristics for C. sinensis can be defined. Thereby, a standard and wide-acceptable distinguishing method for C. sinensis can be provided.

According to the present invention, the rRNA gene is as an operational target for the distinguishing method, in which the rRNA is the most popular gene in species classification. The rRNA has advantages in a high copy number and a high-degree conservability of evolution. The method of the present invention is first to locate the 18SrRNA of the DNA extracted from a specimen or a fungus, and then to analysis the 18SrRNA by the PCR-RFLP technique widely used in identifying fungi. By using restriction enzyme Cfo I and digesting NS3 and NS6 as the primers, it is found in the digesting polymorphism of the amplification of 18S rRNA gene that C. sinensis opposes a total different DNA segment to any other sinensis. This finding can be applied to distinguish C. sinensis from other sinensises. Also, thereby, a distinguishing method for C. sinensis can be proposed. The method for distinguishing C. sinensis in accordance with the present invention can then become a standard and accurate guideline of identifying C. sinensis to the professionals and the related bureau.

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

Table 1 lists the source and collection number of Cordyceps specimens in this specification;

Table 2 lists the collection number of Cordyceps strains in this specification;

Table 3 lists the primers used in this specification. NS 1 =SEQ ID NO: 1. NS3 =SEQ ID NO:2. NS4 =SEQ ID NO:3. NS5 =SEQ ID NO:4. NS6 =SEQ ID NO:5.

Table 4 lists the restriction fragment sizes for amplification of Table 1 specimens' 18S rRNA gene by polymerase chain reaction (PCR) using primer pair NS1 and NS4, in which the product was further digested with restriction enzymes Hpa II, Rsa I, Hae III, Sau 3A and Cfo I;

Table 5 lists the restriction fragment sizes for amplification of Table 2 specimens' 18S rRNA gene by polymerase chain reaction (PCR) using primer pair NS1 and NS4, in which the product was further digested with restriction enzymes Hpa II, Rsa I, Hae III, Sau 3A and Cfo I;

Table 6 lists the restriction fragment sizes for amplification of Table 1 specimens' 18S rRNA gene by polymerase chain reaction (PCR) using primer pair NS3 and NS6, in which the product was further digested with restriction enzyme Cfo I;

Table 7 lists the restriction fragment sizes for amplification of Table 2 specimens' 18S rRNA gene by polymerase chain reaction (PCR) using primer pair NS3 and NS6, in which the product was further digested with restriction enzyme Cfo I; and

FIG. 1 shows the locations on nuclear rDNA of PCR primers, in which the arrowhead represent the 3′ end of each primer.

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.

To show the objectiveness of the method in accordance with the present invention, specimens for Cordyceps testing are collected from various sources. Except for C. sinensis, C. militaris and other Cordyceps, proved to have medical ingredients, are also collected for testing as references. It is believed that the specimen selection is justified and objective. Those specimens are listed in Tables 1 and 2. In Table 1 , C. sinensis and C. militaris are collected from different sources. Those specimens from the same source are further assigned to different numbers according to different tissues or fragments for extracting DNA; so that the nature of the sclerotium and the stroma from the same C. sinensis ' specimen can be identified. The intention of collecting a Saccharomyces cerevisiae as a reference in Table 1 is to determine the reliability of the DNA fragment in polymorphism for Cordyceps after restricted digestion, by referring to the well-known DNA fragment of the Saccharomyces cerevisiae. Table 2, as a comparison to Table 1, is organized to help understanding the nature difference among wild Cordyceps, cultivated Cordyceps, and Cordyceps strains from unknown source.

The target of the testing in accordance with the present invention is the DNA extracted from the specimens in Tables 1 and 2. However, some testing, are invalid due to too short fragments extracted; for example, the 5.8 S rRNA gene in FIG. 1 . Also, some testing are discarded, because the extracted fragments are too long to carry effectively amplification of PCR (Polymerase chain reaction). Therefore, the valid testing of the present invention are from those whose extracted fragments are more complete and appropriate in length for 18S rRNA analysis. Further, in each PCR, only limited primers are used for amplification; for example, NS 1 and NS4 in FIG. 1 . The gene sequences and position of each primer are listed in Table 3. In the following description, the testing of the present invention will focus at the PCR of the primer pairs, (NS 1+NS4) and (NS3+NS6).

