一种检测猪圆环病毒不同型别的三重荧光定量pcr引物组、试剂盒及应用

Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application

文档序号：3432 发布日期：2021-09-17 浏览：34次中文

1. A triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus is characterized by comprising specific primer pairs and probes respectively aiming at the porcine circovirus type 2, the porcine circovirus type 3 and the porcine circovirus type 4;

the primer pair specific to the porcine circovirus type 2 comprises PCV2-2020F2 and PCV2-2020R1, the nucleotide sequence of the PCV2-2020F2 is shown as SEQ ID NO.1, the nucleotide sequence of the PCV2-2020R1 is shown as SEQ ID NO.2, and the nucleotide sequence of the corresponding probe PCV2-2020probe is shown as SEQ ID NO. 3;

the specific primer pair aiming at the porcine circovirus type 3 comprises PCV3-real-F2 and PCV3-real-R2, the nucleotide sequence of the PCV3-real-F2 is shown as SEQ ID NO.4, the nucleotide sequence of the PCV3-real-R2 is shown as SEQ ID NO.5, and the nucleotide sequence of a corresponding probe PCV3 probe is shown as SEQ ID NO. 6;

the primer pair specific to the porcine circovirus type 4 comprises PCV4-R21-R3 and PCV4-R21-F3, the nucleotide sequence of the PCV4-R21-R3 is shown as SEQ ID NO.7, the nucleotide sequence of the PCV4-R21-F3 is shown as SEQ ID NO.8, and the nucleotide sequence of the corresponding probe PCV4 probe3 is shown as SEQ ID NO. 9.

2. The primer set of claim 1, wherein the probe is further modified at the 3 'end with a quencher group and at the 5' end with a fluorescent group.

3. The primer set of claim 2, wherein the quencher group comprises BHQ 1; the fluorophore comprises FAM, HEX or Cy 5.

4. The primer set according to claim 2 or 3, wherein the 5' -end of each of the probes is modified with a different fluorophore.

5. A kit for detecting different types of porcine circovirus, comprising the primer set of any one of claims 1 to 4.

6. The kit of claim 5, wherein the working concentration of the specific primer pair for porcine circovirus type 2 is 0.4 μ M, the working concentration of the specific primer pair for porcine circovirus type 3 is 0.4 μ M, and the working concentration of the specific primer pair for porcine circovirus type 4 is 0.6 μ M;

the working concentration of the probe PCV2-2020probe is 0.25. mu.M, the working concentration of the probe PCV3 probe is 0.25. mu.M, and the working concentration of the probe PCV4 probe3 is 0.4. mu.M.

7. Use of the primer set according to any one of claims 1 to 4 or the kit according to claim 5 or 6 for swine plague monitoring.

8. The use according to claim 7, wherein, in the detection of swine plague, a reaction system is constructed by using the primer set according to any one of claims 1 to 4 or the kit according to claim 5 or 6, and the reaction system is 20 μ l and comprises: 4 × Multiplex PCR Master Mix 5 μ l, PCV2-2020F 20.4 μ l, PCV2-2020R 10.4 μ l, PCV 3-real-F20.4 μ l, PCV 3-real-R20.4 μ l, PCV 4-R21-F30.6 μ l, PCV 4-R21-R30.6 μ l, PCV2-2020probe 0.5 μ l, PCV3 probe 0.5 μ l, PCV4 probe 30.8 μ l, nucleic acid template 4 μ l and RNase-free water 6.4 μ l.

9. The application of claim 7 or 8, wherein in the detection of the swine plague, the fluorescent quantitative PCR is performed based on the reaction system, and the reaction procedure of the fluorescent quantitative PCR comprises: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 45 cycles.

Background

Porcine Circovirus (PCV) is a circular single-stranded DNA virus that is capable of autonomous replication in mammalian cells. PCV there are currently found 4 types, porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3) and porcine circovirus type 4 (PCV4), respectively. PCV1 is a virus found during culture of PK15 cells, and is considered to be nonpathogenic to pigs; PCV2 is pathogenic, mainly infects domestic pigs and wild pigs, and is mostly infected at the age of 4-11 weeks; PCV3 was first reported to be associated with myocarditis and glomerulonephritis in 2016; the research group of veterinary virology, institute of biology, Hunan university, discovered a novel porcine circovirus (tentative PCV4) that was found in pigs with severe clinical symptoms.

