CN110904242B - Primer composition and application thereof in identification of Hirudinaria manillensis - Google Patents
Primer composition and application thereof in identification of Hirudinaria manillensis Download PDFInfo
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- CN110904242B CN110904242B CN201911127667.7A CN201911127667A CN110904242B CN 110904242 B CN110904242 B CN 110904242B CN 201911127667 A CN201911127667 A CN 201911127667A CN 110904242 B CN110904242 B CN 110904242B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The embodiment of the invention provides a primer composition for LAMP amplification, a method for identifying Hirudinaria manillensis by using the primer composition, and a kit for identifying Hirudinaria manillensis. By adopting the primer composition provided by the embodiment of the invention, the DNA fragment in the Hirudinaria manillensis can be specifically amplified, so that the Hirudinaria manillensis can be accurately identified.
Description
Technical Field
The invention relates to the technical field of identification of Hirudo manillensis, in particular to a primer composition and application thereof in identification of Hirudo manillensis.
Background
Leech is dried whole of leech Whitmania Pigra Whitman, leech Hirudo nipponica Whitman or leech Whitmania acranutata Whitman. Leech is a common traditional Chinese medicine, and in recent years, leech is used as a raw material medicine for developing a traditional Chinese medicine preparation in a large amount, so that the price of the traditional Chinese medicine of the leech is increased. The main circulation variety in the current market is leech, also called Hirudo manillensis, which is a good variety for medical use, and a method for rapidly and accurately identifying Hirudo manillensis is needed to reduce adulterated sales of traditional Chinese medicinal materials of the Hirudo.
Disclosure of Invention
The embodiment of the invention aims to provide a primer composition for realizing the specific amplification of DNA fragments of Hirudinaria manillensis, so as to accurately identify the Hirudinaria manillensis. The specific technical scheme is as follows:
the first aspect of the present invention provides a primer composition comprising four primers, the nucleotide sequences of the four primers being as follows:
upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3';
upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3';
downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3';
downstream external primer: 5'-CCTGAATGAGATACGGAGTC-3'.
In a second aspect, the invention provides the use of a primer composition according to the first aspect of the invention for identifying Hirudinaria manillensis.
In a third aspect, the present invention provides a method for identifying Hirudinaria manillensis by LAMP amplification technique, using the primer composition provided in the first aspect of the present invention.
In some embodiments of the third aspect of the invention, the identification of the broadbody Hirudinaria by LAMP amplification technique comprises the steps of:
1) Respectively extracting total DNA of a sample to be detected;
2) Performing LAMP amplification by using the primer composition and the total DNA of each sample to be detected as a template; the sample to be detected with positive amplification result is a wide body Hirudinaria manillensis.
In some embodiments of the third aspect of the invention, in step 2), each 25 μl of LAMP amplification system comprises:
2.5. Mu.L of 10 Xisothermal amplification reaction buffer;
0.2. Mu.M upstream outer primer;
0.2. Mu.M downstream outer primer;
1.6. Mu.M upstream inner primer;
1.6. Mu.M downstream inner primer;
50-500ng total DNA;
1.4mM dNTPs；
6mM MgSO 4 ；
8U strand displacement DNA polymerase.
In some embodiments of the third aspect of the present invention, in step 2), the LAMP amplification conditions are:
incubation at 64℃for 30-60 min and at 80℃for 10 min.
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 Hirudinaria manillensis, comprising the primer composition provided in the first aspect of the invention.
In some embodiments of the fourth aspect of the invention, the kit further comprises isothermal amplification reaction buffer, DEPC water, strand displacement DNA polymerase, dNTPs, mgSO 4 Aqueous solutions, and instructions for operation.
By adopting the primer composition provided by the embodiment of the invention, the DNA fragment in the Hirudinaria manillensis can be specifically amplified, so that the Hirudinaria manillensis can be accurately identified.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the results of gel electrophoresis of LAMP amplification of a sample to be tested using a primer composition of the present invention;
FIG. 2 shows the result of fluorescent staining (under natural light) of nucleic acid for LAPM amplification of a sample to be tested using the primer composition of the present invention;
FIG. 3 shows the result of fluorescent staining (under ultraviolet light) of nucleic acid by LAMP amplification of a sample to be tested using the primer composition of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The first aspect of the present invention provides a primer composition comprising four primers, the nucleotide sequences of the four primers being as follows:
upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3' (SEQ ID No: 1);
upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3' (SEQ ID No: 2);
downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3' (SEQ ID No: 3);
downstream external primer: 5'-CCTGAATGAGATACGGAGTC-3' (SEQ ID No: 4).
The four primers are named 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 Hirudinaria manillensis.
