CN117487968B - Primer and probe for detecting muscovy duck Cha Pama virus and virus separation culture method - Google Patents
Primer and probe for detecting muscovy duck Cha Pama virus and virus separation culture method Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a TaqMan real-time fluorescent quantitative PCR detection primer and a probe for detecting a muscovy duck Cha Pama virus, wherein the primer comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3; the nucleotide sequence of the probe is shown as SEQ ID No. 4. The detection method based on the primer and the probe is quick and simple, and has strong specificity and high sensitivity. Meanwhile, the invention also provides a method for culturing the strain, which discovers that the virus can proliferate in the primary cells of duck embryo kidney without generating obvious cytopathy, and can provide a basis for subsequent proliferation of the strain and vaccine preparation based on the method.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer, a probe and a virus separation culture method for detecting a muscovy duck Cha Pama virus.
Background
The muscovy duck Cha Pama virus is a member of the subfamily hamamarovirinae, the subfamily hamamapar vorvirus, and little research is currently done on the muscovy duck Cha Pama virus.
The applicant has separated a new strain, and after researching the genome characteristics and genome analysis evolutionary tree, the strain is found to be singly gathered into one strain.
Since the strain has formed a new branch, no data has been reported in the art for the accurate detection and efficient culture of the strain.
Disclosure of Invention
The invention aims to provide a primer and a probe for detecting the muscovy duck Cha Pama virus, and the TaqMan real-time fluorescent quantitative PCR detection method adopting the primer and the probe has the characteristics of rapidness, simpleness, strong specificity and high sensitivity.
Meanwhile, the invention also provides a method for culturing the strain, which discovers that the virus can proliferate in the primary cells of duck embryo kidney without generating obvious cytopathy, and can provide a basis for subsequent proliferation of the strain and vaccine preparation based on the method.
In order to achieve the above purpose, the present invention provides the following technical solutions: taqMan real-time fluorescent quantitative PCR detection primer and probe for detecting muscovy duck Cha Pama virus, wherein the classification name of the muscovy duck Cha Pama virus is muscovy duck Cha Pama virus @ChaphamaparvovirusMuChPv-GD 2022), accession number: CCTCC NO: V202311; preservation date: 2023, 3, 1, deposit unit: china center for type culture Collection; preservation address: university of martial arts in chinese;
the primer comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is shown as SEQ ID No. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3;
the nucleotide sequence of the probe is shown as SEQ ID No. 4.
In the TaqMan real-time fluorescence quantitative PCR detection primer and the probe for detecting the muscovy duck Cha Pama virus, the 5 '-end of the probe is marked with a fluorescence reporter group FAM, and the 3' -end of the probe is marked with BHQ.
Meanwhile, the invention also discloses a TaqMan real-time fluorescent quantitative PCR detection method of the muscovy duck Cha Pama virus, which adopts the primer and the probe to detect through the TaqMan real-time fluorescent quantitative PCR.
In the TaqMan real-time fluorescent quantitative PCR detection method of the muscovy duck Cha Pama virus, the TaqMan real-time fluorescent quantitative PCR reaction system is as follows: 2 XProbe RT-qPCR Mix 5.0 [ mu ] L,10 [ mu ] mol/L upstream primer F0.4 [ mu ] L,10 [ mu ] mol/L downstream primer R0.4 [ mu ] L,10 [ mu ] mol/L Probe P0.2 [ mu ] L, and nucleic acid template 1.0 [ mu ] L, ddH O3.0 [ mu ] L;
the TaqMan real-time fluorescence quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 47℃for 20s, extension at 72℃for 20s; cycling was performed 45 times.
Finally, the invention also discloses a separation culture method of the muscovy duck Cha Pama virus strain, which comprises the following steps:
step 1: screening a muscovy duck Cha Pama virus positive sample from the samples by adopting the TaqMan real-time fluorescent quantitative PCR detection method;
step 2: grinding selected muscovy duck Cha Pama virus sample with PBS, adding supernatant into cultured duck embryo kidney primary cells, culturing in incubator, culturing and harvesting culture.
