CN111850173A - Primer and kit for efficiently detecting porcine circovirus type 2 - Google Patents
Primer and kit for efficiently detecting porcine circovirus type 2 Download PDFInfo
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- CN111850173A CN111850173A CN202010786282.8A CN202010786282A CN111850173A CN 111850173 A CN111850173 A CN 111850173A CN 202010786282 A CN202010786282 A CN 202010786282A CN 111850173 A CN111850173 A CN 111850173A
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Abstract
The invention discloses a primer and a kit for efficiently detecting porcine circovirus type 2.A modified LAMP technology is used as a gene amplification reaction principle, gold nanoparticles are added into a reaction system to adsorb ssDNA and protease, so that the nonspecific reaction in the heating process is inhibited, the purpose of hot start is achieved, and the nonspecific reaction in the heating process is avoided; 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.
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 circovirus type 2.
Background
Porcine circovirus is classified into type 1 and type 2. There is no evidence to suggest that porcine circovirus type 1 can cause disease in pigs, but extensive contamination in PK-15 cells and high infection rates in swine herds have attracted much attention, whereas porcine circovirus type 2 has compromised the ability to compromise the immune function of infected pigs, a virus that causes immunosuppression in the body, and is often overlooked due to its frequent occurrence as a subclinical infection. In order to better control the infection of porcine circovirus, it is urgently needed to find a suitable rapid and accurate diagnosis method.
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 circovirus type 2 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 circovirus type 2 specifically comprises the following components:
PCV22-F3-8: ATTACTTCCAACCAAACAACA；
PCV22-B3-8: GGTTAAGTGGGGGGTCTT；
PCV22-FIP-8:CGAGGCCTACGTGGTCTACA-AAAGAAATCAGCTGTGGCT；
PCV22-BIP-8:ACTGCGTTCGAAAACAGTATATACG-TCTGAATTGTACATACATGGTTAC。
the invention provides a kit for efficiently detecting porcine circovirus type 2, which comprises the primers and a reaction solution, wherein the reaction solution comprises the following components:
20mM Tris-HCl 0.18μL；
10mM KCl 6.8μL；
10mM （NH4）2SO41.8μL；
8mM MgSO41.8μL；
0.1% Tween-20 1.8μL；
1.4mM dNTPs 0.9μL；
800U/mL Bst enzyme 1.8. mu.L;
0.9 μ L of 50uM SYBRGREEN fluorescent dye;
4.0x10-61.8 mu L of gold nanoparticles in mol/L;
0.22 μ L of water.
The concentration and volume of the primer are as follows: PCV 22-F3-8: 90 μ M0.5 μ L, PCV 22-B3-8: 90 μ M0.5 μ L, PCV 22-FIP-8: 180. mu.M 2. mu. L, PCV 22-BIP-8: 180 μ M2 μ L.
The kit selects 8 sample chips, namely 1 sample adding hole corresponds to 4 detection holes, the 1 st detection hole and the 2 nd detection hole are respectively embedded with primers for amplifying the porcine circovirus type 2 sequence, the 3 rd detection hole is blank, and the 4 th detection hole is embedded with an internal reference primer and an internal reference plasmid and is freeze-dried.
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 shows a verification target plasmid 10 for sensitivity and detection limit in examples5Schematic representation of amplification results for copies/. mu.L.
FIG. 2 shows the validation target plasmid 10 for sensitivity and detection limit in the examples4Schematic representation of amplification results for copies/. mu.L.
FIG. 3 shows sensitivity and detection in examplesLimited validation target plasmid 103Schematic representation of amplification results for copies/. mu.L.
FIG. 4 shows the validation target plasmid 10 for sensitivity and detection limit in examples2Schematic representation of amplification results for copies/. mu.L.
FIG. 5 shows the validation target plasmid 10 for sensitivity and detection limit in examples1Schematic representation of amplification results for copies/. mu.L.
FIG. 6 shows the validation target plasmid 10 for sensitivity and detection limit in examples0Schematic representation of amplification results for copies/. mu.L.
FIG. 7 is a diagram showing the results of the positive control test for verifying sensitivity and detection limit in the examples.
FIG. 8 is a diagram showing the results of a negative control test for verifying sensitivity and detection limit in examples.
FIG. 9 shows the target plasmid 10 for verifying the reproducibility of the examples4Schematic representation of amplification results for copies/. mu.L.
FIG. 10 is a graph showing the results of the negative control test for verifying reproducibility in the examples.
FIG. 11 is a graph showing the results of the positive control test for verifying reproducibility in the examples.
FIG. 12 is a diagram showing the results of the specific verification positive control test in the examples.
FIG. 13 is a diagram showing the results of the specific verification negative control test in the examples.
FIG. 14 shows amplification curves of porcine parvovirus live vaccine nucleic acid, porcine erysipelas live vaccine nucleic acid, porcine transmissible gastroenteritis virus live vaccine nucleic acid, porcine Japanese encephalitis virus live vaccine nucleic acid, and swine fever live vaccine nucleic acid in 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 the 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 the porcine circovirus type 2 or not according to the appearance time, the intensity and the position of the real-time fluorescent signal.
