CN114107570A - TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method - Google Patents
TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method Download PDFInfo
- Publication number
- CN114107570A CN114107570A CN202111681560.4A CN202111681560A CN114107570A CN 114107570 A CN114107570 A CN 114107570A CN 202111681560 A CN202111681560 A CN 202111681560A CN 114107570 A CN114107570 A CN 114107570A
- Authority
- CN
- China
- Prior art keywords
- pcr
- primer
- sample
- fluorescent quantitative
- tegv
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a TGEV fluorescent quantitative RT-PCR detection primer probe combination, a kit and a detection method. The primer sequences of the probe compositions used were: forward primer 5'-GCATTTGTAAGGGCTCACC-3', reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively; the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB. The product and the method can realize rapid, high-sensitivity and high-specificity TEGV detection.
Description
Technical Field
The invention belongs to the technical field of biological pathogen detection, and particularly relates to a TGEV fluorescent quantitative RT-PCR detection primer probe combination, a kit and a detection method.
Background
Porcine transmissible gastroenteritis (porcine transmissible gastroenteritis of swine) is a highly contact digestive tract infectious disease of pigs caused by porcine transmissible gastroenteritis virus (TEGV) and is characterized by high mortality of piglets within two weeks of age due to vomiting, severe diarrhea, dehydration. The disease is reported in the Guangdong at the earliest time in China, and the disease occurs all over the country at present. With the development of the intensive pig industry, the occurrence amount of the disease shows a trend of obvious rising, and the disease is often mixed with diarrhea, rotavirus and the like to cause serious disease, thereby causing more serious economic loss to the pig industry.
However, the clinical symptoms caused by TEGV infection of pigs are very similar to those caused by diarrhea virus and rotavirus, and the disease is difficult to be differentially diagnosed by simply depending on the clinical symptoms. And at present, detection and identification methods such as pathogen separation and identification, electron microscope observation, immuno-electron microscope observation, ELISA, neutralization test, nucleic acid probe hybridization, RT-PCR, sequence determination and the like which are commonly used in a laboratory aiming at TGEV are mostly long in time consumption and the sensitivity needs to be improved. Although some studies on TGEV fluorescent quantitative RT-PCR detection are carried out, for example, a pair of specific PCR primers is designed and optimized according to a conserved sequence of a swine transmissible gastroenteritis virus S gene disclosed in Chinese patent No. CN102154516A, and a SYBR Green l real-time fluorescent PCR method for rapidly and quantitatively detecting TGEV is established, the method still has the defects of poor specificity and low accuracy. Therefore, it is urgently needed to establish a rapid, sensitive and high-specificity detection method, which provides a basis for the laboratory diagnosis of TGEV.
Disclosure of Invention
Aiming at the technical problems, the invention provides a TGEV fluorescent quantitative RT-PCR detection primer probe combination, a kit and a detection method, which can realize rapid, high-sensitivity and high-specificity TEGV detection.
In order to achieve the purpose, the invention adopts the technical scheme that:
the first aspect of the invention discloses a primer probe combination for detecting TEGV fluorescent quantitative RT-PCR, which has the following primer sequences:
a forward primer: 5'-GCATTTGTAAGGGCTCACC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively;
the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB.
The second aspect of the invention discloses the application of the primer probe combination in quantitative detection of TEGV.
The third aspect of the invention discloses a TEGV fluorescent quantitative RT-PCR detection kit comprising the primer probe combination.
Preferably, the sensitivity of the kit is lowest, 4 copies/. mu.L of a sample to be detected can be detected, and the repetition coefficient in batches and between batches is less than 2%.
The fourth aspect of the invention discloses a TEGV fluorescent quantitative RT-PCR detection method, which comprises the following steps:
(1) design, synthesis of primers and probes: designing a pair of specific primers and a probe according to the S gene conserved sequence of the TGEV, wherein the primer sequence is as follows:
a forward primer: 5'-GCATTTGTAAGGGCTCACC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively;
the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB;
(2) preparing a recombinant plasmid standard product: performing RT-PCR amplification on the S gene target fragment by using the primer probe in the step (1) to obtain a product with the size of 103bp, connecting the product with a pMD18-T vector, selecting a positive clone for sequencing and identifying, and taking a positive plasmid with successful sequencing as a standard substance for fluorescent quantitative detection;
(3) extracting RNA in a sample to be detected, and setting a negative control;
(4) performing TaqMan fluorescent quantitative RT-PCR amplification reaction on a sample to be detected by using the primer and the probe in the step (1);
(5) and (4) detecting a result: reading the corresponding Ct value by the self-contained software of the fluorescence quantitative PCR instrument, and judging whether the sample to be detected is positive or negative.
