CN112094945A - Primer and kit for efficiently detecting porcine epidemic diarrhea virus - Google Patents
Primer and kit for efficiently detecting porcine epidemic diarrhea virus Download PDFInfo
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- CN112094945A CN112094945A CN202010840373.5A CN202010840373A CN112094945A CN 112094945 A CN112094945 A CN 112094945A CN 202010840373 A CN202010840373 A CN 202010840373A CN 112094945 A CN112094945 A CN 112094945A
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- 241001135549 Porcine epidemic diarrhea virus Species 0.000 title claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000010931 gold Substances 0.000 claims abstract description 7
- 229910052737 gold Inorganic materials 0.000 claims abstract description 7
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 7
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 25
- 238000005516 engineering process Methods 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 abstract description 2
- 108091005804 Peptidases Proteins 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 abstract description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 2
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 abstract description 2
- 206010029719 Nonspecific reaction Diseases 0.000 abstract 2
- 230000003321 amplification Effects 0.000 description 34
- 238000003199 nucleic acid amplification method Methods 0.000 description 33
- 150000007523 nucleic acids Chemical group 0.000 description 19
- 108020004707 nucleic acids Proteins 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 238000007689 inspection Methods 0.000 description 10
- 229960005486 vaccine Drugs 0.000 description 7
- 206010012735 Diarrhoea Diseases 0.000 description 6
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- 238000012795 verification Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 5
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- 241000700605 Viruses Species 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
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- 108090000623 proteins and genes Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 2
- 241001673669 Porcine circovirus 2 Species 0.000 description 2
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- 206010037660 Pyrexia Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
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- 238000004108 freeze drying Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 238000000464 low-speed centrifugation Methods 0.000 description 2
- 235000015277 pork Nutrition 0.000 description 2
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- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
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- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 1
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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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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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
Abstract
The invention discloses a primer for efficiently detecting porcine epidemic diarrhea virus and a kit containing the primer, wherein the kit comprises the primer and a reaction solution, and the reaction solution is prepared by mixing the following solutions: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4)2SO4，8mMMgSO40.1% Tween-20, 1.4 mM dNTPs, 8000U/mL Bst enzyme, 25nM SYTO-9 fluorochrome; gold nanoparticles are added into a reaction system to adsorb ssDNA and protease, so that nonspecific reaction in the temperature rise process is inhibited, the purpose of hot start is achieved, and nonspecific reaction in the temperature rise process is avoided; combining the improved LAMP technology and the micro-fluidic chip technology to obtain the productSo as to quickly give an accurate detection result.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer and a kit for efficiently detecting porcine epidemic diarrhea virus.
Background
Porcine Epidemic diarrhea, abbreviated as ped (porcine Epidemic diarrhea) in english, is a contact intestinal infectious disease caused by porcine Epidemic diarrhea virus and is characterized by vomiting, diarrhea, and dehydration. If the existing PCR technology is adopted for virus detection, the reaction is circulated in 2 different temperature regions, the requirement on instruments is high, the cost is relatively high, and the PCR technology only has 1 pair of amplification primers, is easy to be interfered, has relatively insufficient specificity, has long result output time, high operation professional requirement and more trace addition steps. The LAMP technology has 4 different specific primers, so the detection result accuracy is higher, but the isothermal reaction is adopted, a hot start enzyme similar to PCR is lacked, and nonspecific amplification is easily generated at the temperature rise stage of equipment, so the detection result is influenced.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer and a kit for efficiently detecting porcine epidemic diarrhea virus aiming at the defects of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: a primer for efficiently detecting porcine epidemic diarrhea viruses comprises the following specific steps:
PEDV-F3：GCTTCAAATGTGACGGGTT；
PEDV-B3：CTAAACAAAGCCTGCCAAT；
PEDV-FIP：
GCTGCCAACATAATATAATTGCGC-CACCAGTGTTTTTGTTTACTTCT；
PEDV-BIP：
GCGTTTTGCTGTCGTCTTTCTC-CGCAACAAATAATAGTTGCATCT。
the invention provides a kit for efficiently detecting porcine epidemic diarrhea virus, which comprises the primers and a reaction solution, wherein 18 mu L of the reaction solution is prepared by mixing the following solutions: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4)2SO4，8 mM MgSO40.1% Tween-20, 1.4 mM dNTPs, 8000U/mL Bst enzyme, 25nM SYTO-9 fluorescent dye and gold nanoparticles.