In a third aspect, the present invention provides a method for identifying Hirudinaria manillensis by LAMP amplification technique (i.e., loop-mediated isothermal amplification technique), using the primer composition provided in the first aspect of the present invention.
In some embodiments of the third aspect of the invention, the identification of the broadbody Hirudinaria by LAMP amplification technique comprises the steps of:
1) Respectively extracting total DNA of a sample to be detected;
the sample to be tested may be understood as a leech tissue sample to be identified, such as muscle tissue, and in some embodiments of the third aspect of the present invention, after extracting total DNA of the sample to be tested, the concentration of the total DNA is determined, for example, 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 are common technical means in the art, and the present invention is not limited herein.
2) Performing LAMP amplification by using the primer composition and the total DNA of each sample to be detected as a template; the sample to be detected with positive amplification result is a wide body Hirudinaria manillensis.
The positive result is understood to be that the amplification result is detectable, for example, agarose gel electrophoresis detection is adopted, ladder-shaped bands are observed, and no band is a negative result; and (3) dyeing by using a nucleic acid fluorescent dye, wherein fluorescence can be observed under natural light or ultraviolet light to obtain a positive result. 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; because the primer composition has high specificity to the Hirudinaria manillensis, the Hirudinaria manillensis can be rapidly and accurately identified by combining with the detection of nucleic acid fluorescent dye. 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; other nucleic acid fluorochromes may be selected by those skilled in the art according to the actual circumstances, and the invention is not limited herein.
In some embodiments of the third aspect of the invention, in step 2), each 25 μl of LAMP amplification system comprises:
2.5. Mu.L of 10 Xisothermal amplification reaction buffer;
0.2. Mu.M upstream outer primer;
0.2. Mu.M downstream outer primer;
1.6. Mu.M upstream inner primer;
1.6. Mu.M downstream inner primer;
50-500ng total DNA;
1.4mM dNTPs；
6mM MgSO 4 ；
8U strand displacement DNA polymerase;
the balance is DEPC water, which is a common technical means in the art, and the invention is not described herein.
The strand displacement DNA polymerase is a DNA polymerase for LAMP amplification, such as Bst 2.0WarmStart DNA polymerase (New England Biotechnology Co., ltd.), and other DNA polymerases suitable for LAMP amplification may be selected by those skilled in the art according to specific needs, and the present invention is not limited thereto.
In some embodiments of the third aspect of the present invention, in step 2), the LAMP amplification conditions are:
incubation at 64 ℃ for 30-60 min and at 80 ℃ for 10 min; 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 the division of biological engineering (Shanghai), and reagents other than templates and primers in the amplification system and reagents required for detection of the amplification result are commercially available, and the DEPC water is also called enzyme-free water, i.e., water containing no RNase, DNase and protease, which is a conventional reagent in the art, and the present invention is not limited thereto.
In a fourth aspect, the invention provides a kit for identifying Hirudinaria manillensis, comprising the primer composition provided in the first aspect of the invention.
In some embodiments of the fourth aspect of the invention, the kit further comprises isothermal amplification reaction buffer, DEPC water, strand displacement DNA polymerase, dNTPs, mgSO 4 Aqueous solutions, and instructions for operation.
Examples
1. Reagent(s)
The general primers LCO1490 and HCO2198 and the primers sz-F3, sz-FIP, sz-BIP, sz-B3 are synthesized by the division of biological engineering (Shanghai); dNTP mix (dNTPs) (TaKaRa Co.); DEPC water (Biosharp); agarose (Lonza); absolute ethanol (Tianjin, metallocene chemical reagent plant); 6×loading Buffer (TaKaRa Co.); 10000×sybr Green I nucleic acid dye (beijing solebao technologies limited); duRed nucleic acid dye (Beijing Panbo biochemistry Co., ltd.); d2000 DNAMarker (beijing tiangen biochemical technology limited); TAE (50×) (synfatic macrobiotechnology limited); bst 2.0WarmStart DNA polymerase (Neurolim Biotechnology Co., ltd., kit contains 10 Xisothermal amplification reaction buffer); tks Gflex DNA polymerase and 2 XGflex PCRBufer (2 XGflex PCRBufer contains dNTPs) (TaKaRa).
Species identification of two samples to be tested
1. Obtaining of the COI DNA fragment of the sample to be tested
25 parts of leech samples (namely samples to be detected) of different production places are collected, about 30mg of muscle tissues of each leech sample are respectively cut, total DNA is extracted according to the instruction of an animal tissue genome DNA extraction kit (TIANGEN), and the concentration is measured by a Nanodrop2000 micro nucleic acid quantitative analyzer, wherein the total DNA concentration is about 400 ng/. Mu.L. The PCR amplification is carried out by adopting common primers LCO1490 (SEQ ID No: 5) and HCO2198 (SEQ ID No: 6) of CO I (cytochrome oxidase I) and respectively taking total DNA of each sample to be detected as templates, 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,40 cycles; and (5) continuing to extend at 68 ℃ for 5 minutes after the circulation is completed, so as to obtain the COI DNA fragment of the sample to be detected.