In the method for separating and culturing the muscovy duck Cha Pama virus strain, the step 2 specifically comprises the following steps:
taking a muscovy duck Cha Pama virus positive sample to a 2mL grinding tube, adding PBS into the tube, grinding and centrifuging to obtain a supernatant, and filtering and sterilizing the supernatant to obtain a disease treatment liquid;
inoculating virus solution into primary cells of duck embryo kidney, adsorbing at 37deg.C for 90 min, discarding cell solution, adding 1% FBS-containing complete culture medium, and adding 5% CO at 37deg.C 2 Culturing in an incubator, and collecting cells after 72 hours;
freezing the collected cells in a refrigerator at-80 ℃, then putting the cells into a water bath at 37 ℃ for melting, and repeating the steps for 3 times; centrifuging at 10000r/min for 5min, and collecting supernatant to obtain virus liquid.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, the TaqMan real-time fluorescence quantitative PCR detection method adopting the primer and the probe has the characteristics of rapidness, simpleness, strong specificity and high sensitivity;
the invention provides a cell separation culture technology of the muscovy duck Cha Pama virus for the first time, and discovers that the muscovy duck Cha Pama virus can proliferate in duck embryo kidney primary cells without generating obvious cytopathy, and can provide a basis for subsequent strain proliferation and vaccine preparation based on the culture method.
Drawings
Fig. 1 is a normal liver tissue picture of a muscovy duck;
FIG. 2 is a photograph of diseased liver tissue of a Muscovy duck infected with Muscovy duck Cha Pama virus of the present invention;
FIG. 3 is a diagram showing comparison of homology between viruses of the present invention and other related viruses;
FIG. 4 is a phylogenetic tree analysis of the amino acid sequences of the parvoviral NS1 protein; the black triangle marked strain on FIG. 4 is the NS1 protein position of the virus of the invention;
FIG. 5 is a chart showing the morphology of virus particles observed by electron microscopy after negative staining of the disease supernatant;
FIG. 6 is a standard curve of a real-time fluorescent quantitative PCR detection method for the muscovy duck Cha Pama virus TaqMan in the embodiment of the invention;
FIG. 7 shows the specific detection result of the TaqMan real-time fluorescence quantitative PCR detection method of the muscovy duck Cha Pama virus in the embodiment of the invention;
FIG. 8 shows the detection results of 25 samples of clinical samples suspected of being infected with the Muscovy Duck Cha Pama virus in the example of the present invention;
FIGS. 9 and 10 show the cell state of MuChPV-GD2022 strain before and after infection of duck embryo kidney primary cells, wherein FIG. 9 shows the cell state before infection of duck embryo kidney primary cells, and FIG. 10 shows the cell state after infection of MuChPV-GD 2022.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Virus isolation and identification
For 2 months of 2022, the guangdong part of the muscovy duck farm exploded infectious diseases with hepatitis as the main pathological feature (refer to fig. 1 and 2 of the present invention, fig. 1 is normal liver tissue, fig. 2 is pathological liver tissue), and resulted in a decrease of 10% to 15% in egg yield and about 10% to 30% in hatching rate of the muscovy ducks; the quality of eggs shows a low mortality rate. The diseased duck has no obvious respiratory symptoms, and the feces are green. At necropsy, hepatitis, swelling and bleeding or yellowing are mainly observed in the liver; the liver disease material of the disease-type muscovy duck was ground, the supernatant was collected after repeated freeze thawing for 3 times, and after centrifugation at 10000rpm, the supernatant was collected, and subjected to negative staining, and transmission electron microscopy was performed to see virus particles with a diameter of about 25nm (FIG. 5).
Jing Hong virus group sequencing and sequencing result analysis, a new virus is discovered. Blast analysis of the newly discovered viral nucleic acid showed it to be a new parvovirus, muscovy duck Cha Pama virus. The whole genome sequence of the virus is shown as SEQ ID No. 1.
