CN112725536A - LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system - Google Patents
LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system Download PDFInfo
- Publication number
- CN112725536A CN112725536A CN202110157993.3A CN202110157993A CN112725536A CN 112725536 A CN112725536 A CN 112725536A CN 202110157993 A CN202110157993 A CN 202110157993A CN 112725536 A CN112725536 A CN 112725536A
- Authority
- CN
- China
- Prior art keywords
- lsdv
- mgb probe
- taqman
- quantitative pcr
- pcr detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
-
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an LSDV Taqman MGB probe real-time fluorescence quantitative PCR detection system, and relates to the field of biotechnology detection; the detection system comprises an LSDV upstream primer, an LSDV downstream primer and an LSDV MGB probe, wherein the nucleotide sequence of the LSDV upstream primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the LSDV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the LSDV MGB probe is provided with a fluorescence labeling FAM at the 5 'end and an MGB modifying group connected with the 3' end, and the nucleotide sequence is shown as SEQ ID NO: 3 is shown in the specification; the detection system disclosed by the invention has the advantages of high sensitivity, strong specificity, good repeatability and controllable quality, provides conditions for timely discovering and early treating the bovine sarcoidosis, provides technical support for epidemic disease prevention and control, and has very important significance for guaranteeing the safety and healthy development of the animal husbandry in China.
Description
Technical Field
The invention relates to the field of biotechnical detection, in particular to an LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system.
Background
Bovine sarcoidosis (LSD), a pox virus disease characterized by fever, nodules of the skin, mucous membranes and internal organs, wasting, swollen lymph nodes, edema of the skin and sometimes even death. This disease often results in decreased milk production, temporary or permanent sterility of the bulls, and damage and death of the hide due to secondary bacterial infections. LSD virus (LSDV) is transmitted mainly by arthropods. The disease spread in 2015 and 2016 from africa to southeast europe, bardry and the caucasian region. Bovine sarcoidosis is classified as a legal report entry disease by the world animal health Organization (OIE), and is also classified as a second class of animal infectious diseases in China.
With the continuous improvement of the living standard of people and the rapid development of international trade, only about 20 million imported live cattle (excluding fresh beef and ice fresh beef) are imported in China every year, but bovine sarcoidosis is frequently and explosively transmitted in Europe (Makinton, Bulgaria, Albania, Greece, etc.), Africa (nanometer Bia, Grougiia, Morsbick, etc.) and countries around China (Russia, Mongolia, Hasakstein, etc.). In 2019, 8 months and 10 days, the bovine sarcoidosis is confirmed to occur in Yili Kazak autonomous State in Xinjiang for the first time in China, and the disease becomes a large epidemic disease threatening cattle raising industry in China. In order to prevent and control LSD, the development of a rapid and sensitive bovine sarcoidosis virus detection reagent is imminent.
The existing method for detecting the bovine sarcoidosis virus is a conventional gel PCR method, and is used for detecting the bovine sarcoidosis virus genome in blood, semen and tissue culture samples. However, the fluorescent quantitative PCR method has the characteristics of higher speed, higher sensitivity and the like, the TaqMan technology is a technology for carrying out real-time fluorescent quantitative detection on a single-tube PCR product, and the whole PCR process is monitored in real time by adding a double-fluorescent-labeled probe specifically complementary to a target gene sequence and utilizing the accumulation of fluorescent signals, so that the target gene is quantitatively or qualitatively detected. Although relevant reports of the LSDV TaqMan-MGB fluorescent PCR detection method exist at home and abroad, the sensitivity of the currently reported LSDV TaqMan-MGB fluorescent PCR detection method cannot meet the detection requirement, so that the LSDV detection reagent with high sensitivity is the basis and the premise for effectively preventing LSDV from being input and output through the port of the country and avoiding wide-range outbreak of epidemic situation, and is also the urgent need for the quarantine enforcement and disease monitoring of the port of the country.
