CN114480725A - Real-time fluorescence quantitative PCR (polymerase chain reaction) primer group, probe and kit for canine circovirus - Google Patents
Real-time fluorescence quantitative PCR (polymerase chain reaction) primer group, probe and kit for canine circovirus Download PDFInfo
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- CN114480725A CN114480725A CN202011175288.8A CN202011175288A CN114480725A CN 114480725 A CN114480725 A CN 114480725A CN 202011175288 A CN202011175288 A CN 202011175288A CN 114480725 A CN114480725 A CN 114480725A
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- 241001193170 Canine circovirus Species 0.000 title claims abstract description 24
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 23
- 239000000523 sample Substances 0.000 title claims abstract description 21
- 238000003752 polymerase chain reaction Methods 0.000 title description 7
- 241000700605 Viruses Species 0.000 claims abstract description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 10
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- 238000006243 chemical reaction Methods 0.000 description 6
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- 238000012360 testing method Methods 0.000 description 5
- 241000282465 Canis Species 0.000 description 4
- 241001436113 Canine astrovirus Species 0.000 description 3
- 241000711506 Canine coronavirus Species 0.000 description 3
- 241000712083 Canine morbillivirus Species 0.000 description 3
- 241000701931 Canine parvovirus Species 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
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- 101150066583 rep gene Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
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- 241001260012 Bursa Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241001533384 Circovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
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- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 1
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 1
- 102100024407 Jouberin Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 1
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 206010047124 Vasculitis necrotising Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention belongs to the field of virus molecular biology detection by establishing a canine circovirus real-time fluorescent quantitative PCR primer group, a probe and a kit. The method can be used for rapidly detecting the canine circovirus. The method is simple and rapid, the detection time of a single sample is 60 minutes, the sensitivity can reach 10 copy number/ul, the simultaneous detection can be realized aiming at a larger sample size, the method has good sensitivity, repeatability, specificity and stability, and the aim of rapidly and accurately detecting the canine circovirus can be realized.
Description
Technical Field
The invention relates to a virus detection primer group, a kit containing the primer group and application thereof, in particular to a primer group for detecting canine circovirus, a kit containing the primer group and application thereof. The invention belongs to the technical field of virus detection.
Background
The circovirus is a spherical, non-enveloped virus with circular single-stranded DNA and a genome size of about 2 kb. The genome comprises two Open Reading Frames (ORFs), ORF1 encoding a replicase (V1) and ORF2 encoding the capsid (C1) protein. There are two non-coding Intergenic Regions (IR) between the 5 'and 3' ends of the two major ORFs. In 2012, 6 strains of Canine circovirus type1 (Canine circovirus 1, CaCV1) positive were detected from 205 Canine sera in the United states for the first time, and subsequently the virus was found in diarrhea dogs in Europe, North America, Asia, and so on, demonstrating that it is widely prevalent worldwide. Clinically, canine circovirus (canine circovirus) is found to be a main pathogen causing canine granulomatous lymphadenitis and necrotizing vasculitis, and can cause clinical symptoms such as gastroenteritis, hemorrhagic diarrhea and the like.
Therefore, it has become imperative to establish a rapid diagnostic method that is sensitive and highly specific. The real-time PCR with the fluorescent probe system has the characteristics of rapidness, simplicity and reproducibility, and is widely used for virus detection. However, to date, there have been no reports on the CanineCV diagnostic method based on the TaqMan method. In this study, TaqMan-based real-time PCR was established to detect CanineCV, which was subsequently used to evaluate clinical samples as well.
Therefore, it is urgently required to establish a highly specific detection method for diagnosing canine circovirus.
Disclosure of Invention
The invention aims to provide a primer group and a probe for detecting canine circovirus by real-time fluorescent quantitative PCR.
The technical scheme adopted by the invention is as follows:
a real-time fluorescent quantitative PCR primer group and a probe for canine circovirus comprise CaCV-F: 5'-GAGATCGGGAACAAGGAC-3' (SEQ ID No.1), CaCV-R: 5'-CATAAATTGGGGAACAGGAATA-3' (SEQ ID No.2) and probe CaCV-P: FAM-5'-TCTATCGGCGTCTCACTCTTATCA-3' -BHQ1(SEQ ID No. 3). The primers are designed according to the conserved region sequence of the Rep gene whole genome sequence of CanineCV published on GenBank.
The primer group can be used for preparing a real-time fluorescent quantitative PCR detection kit for the canine circovirus, so that the virus content of the canine circovirus is detected. The kit comprises a positive control standard substance, a PCR reaction solution and a negative control; the positive control standard substance is a recombinant plasmid pMD-19T-Rep-CaCV containing a Rep gene sequence of canine circovirus; the PCR reaction solution includes the extracted template DNA, the primer set and probe as described above, the enzyme and ddH2O。
The canine circovirus real-time fluorescent quantitative PCR detection kit prepared by the primer group has the advantages of simple, convenient and quick detection method, good repeatability, high sensitivity and strong specificity, and can carry out quick and high-sensitivity real-time fluorescent quantitative PCR detection on the canine circovirus.