Preferably, the primer concentration is 10. mu. mol/L, and the probe concentration is 10. mu. mol/L.
Preferably, the amplification reaction system of step (4): 2 × RT-PCR Buffer 10 μ L, RT Enzyme 2 μ L, forward primer 0.4 μ L, reverse primer 0.4 μ L, probe 0.4 μ L, RNase-free water 1.8 μ L.
Preferably, the step (4) includes: adding 5 mul of sample RNA to be detected into a reaction system with the total amount of 20 mul, preheating a PCR tube with the prepared reaction system at 52 ℃ for 10min, pre-denaturing at 95 ℃ for 30s, denaturing at 95 ℃ for 10s, annealing and extending at 60 ℃ for 30s, and extending the denaturation and annealing processes for 40 cycles.
Preferably, the positive or negative judgment criteria of the sample to be tested in the step (5) are as follows:
positive: the Ct value of a detected sample is less than or equal to 35.0, and an amplification curve has an obvious exponential growth period;
and (3) suspicious: the Ct value of the detected sample is more than 35, a typical amplification curve appears, and repeated tests are carried out;
negative: the Ct value of the sample cannot be detected, and no obvious amplification curve exists.
Compared with the prior art, the invention has the advantages and positive effects that: specific primers and probes are designed and synthesized aiming at the S gene of TGEV (transmissible gastroenteritis virus), and the fluorescent quantitative reaction conditions are optimized, so that the kit and the detection method can be used for detecting TGEV with high specificity, have good linear relation in the range of 109-101 copies/mu L for the detection of a sample to be detected with high sensitivity, can be used for detecting the sample to be detected with 4 copies/mu L at the lowest sensitivity, have good repeatability, and have the repetition coefficient of less than 2 percent in batches and among batches. The kit and the detection method can be applied to large-scale and high-flux sample detection.
Drawings
FIG. 1 is a kinetic curve diagram of TGEV TaqMan fluorescence quantitative RT-PCR provided by the embodiment of the invention;
FIG. 2 is a standard curve diagram of TGEV TaqMan fluorescent quantitation RT-PCR provided by the embodiment of the invention;
FIG. 3 is a diagram of the result of the specificity test of TGEV TaqMan fluorescent quantitative RT-PCR provided by the embodiment of the present invention.
FIG. 4 is a diagram of the S gene PCR amplification product of TGEV TaqMan fluorescent quantitative RT-PCR provided by the embodiment of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
A TEGV fluorescent quantitative RT-PCR detection method comprises the following steps:
(1) design, synthesis of primers and probes: designing a pair of specific primers and probes according to the conserved sequence of S gene of TGEV, wherein,
a forward primer: 5'-GCATTTGTAAGGGCTCACC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively;
the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB.
(2) Preparing a recombinant plasmid standard product: performing RT-PCR amplification on the S gene target fragment by using the primer in the step (1) to obtain a product with the size of 103bp (as shown in figure 4, detecting the DNA of the amplification product by using 1.5% agarose gel electrophoresis and observing the result, wherein the amplification result is consistent with the designed target fragment), connecting a pMD18-T vector, selecting a positive clone for sequencing and identifying, and taking a positive plasmid with successful sequencing as a standard product for fluorescent quantitative detection;
(3) extracting RNA in a sample to be detected, and setting a negative control;
(4) performing TaqMan fluorescent quantitative RT-PCR amplification reaction on a sample to be detected by using the primer and the probe in the step (1);
(5) and (4) detecting a result: reading the corresponding Ct value by the self-contained software of the fluorescence quantitative PCR instrument, and judging whether the sample to be detected is positive or negative.
Specifically, the primer concentration is 10. mu. mol/L, and the probe concentration is 10. mu. mol/L.