Complete viral genomes were obtained by macrovirology and genome sequencing. The sequence of MuChPV-GD2022 strain was searched in the GenBank database by BLAST, which shows that MuChPV-GD2022 has the highest sequence similarity to Pacific black duck parvovirus No. 1 (Pacific black duck chaphamaparvovirus, genBank accession number MT 247730) of 76.8% (FIG. 2). The whole genome length of MuChPV-GD2022 was 4407bp and the G+C content was 39.1%. Two different ORFs encode the nonstructural protein NS1 (nt 640-2703) and the structural protein VP1 (nt 2696-4282). The NS1 length of MuChPV-GD2022 is 2064bp, and the amino acid homology of the NS1 of 687 amino acid-encoding non-structural proteins with other members of the parvoviridae family is less than 71.5%. The VP1 length of MuChPV-GD2022 is 1587bp, the capsid protein of 528 amino acids is encoded, and the amino acid homology with other members of parvoviridae is lower than 69.4%. Comparison of homology of the virus of the present invention with other related viruses referring to FIG. 3;
and constructing a phylogenetic tree by adopting a Neighbor-joining (NJ) method. As can be seen from FIG. 4, the MuChPV-GD2022 strain was clustered with the Duck source Chaphamapnovovirus on the phylogenetic tree. The MuChPV-GD2022 strain has 47.1% -76.8% of genome sequence similarity with other avian parvoviruses and 61.8-76.8% of genome sequence similarity with other duck-origin parvoviruses. The amino acid sequence of the NS1 protein is 29.3-71.5% similar to all known parvoviruses. The newly discovered muscovy duck Cha Pama virus has a far evolutionary distance from the known duck Cha Pama virus and duck parvovirus, and forms an independent branch.
Example 2 verification of viral sequences
The genome sequence of the virus is obtained through macrovirology sequencing, and in order to verify the whole genome sequence of the novel Muscovy duck parvovirus MuChPV-GD2022, the sequence of 4407bp is amplified by four pairs of primers, and the sequences of four fragments and two UTRs are assembled together. Finally, a 4407bp MuChPV-GD2022 strain viral genome sequence was obtained.
The primers used can be seen in Table 1:
TABLE 1
The amplification procedure described above was as follows:
50. the reaction system of mu L is: 2 XOne Step Mix 25. Mu.L, one Step Enzyme Mix 2.5.5. Mu.L, 10. Mu. Mol/L upstream and downstream primers 2. Mu.L each, 5. Mu.L positive nucleic acid, DEPC H 2 O13.5. Mu.L. The PCR amplification conditions were: 94 ℃ for 3 min; cycling for 35 times at 94 ℃ for 30 s,52 ℃ for 30 s and 72 ℃ for 1 min/Kb; and at 72℃for 5 min.
Example 3
1. Related test pathogen
The Muscovy duck Cha Pama virus (MuChPV-GD 2022 strain), muscovy Duck Parvovirus (MDPV), tembusu virus (TMUV), duck circovirus (DuCV), muscovy Duck Reovirus (MDRV), muscovy duck reovirus (NDRV), duck astrovirus (DAstV) were all isolated, identified and stored by the national institute of animal health, the university of agricultural sciences, guangdong.
2. Establishment of TaqMan real-time fluorescence quantitative PCR detection method
2.1 Primer and probe design
The method is characterized in that the NS1 gene of the muscovy duck Cha Pama virus is taken as a target gene designed by a primer, and a specific region of the muscovy duck Cha Pama virus is selected for primer design according to the nucleotide sequence comparison result of the NS1 gene of the muscovy duck Cha Pama virus and other parvoviruses of the same genus, so that the specificity of a detection method is ensured. The primers and probes were as follows:
upstream primer F: 5'-CTGACCTCTTTGACTCTAC-3' (SEQ ID NO. 2);
the downstream primer R: 5'-GCTCCATTACATACCTCTC-3' (SEQ ID NO. 3);
probe P:5 '-FAM-TGCTGGACATAATGCTCTGAACC-BHQ-3' (SEQ ID NO. 4).
