CN114369685A - Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for mink circovirus based on Taqman probe - Google Patents
Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for mink circovirus based on Taqman probe Download PDFInfo
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Abstract
A real-time fluorescence quantitative PCR kit of mink circovirus based on a Taqman probe relates to the field of mink circovirus detection, and comprises an upstream primer, a downstream primer, a probe, 2X TaqMan Fast qPCR premix solution and ddH2O, positive quality control material and negative quality control material; upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively; downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively; probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3', respectively; the 5 'end of the probe MiCV-P is marked by a fluorescent group, and the 3' end of the probe MiCV-P is marked by a fluorescence quenching group. The method has the advantages of strong pertinence, strong specificity, high sensitivity and good repeatability, and can be used for rapidly determining the mink circovirus in real time.
Description
Technical Field
The invention relates to the technical field of mink circovirus detection, in particular to a real-time fluorescence quantitative PCR kit for mink circovirus based on a Taqman probe.
Background
Mink fur is one of three major products in the international fur market, and the annual trade volume of the mink fur reaches more than 5000 ten thousand in the world. The mink is introduced in 1956 in China, and the market of the mink is stable and tends to rise at present. As a large export country, the mink breeding industry has wide development prospect and plays an important role in promoting the local economic growth.
Mink circovirus (MiCV) is a novel circovirus discovered in recent years, and symptoms such as diarrhea, lethargy, anorexia, listlessness, rough fur and the like of sick minks can be caused after the MiCV infects the minks. At present, main distribution areas of mink breeding in China include Shandong, Hebei, northeast and the like, most mink breeding areas in China have found that minks suffer from the disease, the MiCV infection rate reaches 44-63%, great threat is brought to the mink breeding industry, and the economic benefit of the mink farm is seriously influenced.
At present, no effective vaccine or medicine has been developed for the prevention and treatment of mink circovirus, no cell culture system for the mink circovirus can be used for virus isolation and identification, the pathogenic mechanism of the mink circovirus is not clear, and in addition, along with the prevalence of diseases, the mink circovirus (MiCV) genome is continuously mutated, which increases certain difficulty for the effective prevention and treatment of the mink circovirus. Therefore, how to rapidly and effectively detect the mink circovirus to reduce the economic loss caused by the disease is important.
In the aspect of virus detection, a conventional PCR method, a Recombinase Polymerase Amplification (RPA) method, an indirect enzyme-linked immunosorbent assay, a fluorescent quantitative PCR method and the like are mainly adopted. Although the methods can be applied to mink circovirus detection, the detected effect is not ideal.
Disclosure of Invention
In order to improve the effect and accuracy of mink circovirus (MiCV) detection, the invention provides a real-time fluorescence quantitative PCR kit for mink circovirus based on a Taqman probe, which mainly comprises a primer and a probe sequence for detecting the mink circovirus and the real-time fluorescence quantitative PCR kit.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the primer and probe sequences for detecting the mink circovirus of the invention have the following information:
upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively;
downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively;
probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3', respectively;
wherein, the 5 'end of the probe MiCV-P is marked by a fluorescent group, and the 3' end of the probe MiCV-P is marked by a fluorescence quenching group.
The invention relates to a real-time fluorescence quantitative PCR kit of mink circovirus based on a Taqman probe, which comprises a reaction total system: MiCV fluorescent quantitative PCR reaction liquid, a positive quality control product and a negative quality control product; the MiCV fluorescent quantitative PCR reaction solution comprises an upstream primer, a downstream primer, a probe, 2XTaqMan Fast qPCR premix solution and ddH2O；
The upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively;
the downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively;
probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3', respectively;
the 5 'end of the probe MiCV-P is marked by a fluorescent group, and the 3' end of the probe MiCV-P is marked by a fluorescence quenching group.
As a preferred embodiment, the fluorophore is FAM or ROX; the fluorescence quenching group is TAMR or BHQ 2.
In a preferred embodiment, the total amount of the MiCV fluorogenic quantitative PCR reaction solution used in each total reaction system is 18 μ L; wherein the dosage of the upstream primer is 0.8 muL, the dosage of the downstream primer is 0.8 muL, the dosage of the probe is 0.4 muL, the dosage of the 2X TaqMan Fast qPCR premix solution is 10 muL, and the ddH is2The amount of O is 6. mu.L.
