Gene sequence and method for distinguishing cordyceps sinensis

Details Gene sequence and method for distinguishing cordyceps sinensis Gene sequence and method for distinguishing cordyceps sinensis

1999-11-30

2001-06-26

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C424S195110, C424S254100, C514S169000

Reexamination Certificate

active

06251606

ABSTRACT:

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.


REFERENCES:
patent: 5582828 (1996-12-01), Lin et al.

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