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Double PCR detection method for authenticity identification of cordyceps sinensis

A technology for authenticity identification of Cordyceps sinensis, applied in the biological field, achieves the effect of simple method, suitable for promotion, and good repeatability

Active Publication Date: 2017-08-25
四川省农业科学院分析测试中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the molecular detection technology of Cordyceps sinensis authenticity identification at present, and the existing related research mainly focuses on the isolation and identification of its anamorphs (Yang Jing, 2005; Liang et al., 2008; Zhong et al., 2010; Leiet al., 2013)

Method used

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  • Double PCR detection method for authenticity identification of cordyceps sinensis
  • Double PCR detection method for authenticity identification of cordyceps sinensis
  • Double PCR detection method for authenticity identification of cordyceps sinensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cordyceps sinensis double PCR identification method, in the same PCR reaction system to simultaneously detect the ITS of Cordyceps fungus and the COI gene nucleic acid fragment of the Cordyceps fungus host, the steps are as follows:

[0055] Step 1: Synthesize an amplification primer set for identifying ITS, the amplification primer set is:

[0056] CITS-F10': GTTGCCTCGGCGGGAC

[0057] CITS-R10'-2: CMTTTGCTTGCTTCTTGACTGAG.

[0058] Synthesis is used to identify the amplification primer group of COI gene nucleic acid fragment, and this amplification primer group is:

[0059] COI-F: GGAAATCCHGGATCTTTAATT

[0060] COI-R: GATGCCCCMGARTGTGCAAT.

[0061] The concentration of both groups of primers was 10 μmol / l.

[0062] Step 2: Prepare DNA dilution;

[0063] DNA diluent of the sample to be tested: extract DNA from the sample to be tested and dilute to 50ng / μl DNA diluent;

[0064] Positive control substance DNA diluent: extract DNA from Cordyceps sinensis positive cont...

Embodiment 2

[0080] specificity experiment. The experimental method steps are as follows:

[0081] Step 1: same as embodiment 1

[0082] Step 2: Prepare DNA dilution;

[0083] DNA diluent of the sample to be tested: extract DNA from the sample to be tested, and dilute it to 50ng / μl DNA diluent; the samples to be tested are Cordyceps sinensis, bat moth larvae, Cordyceps mycelium, Liangshan Cordyceps, Yaxiang stick Cordyceps, Xinjiang Cordyceps, Cordyceps militaris (Cordyceps militaris), molded starch, corn, rice, soybeans, rapeseed, chicken, pigs.

[0084] Positive control substance DNA diluent and blank control are the same as in Example 1.

[0085] Step 3, step 4 are the same as embodiment 1.

[0086] The results of the amplification are attached figure 2 shown. Only two target bands (lanes 1 and 3) were amplified in the Cordyceps positive control substance and samples of Cordyceps sinensis, and there were no target bands in the DNA of the blank control, counterfeit Cordyceps sinen...

Embodiment 3

[0088] Repeatable experiments.

[0089] The method of Example 1 was used to detect the positive control sample of Cordyceps sinensis (the standard substance of Cordyceps sinensis purchased from China Institute for Food and Drug Control) and the samples of Cordyceps sinensis collected from Qinghai, Tibet, Sichuan and other places. The results are attached image 3 shown. All Cordyceps detection samples could amplify two (113bp and 302bp) target bands (lanes 1, 3-16), but no bands were amplified in the blank control (lane 2). And the result can be reproduced many times, which is repeatable. Therefore, the present invention is stable and reliable in distinguishing the authenticity of Cordyceps sinensis.

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Abstract

The invention discloses a double PCR detection method for authenticity identification of cordyceps sinensis, and solves the problem that in the prior art, the identification of cordyceps sinensis is conducted only regarding fungi, and therefore the prior art is short of sufficient accuracy. According to the method, in the same PCR reaction system, ITS of cordyceps militaris and COI gene nucleic acid fragments which are hosts of cordyceps militaris are simultaneously detected, and an amplification primer group for identifying the COI gene nucleic acid fragments is COI-F: GGAAATCCHGGATCTTTAATT and COI-R: GATGCCCCMGARTGTGCAAT; an amplification primer group for identifying the ITS is GITS-F10': GTTGCCTCGGCGGGAC and CITS-R10'-2: CMTTTGCTTGCTTCTTGACTGAG. The double PCR detection method for the authenticity identification of cordyceps sinensis is convenient to operate, high in cordyceps sinensis identification specificity and accuracy, good in repeatability and suitable for popularization.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR detection method for identifying the authenticity of Cordyceps sinensis. Background technique [0002] Cordyceps sinensis (Ophiocordyceps sinensis sym. Cordyceps) is a complex of the sub-seat (fruiting body) and larval corpse of the fungus Cordyceps sinensis of the family Ergotaceae parasitizing on the larvae of the bat moth of the Lepidoptera bat moth family. It is a precious nourishing medicinal material for both medicine and food. It is mainly distributed in the areas above the snow line of the Qinghai-Tibet Plateau at an altitude of 3000m. It has good pharmacological effects in antiviral, antitumor, antioxidative and inflammatory aspects, and has extremely high medicinal and economic value. Due to its special requirements for the growing geographical environment and its strict parasitic nature, it cannot be replaced by artificial cultivation and large-scale producti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2537/143
Inventor 张富丽牛蓓宋君雷绍荣郭灵安常丽娟尹全王东刘文娟
Owner 四川省农业科学院分析测试中心
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