Table 4 lists the DNA fragment sizes in polymorphism for amplification of Table 1 specimens' 18S rRNA gene by PCR using primer pair NS1 and NS4, in which the product was further digested with restriction enzymes Hpa II, Rsa I, Hae III, Sau 3A and Cfo I. As listed, each DNA fragment of the tested S. cerevisiae is shown to be the same as the known respective DNA fragment for the S. cerevisiae . That is to say that the testing results in DNA fragments for those Cordyceps specimens in Table 4 are all believable. Further, two facts are found among the DNA fragments in Table 4. One is that an identical gene polymorphism (i.e. DNA fragment) is found in all C. sinensis ' specimens, though from different source. So, that all those C. sinensis ' specimens belongs to the same species has been proved. Another is that the C. sinensis is originated from a single gene, for the founding that the stroma and the sclerotium of the same C. sinensis ' specimen oppose the same DNA fragment. Yet, in Table 4, it is determine to give up the testing by restriction enzyme Sau3A, for the fact in Table 4 that the restriction enzyme Sau3A cannot digest C. militaris (so, no DNA fragment is formed).

Table 5 lists the DNA fragment sizes in polymorphism for amplification of Table 2 specimens' 18S rRNA gene by PCR using primer pair NS1 and NS4, in which the product was further digested with restriction enzymes Hpa II, Rsa I, Hae III, Sau 3A and Cfo I. By comparing Table 5 with Table 4, it is found that only the Cordyceps numbered ATCC 36337 among all fungi of Table 5 carries the same DNA fragment as that for C. sinensis of Table 4. Therefore, the ATCC 36337 Cordyceps is proved to originate from the same species as C. sinensis does. In addition, it is also found that the polymorphism obtained from digested products by the restriction enzymes Hpa II, Rsa I and Hae III is not sufficient to distinguish the DNA fragments for ATCC 36337 and other specimens. Only the polymorphism obtained from digested products by the restriction enzyme Cfo I can be used to carry the distinguishing. Therefore, in all following testing, the restriction enzyme Cfo I is the only one used to digest the products.

Table 6 lists the DNA fragment sizes in polymorphism for amplification of Table 1 specimens' 18S rRNA gene by PCR using primer pair NS3 and NS6, in which the product was further digested with restriction enzyme Cfo I. Table 7 lists the DNA fragment sizes in polymorphism for amplification of Table 2 specimens' 18S rRNA gene by PCR using primer pair NS3 and NS6, in which the product was further digested with restriction enzyme Cfo I. From Table 5, it is proved that the polymorphism obtained from digested products by the restriction enzyme Cfo I can be used to distinguish accurately wild C. sinensis ' specimens (numbered Cs7528A1, Cs7528A2, Cs7528JF, Cs7528JH, Cs7528Jt, Cs1014df, and Cs1014db) from other Cordyceps. However, the founding that the ATCC36337 opposes the nature of a genuine C. sinensis can't be further confirmed by the testing results from a primer pair NS1 and NS4 in Tables 4 and 5. Therefore, in Tables 6 and 7, the primer pair NS3 and NS6 are used to amplify the 18S rRNA gene, and the PCR product is further digested with Cfo I. By investigating the digested DNA fragments, it is found that all wild C. sinensis have the same DNA fragment, as shown in Table 6. Further evidence from Table 4, it is then confirmed that all wild C. sinensis belong to the same species. On the other hand, in Table 7, the digested DNA fragment of the ATCC36337 by Cfo I is not the same as that of the wild C. sinensis . However, the DNA fragments for C. militaris of Table 7 (ATCC26848) and for C. militaris of Table 6 (Cm824a and LM1207f) are the same. This finding explains that the ATCC36337 is not a pure C. sinensis . Therefore, it is concluded that the difference in DNA fragment among C. sinensis and other Cordyceps can be quickly and accurately located by applying the polymorphism of the digested product with restriction enzyme Cfo I after using primer pair NS3 and NS6 in PCR. Hence, the method for distinguishing C. sinensis in accordance with the present invention can then become a strong reference to identifying the nature of C. sinensis.

