CN116694782A - CDA primer set for amplifying sheep-derived genes, kit and application thereof - Google Patents
CDA primer set for amplifying sheep-derived genes, kit and application thereof Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 claims description 36
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- 230000003321 amplification Effects 0.000 claims description 23
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- 239000003795 chemical substances by application Substances 0.000 claims description 5
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- NXPPAOGUKPJVDI-UHFFFAOYSA-N naphthalene-1,2-diol Chemical compound C1=CC=CC2=C(O)C(O)=CC=C21 NXPPAOGUKPJVDI-UHFFFAOYSA-N 0.000 description 1
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- 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
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- 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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Abstract
The invention discloses a CDA primer set for amplifying sheep-derived component (OA) genes, a kit and application thereof. The CDA primer set is a primer set for amplifying a conserved region fragment of a sheep-derived component, namely OA-MF-4/OA-MR-4; wherein the nucleotide sequence of OA-MF-4 is shown as SEQ ID NO.7, and the nucleotide sequence of OA-MR-4 is shown as SEQ ID NO. 8. The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared by the primer group can rapidly and accurately detect whether a sample to be detected contains sheep-derived components. In addition, the visual kit provided by the invention can provide great convenience for field application of customs, ports, community vegetable fields and the like with low professional degree.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a CDA primer set for amplifying sheep-derived genes, a kit and application thereof.
Background
At present, a method for rapidly detecting mutton components is urgently developed, so that the market is standardized, and the public health is guaranteed.
Existing methods for detecting sheep derived components include detection of amplified signals for viral genome-specific nucleic acid sequences based on RT-PCR. The Shanghai entry and exit inspection quarantine bureau has studied the real-time fluorescence PCR detection method of mutton components. However, the RT-PCR method requires the use of a precise real-time fluorescent PCR instrument in a standard biochemical analysis laboratory, and three independent test areas for reagent preparation, specimen preparation and PCR amplification detection, and is not suitable for on-site rapid screening, so that the development of a simple, rapid, sensitive and high-specificity nucleic acid marker on-site detection technology will provide a rapid and powerful treatment means for diseases caused by sheep-derived component infection.
The closed neck ring-mediated isothermal amplification technology (Closed Dumbbell mediated Isothermal Amplification of nucleic acids, CDA) is a new method developed by the university of Chinese academy of sciences Ningbo life and health industry institute (Chinese patent number: ZL 202110473121.8) and can replace the Japanese LAMP nucleic acid amplification method. The method mainly utilizes 2 different specific primers to identify specific regions of target genes, and carries out amplification reaction under isothermal conditions. Compared with the conventional gene detection means (such as PCR, etc.), the CDA reaction can be completed in a constant-temperature water bath box, has lower requirements on instruments and equipment, is simple to operate, can be accurately completed by non-professionals, and is suitable for basic medical institutions and local inspection and quarantine departments. In addition, the CDA can also shorten the operation time, improve the detection efficiency, reduce the probability of sample pollution and is suitable for rapid diagnosis of sheep-derived components. In addition, the key loop-forming primer used by CDA is about 30bp, which is shorter than LAMP (40 bp), thus saving detection cost.
Disclosure of Invention
The invention aims to provide a CDA primer set for detecting sheep-derived components, a kit and application thereof.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention obtains the primer group with optimal detection effect on sheep-derived components as OA-MF-4/OA-MR-4 by screening the 4 pairs of primer groups in the table 1.
TABLE 1
| Sequence number | Primer name | Nucleotide sequence (5 '-3') |
| SEQ ID NO.1 | OA-MF-1 | CTGTTAGAATCTGCATCATGATGAAACTTTGG |
| SEQ ID NO.2 | OA-MR-1 | GCCTATTCCTAGATAGTTTACGTCTCGGCAA |
| SEQ ID NO.3 | OA-MF-2 | TGTATTGCTAGGCATCATGATGAAACTTTGG |
| SEQ ID NO.4 | OA-MR-2 | CTATACACCTGACATAGTTTACGTCTCGGCAA |
| SEQ ID NO.5 | OA-MF-3 | CCTGTTAGAATCCATCATGATGAAACTTTGG |
| SEQ ID NO.6 | OA-MR-3 | CCTATTCCTAGCATAGTTTACGTCTCGGCAA |
| SEQ ID NO.7 | OA-MF-4 | TGCTAGGAATAGCATCATGATGAAACTTTGG |
| SEQ ID NO.8 | OA-MR-4 | ATACACTATACACATAGTTTACGTCTCGGCAA |
The invention also provides a kit for detecting sheep-derived components, which comprises a CDA primer set OA-MF-4/OA-MR-4.
As a preferred embodiment, the concentration of the primers OA-MF and OA-MR in the CDA primer set in the reaction system is 1-2. Mu.M.
As a preferred embodiment, the kit further comprises Bst DNA polymerase, CDA reaction buffer, ultrapure water, and a color developing agent.
As a preferred embodiment, the color developer is selected from Sybr Green I, EVA Green, hydroxynaphthol blue or chrome Black T.