20mM Tris-HCl 3.5μL；
10mM KCl 2μL；
10mM （NH4）2SO4 2μL；
8mM MgSO4 2μL；
0.1% Tween-20 2μL；
1.4mM dNTPs 2.5μL；
8000U/mL Bst enzyme 2 uL;
1 μ L of 25nM SYTO-9 fluorescent dye;
1 μ L of gold nanoparticles.
The concentration and volume of the primer are as follows:
PEDV-F3：100μM 0.01μL；
PEDV-B3：100μM 0.01μL；
PEDV-FIP：100μM 0.08μL；
PEDV-BIP：100μM 0.08μL。
wherein, this kit chooses 8 sample chips for use, 1 adds the sample hole and corresponds 4 inspection holes promptly, and the 1 st inspection hole and the 2 nd inspection hole bury the primer of amplification pig epidemic diarrhea virus sequence respectively, and the 3 rd inspection hole is blank, and reference primer and reference plasmid are buried to the 4 th inspection hole, freeze-drying.
The nucleic acid sequence of the porcine epidemic diarrhea virus is SEQ NO.1:
ATGTAGTTCCAATTAGACAAGCTTCAAATGTGACGGGTTTTCTTTTCACCAGTGTTTTTGTTTACTTCTTTGCACTGTTTAAAGCGTCTTCTTTGAGGCGCAATTATATTATGTTGGCAGCGCGTTTTGCTGTCGTCTTTCTCTATTGCCCACTTTTATATTACTGTGGTGCACTTTTAGATGCAACTATTATTTGTTGCGCACTTATTGGCAGGCTTTGTTTAGTCTGCTTTTACTCCTGGCGCTATAAAAATGCGCTTTTTATTATCTTTAATACTACGACACTTTCTTTTCTCAATGGTAAAGCAGCTTATTATGACGGCAAATCCATTGTGATTCTAGAAGGTGGCGACCATTACATCACTTTTGGCAACTCTTTTGTTGCTTTCGTTAGTAACATTGACTTGTATCTAGCTATACGTGGGCGGCAAGAAGCTGACCTACATCTGTTGCGAACTGTTGAGCTTCTTGATGGCAAGAAGCTTTATGTCTTTTCGCA。
compared with the prior art, the invention has the following advantages: the invention takes the improved LAMP technology as the gene amplification reaction principle, gold nanoparticles are added into a reaction system to adsorb ssDNA and protease, so as to inhibit the non-specific reaction in the heating process, achieve the purpose of hot start and avoid the non-specific reaction in the heating process; the improved LAMP technology and the improved microfluidic chip technology are combined, so that an accurate detection result can be rapidly given, the aim of joint detection of a plurality of different indexes of the same sample is fulfilled, meanwhile, the reaction reagent is embedded on the microfluidic chip, and a user only needs to add the sample, so that the operation is simple and convenient.
Drawings
FIG. 1 is a diagram showing the amplification results of the verification of the detection limit in the examples.
FIG. 2 is a diagram showing the results of amplification in the verification of reproducibility in examples.
FIG. 3 is a diagram showing the amplification results of the verification of specificity in the examples.
Detailed Description
Examples
Firstly, searching a target sequence through NCBI GenBank, designing primers aiming at the target sequence, respectively fixing the primers at corresponding positions of a microfluidic chip, packaging the microfluidic chip, mixing and reacting the primers with a nucleic acid template extracted from pork and various organs, blood, excrement, an environmental sample and cell culture of the pork, adding the mixture into the packaged microfluidic chip, and then putting the packaged microfluidic chip into a microfluidic chip detector with a centrifugal function, a constant temperature function and real-time fluorescence detection, wherein the instruments and the chips are commercially available products, Ningbo love gene science and technology limited company also has corresponding products, and a sample is driven to enter a reaction hole of the microfluidic chip by using centrifugal force to carry out constant temperature amplification. And if the sample contains the target fragment, performing isothermal amplification, effectively combining an amplification product with a fluorescent substance, capturing a fluorescent signal in real time by using a fluorescent detector, intuitively reacting the generation of the amplification product, and judging whether the sample contains PEDV or not according to the appearance time, intensity and position of the real-time fluorescent signal.
The specific operation steps are as follows:
the composition of 18 mul reaction solution in the micro-fluidic chip detection system is as follows:
20 mM Tris-HCl（pH 8.8），10 mM KCl，10 mM （NH4）2SO4，8mM MgSO40.1% Tween-20, 1.4 mM dNTPs, 8000U/mL Bst DNA polymerase, 25nM SYTO-9 fluorochrome, 1. mu.L of gold nanoparticles.