At present, the detection of porcine circovirus mainly comprises virus separation and identification, agar diffusion test, trace serum neutralization test, ELISA and PCR method. In addition, the conventional etiology separation and serology detection methods have the defects of complicated operation, time and labor waste, low sensitivity and the like, only one type can be detected at a time, particularly PCV4 belongs to a newly found virus, and no relevant research is carried out on the detection of various types of porcine circovirus including PCV 4.

Disclosure of Invention

In view of the above, the invention aims to provide a triple fluorescent quantitative PCR primer set for detecting different types of porcine circovirus, a kit and an application thereof, and establishes triple fluorescent quantitative PCR detection systems for detecting porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4, and the triple fluorescent quantitative PCR detection systems have the advantages of good sensitivity and strong specificity, and can simultaneously identify and detect different types of porcine circovirus with pathogenicity or suspected pathogenicity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, which comprises specific primer pairs and probes respectively aiming at the porcine circovirus type 2, the porcine circovirus type 3 and the porcine circovirus type 4;

Preferably, the 3 'end of the probe is further modified with a quenching group, and the 5' end of the probe is further modified with a fluorescent group.

Preferably, the quencher group comprises BHQ 1; the fluorophore comprises FAM, HEX or Cy 5.

Preferably, the 5' -end modified fluorophore of each probe is different.

The invention also provides a kit for detecting different types of porcine circovirus, which comprises the primer group.

Preferably, the working concentration of the specific primer pair aiming at the porcine circovirus type 2 is 0.4 mu M, the working concentration of the specific primer pair aiming at the porcine circovirus type 3 is 0.4 mu M, and the working concentration of the specific primer pair aiming at the porcine circovirus type 4 is 0.6 mu M;

The invention also provides application of the primer group or the kit in swine epidemic disease detection.

Preferably, when the swine plague is detected, the primer group or the kit is used for constructing a reaction system, and the reaction system is calculated by 20 μ l and comprises: 4 × Multiplex PCR Master Mix 5 μ L, PCV2-2020F2(20 μmol/L)0.4 μ L, PCV2-2020R1(20 μmol/L)0.4 μ L, PCV3-real-F2(20 μmol/L)0.4 μ L, PCV3-real-R2(20 μmol/L)0.4 μ L, PCV4-R21-F3(20 μmol/L)0.6 μ L, PCV4-R21-R3(20 μmol/L)0.6 μ L, PCV2-2020probe (10 μmol/L)0.5 μ L, PCV3 probe (10 μmol/L)0.5 μ L, PCV4 probe3(10 μmol/L)0.8 μ L, nuclease 4 μ L and water-free RNA template 4.6 μ L.

Preferably, when the swine plague is detected, the fluorescent quantitative PCR is performed based on the reaction system, and the reaction procedure of the fluorescent quantitative PCR includes: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 45 cycles.

The invention provides a triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, wherein primers and probes are synthesized according to conserved sequences of porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 in Genbank, and a kit is also constructed and used for detecting the porcine circovirus. In the embodiment of the invention, the established fluorescent quantitative PCR system is used for detecting 83 pig tissue samples from Zhejiang province, and the result shows that the primer set has good specificity and good repeatability and stability, and compared with the common single PCR method, the positive coincidence rate is respectively 100%, 95.82% and 94.51%, and the method has higher consistency and higher accuracy. In the invention, the lowest detection values of PCV2 and PCV3 are both 10 copies/mu l, and the lowest detection value of PCV4 is 1 copy/mu l, thus overcoming the defects of low sensitivity, easy laboratory pollution and the like of the current triple PCR detection methods for porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4.

Drawings

FIG. 1 is a test curve for detecting porcine circovirus type 2 specificity by triple fluorescence quantitative PCR, wherein: 1 represents PCV 2; 2-12 sequentially represent PPV, CSFV, PRRSV, PCV1, PRV, FMDV, PEDV, TGEV, PCV3, PCV4 and negative control;

FIG. 2 is a test curve of the specificity of the triple fluorescent quantitative PCR detection of porcine circovirus type 3, wherein: 10 represents PCV 3; 1-9 sequentially represent PCV2, PPV, CSFV, PRRSV, PCV1, PRV, FMDV, PEDV and TGEV; 11-12 represent PCV4 and a negative control, respectively;

FIG. 3 is a test curve of triple fluorescent quantitative PCR detection of porcine circovirus type 4 specificity, in which: 11 represents PCV 4; 1-10 sequentially represent PCV2, PPV, CSFV, PRRSV, PCV1, PRV, FMDV, PEDV, TGEV and PCV 3; 12 represents a negative control;

