CN112458178A

CN112458178A - Periplaneta americana PCR identification primer and identification method

Info

Publication number: CN112458178A
Application number: CN201910841559.XA
Authority: CN
Inventors: 陈碧莲; 陈勇; 马若飞; 刘力; 于垂亮; 李尚蓉; 李亚楠; 侯建
Original assignee: Shanghai Jingxin Biological Medical Co ltd; Inner Mongolia Jingxin Pharmaceutical Co ltd; Zhejiang Jingxin Pharmaceutical Co Ltd
Current assignee: Shanghai Jingxin Biological Medical Co ltd; Inner Mongolia Jingxin Pharmaceutical Co ltd; Zhejiang Jingxin Pharmaceutical Co Ltd
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2021-03-09

Abstract

The invention provides a periplaneta americana PCR identification primer and a method for identifying the periplaneta americana by using the primer. The method utilizes PCR amplification reaction to detect the specific amplification band of the periplaneta americana, and can distinguish the periplaneta americana from other blattaceae species; and is generally suitable for the identification of the periplaneta americana in different growth periods such as larvae, adults and the like. The method has strong specificity, simple operation and low cost, and is easy to be applied to the quality control of the American cockroach.

Description

Periplaneta americana PCR identification primer and identification method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a periplaneta americana PCR identification primer and a method for identifying the periplaneta americana by using the primer.

Background

Periplaneta americana (Periplaneta americana) belongs to the class Insecta, the order Blattaria, the family Blattaceae, commonly referred to as Blatta Seu Periplaneta. The Chinese medicine is first seen in Shen nong Ben Cao Jing as a medicine in China. Nature and taste: salty taste and cold nature; meridian tropism: the liver, spleen and kidney meridians. Modern pharmacological research shows that the extract contains active ingredients such as various glycoproteins, amino acids, polypeptides and the like, and has the effects of enhancing the immunity of the organism, protecting the liver, promoting tissue repair, resisting tumors widely and the like. At present, the periplaneta americana has been developed into 3 medicaments for sale on the market, and the 3 medicaments are respectively rehabilitation new liquid, liver dragon capsules and heart vessel dragon injection and are used for treating skin mucosa injury, hepatitis and heart failure.

The quality standard of the American cockroach serving as the main raw material of the rehabilitation new liquid is recorded in the Chinese medicinal material standard of Hunan province, the Chinese medicinal material standard of Yunnan province and the Chinese medicinal material standard of Sichuan province. The present local standard adopts thin layer chromatography identification method. The traditional Chinese medicine standard of Hunan province and the traditional Chinese medicine standard of Yunnan province use alanine as a contrast, and the traditional Chinese medicine standard of Sichuan province uses American cockroach as a contrast. Tests show that (a research map can be provided), mixed counterfeit products and certified products can hardly be distinguished, and whether the American cockroach feeding materials can be replaced by insects of other blattaria species or not can not be determined, so that an identification method for the specificity of the American cockroach is urgently needed to be found.

The Polymerase Chain Reaction (PCR) method is a method for enzymatically synthesizing a specific gene or DNA fragment in vitro. The basic principle is similar to the natural DNA replication process, the specificity depends on oligonucleotide primers complementary to both ends of a target sequence, and three basic reactions of denaturation, annealing and extension mainly form a period and are carried out circularly. The DNA molecule is used as a direct carrier of genetic information and is not influenced by environmental factors, organism development stages and organ and tissue differences, and any somatic cell of each individual contains the same genetic information. Therefore, compared with the traditional identification method, the species identification by the PCR method is more accurate and reliable, and the method has unique advantages in the identification of precious and precious medicinal materials.

Disclosure of Invention

The invention aims to provide a primer for identifying Periplaneta americana by PCR and a method for identifying Periplaneta americana by using the primer.

In order to achieve the above object, one aspect of the present invention provides a periplaneta americana PCR identification primer, which has the following sequence:

the upstream primer is as follows: 5'-AGAATATTAATTCGTGCTGAGCTCG-3', respectively;

the downstream primer is: 5'-TGGGTATACTGTTCATCCTGTTCCG-3' are provided.