While processing the PCR in the testing according to the present invention, reaction condition at each step is: to heat the target DNA at 98° C. for 2 minutes to reach the initial transformation stage, then to perform transforming reaction at 95° C. for 45 seconds, to refine at 95° C. for another 45 seconds, and then to reheat to 72° C. for 2-minute amplification; to repeat all above procedures 35 times; and, finally, to amplify again at 72° C. for 10 minutes.

In summary, the present invention provides an objective distinguishing method for C. sinensis , other than the conventional classification methodologies by configuration, by physiology, or by biochemistry. Therefore, by applying the distinguishing method of the present invention, the conventional obscurity in classifying the C. sinensis and other Cordyceps is then resolved.

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 source and collection number of Cordyceps specimens in this study.
Species	Collection No.	Tissue used to prepare DNA	Source
Cordyceps sinensis	Cs7528A1	Sclerotium	Collected in Tibet, China
C. sinensis	Cs7528A2	Stroma	Collected in Tibet, China
C. sinensis	Cs7528JF	Stroma	Purchased at Chinese
			drug store in Taipei
C. sinensis	Cs7528Jh	Sclerotium (head)	Purchased at Chinese
			drug store in Taipei
C. sinensi	Cs7528Jt	Sclerotium (tail)	Purchased at Chinese
			drug store in Taipei
C. sinensis	Cs1014df	Stroma	Obtained from Nantong
			Tonghui edible funguses
			trading center of
			Jiangsu, China
C. sinensis	Cs1014db	Sclerotium	Obtained from Nantong
			Tonghui edible funguses
			trading center of
			Jiangsu, China
C. militaris	Cm824a	Stroma	Purchased at Chinese
			drug store in Taipei
C. militaris	LM1207f	Stroma	Obtained from
			Sericulture Science
			Research Institute of
			Jilin, China
Saccharomyces cerevisiae	Y824a	Cells	This Laboratory

TABLE 2
The collection number of Cordyceps strains in this study.
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 ninchukispora	CCRC 31900	Culture Collection & Research
		Center, Hsinchu, Taiwan.
Aspergillus terreus	686	This laboratory
Ganoderma lucidum	950524	This laboratory

TABLE 3
Primer designation	Primer sequences	Position
N S 1	GTAGTCATATGCTTGTCTC	18S rDNA 1-19
N S 3	GCAAGTGTGGTGCCAGCAGCC	18S rDNA 553-573
N S 4	CTTCCGTCAATTCCTTTAAG	18S rDNA 1131-1150
N S 5	AACTTAAAGGAATTGACGGAAG	18 S rDNA 1129-1150
N S 6	GCATCACAGACCTGTTATTGCCTC	18 S rDNA 1413-1436

TABLE 4
Restriction enzyme
	Hpa II	Rsa I	Hae III	Sau 3A	Cfo I
Collection No.	(C ↓ CGG)	(GT ↓ AC)	(GG ↓ CC)	(↓ GATC)	(GCG ↓ C)
	Restriction fragment size
C. sinensis	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
Cs7528A1	170, 100, 60	180, 60	270	140	270, 150
Cs7528A2	500, 290	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180, 60	270	140	270, 150
Cs7528Jf	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180, 60	270	140	270, 150
Cs7528Jh	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180, 60	270	140	270, 150
Cs7528Jt	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180, 60	270	140	270, 150
Cs1014df	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180,60	270	140	270, 150
Cs1014db	500, 290,	517, 410,	400, 350,	900, 242,	400, 350,
	170, 100, 60	180, 60	270	140	270, 150
C. militaris	500, 290, 100	677, 410, 60	400, 350,	1000	750, 260,
Cm824a			270, 60		160
LM1207f	500, 290, 100	677, 410, 60	400, 350,	1000	750, 260,
			270, 60		160
S. cerevisiae	520, 390, 300	520, 190	653, 290, 170	1000, 150	750, 430
Y824a