As a preferred embodiment, the CDA reaction buffer comprises Tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 And Triton X-100.
As a preferred embodiment, the composition of the kit reaction system is:
2～50mM Tris-HCl pH8.8
2～20mM KCl
2～20mM(NH 4 ) 2 SO 4
2～20mM MgSO 4
0.1～0.5％Triton X-100
0.2-1M betaine
1～1.6mM dNTP
5-10U Bst DNA polymerase
100-150 mu mol/L color developing agent
1-2 mu M primer OA-MF-4
1-2 mu M primer OA-MR-4
The reaction solvent is ultrapure water.
The invention also provides a method for detecting sheep-derived components by using the kit for non-identification purposes, which comprises the following steps of:
step 1, mixing a nucleic acid sample to be detected with a reaction system of a kit to prepare an amplification reaction solution;
and 2, reacting the amplification reaction liquid obtained in the step 1 at 60-65 ℃ for 20-80 min, and judging whether the sample contains sheep-derived components according to a color development result.
The invention also provides application of the CDA primer set in sheep-derived component detection for non-identification.
The invention also provides application of the kit in detection of sheep-derived components for non-identification.
Compared with the prior art, the invention has the beneficial effects that:
1. the CDA primer set provided by the invention is aimed at a sheep-derived component gene specific conserved region (GenBank: MZ 664406.1), consists of 2 primers and is an intra-sequence primer pair (OA-MF-4/OA-MR-4). The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared by the primer group can rapidly and accurately detect whether a sample to be detected contains sheep-derived components.
2. The primer group provided by the invention has extremely high specificity, the time required by CDA amplification is short, the detection time is further shortened, and the operation is simple.
3. The method or the kit provided by the invention can complete related detection without expensive instruments or complicated operations, so that the visual kit provided by the invention can provide great convenience for on-site detection of airports, customs, communities and the like with low professionals, and can realize rapid and accurate detection of sheep-derived components.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with time of the amplification reaction of four pairs of primer sets in example 1 of the present invention.
FIG. 2 is a graph showing the change in fluorescence intensity with time of the amplification reaction of the 4 th primer set in example 2 of the present invention.
FIG. 3 is a graph showing the change of fluorescence intensity with time of amplification reaction of genome of various commercially available mutton products by the 4 th primer set in example 3 of the present invention.
Fig. 4 is a graph showing the endpoint monitoring of a reaction based on color change using HNB in example 4 of the present invention.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and can be carried out with reference to the specific methods in the "molecular cloning Experimental guidelines" (third edition) J.Sam Brookfield, or according to the kit and product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 application of EVA Green to respective verification of amplification reaction of four primer sets on sheep-derived component Gene DNA fragments
EVA Green, like SYBR Green I, is a dye with Green excitation wavelength that binds to all dsDNA double helix minor groove regions, and inhibits nucleic acid amplification reactions such as PCR far less than the latter. The fluorescence signal intensity of EVA Green correlates with the amount of double stranded DNA. In the free state, EVA Green emits weak fluorescence, but after binding to double-stranded DNA, the fluorescence is greatly enhanced. Thus, the amount of double-stranded DNA present in the nucleic acid amplification system can be detected based on the fluorescent signal.
The single tube reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EVA Green(Biotum)
Primer:
1600nM OA-MF-1 (shown in SEQ ID NO. 1)
1600nM OA-MR-1 (shown in SEQ ID NO. 2);
1600nM OA-MF-2 (shown in SEQ ID NO. 3)
1600nM OA-MR-2 (shown in SEQ ID NO. 4);
1600nM OA-MF-3 (shown in SEQ ID NO. 5)
1600nM OA-MR-3 (shown in SEQ ID NO. 6);
1600nM OA-MF-4 (shown in SEQ ID NO. 7)
1600nM OA-MR-4 (shown in SEQ ID NO. 8).
And (3) target: sheep-derived component genomic DNA.
Meanwhile, four pairs of primers are respectively provided with a negative control group without targets.
The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min. The curve of fluorescence intensity with time of reaction is shown in FIG. 1. The results in fig. 1 show that: the 1 st group, the 3 rd group and the 4 th group of the four pairs of primer groups provided by the invention can realize the amplification of the sheep-derived gene specific conserved region, wherein the amplification efficiency of the 4 th primer has obvious advantages. Therefore, the 4 th primer group with higher amplification rate is selected to be applied to the real-time monitoring of sheep-derived component genes, and the result can be judged in advance through a real-time amplification curve.
Example 2 verification of amplification reaction of sheep-derived component Gene DNA by primer set of group 4 Using EVA Green (repeated experiments with 8 sets of negative control and Positive samples, respectively)
The procedure is as in example 1.
The reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EVA Green(Biotum)
Group 4 primers:
1600nM OA-MF-4 (shown in SEQ ID NO. 7);
1600nM OA-MR-4 (shown in SEQ ID NO. 8).
And (3) target: sheep-derived component genomic DNA.
Meanwhile, the primer groups are respectively provided with a control group without targets.