The other aspect of the invention provides a method for identifying the periplaneta americana, which comprises the step of carrying out amplification reaction on an identifier DNA sample by using the upstream primer and the downstream primer as PCR primers through a PCR method, wherein if a 290bp amplification strip exists, the identifier is the periplaneta americana.

In one embodiment of the invention, a method for identifying periplaneta americana comprises the steps of:

1) extracting the total DNA of the object to be identified;

2) taking the total DNA extracted in the step 1) as a template, and taking the upstream primer and the downstream primer as PCR primers to carry out amplification reaction on an identifier DNA sample by a PCR method;

3) detecting the PCR amplification reaction product obtained in the step 2) by using agarose gel electrophoresis, wherein if an amplification band of 290bp exists, the identifier is the periplaneta americana.

The reaction conditions of the PCR amplification reaction in the above steps are: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec, cyclic reaction for 30 times, final extension at 72 ℃ for 10 min, and heat preservation at 4 ℃.

The technical scheme provided by the invention achieves the following beneficial effects: 1) the method selects a mitochondrial cytochrome C oxidase subunit I (COI) sequence with good discriminative ability and suitable for animal identification to carry out PCR primer design, further designs and screens out a primer pair with the best specificity according to the different fragments of COI genes of the American cockroach and other three kinds of pseudo blattaria mixed varieties (German cockroach, Dubia and ground beetle), and can distinguish the American cockroach from other blattaria varieties; 2) the method is generally suitable for identifying the periplaneta americana in different growth periods such as larvae, adults and the like; 3) the method has the advantages of small material quantity, no special tissue requirement, and suitability for identification requirements of periplaneta Americana medicinal materials and medicinal material powder. 4) The method has strong specificity, simple operation and low cost, and is easy to be applied to the quality control of the American cockroach.

Drawings

FIG. 1 is an agarose gel electrophoresis image of example 1 of the present invention.

FIG. 2 is an agarose gel electrophoresis image of example 2 of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The invention is not to be thus limited to the specific embodiments described. The conditions and methods not described in the examples are generally those routinely employed by the experimenter of the art: such as the fourth edition of "the molecular biology laboratory Manual of Olympic and Kingston, eds", or the procedures and conditions as suggested by the commercial instructions.

First, primer design

According to the COI gene sequence of the periplaneta americana, a primer with the best specificity is further designed and screened by searching different fragments of the COI genes of the periplaneta americana and other three kinds of pseudo blattadae (the German cockroach, the Dubia and the ground beetle), and the primer sequence is as follows:

the downstream primer is: 5'-TGGGTATACTGTTCATCCTGTTCCG-3' are provided.

Second, DNA extraction

Taking 0.5g of a sample to be tested of an identifier, placing the sample in a mortar, adding a proper amount of liquid nitrogen, fully grinding the sample into powder, taking 2-3 mg of the sample, placing the sample in a 1.5ml centrifuge tube, extracting DNA by using an animal gene rapid extraction kit, adding 96 mul of lysis solution, 4 mul of mixed enzyme, uniformly mixing by vortex, preserving the heat in a 55 ℃ water bath for 15 minutes, preserving the heat in a 95 ℃ water bath for 5 minutes, adding 100 mul of stop solution into the sample, uniformly mixing by vortex, centrifuging (the rotating speed is 12000 r/min) for 10 minutes, taking supernatant as a sample solution, and placing the sample solution at 20 ℃ below zero for later use. And preparing a control medicinal material of the periplaneta americana by the same method to prepare a template DNA solution of the control medicinal material.

