CN110904242A - Primer composition and application thereof in identification of whitmania pigra - Google Patents
Primer composition and application thereof in identification of whitmania pigra Download PDFInfo
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Abstract
The embodiment of the invention provides a primer composition for LAMP amplification, a method for identifying whitmania pigra, and a kit for identifying whitmania pigra. By adopting the primer composition provided by the embodiment of the invention, the DNA fragment in the whitmania pigra can be specifically amplified, so that the whitmania pigra can be accurately identified.
Description
Technical Field
The invention relates to the technical field of whitmania pigra identification, in particular to a primer composition and application thereof in identification of whitmania pigra.
Background
The Hirudo is dried whole body of Whitmania Pigra Whitman, Hirudo nipponica Whitman or Whitmania acranulata Whitman. Leeches are a common traditional Chinese medicinal material, and in recent years, leeches are used as a raw material medicament for developing a large amount of Chinese medicinal preparations, so that the price of the traditional Chinese medicinal material of the leeches is increased. At present, leeches, also called whitmania pigra, are the main circulating varieties in the market, are excellent medicinal varieties, and a method capable of quickly and accurately identifying whitmania pigra is urgently needed for reducing the adulteration and sales of traditional Chinese medicinal materials of leeches.
Disclosure of Invention
The embodiment of the invention aims to provide a primer composition to realize the specific amplification of a DNA fragment of whitmania pigra, so as to accurately identify the whitmania pigra. The specific technical scheme is as follows:
in a first aspect, the present invention provides a primer composition, comprising four primers, wherein the nucleotide sequences of the four primers are as follows:
an upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3', respectively;
an upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3', respectively;
a downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3', respectively;
downstream outer primer: 5'-CCTGAATGAGATACGGAGTC-3' are provided.
In a second aspect, the invention provides the use of a primer composition according to the first aspect of the invention for identifying whitmania pigra.
In a third aspect, the invention provides a method for identifying whitmania pigra by LAMP amplification technology, and the primer composition provided by the first aspect of the invention is adopted.
In some embodiments of the third aspect of the invention, the identification of whitmania pigra by LAMP amplification comprises the following steps:
1) respectively extracting the total DNA of a sample to be detected;
2) performing LAMP amplification by respectively using the total DNA of each sample to be detected as a template by using the primer composition; the sample to be detected with positive amplification result is whitmania pigra.
In some embodiments of the third aspect of the invention, in step 2), each 25 μ L of the LAMP amplification system comprises:
2.5. mu.L of 10 × isothermal amplification reaction buffer;
0.2. mu.M of the upstream outer primer;
0.2. mu.M downstream outer primer;
1.6 μ M upstream inner primer;
1.6 μ M downstream inner primer;
50-500ng total DNA;
1.4mM dNTPs；
6mM MgSO4；
8U strand displacing DNA polymerase.
In some embodiments of the third aspect of the invention, in step 2), the LAMP amplification conditions are:
incubation was carried out at 64 ℃ for 30-60 minutes and at 80 ℃ for 10 minutes.
In some embodiments of the third aspect of the invention, the amplification result is detected using a nucleic acid fluorescent dye.
In a fourth aspect, the invention provides a kit for identifying whitmania pigra, which comprises the primer composition provided by the first aspect of the invention.
In some embodiments of the fourth aspect of the invention, the kit further comprises an isothermal amplification reaction buffer, DEPC water, strand displacement DNA polymerase, dNTPs, MgSO4Aqueous solutions, and instructions for operation.
By adopting the primer composition provided by the embodiment of the invention, the DNA fragment in the whitmania pigra can be specifically amplified, so that the whitmania pigra can be accurately identified.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a gel electrophoresis result chart of LAMP amplification of a sample to be tested by using the primer composition of the present invention;
FIG. 2 shows the fluorescent staining result (under natural light) of nucleic acid obtained by LAPM amplification of a sample to be tested using the primer composition of the present invention;
FIG. 3 shows the result of fluorescent staining of nucleic acid (under UV light) by LAMP amplification of a sample to be tested using the primer composition of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In a first aspect, the present invention provides a primer composition, comprising four primers, wherein the nucleotide sequences of the four primers are as follows:
an upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3' (SEQ ID No: 1);
an upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3' (SEQ ID No: 2);
a downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3' (SEQ ID No: 3);
downstream outer primer: 5'-CCTGAATGAGATACGGAGTC-3' (SEQ ID No: 4).
The four primers are named as sz-F3, sz-FIP, sz-BIP and sz-B3 in sequence.
In a second aspect, the invention provides the use of a primer composition according to the first aspect of the invention for identifying whitmania pigra.
In a third aspect, the invention provides a method for identifying whitmania pigra whitman by LAMP amplification technology (namely, loop-mediated isothermal amplification technology), and the primer composition provided by the first aspect of the invention is adopted.