20mM Tris-HCl 3.5μL；
10mM KCl 2μL；
10mM （NH4）2SO4 2μL；
8mM MgSO4 2μL；
0.1% Tween-20 2μL；
1.4mM dNTPs 2.5μL；
8000U/mL Bst enzyme 2 uL;
1 μ L of 25nM SYTO-9 fluorescent dye;
1 μ L of gold nanoparticles.
The concentration and volume of the primer are as follows:
wherein, this kit chooses 8 sample chips for use, 1 adds the sample hole and corresponds 4 inspection holes promptly, and the 1 st inspection hole and the 2 nd inspection hole bury the primer of amplification pig epidemic diarrhea virus sequence respectively, and the 3 rd inspection hole is blank, and reference primer and reference plasmid are buried to the 4 th inspection hole, freeze-drying.
The nucleic acid sequence of the porcine epidemic diarrhea virus is SEQ NO.1:
ATGTAGTTCCAATTAGACAAGCTTCAAATGTGACGGGTTTTCTTTTCACCAGTGTTTTTGTTTACTTCTTTGCACTGTTTAAAGCGTCTTCTTTGAGGCGCAATTATATTATGTTGGCAGCGCGTTTTGCTGTCGTCTTTCTCTATTGCCCACTTTTATATTACTGTGGTGCACTTTTAGATGCAACTATTATTTGTTGCGCACTTATTGGCAGGCTTTGTTTAGTCTGCTTTTACTCCTGGCGCTATAAAAATGCGCTTTTTATTATCTTTAATACTACGACACTTTCTTTTCTCAATGGTAAAGCAGCTTATTATGACGGCAAATCCATTGTGATTCTAGAAGGTGGCGACCATTACATCACTTTTGGCAACTCTTTTGTTGCTTTCGTTAGTAACATTGACTTGTATCTAGCTATACGTGGGCGGCAAGAAGCTGACCTACATCTGTTGCGAACTGTTGAGCTTCTTGATGGCAAGAAGCTTTATGTCTTTTCGCA。
and 32 mu L of nucleic acid of the porcine epidemic diarrhea virus is taken, and the sequence of the nucleic acid refers to SEQ NO. 1;
then mixing 18 mu L of reaction liquid with 32 mu L of template nucleic acid, adding the mixture into a sample adding hole of the chip, sealing the sample adding hole by using a sealing film, and putting the chip on the chip;
the temperature was set at 63.5 ℃ and the reaction time was set at 30 min.
2. And (3) performing on-machine amplification on the microfluidic chip:
because the method adopts constant temperature amplification, the temperature-variable processes of denaturation, annealing, extension and the like of PCR amplification are not needed, the whole reaction process is finished under the constant temperature condition, and the amplification program comprises the following steps: the temperature was set at 63.5 ℃ and the reaction time was set at 30 min. And (3) running a program: the low-speed centrifugation rotating speed is 1600r/min, the low-speed centrifugation time is 10sec, the high-speed centrifugation rotating speed is 4600r/min, and the high-speed centrifugation time is 30 sec.
3. And (3) judging the result of the microfluidic chip:
3.1 microfluidic chip Detector threshold line set
The threshold line is set to 800 (which can be adjusted according to the actual situation, the setting principle is that the threshold line just exceeds the highest point of the atypical S-type amplification curve, and the Ct value is displayed as 30), and the instrument matching software automatically analyzes the result.
3.2 quality control
Positive control: ct <30, and the reaction well of the positive control has a remarkable typical S-shaped amplification curve.
Negative control: ct <30, no amplification curve for reaction wells of negative control.
3.3 determination of results
3.3.1 Experimental establishment conditions
Positive control: ct value is less than or equal to 30, and corresponding detection holes have obvious typical S-shaped amplification curves.
Negative control: ct value > 30 or 0, no obvious typical S-type amplification curve corresponding to the detection hole.
3.3.2 criteria of determination
Positive: the Ct of the reaction hole is less than 30, and the reaction hole has an obvious amplification curve and is judged to be positive.
Negative: the Ct of the reaction hole is less than 30, no amplification curve exists, and the reaction hole is judged to be negative.
Carrying out constant-temperature amplification on the microfluidic chip on a microfluidic chip detector, carrying out real-time fluorescence detection by the detector, judging and reading according to an effective amplification curve of the fluorescence detection, and judging that any hole or multiple holes are positive if a standard S-shaped amplification curve exists in the hole, namely the sample contains viral nucleic acid corresponding to the detection hole; wells without amplification curve were judged negative, i.e., the sample did not contain viral nucleic acid corresponding to the detection well.