In a preferred embodiment, the concentration of the forward primer is 10. mu. mol/L.
In a preferred embodiment, the concentration of the downstream primer is 10. mu. mol/L.
In a preferred embodiment, the probe concentration is 10. mu. mol/L.
As a preferred embodiment, the positive quality control product is a pMD18-T vector plasmid containing a sequence conserved in Cap gene of mink circovirus.
In a preferred embodiment, the concentration of the positive quality control substance is 1X 106Copies/. mu.L.
In a preferred embodiment, the negative quality control material is DEPC water.
The invention has the beneficial effects that:
the invention relates to a real-time fluorescence quantitative PCR kit for mink circovirus based on Taqman probes, which mainly comprises an upstream primer, a downstream primer, probes, 2X TaqMan Fast qPCR premix solution and ddH2O, positive quality control material and negative quality control material; upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively; downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively; probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3' are provided.
The real-time fluorescence quantitative PCR kit for the mink circovirus based on the Taqman probe has the characteristics of strong pertinence, strong specificity, high sensitivity and good repeatability.
The method can be used for rapidly determining the mink circovirus (MiCV) in real time, and can provide greater technical support for diagnosis and epidemiological investigation of the mink circovirus (MiCV).
Drawings
FIG. 1 shows the PCR amplification results of Cap gene in example 2. In the figure, M: DL 2000bp DNA Marker; 1: PCR amplifying fragments of Cap gene; 2: and (5) negative control.
FIG. 2 shows the results of optimizing the concentrations of primers and probes in example 3.
FIG. 3 shows the result of the annealing temperature optimization in example 3.
FIG. 4 shows the amplification curve of the recombinant positive plasmid in example 3.
FIG. 5 is a standard curve of the fluorescent quantitative PCR in example 3.
FIG. 6 shows the fluorescent quantitative PCR-specific amplification curve in example 3.
FIG. 7 is a fluorescent quantitative PCR-sensitive amplification curve in example 3.
FIG. 8 shows the results of a general PCR sensitivity test. In the figure, M: DL 2000bp DNA Marker; 1-6: 1.0X 105Copy/. mu.L-1.0X 100Copies/. mu.L of recombinant positive plasmid; 7: and (5) negative control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
Example 1 design and Synthesis of fluorescent quantitative PCR primers and probes
Cap gene sequences of all MiCV strains are obtained from GenBank, are compared by using DNAMAN software, 1 pair of fluorescent quantitative PCR primers, 1 pair of common PCR primers and a probe MiCV-P (table 1) are designed aiming at a conserved region by using Premier 5 software, and are sent to a biological company for synthesis.
TABLE 1 primer information
aPosition: primer positions represent the starting and ending positions of the nucleotides in MiCV (refer to the sequence position of MK561562 in Gen Bank).
The probe MiCV-P is labeled at the 5 'end with a fluorescent group such as FAM (6-carboxyfluorescein) and ROX, etc., and labeled at the 3' end with a fluorescence quenching group such as TAMR (6-carboxytetramethylrhodamine) and BHQ2, etc.