Specifically, the amplification reaction system in the step (4): 2 × RT-PCR Buffer 10 μ L, RT Enzyme 2 μ L, forward primer 0.4 μ L, reverse primer 0.4 μ L, probe 0.4 μ L, RNase-free water 1.8 μ L.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Example 1 establishment of TEGV fluorescent quantitation RT-PCR detection method
The TEGV fluorescent quantitative RT-PCR detection method comprises the following steps:
(1) designing a primer and a probe: according to the sequence comparison result of TGEV (transmissible gastroenteritis virus) S gene DNA Star software, a pair of specific primers and probes are designed by using Primer probe design software Primer Express5.0, and are synthesized by Qingdao Biotechnology Limited, and the sequences of the primers and the probes are detailed in Table 1.
| Primers (Probe) | Sequence (5 'to 3') |
| TGEV-F (Forward primer) | GCATTTGTAAGGGCTCACC |
| TGEV-R (reverse primer) | GAAGGACATATAGGGAACTTATGG |
| TGEV-P (Probe) | CTGCACTCACTACCCCAATTGC |
(2) Preparing a recombinant plasmid standard: extraction of TGEV RNA was performed according to the instructions of the Bori nucleic acid extraction kit. Using specific primer containing fluorescent quantitative target fragment to make RT-PCR amplification, using PCR amplification to make purification to obtain 93bp product, using 1.5% agarose gel electrophoresis to detect amplified product DNA and observing result, and the amplification result is identical to designed target fragment. The positive PCR product was purified, ligated into pMD-18T vector, and transformed into DH 5. alpha. competent cells. And selecting a single colony, performing amplification culture, extracting plasmids, performing PCR identification, performing sequencing detection on positive plasmids, and taking the positive plasmids which are successfully sequenced as standard products of fluorescence quantitative detection.
(3) Extracting RNA in a sample to be detected, and setting a negative control (ultrapure water in the embodiment);
(4) performing TaqMan fluorescent quantitative RT-PCR amplification reaction on a sample to be detected by using the primer and the probe in the step (1), and specifically comprising the following steps: adding 5 mul of sample RNA to be detected into a reaction system with the total amount of 20 mul, preheating a PCR tube with the prepared reaction system at 52 ℃ for 10min, pre-denaturing at 95 ℃ for 30s, denaturing at 95 ℃ for 10s, annealing and extending at 60 ℃ for 30s, and extending in the denaturation and annealing processes for 40 cycles;
(5) and (4) detecting a result: reading corresponding Ct values through self-contained software of a fluorescence quantitative PCR instrument, and judging whether a sample to be detected is positive or negative, wherein the judgment standard is as follows:
positive: the Ct value of a detected sample is less than or equal to 35.0, and an amplification curve has an obvious exponential growth period;
and (3) suspicious: the Ct value of the detected sample is more than 35, a typical amplification curve appears, and repeated tests are carried out;
negative: the Ct value of the sample cannot be detected, and no obvious amplification curve exists.
Example 2 establishment of standard curve:
recombinant plasmids were extracted by a plasmid extraction kit, quantified with an ultraviolet spectrophotometer and the copy number calculated. The amplification kinetics curve of TGEV is shown in figure 1 and the corresponding standard curve is shown in figure 2 by performing fluorescence quantitative PCR with 10-fold gradient dilution (4 × 109-4 copies/. mu.L) of the standard as a template. The standard curve has a good linear relationship, the regression equation is-3.305X +38.24, the correlation coefficient R2 is 0.998, and the amplification efficiency E is 1.007.
Example 3 sensitivity test
Plasmid standards were diluted in 10-fold gradients (4 x 10)9-4 copies/. mu.L) as template for fluorescent quantitative PCR reaction, determining the sensitivity of the method. The result shows that the lowest detectable concentration of the method is 4 copies/mu L, and the disclosed PCR detection method can only detect 10 at the lowest2copies/mL. Therefore, the sensitivity of the kit is 100 times higher than that of the existing PCR.
Example 4 specificity test
Nucleic acid extraction kit is used for extracting nucleic acid of Porcine Epidemic Diarrhea Virus (PEDV), pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV) and epidemic encephalitis B virus (JEV), African Swine Fever Virus (ASFV), Rotavirus (RV) and porcine delta coronavirus (PDCoV) as templates for carrying out fluorescent quantitative PCR reaction, and negative and positive controls are set. The results are shown in FIG. 3, and it can be seen from the results in FIG. 3 that only TGEV showed a distinct amplification curve, and that no amplification curve was observed for any of the other porcine viruses and the negative control. Therefore, the kit has good specificity.
