CN110951916A - Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology - Google Patents
Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology Download PDFInfo
- Publication number
- CN110951916A CN110951916A CN201911228922.7A CN201911228922A CN110951916A CN 110951916 A CN110951916 A CN 110951916A CN 201911228922 A CN201911228922 A CN 201911228922A CN 110951916 A CN110951916 A CN 110951916A
- Authority
- CN
- China
- Prior art keywords
- sads
- cov
- primer
- kit
- real
- 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
Abstract
The invention belongs to the technical field of biological detection, and discloses a primer and a kit for detecting SADS-CoV based on a real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology, the invention can specifically amplify SADS-CoV by designing and utilizing the primer and a probe, real-time fluorescent RT-RPA can monitor the specific combination condition of the probe and an amplification fragment in the amplification process in real time, a portable fluorescence detector is used for collecting fluorescence data, the SADS-CoV can be identified after the amplification is carried out by utilizing the method, and the kit has high accuracy, strong specificity and good repeatability; the method can accurately, quickly and efficiently detect the SADS-CoV, does not need expensive and complex instruments, provides a new technical means for epidemiological detection and safe production of the porcine acute diarrhea syndrome coronavirus, greatly shortens the detection time, is favorable for quickly diagnosing and monitoring the site epidemic situation, has high cost performance and wide market prospect.
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a primer and a kit for detecting SADS-CoV based on a real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology.
Background
Porcine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus, and can cause severe porcine acute diarrhea syndrome. SADS-CoV mainly causes watery diarrhea, severe vomiting, emaciation and dehydration of piglets, wherein the mortality rate of infected piglets below 5 days of age reaches 90%, and the mortality rate of infected piglets above 8 days of age is less than 5%. The study found that SADS-CoV does not match the 3 coronaviruses currently prevalent in swine farms: PEDV (Porcine Epidemic diarrhe Virus), Porcine Deltay coronavirus (PDCoV) and Porcine Transmissible Gastroenteritis Virus (TGEV) antibodies are subjected to cross reaction, so that the existing Porcine coronavirus vaccine can not be effective for SADS-CoV infection, and serious economic loss can be caused to the aquaculture industry once large-scale outbreak occurs.
SADS-CoV is a member of the genus coronavirus A, has a typical coronavirus structure, has protrusions on the surface of the virus, has a diameter of 100-200 nm, has a single-stranded positive-strand RNA genome with a total length of about 27.17kb, comprises 5 '-UTR and 3' -UTR, has 5 nonstructural proteins (ORF1a, ORF1b, NS3a, NS7a and NS7b), and has 4 structural proteins including spike protein (S), envelope protein (E), membrane glycoprotein (M) and nucleocapsid protein (N). At present, the diagnosis method applied to the SADS-CoV mainly comprises the following steps: reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), fluorescent quantitative PCR, immunofluorescence staining, pathogen separation and identification and the like. The detection means has advantages of specificity and sensitivity, but the implementation of the detection methods requires professional personnel to operate, the detection equipment is expensive, and the detection period is long, so the technical requirements are relatively high, and the detection methods cannot be popularized and used. In addition, Point of care testing (POCT) can be used to perform on-site rapid diagnosis of diseases by means of small desktop devices or reagents, and has the advantages of rapidness, convenience, low testing cost, and the like.
The prior art has no POCT detection product for detecting SADS-CoV.
Disclosure of Invention
Aiming at the technical defects that the POCT detection product for detecting the SADS-CoV does not exist in the prior art, the invention firstly provides a primer and a probe for detecting the SADS-CoV based on the real-time fluorescence reverse transcription recombinase polymerase nucleic acid amplification technology.
The second purpose of the invention is to provide the application of the primer and the probe in preparing a detection reagent for specifically detecting the SADS-CoV fluorescent RT-RPA.
The third purpose of the invention is to provide a fluorescent RT-RPA kit containing the primer and the probe.
The purpose of the invention is realized by the following technical scheme:
a primer and a probe for detecting SADS-CoV based on a real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology are disclosed, wherein the primer comprises an upstream primer F and a downstream primer R, and the sequences of the upstream primer F and the downstream primer R are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the probe is shown as SEQ ID NO: 3, respectively.
Preferably, the composition of the probe is: 5 'terminal sequence (THF) 3' terminal sequence (C3-spacer).
More preferably, the probe is designed such that: modifying by using a modifying group, modifying the fluorescent reporter gene at a position 30bp away from the 5 'end and replacing a base T, modifying the fluorescent quenching group at a position 15bp away from the 3' end and replacing the base T, and replacing the fluorescent reporter group and the quenching group by THF at a distance of 1 base; specifically, the fluorescence reporter group is FAM, and the quencher group is BHQ 1.
The invention also provides application of the primers and the probes for detecting the SADS-CoV based on the real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology in preparation of a detection reagent for specifically detecting the SADS-CoV fluorescent RT-RPA, and is characterized in that RNA of a sample to be detected is extracted as a template, and the primers and the probes are used for amplification and real-time fluorescent detection.