EXAMPLE 2 preparation of recombinant Positive plasmid Standard
Viral DNA was extracted from a positive sample of mink circovirus (MiCV HB3 strain, GenBank accession number MK561562 for the whole virus genome) by performing the operation according to the instructions of a commercial viral genome extraction kit, and PCR amplification was performed using MiCV-Cap-F and MiCV-Cap-R as upstream and downstream primers. The PCR reaction system is 25 μ L: ex Taq DNA polymerase 12.5. mu.L (5U/. mu.L), upstream and downstream primers 0.5. mu.L (10. mu. mol/L), DNA template 1. mu.L, ddH2O10.5. mu.L. And (3) PCR reaction conditions: 10s at 98 ℃, 30s at 55 ℃, 1min at 72 ℃ and 30 cycles. Meanwhile, a negative control is set, and a band with the size of about 684bp is obtained through agarose gel electrophoresis detection, as shown in figure 1. The target band was recovered and ligated with pMD18-T vector overnight at 16 ℃ using a gene amplifier. The reaction system was as follows (10. mu.L): PCR recovered 4. mu.L of product, 1. mu.L of pMD18-T vector, and 5. mu.L of Solution I. And then transforming the ligation product into DH5 alpha competent cells for culture, extracting plasmids for sequencing, and obtaining a recombinant positive plasmid, namely pMD18-T vector plasmid, by correctly sequencing. The concentration of the recombinant positive plasmid was measured by a microplate reader, and as a result, it was found that the concentration was 210.47 ng/. mu.L and the OD260/280 value was 1.80. The copy number of the positive standard was converted using a sample copy number calculation formula. The calculation formula is as follows: sample copy number ═ concentration (ng/. mu.L) × African-and-Dreher constant (NA) × 10-9]/(660 times the number of bases of recombinant plasmid DNA), the copy was calculated to be 5.68X 1010Copies/. mu.L.
Example 3 establishment of real-time fluorescent quantitative PCR detection method for mink circovirus (MiCV) based on Taqman probe
1. Optimization of primer concentration, probe concentration, annealing temperature
Primers (MiCV-F and MiCV-R) were diluted to 2. mu. mol/L, 4. mu. mol/L, 6. mu. mol/L, 8. mu. mol/L and 10. mu. mol/L, respectively, and probes MiCV-P were diluted to 2. mu. mol/L, 4. mu. mol/L, 6. mu. mol/L, 8. mu. mol/L and 10. mu. mol/L, respectively, and fluorescent quantitative PCR was performed according to a checkerboard method using the recombinant positive plasmid as a template. The PCR reaction system is 20 μ L: 2 mu L of recombinant positive plasmid, 1 mu L of different concentrations of upstream primer (MiCV-F) (final concentrations in the reaction system are 100nM, 200nM, 300nM, 400nM and 500nM, respectively), 1 mu L of different concentrations of downstream primer (MiCV-R) (final concentrations in the reaction system are 100nM, 200nM, 300nM, 400nM and 500nM, respectively), 1 mu L of different concentrations of probe (MiCV-P) (final concentrations in the reaction system are 100nM, 200nM, 300nM, 400nM and 500nM, respectively), 10 mu L of 2XTaqMan Fast qPCR solution, 10 mu L of ddH2O5. mu.L. PCR reaction procedure: 3min at 95 ℃; 45 cycles: the concentrations of primers and probes were optimized at 95 ℃ for 10s and 55 ℃ for 40 s. The results showed that the Ct value was 10.33 at a minimum at a primer concentration of 400nM and a probe concentration of 200 nM. Therefore, the optimal primer final concentration is determined to be 400nM, the optimal probe final concentration is determined to be 200nM, as shown in FIG. 2, the Ct value is minimum 10.33, the corresponding primer final concentration is 400nM, and the probe final concentration is 200 nM; the maximum Ct value is 15.39, the corresponding primer final concentration is 100nM, and the probe final concentration is 500 nM.
Performing fluorescent quantitative PCR reaction with the above reaction system and recombinant positive plasmid as template at 95 deg.C for 3 min; 45 cycles: the optimal annealing temperature is determined at 95 ℃ for 10s and 55-60 ℃ for 40 s. The results showed that the annealing temperatures were 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ and 60 ℃ respectively, and the CT values were 12.61, 11.55, 10.89, 11.89, 11.91, 12.16 and 12.28, respectively. Therefore, the optimum annealing temperature was determined to be 57 ℃, as shown in fig. 3.
2. Standard curve establishment
The recombinant positive plasmid was diluted 10-fold and 6 dilution gradients (1.0X 10)1Copy/. mu.L-1.0X 106Copies/. mu.L) as template, 3 replicates per dilutionAnd performing fluorescent quantitative PCR amplification according to the optimized reaction system and conditions to generate a cycle threshold (Ct value), and constructing a fluorescent quantitative PCR standard curve by taking the Ct value as a vertical coordinate and the logarithm value of the template concentration as a horizontal coordinate. The results are shown in fig. 4 and fig. 5, the standard equation is that y is-3.49 x +42.12, the slope is-3.49, the intercept is 42.12, the correlation coefficient is 0.9965, and the amplification efficiency is 0.93, thereby demonstrating that the real-time fluorescence quantitative PCR detection method has a good linear relationship in the dilution range.