2.2 Construction of a Positive Standard
Extracting nucleic acid RNA of a Muscovy duck Cha Pama virus (MuChPV-GD 2022 strain) by using a commercial viral nucleic acid extraction kit, and amplifying by using a one-step PCR amplification reagent (PrimeScript 1 Step Enzyme Mix, TAKARA), wherein PrimeScript 1 Step Enzyme Mix 2 [ mu ] L, 2X 1 Step Buffer 25 [ mu ] L,10 [ mu ] mol/L upstream primer F2 [ mu ] L,10 [ mu ] mol/L downstream primer R2 [ mu ] L, an extracted nucleic acid RNA template 2 [ mu ] L, and supplementing deionized water of RNase-free to a final reaction system 50 [ mu ] L, and uniformly mixing and performing PCR amplification under the following amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 47℃for 20s, extension at 72℃for 20s, and final extension at 72℃for 10min after 35 cycles;
after the PCR reaction is finished, the PCR product is identified by 2.0% agarose gel electrophoresis, and the specific target fragment is subjected to gel cutting recovery by using an agarose gel recovery kit. The fragment of interest was cloned into the pMD ™ -T Vector according to the pMD ™ -T-Vector instructions. 10 single colonies were randomly picked and cultured in ampicillin (100. Mu.g/mL) resistant LB liquid medium for 14h, and the corresponding plasmids were extracted using a rapid plasmid miniprep kit. PCR identification is carried out on the extracted plasmid by adopting a pMD ™ -T vector universal PCR detection primer and conditions, and the screened positive recombinant plasmid is sent to a biological engineering (Shanghai) Co., ltd for sequencing. After Blast analysis, positive recombinant plasmids meeting the experimental expectations were used as the standard (p-Chaphamaparvovirus) for the study. The concentration was measured by a spectrophotometer.
3. Establishment of TaqMan real-time fluorescence quantitative PCR detection method
3.1 TaqMan real-time fluorescence quantitative PCR detection reaction condition optimization
Preparing a 10 [ mu ] L real-time fluorescent quantitative PCR reaction system according to a Probe RT-qPCR Mix kit specification, wherein the screened optimal reaction system is as follows: 2 XProbe RT-qPCR Mix 5.0 [ mu ] L,10 [ mu ] mol/L upstream primer F0.4 [ mu ] L,10 [ mu ] mol/L downstream primer R0.4 [ mu ] L,10 [ mu ] mol/L Probe P0.2 [ mu ] L, nucleic acid template 1.0 [ mu ] L, ddH 2 O3.0 [ mu ] L; the optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s, annealing at 47℃for 20s, extension at 72℃for 20s; cycling was performed 45 times.
3.2 Standard curve drawing of TaqMan real-time fluorescent quantitative PCR detection method
The standard (p-Chaphamaparvovirus) was diluted 10-fold to give 2.1X10 0 cobies/[ mu ] L to 2.11X10 10 11 dilutions of positive control samples are taken as amplification templates, real-time fluorescent quantitative PCR (polymerase chain reaction) amplification is carried out by taking 11 dilutions of positive control samples as amplification templates, and 3 parallel samples are taken for each dilution of positive control samples, so that an amplification kinetic curve is obtained. The standard linear regression equation (standard curve, see figure 6) is deduced by taking the common logarithm of the initial copy number of the standard substance as the abscissa and the cycle threshold (Ct value) as the ordinate, the slope of the linear equation of the obtained real-time fluorescence quantitative PCR standard curve is-3.4937, the Y-axis intercept is 40.4, the correlation coefficient is 0.9986, and the amplification efficiency is 93%, which indicates that the standard curve of the established real-time fluorescence quantitative PCR method has good linear relation. The minimum detection limit of the invention can reach 2.11 multiplied by 10 2 copies/µL。
3.3 TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) specific detection
And detecting the Muscovy duck Cha Pama virus (MuChPV), muscovy Duck Parvovirus (MDPV), tembusu virus (TMUV), duCV, muscovy Duck Reovirus (MDRV), novel Duck Reovirus (NDRV) and Duck astrovirus (DAstV) respectively by using optimized real-time fluorescence quantitative PCR conditions. Extracting nucleic acid of the corresponding virus as a template, detecting according to the specification of the Probe qPCR Mix kit by using optimized reaction conditions, and evaluating the specificity of the established real-time fluorescence quantitative PCR method;
FIG. 7 shows the specific detection results of the TaqMan real-time fluorescent quantitative PCR detection method of the Muscovy duck Cha Pama virus in the embodiment of the invention, wherein amplification curves do not appear in Muscovy Duck Parvovirus (MDPV), tembusu virus (TMUV), duCV, muscovy Duck Reovirus (MDRV), muscovy duck reovirus (NDRV), duck astrovirus (DAstV) and negative control, and amplification curves appear in p-Chaphamatarvovirus positive plasmids only;
as can be seen from fig. 7: and (3) respectively carrying out real-time fluorescent quantitative PCR detection on MuChPV, GPV, TMUV, goCV, MDRV, NDRV and GAstV by using optimized real-time fluorescent quantitative PCR conditions, wherein positive amplification is carried out on MuChPV only from the FAM channel, and positive amplification signals are not found on GPV, TMUV, goCV, MDRV, NDRV and GAstV. The above results demonstrate that the TaqMan fluorescent quantitative PCR detection method in this example has strong specificity.