FIG. 4 is a triple fluorescence quantitative PCR detection PCV2 amplification curve, wherein: 1-8 represents a final concentration of 10⁶～10^-1copies/. mu.l, 9 indicates negative control;

FIG. 5 is a triple fluorescence quantitative PCR detection PCV3 amplification curve, wherein: 1-8 represents a final concentration of 10⁶～10^-1copies/. mu.l, 9 indicates negative control;

FIG. 6 is a triple fluorescence quantitative PCR detection PCV4 amplification curve, wherein: 1-8 represents a final concentration of 10⁶～10^-1copies/. mu.l, 9 indicates negative control;

FIG. 7 is a standard curve for porcine circovirus type 2 amplification;

FIG. 8 is a standard curve for porcine circovirus type 3 amplification;

FIG. 9 is a standard curve for porcine circovirus type 4 amplification.

Detailed Description

The invention provides a triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, which comprises specific primer pairs and probes respectively aiming at the porcine circovirus type 2, the porcine circovirus type 3 and the porcine circovirus type 4;

According to the invention, the 3 'end of the probe is preferably modified with a quenching group, and the 5' end of the probe is preferably modified with a fluorescent group. The quencher group of the present invention preferably comprises BHQ 1; the fluorescent group preferably comprises FAM, HEX or Cy5, and the modified fluorescent group on each probe is different.

In the present example, the sequences of the primer pairs and probes used in triple fluorescent quantitative PCR are shown in table 1:

TABLE 1 primer pairs and Probe sequences for triple fluorescent quantitative PCR

In the present invention, it is preferable that the above-mentioned primers and probes are synthesized based on conserved sequences of porcine circovirus type 2, porcine circovirus type 3, and porcine circovirus type 4 in Genbank, and synthesized and labeled by committee of biologies (shanghai) engineering limited.

The invention also provides a kit for detecting different types of porcine circovirus, which comprises the primer group. The Kit of the present invention preferably further comprises reagents for performing fluorescent quantitative PCR, such as QuantiNova Multiplex PCR Kit.

In the kit, the working concentration (namely the concentration in a reaction system) of the specific primer pair aiming at the porcine circovirus type 2 is preferably 0.4 mu M, the working concentration of the specific primer pair aiming at the porcine circovirus type 3 is preferably 0.4 mu M, and the working concentration of the specific primer pair aiming at the porcine circovirus type 4 is preferably 0.6 mu M; the working concentration of the probe PCV2-2020probe is preferably 0.25. mu.M, the working concentration of the probe PCV3 probe is preferably 0.25. mu.M, and the working concentration of the probe PCV4 probe3 is preferably 0.4. mu.M.

The invention also provides application of the primer group or the kit in swine epidemic disease detection.

In the present invention, when the primer set or the kit is used for detecting swine plague, it is preferable that the primer set or the kit is used to construct a reaction system, and the reaction system is calculated by 20 μ l and comprises: 4 × Multiplex PCR Master Mix 5 μ L, PCV2-2020F2(20 μmol/L)0.4 μ L, PCV2-2020R1(20 μmol/L)0.4 μ L, PCV3-real-F2(20 μmol/L)0.4 μ L, PCV3-real-R2(20 μmol/L)0.4 μ L, PCV4-R21-F3(20 μmol/L)0.6 μ L, PCV4-R21-R3(20 μmol/L)0.6 μ L, PCV2-2020probe (10 μmol/L)0.5 μ L, PCV3 probe (10 μmol/L)0.5 μ L, PCV4 probe3(10 μmol/L)0.8 μ L, nuclease 4 μ L, and RNA template-free of water 4.6 μ L.

In the detection of swine plague, the invention preferably performs fluorescent quantitative PCR based on the reaction system, and the reaction procedure of the fluorescent quantitative PCR preferably comprises: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 45 cycles.

The invention also provides a method for detecting different types of porcine circovirus by using the primer group or the kit, and preferably comprises the steps of constructing the reaction system and carrying out fluorescence quantitative PCR. The reaction system and the fluorescent quantitative PCR procedure of the present invention are preferably the same as those described above, and will not be described herein.

The triple fluorescent quantitative PCR primer set, the kit and the application for detecting different types of porcine circovirus provided by the invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.

The experimental materials used in the examples of the present invention were all purchased conventionally unless otherwise specified.