The present invention also provides a fluorescent RT-RPA kit for detecting SADS-CoV, comprising the primers and probes of claim 1.
Preferably, the concentration of the upstream primer F, the concentration of the downstream primer R and the concentration of the probe are respectively 10 μ M.
Preferably, the kit further comprises a positive standard and a fluorescent RT-RPA reagent.
Preferably, the reaction system for carrying out fluorescence RT-RPA detection by the kit is as follows: fluorescent RT-RPA reagent, 2. mu.L of upstream primer F, 2. mu.L of downstream primer R, 0.6. mu.L of Probe Probe, and 2.0. mu.L of RNA template, with a total volume of 50. mu.L.
Specifically, 2. mu.L of the forward primer F, 2. mu.L of the reverse primer R, 0.6. mu.L of the Probe Probe and 2.0. mu.L of the RNA template can be added to a 50. mu.L system recommended by fluorescent RT-RPA reagent (Zhongzhou bioscience technology Co., Ltd.) according to the instruction for use.
More preferably, the preparation method of the positive standard substance comprises the following steps: s1, extracting RNA from a SADS-CoV positive disease material, and performing RNA extraction by using a nucleic acid sequence shown as SEQ ID NO: 4 and SEQ ID NO: 5, specifically amplifying the SADS-CoV N gene by the primer of the RT-PCR to obtain an amplification product; s2, recovering the target fragment, connecting the target fragment with a vector, and obtaining an in vitro transcription positive standard TSADS-RNA through purification and in vitro transcription.
Specifically, the preparation process of the positive standard substance comprises the following steps: RNA is extracted from SADS-CoV positive pathological material, and a specific SADS-CoV N gene amplification primer (the primer sequence is shown as SEQ NO: 4-5) is used for amplifying the SADS-CoV-N gene through RT-PCR to obtain an amplification product with the same size as a target fragment. After the PCR product is recovered, the PCR product is connected with a pGEM-T easy vector, transformed and amplified to obtain a recombinant plasmid containing a target fragment, after bacterial liquid PCR and sequencing identification, the pGEM-T-SADS plasmid is linearized and purified, and the transcription of a target gene is carried out by using the instruction of an in vitro transcription kit Ribomax Large Scale RNA transcription Systems (Promega), so as to obtain an in vitro transcription standard TSASD-RNA for later use.
The invention also provides a method for detecting the fluorescent RT-RPA of the SADS-CoV in the sample by using the detection kit, which comprises the following steps:
(1) extracting sample RNA;
(2) an amplification system: respectively adding 2 muL of an upstream primer F, 2 muL of a downstream primer R, 0.6 muL of a Probe Probe and 2.0 muL of an RNA template into a recommended 50 muL system according to the use instruction of a fluorescent RT-RPA reagent (Hangzhou mass measurement Biotechnology Co., Ltd.), reacting the prepared amplification system at 39 ℃ for 20min, placing the reaction system in a fluorescence detector after reaction, and generating a fluorescence amplification curve according to a detected fluorescence signal;
(3) and (4) judging a result: after the reaction is finished, an obvious amplification curve is generated, namely the sample is judged to contain the SADS-CoV, otherwise, the SADS-CoV is not generated.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains a new SADS-CoV real-time fluorescence RT-RPA detection primer and probe by design, wherein the sequences of the upstream primer, the downstream primer and the probe are shown as SEQ ID NO: 1-3; the primer and the probe can be used for specifically amplifying the SADS-CoV, the condition of specific combination of the probe and an amplification fragment in the amplification process can be monitored in real time by the real-time fluorescence RT-RPA, a portable fluorescence detector is used for collecting fluorescence data, the SADS-CoV can be identified after the amplification is carried out by the method, the method has the advantages of high accuracy, strong specificity and good repeatability, the SADS-CoV can be accurately, quickly and efficiently detected, expensive and complex instruments are not needed, a new technical means is provided for epidemiological detection and safe production of the porcine acute diarrhea syndrome coronavirus, the detection time is greatly shortened, the rapid diagnosis and monitoring of the site epidemic situation are facilitated, the cost performance is high, the large-scale popularization is easy, and the market prospect is wide.
Drawings
FIG. 1 is a graph showing the specific amplification curve of the SADS-CoV real-time fluorescence RT-RPA method of the present invention, wherein 1 is SADS-CoV; 2 is FMDV; 3 is PEDV; 4 is TGEV; 5 is RV; 6 is SVA; 7 is PCV 3; 8 is PRRSV; 9 is PCV 2; 10 is CSFV; 11 is PRV; 12 is JEV; 13 is a negative control.
FIG. 2 is a graph showing the sensitivity amplification curve of the SADS-CoV real-time fluorescence RT-RPA method of the present invention, which is 10 from left to right6copies/μL、105copies/μL、104copies/μL、103copies/μL、 102copies/μL、101copies/μL、100copies/. mu.L, negative control.