EXAMPLE 5 repeatability test
(1) Batch to batch repeatability test: taking 7 samples with different CT values (evenly distributed between 12 and 34), and carrying out 2 repeated tests under the same condition, wherein each sample is repeated for 8 times; the test results are shown in table 2:
TABLE 2 TGEV fluorescent quantitation RT-PCR kit batch-to-batch repeats
The experimental results in table 2 show that the variation coefficient of the batch-to-batch repetition is less than 2%, which indicates that the kit of the present invention has good repeatability among batches.
(2) In-batch repeatability test: taking 7 samples with different CT values (evenly distributed between 12 and 34), and carrying out 8 times of repeated tests in one test; the test results are shown in table 3:
TABLE 3 TGEV fluorescent quantitative RT-PCR kit in-batch replicates
The experimental results in table 3 show that the variation coefficients of the batch repeats are less than 2%, which indicates that the kit of the present invention has good repeatability in the batch.
Example 6 sensitivity comparative test
Randomly selecting commercialized TGEV fluorescent PCR detection kits produced by three different manufacturers, wherein the serial numbers of the TGEV fluorescent PCR detection kits are respectively A, B and C, wherein A is a biological science and technology limited company for testing in Hunan China, a real-time fluorescent RT-PCR detection kit for porcine transmissible gastroenteritis virus, B is a Beijing world Henren animal epidemic prevention technology limited company, a real-time fluorescent RT-PCR detection kit for porcine transmissible gastroenteritis virus, and C is a Beijing Yishengbao biological science and technology limited company and a single fluorescent PCR detection kit for porcine transmissible gastroenteritis virus. 24 parts of TGEV clinical samples (including negative samples and positive samples) are selected, and the 24 parts of samples are subjected to fluorescence PCR test detection by using the three kits and the kit adopting the detection method in the example 1. The test results are shown in table 4:
TABLE 4 TGEV fluorescence quantitative RT-PCR kit sensitivity contrast test
As can be seen from the experimental results in Table 4, the detection rate of the kit is higher than that of a commercial kit, and the kit has smaller CT and higher sensitivity.
Sequence listing
<110> Guidao Biotechnology, Inc. of Qingdao Jiazhi, Xinkui Liu Ju, and Xinkui Liu Ju, Inc
Primer and probe combination, kit and detection method for TGEV fluorescent quantitative RT-PCR detection
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcatttgtaa gggctcacc 19
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaaggacata tagggaactt atgg 24
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctgcactcac taccccaatt gc 22
<210> 4
<211> 103
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcatttgtaa gggctcacca cctactacca ccacagaatc tagtttgact tgcaattggg 60
gtagtgagtg caggttaaac cataagttcc ctatatgtcc ttc 103
Claims (9)
1. A primer probe combination for detecting TEGV fluorescent quantitative RT-PCR is characterized in that the primer sequence is as follows:
a forward primer: 5'-GCATTTGTAAGGGCTCACC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively;
the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB.
2. Use of the primer probe combination of claim 1 for quantitative detection of TEGV.
3. A kit for TEGV fluorescent quantitative RT-PCR detection comprising the primer probe combination of claim 1.
4. The TEGV fluorescent quantitative RT-PCR detection kit according to claim 3, characterized in that the lowest sensitivity can detect 4copies/μ L of the sample to be detected, and the intra-batch and inter-batch repetition coefficients are both less than 2%.
5. A TEGV fluorescence quantitative RT-PCR detection method is characterized by comprising the following steps:
(1) design, synthesis of primers and probes: designing a pair of specific primers and a probe according to the S gene conserved sequence of the TGEV, wherein the primer sequence is as follows:
a forward primer: 5'-GCATTTGTAAGGGCTCACC-3' the flow of the air in the air conditioner,
reverse primer: 5'-GAAGGACATATAGGGAACTTATGG-3', respectively;
the probe sequence is as follows: FAM-CTGCACTCACTACCCCAATTGC-MGB.