Disclosure of Invention
The invention aims to provide an LSDV Taqman MGB probe real-time fluorescence quantitative PCR detection system, so as to solve the problems in the prior art, provide conditions for timely discovering and early treating bovine sarcoidosis, provide technical support for epidemic disease prevention and control, and have very important significance for guaranteeing the safety and healthy development of animal husbandry in China.
In order to realize the purpose, the invention provides an LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection system, which comprises a primer and a probe; wherein, the primer comprises an LSDV upstream primer and an LSDV downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the probe is an LSDV MGB probe, and the nucleotide sequence is shown as SEQ ID NO: 3, the 5 'end of the LSDV MGB probe is provided with a fluorescence labeling FAM, and the 3' end is connected with an MGB modifying group.
The invention also provides an application of the LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection system in preparation of products for detecting LSDV.
Furthermore, the product is a kit, a PCR detection reagent or a chip.
The invention also provides a kit for detecting the LSDV Taqman-MGB probe by real-time fluorescence quantitative PCR, which comprises the LSDV upstream primer, the LSDV downstream primer and the LSDV MGB probe.
Further, the kit specifically comprises: LSDV upstream primer, LSDV downstream primer, LSDV MGB probe, sterile nuclease-free water, PCR reaction solution, positive control, negative control and sterilized deionized water; wherein the positive control is a recombinant bovine sarcoidosis virus plasmid pUC 57-LSDV; the negative control was DEPC water.
Further, the PCR reaction solution comprises Taq DNA polymerase, dNTPs and buffer solution.
Further, the reaction conditions of the real-time fluorescence quantitative PCR of the Taqman-MGB probe are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s and annealing at 58 ℃ for 30s for 40 cycles, and FAM fluorescence signals were collected at the end of each cycle of annealing.
The invention discloses the following technical effects: the invention adopts a new generation of TaqMan-MGB probe, the quenching group of the TaqMan-MGB probe adopts a non-fluorescent quenching group, the non-fluorescent quenching group does not generate fluorescence, the intensity of a fluorescence background signal of PCR reaction is reduced compared with the common TaqMan probe, and the specificity of the method is further enhanced; meanwhile, the tail end of the probe is also connected with an MGB modifying group, the Tm temperature of the probe can be increased by about 10 ℃, and under the condition of obtaining the same Tm value, the base needed by the MGB probe is shorter than that of a common TaqMan probe, and the selectable range in a target sequence is wider, so that the MGB probe is particularly beneficial to designing a probe with better sensitivity and specificity in the target sequence. The detection method and the kit disclosed by the invention have the advantages of high sensitivity, strong specificity, good repeatability and controllable quality, provide conditions for timely discovering and early treating the nodular skin disease of the cattle, provide technical support for epidemic disease prevention and control, and have very important significance for guaranteeing the safe and healthy development of animal husbandry in China.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a recombinant bovine sarcoidosis virus plasmid pUC57-LSDV amplification map, wherein M is DL2000 molecular mass standard, and 1 is a negative control; 2. 3 is positive plasmid;
FIG. 2 shows the result of nucleic acid homology comparison of recombinant bovine sarcoidosis virus plasmid pUC57-LSDV and LSDV;
FIG. 3 shows the results of the LSDV sensitivity assays, where 1-9 are 1.16X 108copies/μl、1.16×107copies/μl、1.16×106copies/μl、1.16×105copies/μl、1.16×104copies/μl、1.16×103copies/μl、1.16×102copies/μl、1.16×101Negative quality control is performed on copies/mu l and 1.16 multiplied by 100 copies/mu l, 10;
FIG. 4 is an LSDV standard curve;
FIG. 5 shows the results of the detection of LSDV clinical samples, where 1-2 is 1.37X 10-5ng/μ l, 3-4 is 1.37X 10-6ng/μ l, 5-6 is 1.37X 10-7ng/ul, 7-8 is 1.37X 10-8ng/μ l, 9-10 is 1.37 × 10-9ng/. mu.l, 11 is a negative control;
FIG. 6 shows the specific detection results of LSDV, wherein 1 is positive quality control, 2-4 are antigens of agglutination test of sheeppox, goat pox virus nucleic acid, bovine infectious rhinotracheitis virus and brucellosis tiger red plate, and 5 is 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 drawings in 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.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Example 1LSDV Taqman MGB Probe real-time fluorescent quantitative PCR detection method establishment
1.1 specific primer and Probe design
According to the south Africa wild strain bovine sarcoidosis virus (LSDV) whole gene sequence published in GenBank (accession number: MN636843.1), the primers and the fluorescent probe are analyzed and compared, and the gene LSDV071 (coding RNA polymerase subunit) is designed, and the MGB probe technology is utilized to establish the rapid, specific and sensitive bovine sarcoidosis virus fluorescent PCR detection method.