3.4 Clinical application of TaqMan real-time fluorescence quantitative PCR detection method
As can be seen from table 2 and fig. 8: the established real-time fluorescent quantitative PCR detection method for the TaqMan Cha Pama virus of the Muscovy ducks is used for detecting 193 parts of anus swabs and viscera tissues (liver, spleen, heart and the like) suspected to be infected with the Muscovy ducks Cha Pama virus, and 126 parts of positive samples appear in FAM channels, which shows that the MuChPV positive rate is 65.3%.
Table 2 muscovy duck Cha Pama virus TaqMan real-time fluorescent quantitative PCR detection method clinical sample detection results
| Sample species | Quantity of | Number of positive samples | Negative sampleNumber of products | Positive rate (%) |
| Anal swab for 31-week-old laying-breed muscovy ducks | 47 | 26 | 21 | 55.3 |
| 45-week-old muscovy duck anus swab for egg-laying | 56 | 35 | 21 | 62.5 |
| Anal swab for 56-week-old laying ducks | 51 | 33 | 18 | 64.7 |
| Male muscovy duck anus swab | 22 | 15 | 7 | 68.2 |
| Liver tissue of diseased laying muscovy duck | 17 | 17 | 0 | 100 |
| Totalizing | 193 | 126 | 67 | 65.3% |
Example 4 isolation culture
1. Preparation of duck embryo kidney primary cells
1.1 Materials and reagents
Sterile plates, autoclaved ophthalmic forceps, scissors, small beakers, 70 mesh cell sieves, 50ml centrifuge tubes, 15ml centrifuge tubes. Complete medium DMEM (5% fbs, 1% diabody), pancreatin (0.25%), etc.
1.2 Method of
(1) Cleaning 14-20 day old duck embryo with warm water, and wiping and sterilizing the surface with alcohol; taking out duck embryo from the air chamber end in the cell table, cutting off abdominal cavity of duck embryo in supine mode, taking out kidney, cleaning twice in sterile PBS and removing redundant connective tissue;
(2) Washing duck embryo kidney tissue twice with sterile PBS, and placing in a small beaker; washing with a small amount of pancreatin, standing for 2min, and sucking away excessive base solution; cutting kidney tissue into paste with small scissors;
(3) Transferring the sheared kidney tissue into a 15ml centrifuge tube, adding pancreatin preheated to 37 ℃ (about 2ml pancreatin is added into 1 duck embryo), and digesting for 15min, and shaking continuously during the period;
(4) An equal volume of complete medium was added and pancreatin digestion was stopped. And gently blow off the cells. And filtering with a 70 mesh cell sieve;
(5) Centrifuging at 800r/min for 5min, and discarding the waste liquid. Adding a complete culture medium for resuspension, and taking a small amount of cell suspension for counting;
(6) Diluting the cells to 5-8X10 9 In the range of/L, the flask was separated and placed in a cell culture vessel for culturing. After the cells grew to 70% -80%, the complete medium containing 5% fbs was changed to complete medium containing 1% fbs.