Third, PCR amplification reaction

The reaction system was carried out in a 200. mu.l centrifuge tube in a total volume of 20. mu.l, 10. mu.l of each of premixed reagent 2X (containing 2 Xcommon Taq DNA polymerase, 2 XPCR buffer, 2 XdNTP and 2 Xloading buffer), identifying primers (10. mu. mol/L) 0.8. mu.l, template 1. mu.l and sterile double distilled water 7.4. mu.l. Placing the centrifugal tube in a PCR instrument, wherein the PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec, cyclic reaction for 30 times, final extension at 72 ℃ for 10 min, and heat preservation at 4 ℃.

Fourth, electrophoresis detection

The gel concentration was 1.5% by agarose gel electrophoresis (0541, the four-part general rule of the Chinese pharmacopoeia 2015 edition), to which was added the nucleic acid gel stain GelRed. The sample loading amounts of the PCR reaction solutions of the test sample and the control drug are respectively 4 μ l, and the sample loading amount of the DNA molecular weight marker is 4 μ l (90ng/μ l). And after the electrophoresis is finished, taking the gel sheet to inspect on a gel imager or an ultraviolet transilluminator.

Example 1

The American cockroach PCR identification primer and the identification method thereof provided by the invention are adopted to identify the American cockroach on DNA samples of the American cockroach, the German cockroach, the Dubia and the ground beetle according to the specific experimental method, and an agarose gel electrophoresis chart is shown in figure 1. From right to left in fig. 1 are: M-DL500 Marker; 1-Periplaneta americana reference drug; 2-periplaneta americana; 3-german cockroach; 4-dubia; 5-ground beetle; 6-blank.

And (3) analyzing the electrophoresis chart of the figure 1, and judging the authenticity of the periplaneta americana according to the size of the target fragment. DNA (shown as

marks

1 and 2 in figure 1) extracted from a periplaneta americana control medicinal material and a periplaneta americana medicinal material is amplified by PCR, and a single amplification band appears at 290bp of a 1.5% agarose gel electrophoresis gel plate. And from other blattaceae, such as: after the DNA extracted from the German cockroach (shown as a mark 3 in figure 1), the Dubia (shown as a mark 4 in figure 1) and the ground beetle (shown as a mark 5 in figure 1) is amplified by PCR, a single amplification band does not appear at 290bp of a 1.5% agarose gel electrophoresis gel plate. The results show that the method can be used for identifying the authenticity of the periplaneta americana medicinal material.

Example 2

The primers and the method for identifying the American cockroach PCR provided by the invention are adopted to identify the American cockroach control medicinal material, the rest 26 batches of American cockroaches with different production areas and different growth cycles and 3 batches of adulterants (German cockroach, Dubia and ground beetle), and an agarose gel electrophoresis chart is shown in figure 2. In FIG. 2, M is DL500Marker, and the rest numbers and corresponding sample information are shown in the following table:

the result shows that the periplaneta americana reference medicinal material, the periplaneta americana with different production areas and different growth cycles have single amplification bands at 290bp, and 3 different mixed counterfeit products have no single amplification band at 290bp, so that the method can stably and accurately identify the periplaneta americana with different production areas and different growth cycles and the mixed counterfeit products.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of this invention without departing from the spirit thereof.

Claims

1. The primers for the Periplaneta americana PCR identification are characterized by comprising the following sequences:

the downstream primer is: 5'-TGGGTATACTGTTCATCCTGTTCCG-3' are provided.

2. A method for identifying Periplaneta americana is characterized in that the upstream primer and the downstream primer in claim 1 are used as PCR primers to perform amplification reaction on an identifier DNA sample by a PCR method, and if a 290bp amplification band exists, the identifier is the Periplaneta americana.

3. The method of claim 2, comprising the steps of:

1) extracting the total DNA of the object to be identified;

2) taking the total DNA extracted in the step 1) as a template, and taking the upstream primer and the downstream primer claimed in the claim 1 as PCR primers to carry out amplification reaction on an identifier DNA sample by a PCR method;

4. The method of claim 3, wherein the reaction conditions of the PCR amplification reaction are: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec, cyclic reaction for 30 times, final extension at 72 ℃ for 10 min, and heat preservation at 4 ℃.