1. Pathogenic samples and viral strains

Porcine foot-and-mouth disease virus inactivated vaccine, Classical Swine Fever Virus (CSFV) live vaccine, Porcine parvovirus ((PPV) inactivated vaccine (WH-1 strain) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccine (JXA 1-R strain) from Central farm industries, Inc., Porcine circovirus type 2 (PCV2) inactivated vaccine from Zhejiang Huanbao Wei Biotech Co., Ltd, which all contain the corresponding virus and a higher concentration of nucleic acid detected Porcine epidemic diarrhea virus (Porcine epidemic diarrheal virus 748, PEDV, GenBank No. MZ403746), Porcine infectious gastroenteritis virus (MZBank virus 40319, Porcine infectious Porcine transmissible Porcine virus 4034, Porcine reproductive and respiratory syndrome virus 4034 (PCV 744, PCV) live vaccine, Porcine parvovirus and Porcine reproductive and respiratory syndrome virus (PRFV 4034, PCV) live vaccine (PCV) from Porcine reproductive and respiratory syndrome virus), PCV4) and Porcine circovirus type 1 (PCV1, GenBank number MZ463060) virus positive samples with high nucleic acid content were identified and stored in the laboratory.

2. Primary reagents and instruments

pGEM-T Easy Vector Systems are available from Promega, isopropyl-. beta. -D-thiogalactoside (IPTG) is available from Biotechnology engineering (Shanghai) Co., Ltd., Plasmid Extraction Kit Mini BEST Plasmid Purification Kit, DNA Gel recovery Kit Mini BEST Agarose Gel DNA Extraction Kit, Ex Taq enzyme, DL2000 DNA Marker, DH 5. alpha. sensory cells and X-gal are all products of Dalian Biotechnology Ltd., QuantiNova Multiplex PCR Kit is Qiagen. The virus nucleic acid extraction Kit MagNApure 96 DNA and viral NA Small Voume Kit is a product of Roche. The fluorescent quantitative PCR instrument (model Lightcycler480) was manufactured by Roche, the PCR instrument (model TProfessional) was manufactured by Biometra, Germany, the refrigerated high-speed centrifuge (model 5430R) was manufactured by eppendorf, and the ultraminiUV spectrophotometer NanoDrop2000 was manufactured by Thermo.

Example 1

1. Primers and probes shown in Table 1 were synthesized and labeled by Venezetian engineering Co.Ltd.

2. Viral nucleic acids were extracted using the MagNA pure 96 DNA and viral NA Small Voume Kit using the viral nucleic acid extraction Kit according to the protocol, and the obtained viral nucleic acids were stored in a freezer at-80 ℃ until use.

3. Preparation of porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 recombinant plasmid standard substance

According to target gene segments detected in a triple fluorescent quantitative detection method for porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4, primers (shown in a table 2) are designed to amplify nucleic acid segments containing porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 detection genes respectively, and the specific operation is as follows: PCR reactions were carried out using the viral nucleic acids as templates, respectively, in the following reaction system (50. mu.l): 5. mu.l of 10 XEx Taq Buffer, 4. mu.l of dNTP, 0.25. mu.l of Ex Taq (5U/. mu.l), 1. mu.l of upstream primer (20. mu. mol/L), 1. mu.l of downstream primer (20. mu. mol/L), 5. mu.l of template, and 33.75. mu.l of RNase-free water. The reaction conditions were as follows: at 95 ℃ for 2 min; 10s at 98 ℃, 30s at 52 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃.

And recovering the amplified target fragment, cloning the amplified target fragment into a pGEM-T Easy vector to construct a recombinant plasmid containing a target gene, wherein the constructed recombinant plasmids are respectively named as T-PCV2, T-PCV3 and T-PCV4 and are sent to Shanghai Bioengineering technology Limited company for sequencing identification. The recombinant plasmid concentration was determined using an ultraviolet spectrophotometer and the copy number was calculated.

TABLE 2 primers for amplifying the detection gene fragments of different types of porcine circovirus

4. The reaction system is optimized by taking the recombinant plasmid as a detection template and optimizing the concentration of the probe and the primer by using a matrix method.

And optimizing the concentrations of the primers and the probes by adopting a matrix method, wherein the optimized final concentrations of the primers and the probes are respectively as follows: the optimal final concentrations of the porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 primers in the reaction system are respectively 0.4 mu M, 0.4 mu M and 0.6 mu M, and the optimal final concentrations of the porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 probes in the reaction system are respectively 0.25 mu M, 0.25 mu M and 0.4 mu M. The reaction conditions are as follows: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 45 cycles. Fluorescence was collected at 60 ℃.