The sequences of the primers and the probes are as follows:
an upstream primer: 5'-tggtaggatagtcgcaaatgatat-3', respectively;
a downstream primer: 5'-tgtgaccccaaatccataaatact-3', respectively;
a fluorescent probe: FAM-atgcgtggcatgtc-MGB.
1.2 establishment of LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection method
1.2.1 Positive quality control construction
PCR amplification was carried out using the primers designed in 1.1 and an artificially synthesized bovine sarcoidosis virus-specific gene as a template.
The PCR reaction system (total volume 25uL) was: upstream primer (10umol)1 uL; downstream primer (10umol)1 uL; template 1 uL; 2 × EasyTaq PCR Supermix (+ dye)12.5 uL; 9.5uL of water.
The reaction conditions are as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30 s; storing at 72 deg.C for 7min and 4 deg.C.
Recovering the PCR product, connecting the PCR product to a pUC57 vector, constructing a recombinant bovine sarcoidosis virus plasmid pUC57-LSDV, transforming the recombinant plasmid into a competent cell of Escherichia coli DH5 alpha, constructing a recombinant Escherichia coli pUC57-LSDV-DH5 alpha strain of the bovine sarcoidosis virus, and identifying the recombinant bacterium. The result shows that the recombinant bacteria are gram-negative bacilli, and round colonies with the size of 1-2mm, neat edges, glossy, moist, smooth and grey-white surfaces are formed on LB agar plates after the recombinant bacteria are cultured; the amplified fragment was amplified to a band of about 172bp by using bovine sarcoidosis virus primers (FIG. 1), and the PCR product was aligned with the reference target gene sequence of bovine sarcoidosis virus nucleic acid registered in GenBank to show 100% identity (FIG. 2). The result shows that the plasmid constructed by the PCR product is suitable for positive quality control of nucleic acid detection.
1.2.2 negative quality control construction
DEPC water (DNase, RNase free) of Biosharp company is purchased as negative quality control, the batch of DEPC water is colorless clear liquid by observing the color and the properties of the solution, and the batch of DEPC water is tested according to the appendix of the three parts of the version 2015 of Chinese veterinary pharmacopoeia, and grows aseptically. The bovine sarcoidosis virus nucleic acid is negative as a result of detection by a bovine sarcoidosis virus fluorescent PCR detection method.
1.3 LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection method establishment
PCR amplification was performed using the primers and probes designed in 1.1, and the DNA of the sample to be examined as a template.
The reaction system is as follows: upstream primer (10. mu. mol) 1. mu.L; downstream primer (10. mu. mol) 1. mu.L; probe (10. mu. mol) 0.5. mu.L; 5 mu L of template; 2 XQuantiNova Probe PCR Master Mix 10. mu.L; 2.5 μ L of water, total volume 20 μ L.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s, and annealing at 58 ℃ for 30s for 40 cycles; FAM fluorescence signals were collected at the end of each cycle of annealing.
Conditions for test establishment: and the Ct values of the positive controls are all less than or equal to 30, and the Ct values of the negative controls are all No Ct, and the test is determined to be established.