Drawings
FIG. 1 is a fluorescent quantitative PCR standard curve;
FIG. 2 is a diagram of the fluorescent quantitative PCR specificity assay, in which reference numeral 1 is a positive standard, 2-8 are negative controls, canine distemper virus, canine parvovirus, canine coronavirus, canine kubuvirus and canine astrovirus, respectively;
FIG. 3 is a graph of sensitivity experimental amplifications of serial 10 dilutions of a standard sample.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention and are not to be construed as limiting the scope of the invention.
Example 1
1. Design of real-time fluorescent quantitative PCR primer group and probe
Designing a specific primer sequence aiming at the Rep gene of the canine circovirus:
an upstream primer: 5'-GAGATCGGGAACAAGGAC-3'
A downstream primer: 5'-CATAAATTGGGGAACAGGAATA-3'
And (3) probe: FAM-5'-TCTATCGGCGTCTCACTCTTATCA-3' -BHQ1
The upstream and downstream primers and probes were synthesized by Shanghai Biotechnology engineering Co., Ltd.
2. Strain
The canine circovirus samples used in the experiment were collected in some animal hospitals in Hefei city, Anhui province and Maanshan city.
3. Extraction of viral nucleic acids
The collected sample was subjected to extraction of viral genome according to the instructions of the Tiangen genome kit (Tiangen Biotechnology Co., Ltd.).
Example 2
Real-time fluorescent quantitative PCR reaction and standard curve drawing
Measuring the concentration of recombinant plasmid pMD-19T-Rep-CaCV by a nucleic acid concentration measuring instrument, and calculating to obtain the copy number of the recombinant plasmidIs 2.056X 1011Diluting the recombinant plasmid by 10-fold concentration gradient to establish a standard curve, wherein the abscissa represents the logarithm (X) of the copy number of the plasmid, and the ordinate is a Ct value (Y); the corresponding regression equation obtained on the basis is that y is-3.129 x +38.432, and the correlation coefficient R of the regression equation and the standard curve20.998, and the results are shown in FIG. 1. And no primer dimer and non-specific amplification product appear in the amplification process, and the primer specificity of the established PCR method is good.
Example 3
Specificity, sensitivity and reproducibility test
1. Experiment of specificity
The established real-time fluorescence quantitative PCR method is adopted to amplify the nucleic acids of canine circovirus positive standard, Canine Distemper Virus (CDV), Canine Parvovirus (CPV), Canine Coronavirus (CCV), canine bursa virus (CaKoV) and Canine Astrovirus (CASTV), and meanwhile, a negative control is set, the result is shown in figure 2, and no amplification curve exists except the positive standard, which indicates that the method has good specificity.
2. Sensitivity test
The recombinant plasmids with the calculated copy number are diluted in a 10-fold gradient manner, the recombinant plasmids with each gradient concentration are respectively used as templates, and the fluorescence quantitative PCR amplification is carried out under the optimal reaction condition, and the result is shown in figure 3. As can be seen from FIG. 3, the lowest copy number detected was 1X 101copies/μL。
3. Repeatability test
At 107、105And 103The recombinant control samples with 3 concentrations of copies/mu L are used as templates, and the results of the repeated experiments of real-time fluorescent quantitative PCR are shown in Table 1 by using the optimal reaction conditions. As can be seen from Table 1, the coefficient of variation was less than 5% in the reproducibility test, indicating that the established real-time fluorescent quantitative PCR method has good reproducibility.
TABLE 1 fluorescent quantitative PCR repeatability test
The fluorescence quantitative PCR is to continuously monitor the intensity of a fluorescence model in the PCR reaction process to measure the amount of a specific product in real time, and accordingly deduces and monitors the initial toxic content of a pathological material, so that the method not only has the characteristic of high amplification efficiency of the conventional PCR technology, but also has the characteristics of high specificity, high sensitivity and accuracy of the spectral technology and the like, simultaneously avoids the steps of subsequent electrophoresis and the like, and reduces the errors caused by pollution and manual operation. In the experimental process, the method is proved to have high sensitivity and specificity and good repeatability. Meanwhile, the accurate quantification can be carried out according to the standard curve, namely, the initial virus amount contained in the sample is detected.
Claims (5)
1. A fluorescent quantitative PCR primer group and a probe for detecting canine circovirus, wherein the primer consists of an upstream primer, a downstream primer and a probe, and the nucleotide sequences of the upstream primer, the downstream primer and the probe are shown in a readable sequence table.
2. The fluorescent quantitative PCR primer group and the probe of claim 1 are used for preparing reagents for detecting canine circovirus.
3. A fluorescent quantitative PCR kit for detecting canine circovirus, characterized by comprising the primer set and the probe of claim 1.
4. Preparation of a standard curve: a series of positive standard instruments with different concentration gradients are used as templates, the logarithm of the copy number of the standard product is used as an X axis, and the Ct value is used as a Y axis to draw a standard curve.
5. Calculation of virus content in samples: and respectively calculating the virus copy number in the sample according to the established standard curve.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116083646A (en) * | 2022-11-04 | 2023-05-09 | 青岛农业大学 | Real-time fluorescent quantitative PCR primer for detecting chicken circovirus, kit and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116083646A (en) * | 2022-11-04 | 2023-05-09 | 青岛农业大学 | Real-time fluorescent quantitative PCR primer for detecting chicken circovirus, kit and application |
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Application publication date: 20220513 |