(2) Preparing a recombinant plasmid standard product: amplifying an S gene target fragment by PCR, connecting a pMD18-T vector, selecting a positive clone, sequencing and identifying, and taking a positive plasmid with successful sequencing as a standard substance for fluorescent quantitative detection;
(3) extracting RNA in a sample to be detected, and setting a negative control;
(4) performing TaqMan fluorescent quantitative RT-PCR amplification reaction on a sample to be detected by using the primer and the probe in the step (1);
(5) and (4) detecting a result: reading the corresponding Ct value by the self-contained software of the fluorescence quantitative PCR instrument, and judging whether the sample to be detected is positive or negative.
6. The TEGV fluorescent quantitative RT-PCR detection method as claimed in claim 5, wherein the primer concentration is 10 μmol/L and the probe concentration is 10 μmol/L.
7. The TEGV fluorescent quantitative RT-PCR detection method as claimed in claim 5, wherein the amplification reaction system of step (4): 2 × RT-PCR Buffer 10 μ L, RT Enzyme 2 μ L, forward primer 0.4 μ L, reverse primer 0.4 μ L, probe 0.4 μ L, RNase-free water 1.8 μ L.
8. The TEGV fluorescent quantitative RT-PCR detection method according to claim 5, characterized in that the step (4) comprises: adding 5 mul of sample RNA to be detected into a reaction system with the total amount of 20 mul, preheating a PCR tube with the prepared reaction system at 52 ℃ for 10min, pre-denaturing at 95 ℃ for 30s, denaturing at 95 ℃ for 10s, annealing and extending at 60 ℃ for 30s, and extending the denaturation and annealing processes for 40 cycles.
9. The TEGV fluorescent quantitative RT-PCR detection method according to claim 5, characterized in that the positive or negative judgment criteria of the sample to be tested in step (5) are:
positive: the Ct value of a detected sample is less than or equal to 35.0, and an amplification curve has an obvious exponential growth period;
and (3) suspicious: the Ct value of the detected sample is more than 35, a typical amplification curve appears, and repeated tests are carried out;
negative: the Ct value of the sample cannot be detected, and no obvious amplification curve exists.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111681560.4A CN114107570A (en) | 2021-12-30 | 2021-12-30 | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111681560.4A CN114107570A (en) | 2021-12-30 | 2021-12-30 | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114107570A true CN114107570A (en) | 2022-03-01 |
Family
ID=80363228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111681560.4A Pending CN114107570A (en) | 2021-12-30 | 2021-12-30 | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114107570A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009009A1 (en) * | 2003-03-24 | 2005-01-13 | Peiris Joseph S.M. | Diagnostic assay for the human virus causing severe acute respiratory syndrome (SARS) |
| CN104293973A (en) * | 2013-07-15 | 2015-01-21 | 国家纳米科学中心 | Swine fever, porcine reproductive and respiratory syndrome and swine transmissible gastroenteritis virus detection kit and detection method |
| CN104673936A (en) * | 2015-02-26 | 2015-06-03 | 广东大华农动物保健品股份有限公司 | RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene |
| CN105154583A (en) * | 2015-07-14 | 2015-12-16 | 天津瑞普生物技术股份有限公司 | Double-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously identifying TGEV (transmissible gastroenteritis viruses) and PEDV (porcine epidemic diarrhea viruses) |
| CN106435023A (en) * | 2016-09-23 | 2017-02-22 | 湖南新南方养殖服务有限公司 | Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof |
| CN107236824A (en) * | 2017-06-29 | 2017-10-10 | 新希望六和股份有限公司 | A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection |
| CN109762931A (en) * | 2018-09-19 | 2019-05-17 | 天津瑞普生物技术股份有限公司 | Primer, probe and the method for transmissible gastro-enteritis virus fluorescence quantitative RT-RCR detection |
| CN113462820A (en) * | 2021-07-21 | 2021-10-01 | 河北工程大学 | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof |
-
2021
- 2021-12-30 CN CN202111681560.4A patent/CN114107570A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050009009A1 (en) * | 2003-03-24 | 2005-01-13 | Peiris Joseph S.M. | Diagnostic assay for the human virus causing severe acute respiratory syndrome (SARS) |
| CN104293973A (en) * | 2013-07-15 | 2015-01-21 | 国家纳米科学中心 | Swine fever, porcine reproductive and respiratory syndrome and swine transmissible gastroenteritis virus detection kit and detection method |