3. Fluorescent quantitative PCR specificity test
The method is characterized in that mink circovirus (MiCV), mink Aleutian disease virus (AMDV), porcine circovirus type 2 (PCV-2), Mink Enteritis Virus (MEV), Canine Distemper Virus (CDV) and porcine pseudorabies virus (PRV) are used as templates to carry out fluorescence quantitative PCR reaction, and the result shows that only mink circovirus (MiCV) can amplify a specificity curve, and the rest viruses have no specificity amplification, as shown in figure 6, the result shows that the real-time fluorescence quantitative PCR detection method has good specificity.
4. Fluorescent quantitative PCR sensitivity test
Recombinant positive plasmids were diluted 10-fold (from 1.0X 10)0Copy/. mu.L-1.0X 105Copy/. mu.L) as a template, and performing a fluorescent quantitative PCR reaction and a general PCR reaction to compare the sensitivity. The results showed that the minimal copy number detected by fluorescent quantitative PCR was 101Copies/. mu.L, as shown in FIG. 7, and the minimum number of copies detected by ordinary PCR was 103The result of copying/. mu.L is shown in FIG. 8, which shows that the fluorescent quantitative PCR detection method has good sensitivity.
5. Fluorescent quantitative PCR repeatability test
Respectively used at a concentration of 1.0 × 102Copy/. mu.L, 1.0X 104Copies/. mu.L and 1.0X 106Copies/. mu.L of the recombinant positive plasmid were used as template, and 3 experiments were performed under the same conditions as an inter-batch reproducibility test, while an intra-batch reproducibility test was performed in triplicate for each concentration. The inter-lot and intra-lot variation Coefficients (CVs) were calculated by dividing the Standard Deviation (SD) of each test sample by the average value, and were evaluated for reproducibility. The results show thatThe range of the inner CV is 0.31-0.67%, the range of the batch CV is 0.58-0.94%, and the ranges are all less than 1% (table 2), and the results show that the repeatability of the fluorescence quantitative PCR detection method is good.
TABLE 2 Intra-and inter-batch repeatability tests
6. Determination of results
When the result is judged, analyzing the amplification curve, and judging that the MiCV is positive when the Ct value is less than or equal to 5 and the amplification curve is S-shaped; when there is no Ct value, determining the MiCV is negative; when the Ct value is more than 35, detecting the gray area, detecting for 2 times again, and judging as negative when the Ct value is still more than 35.
Example 4 methods of assembling and Using a real-time fluorescent quantitative PCR kit for mink circovirus (MiCV) based on Taqman probes
The fluorescent quantitative PCR kit comprises the following components:
1. MiCV fluorescent quantitative PCR reaction solution: 1.0 ml/tube. Each reaction system included 0.8. mu.L (10. mu. mol/L) of the upstream primer, 0.8. mu.L (10. mu. mol/L) of the downstream primer, 0.4. mu.L (10. mu. mol/L) of the probe, 10. mu.L of 2XTaqMan Fast qPCR premix, ddH2O6. mu.L, total 18. mu.L.
| Primer name | Primer sequences (5'to3') |
| Upstream primer MiCV-F | AGGGCCTTTGGGCATCATTG |
| Downstream primer MiCV-R | CCCGCCTGCAAACTGAAGAA |
| Probe MiCV-P | FAM-ACGGAGTTGCTGCAGATGCCACGGT-TAMR |
2. Positive quality control product: pMD18-T vector plasmid (standard recombinant positive plasmid in example 2) containing conserved sequence of Cap gene of mink circovirus (concentration 1X 10)6One copy/. mu.L), 100. mu.L/tube.
3. Negative quality control product: DEPC water, 200. mu.L/tube.
The method for using the fluorescent quantitative PCR kit comprises the following steps:
1. extraction of viral DNA
And (3) extracting virus DNA in a sample to be detected by using a commercial virus nucleic acid extraction kit, and operating according to a kit instruction method.