The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min. The results in fig. 2 show that: the 4 th primer group provided by the invention can realize rapid amplification of sheep-derived component genes, no false positive appears in a negative control group, the fluorescent detection is applied to the primer group, real-time monitoring can be performed, and the result can be judged in advance through a real-time amplification curve.
Example 3 use of EVA Green to verify group 4 primers for amplification reactions on genomes of various commercially purchased mutton products
The procedure is as in example 1.
The reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EVA Green(Biotum)
Group 4 primers:
1600nM LM-MF (shown in SEQ ID NO. 7)
1600nM LM-MR (shown in SEQ ID NO. 8)
Target nucleic acid 1 sheep-derived component DNA
Target nucleic acid 2 non-sheep derived component DNA
A no-target control group was also set as a negative control.
8 replicates were set for each set of amplification reactions. The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min. The curve of fluorescence intensity with time of reaction is shown in FIG. 3. The results in fig. 3 show that: the 4 th primer group provided by the invention can only amplify the sheep source component DNA of the positive sample group, and can not amplify other bacteria; thus, it is possible to distinguish between sheep-derived and non-sheep-derived component DNA. The experimental result further illustrates the specificity of the CDA primer set for detecting sheep-derived components.
EXAMPLE 4 sheep derived component CDA amplification reaction end-point monitoring Using Hydroxy Naphthol Blue (HNB)
Hydroxyl Naphthol Blue (HNB) belongs to a metal ion indicator, and aims at the change of magnesium ions or manganese ions combined with byproduct pyrophosphates in the reaction, so that different indication colors are displayed to judge the result.
The combinations of the reaction solutions for amplifying sheep-derived component CDA using hydroxynaphthol blue (HNB) are shown below.
The reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
120μM HNB
Primer:
1600nM LM-MF (shown in SEQ ID NO. 7)
1600nM LM-MR (shown in SEQ ID NO. 8)
Target nucleic acid 1 sheep-derived component DNA
Target nucleic acid 2 non-sheep derived component DNA
A no-target control group was also set as a negative control.
8 replicates were set for each set of amplification reactions. The reaction temperature of the constant temperature water bath kettle is set to be 63 ℃ and the reaction time is set to be 60 minutes. The end point results of the yin-yang reaction are shown in fig. 4, wherein the color of violet is negative, and the sky blue is positive. The experimental results show that: the HNB can intuitively judge the reaction result through the color, and can judge without the assistance of an instrument.
The foregoing is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.
Claims (10)
1. A CDA primer set for detecting sheep-derived components for non-identification purposes, characterized in that: the primer group is OA-MF-4/OA-MR-4, wherein the nucleotide sequence of the OA-MF-4 is shown as SEQ ID NO.7, and the nucleotide sequence of the OA-MR-4 is shown as SEQ ID NO. 8.
2. A kit for detecting sheep-derived components is characterized in that: the kit comprises the CDA primer set of claim 1.
3. A kit for detecting sheep-derived components as claimed in claim 2, wherein: the concentration of the primers OA-MF-4 and OA-MR-4 in the CDA primer set in the reaction system is 1-2 mu M.
4. A kit for detecting sheep-derived components as claimed in claim 2, wherein: the kit also comprises Bst DNA polymerase, CDA reaction buffer, ultrapure water and a color developing agent.
5. A kit for detecting a sheep derived composition as claimed in claim 4, wherein: the color developing agent is selected from one of Sybr green I, EVA green, hydroxynaphthol blue and chrome black T.
6. A kit for detecting a sheep derived composition as claimed in claim 4, wherein: the CDA reaction buffer comprises Tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 And Triton X-100.
7. A kit for detecting sheep-derived components as claimed in claim 2, wherein: the kit reaction system comprises the following components:
2～50mM Tris-HCl pH8.8
2～20mM KCl
2～20mM(NH 4 ) 2 SO 4
2～20mM MgSO 4
0.1～0.5％Triton X-100
0.2-1M betaine
1～1.6mM dNTP
5-10U Bst DNA polymerase
100-150 mu mol/L color developing agent
1-2 mu M primer OA-MF-4
1-2 mu M primer OA-MR-4
The reaction solvent is ultrapure water.
8. A method of detecting sheep derived components using the kit of claim 7 for non-identification purposes, comprising the steps of:
step 1, mixing a nucleic acid sample to be detected with a reaction system of a kit to prepare an amplification reaction solution;
and 2, reacting the amplification reaction solution prepared in the step 1 at 60-65 ℃ for 20-80 min, and judging whether the sample contains sheep-derived components according to a color development result.
9. Use of the CDA primer set of claim 1 for detection of sheep-derived components for non-identification purposes.
10. Use of a kit according to any one of claims 2 to 7 for the detection of sheep derived components for non-identification purposes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310815887.9A CN116694782A (en) | 2023-07-05 | 2023-07-05 | CDA primer set for amplifying sheep-derived genes, kit and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310815887.9A CN116694782A (en) | 2023-07-05 | 2023-07-05 | CDA primer set for amplifying sheep-derived genes, kit and application thereof |
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Application publication date: 20230905 |