And (3) judging a detection result: the Ct value of the FAM channel of the sample to be detected is less than or equal to 30, and the sample is judged to be positive by the bovine sarcoidosis virus nucleic acid; the FAM channel Ct value of the sample to be detected is more than 35 or is shown as No Ct, and the sample is judged to be negative to bovine sarcoidosis virus nucleic acid; if the Ct value of the FAM channel 30 of the sample to be detected is more than or equal to 35, the sample is judged to be suspicious and needs to be detected again, and if the Ct value of the FAM channel 30 of the sample is more than or equal to 35 and an obvious amplification curve exists, the sample is judged to be positive by the bovine sarcoidosis virus nucleic acid.
Example 2 LSDV Taqman MGB Probe real-time fluorescent quantitative PCR detection kit
The LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection kit comprises: LSDV upstream primer, LSDV downstream primer, LSDV MGB probe, sterile nuclease-free water, PCR reaction solution, positive control, negative control and sterilized deionized water;
wherein the positive control is a recombinant bovine sarcoidosis virus plasmid pUC 57-LSDV; negative control is DEPC water; the PCR reaction solution is Taq DNA polymerase, dNTPs and buffer solution).
Example 3 evaluation of sensitivity
When the kit is developed, viruses are taken as a sensitive quality control sample of the kit, but the bovine sarcoidosis is a second type of disease, and the biohazard of live viruses is large, so that a bovine sarcoidosis virus plasmid is taken as a sensitive test standard substance.
Sensitivity is measured by copy number, and the lowest detection limit of the kit on bovine nodular skin disease virus plasmids is 1.16 multiplied by 100copies/. mu.l (sensitivity measured in nucleic acid concentration, minimum detection limit 2.74X 10)-9ng/. mu.l), therefore the constructed pUC57-LSDV positive plasmid was selected to be 10-fold diluted (1.16X 10) with sterile nuclease-free water8copies/μl-1.16×100copies/. mu.l) as a kit sensitivity quality control sample group, and performing fluorescent quantitative PCR amplification.
The reaction system is as follows: upstream primer (10. mu. mol) 1. mu.L; downstream primer (10. mu. mol) 1. mu.L; probe (10. mu. mol) 0.5. mu.L; 5 mu L of template; 2 XQuantiNova Probe PCR Master Mix 10. mu.L; 2.5 μ L of water, total volume 20 μ L.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s and annealing at 58 ℃ for 30s for 40 cycles.
The results show that from 1.16X 108copies/μl～1.16×100The copies/. mu.l PCR amplification curves were all valid (FIG. 3). 10-fold gradient dilution from 1.16X 108copies/μl～1.16×100copies/. mu.l, 9 concentration gradients, each concentration repeated 5 times, 5 mean values were taken, and standard curves were plotted with the logarithm of fluorescence intensity as abscissa and the number of cycles as ordinate. The resulting correlation R2The amplification efficiency was 102% (fig. 4), 0.9956.
Meanwhile, in south Africa agriculture research council, the bovine nodular skin disease virus inactivated vaccine provided by the south Africa agriculture research council is verified by adopting the bovine nodular skin disease virus fluorescent PCR detection method established in the research, and the detection result is positive for bovine nodular skin disease virus nucleic acid (figure 5). The sensitivity of the quantitative fluorescent PCR method for LSDV detection recommended by the OIE reference is 37.39copies (Lamien et al, 2011).
Example 4 evaluation of specificity
The kit of embodiment 2 and the LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection method of embodiment 1 are selected to detect sheep pox, goat pox virus nucleic acid, infectious bovine rhinotracheitis virus and brucellosis tiger red plate agglutination test antigens for evaluating the specificity of the detection method and the kit, and the result shows that effective amplification curves do not appear in other viruses except the bovine sarcoidosis virus, which indicates that the kit and the detection method have good specificity (figure 6).
