Specific endogenous gene of Fritillaria sichuanensis and its authenticity rapid detection primer set and method

An endogenous gene, Fritillary fritillary technology, applied in the field of biotechnology analysis, can solve the problems of high price, false positive and false negative, poor timeliness, etc. , the effect of less process

Active Publication Date: 2019-04-23
四川省农业科学院分析测试中心
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Problems solved by technology

[0006] The technical problem solved by the present invention is: provide the endogenous gene of Fritillaria sichuanensis for its authenticity detection, and solve the problem of high price, poor timeliness, fuzzy map, unfavorable judgment, false positives and false negatives of the molecular identification technology of Fritillaria sichuanensis in the prior art And other issues

Method used

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  • Specific endogenous gene of Fritillaria sichuanensis and its authenticity rapid detection primer set and method
  • Specific endogenous gene of Fritillaria sichuanensis and its authenticity rapid detection primer set and method
  • Specific endogenous gene of Fritillaria sichuanensis and its authenticity rapid detection primer set and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] This example provides the acquisition and species-specific verification of the endogenous gene sequence of Fritillaria sichuanensis.

[0054] We intend to screen the specific sequences of genes related to the synthesis of saponins in Fritillaria sichuanensis as specific endogenous gene markers for species identification, and establish a new technical system for authenticity identification of Fritillaria sichuanensis on this basis. During the project, the project team cloned 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the study of the key synthetases in the synthesis pathway of steroidal saponins, the characteristic metabolites of Fritillaria chinensis Gene, a 342bp unknown functional gene sequence (such as figure 2 Shown in A), the band was recovered and sequenced, and compared with BLAST in the NCBI GenBank database, no homologous sequence was found, so it was determined as a new unique gene sequence of Fritillaria sichuanensis different from the HMGR gene. Ba...

Embodiment 2

[0056] The present embodiment provides the specificity test of the method of the present invention, and concrete steps are as follows:

[0057] (1) Synthetic PCR detection primer set, the nucleotide sequence of the primer set is as follows:

[0058] BMH-YF: CAGCAGGAATCCCAAGC

[0059] BMH-YR: GGTTGGCACAGTTGGAGG.

[0060] The working concentration of the primers in this embodiment is 10 μmol / l.

[0061] The nucleotide sequence of the above primers is designed based on the specific site of the specific endogenous gene in Fritillaria sichuan, and the nucleotide sequence of the endogenous gene is shown in SEQ ID NO.1. Through this design, the target fragments of 234bp size can be amplified respectively, and all Fritillaria chinensis and products containing Fritillaria chinensis can be accurately and effectively detected.

[0062] (2) Preparation of DNA template solution; DNA extracted from samples such as Fritillaria, Guangzigu, and Fritillaria, as well as DNA dilutions from co...

Embodiment 3

[0078] This example provides a sensitivity test of the method of the present invention.

[0079] The 50ng / μl Fritillaria DNA solution was diluted 5 times with TE buffer into 7 different concentration gradients. Take 1 μl as a template, which is equivalent to the amount of DNA template in the reaction system, respectively 50ng, 10ng, 2ng, 400pg, 80pg, 16pg, 3.2pg, and use the primers and PCR technology in the present invention to amplify. see results Figure 4 , "M" in the attached figure refers to a DNA marker (DNA marker), which has multiple bands, respectively used to represent DNA fragments of different sizes. The results of the sensitivity experiment showed that the DNA content of Fritillaria fringiosa in the reaction system can be detected as low as 16 pg (swimming lane 6). This result is a good indication of the high sensitivity of the method.

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Abstract

The invention discloses an endogenous gene of Fritillaria cirrhosa, and solves the problems such as high price of the molecular identification technology of Fritillaria cirrhosa, poor timeliness, fuzzy graph unfavorable to determine, false positive false negative and the like in the prior art. The specific endogenous gene comprises a nucleotide sequence, such as SEQ ID NO. 1. The invention also provides a primer set for accurately detecting the authenticity of Fritillaria cirrhosa and a method for detecting the authenticity of Fritillaria cirrhosa by using the primer set. The sequence of upstream and downstream primers in the primer set is as follows: BMH-YF:CAGCAGGAATCCCAAGC; BMH-YR: GGTTGGCACAGTTGGAGG. The specific primer is designed and a high-specific PCR detection technology system isestablished based on the specific endogenous gene sequence of Fritillaria cirrhosa obtained in the first cloning, thereby establishing a simple, efficient and accurate high-specific PCR molecular detection technology for the authenticity identification of Fritillaria cirrhosa.

Description

technical field [0001] The invention relates to the field of biotechnology analysis methods, in particular to a specific endogenous gene of Fritillaria sichuanensis and a primer set and method for authenticity detection. Background technique [0002] Fritillaria is a variety of plants of the genus Fritillaria L. in the family Liliaceae (Fritillaria cirrhosa D.Don), Fritillaria unibracteata Hsiaoet K.C.Hsia, Fritillaria gansu (Fritillariaprzezvalskii Maxim), Fritillaria delavayi Franch, Fritillaria taipaiensis P.Y.Li or Fritillaria unibracteata Hsiao et K.C Hsia van wabuensis(Y.Tang et S.C.Yue) Z.D.Liu, S.Wang et S.C.Chen) general term for dry bulbs. It grows in vast high-altitude areas, on the border of the Qinghai-Tibet Plateau. It tastes slightly bitter, has the effects of clearing away heat and moistening the lungs, relieving cough and reducing phlegm, and is a rare and precious medicinal material commonly used in traditional Chinese medicine. An important raw material f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/166C12Q2565/125
Inventor 张富丽牟梦雪郭灵安雷绍荣宋君
Owner 四川省农业科学院分析测试中心
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