5. Specificity test

The built fluorescent quantitative PCR method is used for detecting PCV1, PCV2, PCV3, PCV4, CSFV, PEDV, TGEV, FMDV, PPV, PRRSV and PRV nucleic acid, and meanwhile, a negative control is arranged, and the specificity of the method is evaluated.

As shown in FIGS. 1 to 3, PCV2, PCV3 and PCV4 show amplification curves at wavelengths 618 to 660, 533 to 580 and 455 to 510, respectively. And no amplification curve appears in other virus nucleic acids and negative control in the wavelength intervals, which indicates that the established porcine circovirus fluorescent quantitative PCR detection method has no cross reaction with PCV1, CSFV, PEDV, TGEV, FMDV, PPV, PRRSV and PRV, and has no cross reaction with PCV2, PCV3 and PCV 4. Has good specificity.

6. Triple fluorescent quantitative PCR detection method for porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 sensitivity evaluation and standard curve establishment

Diluting the prepared recombinant plasmids of T-PCV2, T-PCV3 and T-PCV4 by 10 times, detecting by adopting established fluorescent quantitative PCR, and determining the lowest detection concentration of each template in a fluorescent quantitative PCR reaction system. And according to the detection result, respectively drawing standard curves by taking the logarithm of the final concentration of the recombinant plasmid in the reaction system and the Ct value as an X axis and a Y axis respectively.

Carrying out fluorescent quantitative PCR amplification by taking recombinant plasmids (T-PCV2, T-PCV3 and T-PCV4) with different dilutions as templates to obtain an amplification curve (see figures 4-6), establishing a fluorescent quantitative PCR standard curve (see figures 7-9), and displaying the correlation coefficient R of the fluorescent quantitative PCR standard curves of PCV2, PCV3 and PCV4 by results²0.9959, 0.9928, and 0.9958, respectively, all have good linearity. The result shows that the lowest detection values of PCV2 and PCV3 are both 10 copies/. mu.l, and the lowest detection value of PCV4 is 1 copy/. mu.l.

7. Repeatability test

Respectively taking the final concentration of the reaction system as 10⁶、10⁵、10⁴、10³The detection was performed by the established fluorescent quantitative PCR method using copies/. mu.l of recombinant plasmids (T-PCV2, T-PCV3 and T-PCV4) as templates, and the same template at each dilution was subjected to 4 batch-to-batch repeated experiments, and the mean, standard deviation and coefficient of variation were calculated both batch-to-batch.

Respectively taking the final concentration of the reaction system as 10⁶、10⁵、10⁴、10³、10²Mu.l copies of the recombinant plasmids (T-PCV2, T-PCV3 and T-PCV4) were used as templates, and the template dilution was performed 4 times for each dilution, respectivelyThe results of the repeated detection in batches and among batches are shown in tables 3 to 5, and the variation coefficients in batches and among batches of the same template with different concentrations are within 3 percent, which indicates that the established fluorescence quantitative PCR method has good repeatability and stability.

TABLE 3 repeatability test for porcine circovirus type 2 by fluorescent quantitative PCR method

TABLE 4 fluorescent quantitative PCR method for porcine circovirus type 3 repeatability test

TABLE 5 fluorescent quantitative PCR method for porcine circovirus type 4 repeatability test

8. Clinical sample testing

Diluting and grinding the pig tissue sample with PBS, centrifuging, taking supernatant, extracting virus nucleic acid by using a virus nucleic acid extraction kit, and detecting 83 pig tissue samples from Zhejiang province by using the established fluorescent quantitative PCR and common single PCR method (amplification primers are shown in Table 6).

TABLE 6 common simple PCR detection primers for different types of porcine circovirus

The detection results are shown in tables 7-9, and the established triple fluorescent quantitative PCR detection method for porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4 has positive coincidence rates of 100%, 95.82% and 94.51% respectively with the single PCR detection method for porcine circovirus type 2, porcine circovirus type 3 and porcine circovirus type 4, and has higher consistency and higher accuracy.

TABLE 7 detection results of triple fluorescent quantitative PCR method and PCV2 PCR method

TABLE 8 detection results of triple fluorescent quantitative PCR method and PCV3 PCR method

TABLE 9 detection results of triple fluorescent quantitative PCR method and PCV4 PCR method

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.

Sequence listing

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