2. Sanger sequencing to determine sample species to be tested
And (3) carrying out Sanger sequencing on the amplified products of the primers LCO1490 and HCO2198, wherein the sequencing work is completed by the Beijing Hua megagene. 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 of the invention is adopted to identify the sample to be detected
The total DNA of the sample to be detected is taken as a template, the primer composition is adopted to carry out LAMP amplification, and an amplification system is as follows:
2.5. Mu.L of 10 Xisothermal amplification reaction buffer
0.5μL sz-F3(10μM)
0.5μL sz-B3(10μM)
4μL sz-FIP(10μM)
4μL sz-BIP(10μM)
1 mu L of total DNA
3.5μL dNTPs(10mM)
1.5μL MgSO 4 (100mM)
1 μL Bst 2.0WarmStart DNA polymerase (8U)
6.5. Mu.L DEPC Water
The amplification conditions were: 64 ℃,60 minutes; 80℃for 10 min.
5. Mu.L of the reaction product was added to 5. Mu.L of a 6×loading buffer (Takara Co.) and mixed, followed by detection by electrophoresis on a 1.5% agarose gel stained with DU Red nucleic acid dye, 110V, for 30 minutes, and imaging by a gel imaging system, the results of which are shown in FIG. 1. M in FIG. 1 is represented as a marker (molecular weight marker), the numbering in the figure is consistent with the sample number in Table 1, and N is the negative control (DEPC water). As can be seen from fig. 1, the samples corresponding to the reference numbers K1, K2 and K3 can observe ladder-shaped bands, namely positive results, which indicate that the samples to be tested corresponding to the sample numbers are the broadbody hirudinaria manillensis, and the results are consistent with the Sanger sequencing results, which indicate that the primer composition of the invention can accurately identify the broadbody hirudinaria manillensis.
20. Mu.L of the reaction product was added to 0.2. Mu.L of 10000 XSYBR Green I, and the result was observed under natural light, as shown in FIG. 2, wherein the numbers were the same as those of the samples in Table 1, and N was negative control (DEPC water). The samples corresponding to the marks K1, K2 and K3 are green, namely positive results, and the results are consistent with electrophoresis results; the reaction product to which SYBR Green I was added was observed under a 254nm UV lamp, and the results are shown in FIG. 3, in which Green fluorescence was observed for the samples corresponding to the numbers K1, K2, K3, which were also consistent with the electrophoresis results. The use of nucleic acid fluorescent dyes is illustrated to enable accurate detection of amplification results.
In this specification, each embodiment is described in a related manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments. In particular, for system embodiments, since they are substantially similar to method embodiments, the description is relatively simple, as relevant to see a section of the description of method embodiments.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.
Sequence listing
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Tianjin University of Traditional Chinese Medicine
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Claims (9)
1. A primer composition comprising four primers, wherein the nucleotide sequences of the four primers are as follows:
upstream outer primer: 5'-GTGGGTTTGGTAATTGACTC-3';
upstream inner primer: 5'-AAGGGGGCAGTAACCAAAATCTTAACTACCATTAATGGTAGGGGC-3';
downstream inner primer: 5'-TTGCTTAGGTCATCCTTAATTGAGGTGATAGTGGAGGATAAAGGGTT-3';
downstream external primer: 5'-CCTGAATGAGATACGGAGTC-3'.
2. Use of the primer composition of claim 1 for identifying a broadbody Hirudinaria manillensis.
3. A method for identifying Hirudinaria manillensis by LAMP amplification technique, which is characterized by using the primer composition according to claim 1.
4. A method as claimed in claim 3, comprising the steps of:
1) Respectively extracting total DNA of a sample to be detected;
2) Performing LAMP amplification by using the primer composition and the total DNA of each sample to be detected as a template; the sample to be detected with positive amplification result is a wide body Hirudinaria manillensis.
5. The method of claim 4, wherein in step 2), each 25 μl of LAMP amplification system comprises:
6. the method of claim 4, wherein in step 2), LAMP amplification conditions are:
incubation at 64℃for 30-60 min and at 80℃for 10 min.
7. The method of claim 4, wherein the amplification result is detected using a nucleic acid fluorescent dye.
8. A kit for identifying a broadbody Hirudinaria manillensis, comprising the primer composition of claim 1.
9. The kit according to claim 8, wherein the kit further comprises an isothermal amplification reaction buffer, DEPC water, a strand displacement DNA polymerase, dNTPs, mgSO 4 Aqueous solutions, and instructions for operation.
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