2. Separation of muscovy duck Cha Pama virus
2.1 Proliferation of Muscovy Duck Cha Pama Virus
Taking a muscovy duck tissue positive to detection of a muscovy duck Cha Pama virus, adding 900 mu L PBS (containing 100U/mL penicillin and 100 g/mL streptomycin) into a 2mL grinding tube, grinding, centrifuging at 7000r/min for 5min, filtering and sterilizing the obtained supernatant through a 0.22 mu m filter membrane to obtain a disease treatment liquid, and storing in a refrigerator at-80 ℃;
inoculating the above virus solution into duck embryo kidney primary cells, adsorbing at 37deg.C for 90 min, discarding cell solution, adding 1% FBS-containing complete culture medium, and adding 5% CO at 37deg.C 2 Culturing in an incubator, and collecting cells and culture solution after 72 hours;
freezing the collected cells in a refrigerator at-80 ℃, then putting the cells into a water bath at 37 ℃ for melting, and repeating the steps for 3 times. Centrifuging at 10000r/min for 5min, and collecting supernatant to obtain virus liquid.
2.2 Muscovy duck Cha Pama virus proliferation detection
After the Muscovy duck Cha Pama virus infects the primary cells of duck embryo kidney, no obvious cytopathy is produced.
After virus (500. Mu.L/well) was inoculated into cells (six well plates), cells and culture broth were collected every 12 hours, and the proliferation of the virus was determined by detecting the virus stock solution of the inoculated virus and the virus content in the cells at each time point after the inoculation by the TaqMan qPCR detection method in example 3. As can be seen from Table 3, the Chapama virus was able to proliferate from duck embryo kidney primary cells.
TABLE 3 determination of viral nucleic acid content before and after Vaccination of the cells with Muscovy Duck Cha Pama Virus
| Detecting a sample | qPCR detection of Ct value | Detecting a sample | qPCR detection of Ct value |
| Virus stock solution for inoculation | 18.3683 | Cells 48 hours post inoculation | 18.6492 |
| Cells 12 hours after inoculation | 29.5785 | Cells 60 hours after inoculation | 15.8938 |
| Cells 24 hours after inoculation | 26.9041 | Cells 72 hours post inoculation | 15.0694 |
| Cells 36 hours after inoculation | 23.0893 |
Referring specifically to FIGS. 9 and 10, FIG. 9 shows the cell state of MuChPV-GD2022 strain before infection of duck embryo kidney primary cells, and FIG. 10 shows the cell state of MuChPV-GD2022 after infection, with no obvious lesions after infection.
From the above experiments, it can be found that:
1. the muscovy duck Cha Pama virus is singly gathered into one strain, and the acquisition of the strain enriches pathogenic species causing muscovy duck diseases.
2. The discovery of the strain and the inactivated or attenuated vaccine prepared by the strain are of great significance for the high-speed development of the Muscovy duck breeding industry.
3. The invention provides a detection technology of the muscovy duck Cha Pama virus for the first time, and the detection method provided by the invention is rapid and simple, and has strong specificity and high sensitivity.
4. The invention provides a cell separation culture technology of the muscovy duck Cha Pama virus for the first time, and discovers that the muscovy duck Cha Pama virus can proliferate in duck embryo kidney primary cells without generating obvious cytopathy, and can provide a basis for subsequent strain proliferation and vaccine preparation based on the culture method.
Claims (2)
1. The TaqMan real-time fluorescent quantitative PCR detection primer and probe for detecting the muscovy duck Cha Pama virus are characterized in that the preservation number of the muscovy duck Cha Pama virus is as follows: CCTCC NO: V202311; preservation date: 2023, 3, 1, deposit unit: china center for type culture Collection; preservation address: university of martial arts in chinese;
the primer comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is shown as SEQ ID No. 2;
the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3;
the nucleotide sequence of the probe is shown as SEQ ID No. 4.
2. The TaqMan real-time fluorescent quantitative PCR detection primer and probe for detecting the muscovy duck Cha Pama virus according to claim 1, wherein the probe is labeled with a fluorescent reporter group FAM at the 5 '-end and BHQ at the 3' -end.
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| Metagenomic characterisation of avian parvoviruses and picornaviruses from Australian wild ducks;Jessy Vibin 等;《Scientific Reports》;20200730;第10卷;摘要 * |
| Unravelling Bile Viromes of Free-Range Laying Chickens Clinically Diagnosed with Spotty Liver Disease: Emergence of Many Novel Chaphamaparvoviruses into Multiple Lineages;Subir Sarker 等;《Viruses》;20221117;第14卷(第11期);摘要 * |
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