In some embodiments of the third aspect of the invention, the identification of whitmania pigra by LAMP amplification comprises the following steps:
1) respectively extracting the total DNA of a sample to be detected;
the sample to be tested can be a leech tissue sample to be identified, such as muscle tissue, in some embodiments of the third aspect of the present invention, after the total DNA of the sample to be tested is extracted, the concentration of the total DNA is determined, for example, the concentration of the total DNA is determined by using a Nanodrop2000 micro nucleic acid quantitative analyzer, and the DNA purity is determined according to the values of absorbance a260/a280 and a260/a230, which is a common technical measure in the art, and the present invention is not limited herein.
2) Performing LAMP amplification by respectively using the total DNA of each sample to be detected as a template by using the primer composition; the sample to be detected with positive amplification result is whitmania pigra.
The "positive" result can be understood as that the amplification result is detectable, for example, a ladder-shaped band is observed by agarose gel electrophoresis, and a negative result is obtained when no band is detected; the nucleic acid fluorescent dye is adopted for dyeing, and the positive result can be observed when the fluorescence is observed under natural light or ultraviolet light. In some embodiments of the third aspect of the invention, the amplification result is detected using a nucleic acid fluorescent dye. The nucleic acid fluorescent dye has high sensitivity for detecting the amplification result, so that the specificity of the primer is required, otherwise, false positive results can occur; the primer composition has high specificity to the whitmania pigra, so that the whitmania pigra can be quickly and accurately identified by combining nucleic acid fluorescent dye detection. The nucleic acid fluorescent dye is a reagent commonly used in the field, such as SYBR Green I nucleic acid dye, the positive result is Green under natural light, and the negative result is orange; one skilled in the art can select other nucleic acid fluorescent dyes according to the actual situation, and the invention is not limited herein.
In some embodiments of the third aspect of the invention, in step 2), each 25 μ L of the LAMP amplification system comprises:
2.5. mu.L of 10 × isothermal amplification reaction buffer;
0.2. mu.M of the upstream outer primer;
0.2. mu.M downstream outer primer;
1.6 μ M upstream inner primer;
1.6 μ M downstream inner primer;
50-500ng total DNA;
1.4mM dNTPs；
6mM MgSO4；
8U strand displacement DNA polymerase;
the balance is filled with DEPC water, which is a common technical means in the field, and the invention is not described herein.
The strand displacement DNA polymerase is a DNA polymerase used for LAMP amplification, such as Bst 2.0WarmStart DNA polymerase (british biotechnology limited), and those skilled in the art can select other DNA polymerases suitable for LAMP amplification according to specific needs, and the invention is not limited herein.
In some embodiments of the third aspect of the invention, in step 2), the LAMP amplification conditions are:
incubating at 64 ℃ for 30-60 minutes and at 80 ℃ for 10 minutes; wherein 64 ℃ is the amplification temperature and 80 ℃ is the termination temperature.
The primers sz-F3, sz-FIP, sz-BIP, and sz-B3 used in the present invention are synthesized by Biotechnology, Inc. (Shanghai), and reagents other than the template and the primer in the amplification system and reagents required for detecting the amplification result are commercially available.
In a fourth aspect, the invention provides a kit for identifying whitmania pigra, which comprises the primer composition provided by the first aspect of the invention.
In some embodiments of the fourth aspect of the invention, the kit further comprises an isothermal amplification reaction buffer, DEPC water, strand displacement DNA polymerase, dNTPs, MgSO4Aqueous solutions, and instructions for operation.
Examples
Reagent
The universal primers LCO1490 and HCO2198 and the primers sz-F3, sz-FIP, sz-BIP, sz-B3 were synthesized by Biotechnology engineering (Shanghai) Ltd; dNTP mix (i.e., dNTPs) (TaKaRa Co.); DEPC water (Biosharp); agarose (Lonza); absolute ethanol (mao chemical reagents works, Tianjin); 6 × Loading Buffer (TaKaRa Co.); 10000 XSYBR Green I nucleic acid dye (Beijing Solebao technologies, Inc.); DuRed nucleic acid dye (beijing panbo biochemistry ltd); d2000 DNAarker (Beijing Tiangen Biochemical technology Co., Ltd.); TAE (50X) (Hefeizhizhi Macro Biotech Co., Ltd.); bst 2.0WarmStart DNA polymerase (New England Biotechnology limited, kit contains 10 x isothermal amplification reaction buffer); tks Gflex DNA polymerase and 2 XGflex PCRbuffer (2 XGflex PCRbuffer containing dNTPs) (TaKaRa).
Species identification of two samples to be tested
1. Obtaining CO I DNA fragment of sample to be detected
Collecting 25 parts of leech samples (samples to be detected) from different producing areas, respectively shearing about 30mg of muscle tissue of each leech sample, extracting total DNA according to the instruction of an animal tissue genome DNA extraction kit (TIANGEN), and determining the concentration by using a Nanodrop2000 micro nucleic acid quantitative analyzer, wherein the concentration of the total DNA is about 400 ng/mu L. Adopting a CO I (cytochrome oxidase I) universal primer LCO1490(SEQ ID No:5) and HCO2198(SEQ ID No:6) to respectively take the total DNA of each sample to be detected as a template to carry out PCR amplification, wherein the amplification system is as follows:
12.5μL 2×Gflex PCRBuffer，
0.5μL LCO1490(10μM)，
0.5μL HCO2198(10μM)，
0.5. mu.L of total DNA,
1.25U of Tks Gflex DNA polymerase,
10.5. mu.L of DEPC water,
the amplification conditions were: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, extension at 68 ℃ for 30s, and 40 cycles; and (4) after the circulation is finished, continuing to extend for 5 minutes at 68 ℃ to obtain a CO I DNA fragment of the sample to be detected.