4. Verification of sensitivity and detection limits
4.1 Experimental materials
Reagent: reaction solution; 1X 106 copies/µL、1×105 copies/µL、1×104 copies/µL、1×103copies/µL、1×102 copies/µL、1×101 copies/µL、1×100A plasmid of copies/μ L with a porcine epidemic diarrhea virus gene fragment; negative control; and (4) positive control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
4.2 detection System
Performing experiment operation with reference to the above detection system, placing the chip into a constant temperature amplification instrument for test detection, wherein the amplification result can be seen as the result of detection limit in FIG. 1, and the lowest detection limit is 1 × 101The Ct of the plasmid with copies/mu L is less than 30min, which indicates that the sensitivity is very high.
5. Verification of repeatability
5.1 Experimental materials
Reagent: reaction solution; 1X 104 Plasmid of copies/μ L; negative control; and (4) positive control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
5.2 detection System
And (3) carrying out experimental operation by referring to the detection system, and then putting the chip into a constant-temperature amplification instrument for experimental detection.
5.3 amplification results
| Repetition of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Ct value | 8.05 | 7.88 | 7.99 | 8.35 | 8.43 | 8.63 | 8.49 | 8.73 |
Through calculation, the coefficient of variation (CV,%) of the Ct value is 3.5%, the repeatability is good and is less than 5%, and the requirements are met.
6. Verification of specificity
6.1 Experimental materials
Reagent: reaction solution; swine fever live vaccine nucleic acid; porcine reproductive and respiratory syndrome live vaccine nucleic acid; porcine pseudorabies live vaccine nucleic acid; porcine circovirus type 2 culture fluid nucleic acid; porcine parvovirus cell culture fluid nucleic acid; negative control; and (4) positive control.
The instrument comprises the following steps: a constant temperature amplification instrument; a palm centrifuge; a pipette.
6.2 detection System
The experimental operation was carried out with reference to the detection system in the above 1, and then the chip was put into an isothermal amplification apparatus for experimental detection.
6.3 amplification results
The amplification results can be referred to fig. 2, and from the results in fig. 2, it can be seen that the nucleic acid of the swine fever live vaccine; porcine reproductive and respiratory syndrome live vaccine nucleic acid; porcine pseudorabies live vaccine nucleic acid; porcine circovirus type 2 culture fluid nucleic acid; the porcine parvovirus cell culture solution nucleic acid and the porcine foot-and-mouth disease vaccine nucleic acid have no amplification curve, which indicates that the primer can only specifically amplify and detect the porcine epidemic diarrhea, has good specificity and generally does not generate cross reaction with other pathogenic bacteria.
The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by equivalent exchanges or equivalent substitutions fall within the protection scope of the present invention.
Claims (4)
1. A primer for efficiently detecting porcine epidemic diarrhea virus is characterized by comprising the following specific steps:
PEDV-F3：GCTTCAAATGTGACGGGTT；
PEDV-B3：CTAAACAAAGCCTGCCAAT；
PEDV-FIP：
GCTGCCAACATAATATAATTGCGC-CACCAGTGTTTTTGTTTACTTCT；
PEDV-BIP：
GCGTTTTGCTGTCGTCTTTCTC-CGCAACAAATAATAGTTGCATCT。
2. the kit for efficiently detecting the porcine epidemic diarrhea virus is characterized by comprising the primers and a reaction solution, wherein the reaction solution is packagedThe preparation method comprises the following steps of: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4)2SO4，8mM MgSO40.1% Tween-20, 1.4 mM dNTPs, 8000U/mL Bst enzyme, 25nM SYTO-9 fluorescent dye.
3. The kit for efficiently detecting porcine epidemic diarrhea virus according to claim 2, wherein the reaction solution specifically comprises:
20mM Tris-HCl 3.5μL；
10mM KCl 2μL；
10mM （NH4）2SO4 2μL；
8mM MgSO4 2μL；
0.1% Tween-20 2μL；
1.4mM dNTPs 2.5μL；
8000U/mL Bst enzyme 2 uL;
1 μ L of 25nM SYTO-9 fluorescent dye;
1 μ L of gold nanoparticles.
4. The kit for efficiently detecting porcine epidemic diarrhea virus of claim 2, wherein the concentration and volume of the primers are as follows:
PEDV-F3：100μM 0.01μL；
PEDV-B3：100μM 0.01μL；
PEDV-FIP：100μM 0.08μL；
PEDV-BIP：100μM 0.08μL。
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Application publication date: 20201218 |