Example 5 repeatability evaluation
Using recombinant plasmid standard 1.16X 105Copies/. mu.l-1.16X 103The copy/mul is used as a template, the kit of the embodiment 2 and the LSDV Taqman MGB probe real-time fluorescence quantitative PCR detection method of the embodiment 1 are adopted to carry out a repeatability test, as shown in Table 1, the Coefficient of Variation (CV) in standard substance groups with different concentrations is between 0.05% and 0.15%, and the Coefficient of Variation (CV) between the groups is between 1.63% and 2.00%, which indicates that the method has better repeatability.
TABLE 1
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> scientific research institute of Chinese inspection and quarantine
<120> LSDV Taqman MGB probe real-time fluorescence quantitative PCR detection system
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggtaggata gtcgcaaatg atat 24
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgtgacccca aatccataaa tact 24
<210> 3
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcgtggca tgtc 14
Claims (7)
- The LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection system is characterized in that: the LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection system comprises a primer and a probe; wherein, the primer comprises an LSDV upstream primer and an LSDV downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the probe is an LSDV MGB probe, and the nucleotide sequence is shown as SEQ ID NO: 3, the 5 'end of the LSDV MGB probe is provided with a fluorescence labeling FAM, and the 3' end is connected with an MGB modifying group.
- 2. Use of the LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection system according to claim 1 in preparation of products for detecting LSDV.
- 3. Use according to claim 2, characterized in that: the product is a kit, a PCR detection reagent or a chip.
- 4. A kit for LSDV Taqman-MGB probe real-time fluorescence quantitative PCR detection is characterized in that: the kit comprises the LSDV upstream primer, the LSDV downstream primer and the LSDV MGB probe as described in claim 1.
- 5. The LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection kit according to claim 4, which is characterized in that: the kit specifically comprises: LSDV upstream primer, LSDV downstream primer, LSDV MGB probe, sterile nuclease-free water, PCR reaction solution, positive control, negative control and sterilized deionized water;wherein the positive control is a recombinant bovine sarcoidosis virus plasmid pUC 57-LSDV; the negative control was DEPC water.
- 6. The LSDV Taqman-MGB probe real-time fluorescent quantitative PCR detection kit according to claim 5, which is characterized in that: the PCR reaction solution comprises Taq DNA polymerase, dNTPs and buffer solution.
- 7. The kit for real-time fluorescent quantitative PCR detection of LSDV Taqman-MGB probe according to any one of claims 4-6, wherein: the reaction conditions of the Taqman-MGB probe real-time fluorescence quantitative PCR are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5s and annealing at 58 ℃ for 30s for 40 cycles, and FAM fluorescence signals were collected at the end of each cycle of annealing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110157993.3A CN112725536A (en) | 2021-02-04 | 2021-02-04 | LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110157993.3A CN112725536A (en) | 2021-02-04 | 2021-02-04 | LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112725536A true CN112725536A (en) | 2021-04-30 |
Family
ID=75595818
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110157993.3A Pending CN112725536A (en) | 2021-02-04 | 2021-02-04 | LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725536A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736918A (en) * | 2021-05-19 | 2021-12-03 | 中国动物卫生与流行病学中心 | Cyclic fluorescent PCR (polymerase chain reaction) method for detecting bovine nodular dermatosis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030054356A1 (en) * | 2000-09-21 | 2003-03-20 | Jacobson James W. | Multiple reporter read-out for bioassays |
| US20120214155A1 (en) * | 2009-11-03 | 2012-08-23 | Yu Li | Diagnostic assays for chordopoxviruses |
