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PCR (polymerase chain reaction) primer and method for identifying authenticity of bulbus fritillariae cirrhosae or detecting degree of adulteration of bulbus fritillariae cirrhosae

A fritillary, authenticity technology, applied in the field of PCR primers for detecting the degree of adulteration of fritillary, can solve the problem of difficulty in detecting the degree of adulteration of mixed powder samples, inability to control the quality of fritillary related products, and inability to accurately respond to samples Adulteration degree and other problems, to achieve the effect of rapid and accurate detection of adulteration degree, excellent amplification specificity, economical and time cost saving

Active Publication Date: 2019-06-21
GUIZHOU MEDICAL UNIV
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  • Claims
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Problems solved by technology

However, there are about 50 to 100 fritillaria of about 25 to 50 g. If the PCR-RFLP method is used for identification, 50 to 100 PCR-RFLP reactions need to be performed, which has a large workload, long experiment time and high cost. question
Therefore, in production, only about 10 fritillaria are often taken for authenticity identification, but this sampling method cannot reflect the overall situation of the sample, and cannot accurately reflect the degree of adulteration of the sample
Especially in clinical practice, Fritillaria is often used in the form of powder, which is more convenient for criminals to use counterfeit products to impersonate or mix Fritillaria; and mixed samples are difficult to use due to the impure DNA of the Pharmacopoeia PCR-RFLP method to detect the degree of adulteration in mixed powder samples
The above problems lead to the inability to effectively control the quality of Fritillaria fritillaria related products in actual production

Method used

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  • PCR (polymerase chain reaction) primer and method for identifying authenticity of bulbus fritillariae cirrhosae or detecting degree of adulteration of bulbus fritillariae cirrhosae
  • PCR (polymerase chain reaction) primer and method for identifying authenticity of bulbus fritillariae cirrhosae or detecting degree of adulteration of bulbus fritillariae cirrhosae
  • PCR (polymerase chain reaction) primer and method for identifying authenticity of bulbus fritillariae cirrhosae or detecting degree of adulteration of bulbus fritillariae cirrhosae

Examples

Experimental program
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Effect test

Embodiment 1

[0063] A kind of PCR method that present embodiment is used to distinguish the authenticity of Fritillaria sichuanensis adopts respectively following primer pair 1, primer pair 2, primer pair 3, primer pair 4 pairs of Fritillaria sichuanensis base (Fritillaria sichuanensis, Fritillaria gansuensis) , Dark Purple Fritillaria, Wabu Fritillaria, Suosha Fritillaria and Taibai Fritillaria), Chuan Fritillaria fakes (Zhejiang Fritillaria, Flat Fritillaria, Yibeimu and Hubei Fritillaria), the primer pairs The upstream primers introduced base mismatches at the 3rd, 9th, and 10th bases at the 3' end (3'->5' direction). Identification methods include:

[0064] (1) Extraction of genomic DNA: same as step (1) in Example 1;

[0065] (2) PCR amplification

[0066] Using the genomic DNA obtained in step (1) as a template, the following primer pairs were used for PCR amplification:

[0067] Wherein, primer pair 1 includes: upstream primer 1: 5'-GGCACTATGCTAGCCCTCCC-3' (SEQ ID No.1);

[0068...

Embodiment 2

[0078] According to the method of Comparative Example 1, 5 pairs of 10 groups of commercially available Chuan Fritillaria samples CBM-1 to CBM-10 were used for authenticity identification by using primer pairs, and the results were as follows Figure 11 shown. Simultaneously according to the method of embodiment 1, adopt primer pair 1~4 to carry out authenticity identification to 10 groups of commercially available Chuan Fritillaria samples CBM-1 to CBM-10 respectively, its result is respectively as follows Figure 12 , Figure 13 , Figure 14 with Figure 15 shown.

[0079] According to the provisions of the Chinese Pharmacopoeia, two new bands were produced after the standard product of Fritillaria sichuanensis was digested with Sma I, and the size was between 100 and 250 bp. from Figure 11 It can be seen that there are two bands between 100 and 250bp in lane 1 (standard product of Fritillaria sichuanensis), proving that it is an authentic product of Fritillaria sichua...

Embodiment 3

[0153] In this embodiment, a real-time fluorescent quantitative PCR method for detecting the degree of adulteration of Fritillaria sichuanensis comprises the following steps:

[0154] (1) Preparation of true and false mixed samples

[0155] Take Chuan Fritillaria, Zhejiang Fritillaria and Ping Fritillaria standard powders, and mix Chuan Fritillaria with Zhejiang Fritillaria and Flat Fritillaria respectively to obtain two mixed samples of true and false, each of which contains 9 Authentic and false mixed samples, the counterfeit content (mass percentage) in 9 genuine and false mixed samples were 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% in turn.

[0156] (2) Extraction of genomic DNA

[0157] According to the same method as in step (1) of Example 1, the genomic DNA of each true and false mixed sample was extracted.

[0158] (3) Fluorescent quantitative PCR amplification

[0159] Using the genomic DNA extracted in step (2) as a template, use primer pair 1 and primer pair 3 ...

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Abstract

The invention discloses a PCR (polymerase chain reaction) primer and a method for identifying the authenticity of bulbus fritillariae cirrhosae or detecting the degree of adulteration of the bulbus fritillariae cirrhosae. The PCR primer comprises an identification primer for authentic bulbus fritillariae cirrhosae and / or an identification primer for counterfeit bulbus fritillariae cirrhosae. The 75th base 'C' of an ITS1 spacer of the bulbus fritillariae cirrhosae serves as the 3' end of the identification primer for the authentic bulbus fritillariae cirrhosae, and the 75th base 'T' of the ITS1spacer of the bulbus fritillariae cirrhosae serves as the 3' end of the identification primer for the counterfeit bulbus fritillariae cirrhosae. The base mismatch is introduced to third, ninth and tenth bases at the 3' ends of the two primers to obtain the identification primer for the authentic bulbus fritillariae cirrhosae and the identification primer for the counterfeit bulbus fritillariae cirrhosae, wherein the identification primer for the authentic bulbus fritillariae cirrhosae is represented by SEQ ID No. 1 or 2, and the identification primer for the counterfeit bulbus fritillariae cirrhosae is represented by SEQ ID No. 3 or 4. When the PCR primer is used for authenticating bulbus fritillariae cirrhosae samples, the authenticity of the bulbus fritillariae cirrhosae or the degree of adulteration of the bulbus fritillariae cirrhosae can be accurately and quickly identified.

Description

technical field [0001] The invention belongs to the technical field of identification of Fritillaria sichuanensis, and specifically relates to a PCR primer and a method for identifying the authenticity of Fritillaria sichuanensis or detecting the degree of adulteration of Fritillaria sichuanensis. Background technique [0002] The traditional Chinese medicine Chuan Fritillaria belongs to the Liliaceae Fritillaria plant. It is the dried bulb of Chuan Fritillaria, Gansu Fritillaria, Dark Purple Fritillaria, Wabu Fritillaria, Suosha Fritillaria or Taibai Fritillaria. It is mainly produced in Sichuan and its surrounding areas. The provinces have been included in the Pharmacopoeia of the People's Republic of China over the years. The high medicinal value of Fritillaria sichuanensis, the large market demand, and over-harvesting and overharvesting have led to a sharp decline in wild plant resources of Fritillaria sichuanensis, which has led to an increase in the price of Fritillari...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
Inventor 刘亭薛维娜杨畅陆定艳刘香香李靖张煜彬李勇军兰燕宇王永林
Owner GUIZHOU MEDICAL UNIV
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