| CN104673936A (en) * | 2015-02-26 | 2015-06-03 | 广东大华农动物保健品股份有限公司 | RT-qPCR detection method and primers and TaqMan probe for detecting swine transmissible gastroenteritis virus N gene |
| CN105154583A (en) * | 2015-07-14 | 2015-12-16 | 天津瑞普生物技术股份有限公司 | Double-RT-PCR (reverse transcription-polymerase chain reaction) method for simultaneously identifying TGEV (transmissible gastroenteritis viruses) and PEDV (porcine epidemic diarrhea viruses) |
| CN106435023A (en) * | 2016-09-23 | 2017-02-22 | 湖南新南方养殖服务有限公司 | Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof |
| CN107236824A (en) * | 2017-06-29 | 2017-10-10 | 新希望六和股份有限公司 | A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection |
| CN109762931A (en) * | 2018-09-19 | 2019-05-17 | 天津瑞普生物技术股份有限公司 | Primer, probe and the method for transmissible gastro-enteritis virus fluorescence quantitative RT-RCR detection |
| CN113462820A (en) * | 2021-07-21 | 2021-10-01 | 河北工程大学 | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 任玉鹏等: "一种猪伪狂犬病病毒gE基因TaqMan荧光定量PCR检测方法的建立", 《养猪》 * |
| 张云等: "猪传染性胃肠炎病毒S基因的SYBR Green Ⅰ荧光定量RT-PCR检测", 《湖南农业大学学报(自然科学版)》 * |
| 李子祥等: "猪传染性胃肠炎病毒实时荧光定量PCR检测方法的建立及应用", 《河南农业科学》 * |
| 李自刚等: "《生物检测技术》", 31 August 2016, 中国轻工业出版社 * |
| 王建华等: "一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立", 《中国畜牧兽医》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN106957927B (en) | African swine fever fluorescent PCR detection reagent, African swine fever fluorescent PCR detection kit and application thereof | |
| CN111020062A (en) | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN112430686B (en) | Kit, primer and probe for simultaneously detecting BVDV-1, BVDV-2 and BVDV-3 | |
| CN112176105B (en) | Special primer for virus BVDV, BRV and BCV one-tube multiplex fluorescence PCR detection and application thereof | |
| CN113684309A (en) | 7 primer probe and kit for detecting viruses related to porcine reproductive disorder diseases based on liquid chip technology and application of primer probe and kit | |
| CN110904270A (en) | Multiplex RT-PCR detection method for porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus and application | |
| Zhang et al. | Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China | |
| CN113943831A (en) | Multiple fluorescent quantitative primer and probe combination capable of diagnosing three high-incidence pathogens of porcine diarrhea disease simultaneously and application thereof | |
| CN112458208B (en) | Kit and method for detecting bovine sarcoidosis virus | |
| CN116875743B (en) | Fluorescent quantitative PCR kit for detecting two cat enteroviruses at one time and application thereof | |
| CN113913559A (en) | Reagent for fluorescence quantitative detection of PRRSV and detection method for PRRSV typing | |
| CN112553372A (en) | Porcine pseudorabies virus and porcine circovirus type 3 dual-fluorescence quantitative PCR detection primer, probe, kit and method | |
| CN112646931A (en) | Primer pair, probe, kit and detection method for detecting porcine circovirus type 4 | |
| CN111471802A (en) | Porcine delta coronavirus rapid detection primer, kit and application thereof | |
| CN116814859A (en) | Primer probe composition, kit and method for identifying African swine fever virus genes I and II | |
| CN110607398A (en) | RT-LAMP kit for fluorescent visual rapid detection of porcine epidemic diarrhea virus | |
| CN109762931A (en) | Primer, probe and the method for transmissible gastro-enteritis virus fluorescence quantitative RT-RCR detection | |
| CN111500774B (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN114107570A (en) | TGEV fluorescent quantitative RT-PCR detection primer probe combination, kit and detection method | |
| CN111647692A (en) | Primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and using method thereof | |
| CN111961757A (en) | Double-gene probe method real-time fluorescence quantitative PCR kit for detecting duck tembusu virus and application | |
| CN112410466A (en) | Primer, probe and detection method for porcine circovirus type 2 and porcine circovirus type 4 dual real-time fluorescent quantitative PCR detection | |
| CN115976273B (en) | Dual fluorescence PCR detection kit for identifying African swine fever virus genes type I and type II |
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 |
Application publication date: 20220301 |
|
| RJ01 | Rejection of invention patent application after publication |