	TABLE 5
	Restriction enzyme
	Hpa II	Rsa I	Hae III	Cfo I
Collection No.	(C ↓ CGG)	(GT ↓ CC)	(GG ↓ CC)	(GCG ↓ C)
	Restriction Fragment Size
C. memorabilis	500, 290,	517, 410,	400, 350	750, 260,
ATCC 36743	100	180, 60	270	160
C. militaris	500, 290, 100	677, 410, 60	400, 350, 270	750, 260, 160
ATCC 26848
C. ophioglossoides	500, 290, 100	517, 410, 180,	400, 350, 270	750, 260, 160
ATCC 36865		60
Cordyceps. sp.	500, 290, 100	517, 410, 180,	400, 350, 270	400, 350, 270,
ATCC 36337		60		150
C. sinensis	500, 290, 100	517, 410, 180,	400, 350, 270	750, 260, 160
CCRC 36421		60
P. ninchukispora	500, 290, 100	517, 410, 180,	400, 350, 270	750, 260, 160
CCRC 31900		60
A. terreus	500, 290, 100	517, 410, 180,	400, 350, 270	750, 260, 150
686	60	60
G. lucidum	500, 290, 170,	517, 410, 190,	400, 350, 270	750, 260, 150
950524	100, 80	50	100

	TABLE 6
			Cfo I digested restriction
	Collection No.	PCR product size	fragment size
	C. sinensis
	Cs7528A1	920	570, 300, 50
	Cs7528A2	920	570, 300, 50
	Cs7528Jf	920	570, 300, 50
	Cs7528Jh	920	570, 300, 50
	Cs7528Jt	920	570, 300, 50
	Cs1014df	920	570, 300, 50
	Cs1014db	920	570, 300, 50
	C. militaris
	Cm824a	1300	630, 310, 184, 90, 50
	LM1207f	1300	630, 310, 184, 90, 50
	S. cerevisiae	920	920
	Y824a

	TABLE 6
			Cfo I digested restriction
	Collection No.	PCR product size	fragment size
	C. sinensis
	Cs7528A1	920	570, 300, 50
	Cs7528A2	920	570, 300, 50
	Cs7528Jf	920	570, 300, 50
	Cs7528Jh	920	570, 300, 50
	Cs7528Jt	920	570, 300, 50
	Cs1014df	920	570, 300, 50
	Cs1014db	920	570, 300, 50
	C. militaris
	Cm824a	1300	630, 310, 184, 90, 50
	LM1207f	1300	630, 310, 184, 90, 50
	S. cerevisiae	920	920
	Y824a

5 1 19 DNA Artificial Sequence Description of Artificial Sequenceprimer NS1 1gtagtcatat gcttgtctc 19 2 21 DNA Artificial Sequence Description of Artificial Sequenceprimer NS3 2gcaagtctgg tgccagcagc c 21 3 20 DNA Artificial Sequence Description of Artificial Sequenceprimer NS4 3cttccgtcaa ttcctttaag 20 4 22 DNA Artificial Sequence Description of Artificial Sequenceprimer NS5 4aacttaaagg aattgacgga ag 22 5 24 DNA Artificial Sequence Description of Artificial Sequenceprimer NS6 5gcatcacaga cctgttattg cctc 24

Claims

1. A method for identifying Cordyceps sinensis, which comprisesextracting DNA from a Cordyceps specimen; amplifying the DNA encoding 18S rRNA by PCR wherein the DNA contains a restriction fragment length polymorphism and wherein the PCR is performed using primer pair NS3 and NS6; digesting the amplified DNA fragments with restriction enzyme Cfo I; determining the restriction fragment lengths of the digested DNA; and identifying the specimen as Cordyceps sinensis by the presence of polymorphic restriction fragments lengths of 570bp, 300bp and/or 50bp.

2. The method for identifying Cordyceps sinensis of claim 1, wherein said PCR is performed using the following reaction conditions:initial transformation 98° C. for 2 minutes; transformation, 95° C. for 45 seconds; refining, 52° C. for 45 seconds; amplifying, 72° C. for 2 minutes; repeating transformation, refining and amplification for 35 cycles; and final amplification, 72° C. for 10 minutes.