2. Fluorescent quantitative PCR amplification
Taking out the MiCV fluorescent quantitative PCR reaction solution, melting at room temperature, reversing and uniformly mixing, respectively adding 18 mu L of PCR reaction solution into each PCR reaction tube, respectively adding 2 mu L of the extracted sample DNA to be detected, the negative quality control product and the diluted positive quality control product into the set PCR reaction tube, tightly covering the tube cap, placing on a fluorescent PCR instrument, and setting a reaction program as follows: 3min at 95 ℃; 45 cycles: 95 ℃ for 10s and 57 ℃ for 40 s.
Example 5 clinical sample testing
Detection of MiCV was performed on 116 clinically collected tissue samples using an assembled Taqman probe-based MiCV fluorescent quantitative PCR detection kit and a conventional PCR method. The result shows that 44 samples detected by the MiCV fluorescence quantitative PCR detection kit are MiCV positive with the detection rate of 37.9 percent, while 35 samples detected by the conventional PCR are MiCV positive with the detection rate of 30.2 percent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The primer and probe sequence for detecting the mink circovirus is characterized in that the information of the primer and the probe is as follows:
upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively;
downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively;
probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3', respectively;
wherein, the 5 'end of the probe MiCV-P is marked by a fluorescent group, and the 3' end of the probe MiCV-P is marked by a fluorescence quenching group.
2. Real-time fluorescence quantitative PCR kit of mink circovirus based on Taqman probe, its characterized in that, the reaction total system of this kit includes: MiCV fluorescent quantitative PCR reaction liquid, a positive quality control product and a negative quality control product; the MiCV fluorescent quantitative PCR reaction solution comprises an upstream primer, a downstream primer, a probe, 2X TaqMan Fast qPCR premix solution and ddH2O；
The upstream primer MiCV-F: 5'-AGGGCCTTTGGGCATCATTG-3', respectively;
the downstream primer MiCV-R: 5'-CCCGCCTGCAAACTGAAGAA-3', respectively;
probe MiCV-P: 5'-ACGGAGTTGCTGCAGATGCCACGGT-3', respectively;
the 5 'end of the probe MiCV-P is marked by a fluorescent group, and the 3' end of the probe MiCV-P is marked by a fluorescence quenching group.
3. The real-time fluorescence quantitative PCR kit for mink circovirus based on Taqman probes according to claim 2, wherein the fluorophore is FAM or ROX; the fluorescence quenching group is TAMR or BHQ 2.
4. The real-time fluorescence quantitative PCR kit for mink circovirus based on Taqman probes according to claim 2, wherein the total amount of the MiCV fluorescence quantitative PCR reaction solution in each reaction total system is 18 μ L; wherein the dosage of the upstream primer is 0.8 mu L, and the downstream primer isThe dosage of the primer is 0.8 mu L, the dosage of the probe is 0.4 mu L, the dosage of the 2X TaqMan Fast qPCR premix solution is 10 mu L, and the dosage of the ddH2The amount of O is 6. mu.L.
5. The Taqman probe-based real-time fluorescence quantitative PCR kit for mink circovirus according to claim 4, wherein the concentration of the upstream primer is 10 μmol/L.
6. The Taqman probe-based real-time fluorescence quantitative PCR kit for mink circovirus according to claim 4, wherein the concentration of the downstream primer is 10 μmol/L.
7. The Taqman probe-based real-time fluorescence quantitative PCR kit for mink circovirus according to claim 4, wherein the probe concentration is 10 μmol/L.
8. The real-time fluorescence quantitative PCR kit for mink circovirus based on the Taqman probe according to claim 2, wherein the positive quality control product is pMD18-T vector plasmid containing a Cap gene conserved sequence of the mink circovirus.
9. The Taqman probe-based real-time fluorescence quantitative PCR kit for mink circovirus according to claim 2, wherein the concentration of the positive quality control substance is 1 x 106Copies/. mu.L.
10. The real-time fluorescence quantitative PCR kit for mink circovirus based on Taqman probes according to claim 2, wherein the negative quality control substance is DEPC water.
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