Mitochondrial gene sequence for identifying Cordyceps sinensis and method
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A technology of mitochondrial gene and mitochondrial genome, which is applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of indistinguishable and inability to actually identify the quality of Cordyceps sinensis, and achieve easy operation and reduce sample loss , the effect of less sample amount
Inactive Publication Date: 2013-08-21
上海立得生物科技有限公司
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[0003] At present, the patents for identifying the authenticity of Cordyceps sinensis are mainly based on the nucleotide sequence of Cordyceps sinensis to design primers. Although the authenticity of Cordyceps sinensis can be identified, it is impossible to distinguish whether it is the best quality Cordyceps sinensis produced in Naqu or another place of origin pretending to be Naqu The inferior quality of Cordyceps produced, so it is impossible to actually identify the quality of Cordyceps
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Embodiment 1
[0025] 1) Take the sample of Cordyceps sinensis to be tested, select a dry specimen that is well preserved and free from moth-eaten and mildew, cut a short section of the dry specimen, cut it open and take out the content, and wipe the inside and outside of the dry specimen body wall with 75% alcohol 2- 3 times to eliminate contamination from foreign substances.
[0026] 2) After immersing the sample in TE (10mM Tris-HCL, 200mM EDTA, 50mM NaCl, pH 9.0) for 48 hours, take out the soaked and softened material and use the phenol / chloroform method to extract the total DNA of the bat moth.
[0027] 3) Three pairs of specific primers designed by the present invention are used to carry out PCR amplification, and the primer sequences are:
[0028] ND2F: ATTTTCGTGCTTCTTA and ND2R: TTGAAGATTATTAGTT;
[0029] ATP6F: GTAAGTATTGGTCTCT and ATP6R: ATGGGTGATTTTGATT;
[0030] COX3F:CCAAGGAACACCAAAT and COX3R:GTAATGAAAATGGAAT.
[0031] 4) The amplification system of the three pairs of primer...
Embodiment 2
[0036] 1) with embodiment 1.
[0037] 1) with embodiment 1.
[0038] 2) with embodiment 1.
[0039] 3) with embodiment 1.
[0040] 4) With embodiment 1.
[0041] 5) With embodiment 1.
[0042] 6) With embodiment 1.
[0043] 7) Take the tested sample to extract genomic DNA, and use the above three pairs of specific primers for PCR amplification. If the sequences of the three protein-coding genes can be obtained respectively, and the nucleotide sequence differences in the three gene joint data sets are calculated by Mega software> 6%, which is the variation rate between different species of the same genus, shows that it is a true Cordyceps sinensis, but its host insects are insects of the genus Hematodes other than the male bat moth.
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Abstract
The invention provides a mitochondrial gene sequence for identifying authenticity and quality of Cordyceps sinensis and an operation method thereof. According to the method, differences of mitochondrial genes between ghostmoth and other lepidopteran insects are compared and thus high-variability areas can be searched. Specific primers of the ghostmoth are designed to obtain sequences of corresponding sections of mitochondria of host insects; if a nucleotide difference rate is less than 1-1.5 %, the insects are determined to be a same species but different individuals, and if the nucleotide difference rate is more than 6 %, the insects are determined to be a same genus but different species, thereby not only identifying whether to-be-detected samples are the Cordyceps sinensis formed with Hepialus larvae as the host insects, but also determining producing areas of the to-be-detected samples, and thus determining the authenticity and grades of the to-be-detected samples The method is simple in steps, easy to operate, and few in sample amount, and can minimize sample loss. According three pairs of the specific primers provided by the invention, the authenticity and the grades of the samples can be rapidly and conveniently detected through detecting whether the samples have same or similar nucleotide sequences as well as similarity of the nucleotide sequences.
Description
technical field [0001] The invention relates to a mitochondrial gene sequence and a method for identifying Cordyceps sinensis, in particular to a mitochondrial gene sequence that can be used to identify the authenticity and quality of Cordyceps sinensis and an operation method thereof. Background technique [0002] Cordyceps sinensis (Berk.) Sacc., also known as Cordyceps sinensis, is one of the more commonly used precious Chinese medicinal materials. It is parasitic in the alpine meadow soil by Clavicipitaceae Clavicipitaceae Cordyceps Cordyceps larvae in the bat moth larvae of Hepialidae insects of the Lepidoptera bat moth family. After vegetative growth and reproductive growth, it forms the fruiting bodies and dead worms of Cordyceps sinensis A complex composed of sclerotia. Cordyceps sinensis is sweet and flat, has the functions of tonifying deficiency, benefiting essence, nourishing yin and moistening lung, stopping bleeding and resolving phlegm, strengthening the kidn...
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