The specific operation steps are as follows:
1. the composition of 18 mul reaction solution in the micro-fluidic chip detection system is as follows:
20mM Tris-HCl 0.18μL；
10mM KCl 6.8μL；
10mM （NH4）2SO41.8μL；
8mM MgSO41.8μL；
0.1% Tween-20 1.8μL；
1.4mM dNTPs 0.9μL；
800U/mL Bst enzyme 1.8. mu.L;
0.9 μ L of 50uM SYBRGREEN fluorescent dye;
4.0x10-61.8 mu L of gold nanoparticles in mol/L;
0.22 microliter of ultrapure water;
the concentration and volume of the primer are as follows: PCV 22-F3-8: 90 μ M0.5 μ L, PCV 22-B3-8: 90 μ M0.5 μ L, PCV 22-FIP-8: 180. mu.M 2. mu. L, PCV 22-BIP-8: 180 μ M2 μ L. The kit selects 8 sample chips, namely 1 sample adding hole corresponds to 4 detection holes, the 1 st detection hole and the 2 nd detection hole are respectively embedded with primers for amplifying the porcine circovirus type 2 sequence, the 3 rd detection hole is blank, and the 4 th detection hole is embedded with an internal reference primer and an internal reference plasmid and is freeze-dried.
And 32 mu.L of nucleic acid of porcine circovirus type 2 is taken, and the sequence of the nucleic acid is referred to DQ235696.1
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 105copies/µL、1×104copies/µL、1×103copies/µL、1×102copies/µL、1×101copies/µL、1×100A plasmid of copies/μ L with a porcine circovirus type 2 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 the above detection system, placing the chip into a constant temperature amplification instrument for test detection, wherein the amplification result is shown in FIGS. 1-8, and the lowest detection limit is 1 × 102The 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 104Plasmid 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
As shown in fig. 9-11;
table 1:
| repetition of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| CT value | 14.89 | 15.28 | 15.24 | 14.27 | 14.23 | 14.68 | 13.94 | 14.79 | 14.60 | 14.91 |
Through calculation, the coefficient of variation (CV,%) of the Ct value is 2.96%, 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; porcine parvovirus live vaccine nucleic acid; swine erysipelas live vaccine nucleic acid; porcine transmissible gastroenteritis virus live vaccine nucleic acid; porcine Japanese encephalitis virus live vaccine nucleic acid; swine fever live vaccine 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 results of amplification are shown in FIGS. 12 to 14, from which it can be seen that the porcine parvovirus live vaccine nucleic acid; swine erysipelas live vaccine nucleic acid; porcine transmissible gastroenteritis virus live vaccine nucleic acid; porcine Japanese encephalitis virus live vaccine nucleic acid; the nucleic acid of the swine fever live vaccine has no amplification curve, which indicates that the primer can only specifically amplify and detect the porcine circovirus type 2 nucleic acid, has good specificity and generally does not produce 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 (3)
1. A primer for efficiently detecting porcine circovirus type 2 is characterized by comprising the following specific components:
PCV22-F3-8: ATTACTTCCAACCAAACAACA；
PCV22-B3-8: GGTTAAGTGGGGGGTCTT；
PCV22-FIP-8:CGAGGCCTACGTGGTCTACA-AAAGAAATCAGCTGTGGCT；
PCV22-BIP-8:ACTGCGTTCGAAAACAGTATATACG-TCTGAATTGTACATACATGGTTAC。
2. the kit for efficiently detecting the porcine circovirus type 2 is characterized by comprising the primers and a reaction solution, wherein the reaction solution comprises the following components:
20mM Tris-HCl 0.18μL；
10mM KCl 6.8μL；
10mM （NH4）2SO41.8μL；
8mM MgSO41.8μL；
0.1% Tween-20 1.8μL；
1.4mM dNTPs 0.9μL；
800U/mL Bst enzyme 1.8. mu.L;
0.9 μ L of 50uM SYBRGREEN fluorescent dye;
4.0x10-61.8 mu L of gold nanoparticles in mol/L;
0.22 μ L of water.
3. The kit for efficiently detecting porcine circovirus type 2 according to claim 2, wherein the concentration and volume of the primers are as follows: PCV 22-F3-8: 90 μ M0.5 μ L, PCV 22-B3-8: 90 μ M0.5 μ L, PCV 22-FIP-8: 180. mu.M 2. mu. L, PCV 22-BIP-8: 180 μ M2 μ L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322349A (en) * | 2021-04-23 | 2021-08-31 | 华南农业大学 | Primer and kit for detecting porcine circovirus type 2 by combining centrifugal microfluidic chip with loop-mediated isothermal amplification technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307367A (en) * | 2008-02-20 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Technology for rapidly detecting porcine circovirus type2 |
| CN110791592A (en) * | 2019-12-04 | 2020-02-14 | 宁波爱基因科技有限公司 | Primer and kit for rapidly detecting African swine fever virus |
| CN111270007A (en) * | 2019-12-09 | 2020-06-12 | 宁波爱基因科技有限公司 | Primer, micro-fluidic chip and system for detecting classical swine fever virus and application of primer, micro-fluidic chip and system |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307367A (en) * | 2008-02-20 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Technology for rapidly detecting porcine circovirus type2 |
| CN110791592A (en) * | 2019-12-04 | 2020-02-14 | 宁波爱基因科技有限公司 | Primer and kit for rapidly detecting African swine fever virus |
| CN111270007A (en) * | 2019-12-09 | 2020-06-12 | 宁波爱基因科技有限公司 | Primer, micro-fluidic chip and system for detecting classical swine fever virus and application of primer, micro-fluidic chip and system |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322349A (en) * | 2021-04-23 | 2021-08-31 | 华南农业大学 | Primer and kit for detecting porcine circovirus type 2 by combining centrifugal microfluidic chip with loop-mediated isothermal amplification technology |
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