| CN108950069A (en) * | 2018-07-23 | 2018-12-07 | 重庆出入境检验检疫局检验检疫技术中心 | Ox lumpy skin disease virus street strain TaqMan-MGB real-time fluorescence quantitative PCR detection primer, kit and detection method |
| CN110964857A (en) * | 2019-12-24 | 2020-04-07 | 北京森康生物技术开发有限公司 | Kit for detecting bovine sarcoidosis virus by excluding capripoxvirus, preparation method and application thereof |
-
2021
- 2021-02-04 CN CN202110157993.3A patent/CN112725536A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030054356A1 (en) * | 2000-09-21 | 2003-03-20 | Jacobson James W. | Multiple reporter read-out for bioassays |
| US20120214155A1 (en) * | 2009-11-03 | 2012-08-23 | Yu Li | Diagnostic assays for chordopoxviruses |
| CN108950069A (en) * | 2018-07-23 | 2018-12-07 | 重庆出入境检验检疫局检验检疫技术中心 | Ox lumpy skin disease virus street strain TaqMan-MGB real-time fluorescence quantitative PCR detection primer, kit and detection method |
| CN110964857A (en) * | 2019-12-24 | 2020-04-07 | 北京森康生物技术开发有限公司 | Kit for detecting bovine sarcoidosis virus by excluding capripoxvirus, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| E. R. TULMAN等: "Genome of Lumpy Skin Disease Virus", 《JOURNAL OF VIROLOGY》 * |
| 聂福平等: "牛结节性皮肤病病毒TaqMan-MGB 荧光PCR 检测方法的建立", 《中国兽医科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736918A (en) * | 2021-05-19 | 2021-12-03 | 中国动物卫生与流行病学中心 | Cyclic fluorescent PCR (polymerase chain reaction) method for detecting bovine nodular dermatosis |
| CN113736918B (en) * | 2021-05-19 | 2022-08-19 | 中国动物卫生与流行病学中心 | Cyclic fluorescent PCR (polymerase chain reaction) method for detecting bovine nodular dermatosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN110894556A (en) | PCR primer, probe, kit and detection method for detecting African swine fever virus infectivity | |
| CN107034309B (en) | Real-time fluorescent RPA kit and test strip RPA kit for rapidly detecting porcine pseudorabies virus and application thereof | |
| CN102146466A (en) | Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method | |
| CN110735006A (en) | African swine fever TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and kit | |
| CN113913543B (en) | EHP real-time fluorescence quantitative PCR detection primer probe combination, kit and method | |
| CN111876502A (en) | Method for identifying Brucella S2 vaccine strain by dual real-time fluorescent quantitative PCR and kit used by same | |
| CN117070673B (en) | LAMP detection primer group for pangolin alpha coronavirus and application thereof | |
| CN112458208B (en) | Kit and method for detecting bovine sarcoidosis virus | |
| CN113046484B (en) | Primer probe, kit and method for detecting African swine fever virus p72 gene | |
| CN112725536A (en) | LSDV Taqman MGB probe real-time fluorescent quantitative PCR detection system | |
| CN110699485A (en) | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus | |
| CN102676697B (en) | Primers and probe for detecting peste des petits ruminants virus and kit | |
| CN108866164A (en) | The streptococcic detection method of B race and kit | |
| He et al. | Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats | |
| CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
| CN107604101A (en) | One breeding pigeon New-type adenovirus real-time fluorescence quantitative PCR detection kit | |
| CN104404132B (en) | A kind of SS2-LAMP detection kit of streptococcus suis 2-type and application | |
| CN110157836B (en) | Primer, probe and method for detecting IBRV and BVDV | |
| CN112695137A (en) | PMA-qPCR detection method of porcine pseudorabies virus | |
| Ji et al. | Simple and visible detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription polymerase spiral reaction method | |
| CN107164511B (en) | Method for rapidly detecting haemophilus parasuis serotype 4 | |
| CN112458202A (en) | PCR detection primer and kit for riemerella anatipestifer virulent phage | |
| CN111593137A (en) | Fluorescent quantitative PCR detection reagent and kit for African swine fever virus | |
| CN111172306A (en) | Composition and kit for detecting mycobacterium tuberculosis and/or brucella and application of composition and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210430 |