2. Sanger sequencing for determining species of sample to be tested
Sanger sequencing is carried out on products amplified by primers LCO1490 and HCO2198, and the sequencing work is completed by Beijing Huada gene. Sequencing results Blast identification of species was performed at NCBI and the identification results are shown in table 1.
TABLE 1
3. The primer composition is adopted to identify a sample to be detected
Taking the total DNA of a sample to be detected as a template, and carrying out LAMP amplification on the sample by adopting the primer composition, wherein the amplification system is as follows:
2.5. mu.L of 10 × isothermal amplification reaction buffer
0.5μL sz-F3(10μM)
0.5μL sz-B3(10μM)
4μL sz-FIP(10μM)
4μL sz-BIP(10μM)
1 μ L of total DNA
3.5μL dNTPs(10mM)
1.5μL MgSO4(100mM)
mu.L of Bst 2.0WarmStart DNA polymerase (8U)
6.5 μ L DEPC Water
The amplification conditions were: 60 minutes at 64 ℃; 80 ℃ for 10 minutes.
mu.L of the reaction product was taken out, and 5. mu.L of 6 Xloading buffer (Takara Co.) was added thereto, and after mixing, the mixture was subjected to electrophoresis on 1.5% agarose gel stained with DU Red nucleic acid dye at 110V for 30 minutes, and imaged by a gel imaging system, and the results are shown in FIG. 1. In FIG. 1, M represents marker (molecular weight marker), the numbers of which are identical to those of the samples in Table 1, and N is a negative control (DEPC water). As can be seen from FIG. 1, ladder-like bands can be observed in the samples corresponding to the numbers K1, K2 and K3, which are positive results, which indicate that the samples to be tested corresponding to the numbers of the samples are Whitmania pigra Whitman, and the results are consistent with the Sanger sequencing results, and indicate that the primer composition can accurately identify the Whitmania pigra Whitman.
20. mu.L of the reaction product was taken, and 10000 XSSYBR Green I was added in an amount of 0.2. mu.L, and the results of observation under natural light are shown in FIG. 2, in which the numbers are the same as those of the samples in Table 1, and N is a negative control (DEPC water). Wherein the samples corresponding to the labels K1, K2 and K3 are green, namely positive results are obtained, and the results are consistent with the electrophoresis results; the reaction product added with SYBR Green I is observed under an ultraviolet lamp of 254nm, and the result is shown in FIG. 3, wherein Green fluorescence is observed in samples corresponding to the labels K1, K2 and K3, and the result is also consistent with the electrophoresis result. Thus, the nucleic acid fluorescent dye can accurately detect the amplification result.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Sequence listing
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Tianjin Chinese medicine university
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Claims (9)
1. A primer composition comprising four primers, wherein the nucleotide sequences of the four primers are as follows:
an upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3', respectively;
an upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3', respectively;
a downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3', respectively;
downstream outer primer: 5'-CCTGAATGAGATACGGAGTC-3' are provided.
2. Use of the primer composition of claim 1 for identifying whitmania pigra.
3. A method for identifying whitmania pigra by using LAMP amplification technology, which is characterized in that the primer composition of claim 1 is adopted.
4. The method of claim 3, comprising the steps of:
1) respectively extracting the total DNA of a sample to be detected;
2) performing LAMP amplification by respectively using the total DNA of each sample to be detected as a template by using the primer composition; the sample to be detected with positive amplification result is whitmania pigra.
5. The method of claim 4, wherein in step 2), each 25 μ L LAMP amplification system comprises:
6. the method of claim 4, wherein in step 2), the LAMP amplification conditions are:
incubation was carried out at 64 ℃ for 30-60 minutes and at 80 ℃ for 10 minutes.
7. The method of claim 4, wherein the amplification is detected using a nucleic acid fluorescent dye.
8. A kit for identifying whitmania pigra, comprising the primer composition of claim 1.
9. The kit of claim 8, further comprising an isothermal amplification reaction buffer, DEPC water, strand displacement DNA polymerase, dNTPs, MgSO4Aqueous solutions, and instructions for operation.
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Cited By (1)
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| CN112481394A (en) * | 2020-12-15 | 2021-03-12 | 泸州百草堂健康产品有限公司 | PCR detection kit for identifying whitmania pigra |
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| CN112481394B (en) * | 2020-12-15 | 2023-09-15 | 泸州百草堂健康产品有限公司 | PCR detection kit for identifying Hirudinaria manillensis |
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| CN110904242B (en) | 2023-05-02 |
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