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. 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.
EXAMPLE 1 preparation of Positive Standard
Magnetic bead method for extracting sample RNA
The following protocol was used with the Magnetic Viral DNA/RNA Kit from TIANGEN. 200 μ L of the positive pathogen grinding fluid supernatant and 15 μ L of the magnetic bead suspension G were added to a 1.5 mLeppendorf tube; adding 20 mu L of proteinase K; adding 300 mu L of Carrier RNA working solution into the sample, covering a tube cover, shaking and uniformly mixing for 10sec, incubating at room temperature for 10min, and uniformly mixing by turning upside down every 3min for 10sec so as to fully combine the magnetic beads and the nucleic acid. Placing the centrifuge tube on a magnetic frame, standing for 1min, and carefully removing liquid when the magnetic beads are completely adsorbed. The centrifuge tube was removed from the magnetic stand and 500. mu.l of the rinsing solution PWC was added. Placing the centrifugal tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and carefully absorbing the liquid. The centrifuge tube was removed from the magnetic stand and 500. mu.l of the rinsing solution PWE was added. Placing the centrifugal tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and carefully absorbing the liquid. Centrifuging the tube on a magnetic frame, and air drying at 56 deg.C for 5-10 min. The centrifuge tube was removed from the magnetic stand and 100. mu.l RNase-Free ddH was added2O, shaking and mixing evenly at 56 ℃ for 5 min. Placing the centrifuge tube on a magnetic frame, standing for 2min, and carefully adsorbing magnetic beadsThe nucleic acid solution was transferred to a fresh centrifuge tube and stored under appropriate conditions.
Second, primer design and amplification
According to the N gene sequence of SADS-CoV in GenBank (Accession number: MF094681-MF094684), positive standard primers are designed:
upstream primer P1: 5'-ATGGCCACTGTTAATTGGG-3', respectively;
the downstream primer P2: 5'-CTAATTAATAATCTCATCCACCATC-3' are provided.
Real-time fluorescent RT-RAA primers:
an upstream primer F: 5'-CCAACAATTGCTGAGTTGCTTCCGACACAAGCTGC-3', respectively;
a downstream primer R: 5'-CATCAGCATTTAGGTCTTGTTGAGATTGAG-3', respectively;
probe Probe:
5'-AGTGAAATCACAACCAAAGAGTCTGGTGAA(FAM-dT)(THF)(BHQ1 -dT)GTAGAAGTCACCTAT(C3 spacer)-3'。
the SADS-CoV N gene is amplified by using a positive standard primer, and the reaction system is as follows: 2 × RT-PCR Buffer 25 μ L, upstream primer P12 μ L, downstream primer P22 μ L, template RNA2 μ L, Enzyme Mix 2 μ L, and RNase FreeH2O make up the total volume of the reaction system to 50. mu.L. The amplification procedure was: (1) reverse transcription at 50 ℃ for 30min, (2) pre-denaturation at 94 ℃ for 4min, (3) denaturation at 94 ℃ for 30sec, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 1min for 30s for 35 cycles; (3) extension at 72 ℃ for 5 min.
The PCR product was recovered (see the instruction manual of the Omega E.Z.N.A.gel Extraction Kit), and subjected to agarose gel electrophoresis to identify the correct target fragment.
Three, in vitro transcription
And (3) connecting the target fragment with a pGEM-T Easy vector after purification, converting the target fragment into an escherichia coli competence, selecting a single clone for culture, extracting and identifying plasmids, and sending the positive plasmid pGEM-T-SADS for sequencing verification. pGEM-T-SADS plasmid is linearized and purified, transcription of the target gene is performed using the in vitro transcription kit Ribomax Large Scale RNA transcription Systems (Promega) instructions to obtain an in vitro transcript TSASD-RNA as a positive standard, and the copy number is calculated from the RNA concentration determined by a spectrophotometer.
Example 2 real-time fluorescent RT-RPA amplification
RT-RPA reaction System: according to the instructions of the fluorescent RT-RPA reagent (Hangzhou Zhongzhiyu Biotech Co., Ltd.), 2. mu.L of the forward primer F, 2. mu.L of the reverse primer R, 0.6. mu.L of the Probe Probe and 2.0. mu.L of the RNA template were added to the recommended 50. mu.L system.
The reaction conditions are as follows: the reaction was carried out at 39 ℃ for 20 min.
And (4) analyzing results: the reaction tube after the configuration is placed in a fluorescence detector (a Bio-Rad fluorescence quantitative instrument is used in the implementation process of the invention), and whether a fluorescence signal curve is generated or not is observed according to a fluorescence signal detected by the instrument.
And (4) interpretation of results: within 20min, an obvious amplification curve is generated, and the sample is judged to be a positive sample; and if the amplification curve is not available, judging the sample as a negative sample.
Example 3 specificity assay for real-time fluorescent RT-RPA
Real-time fluorescent RT-RPA amplification was performed using the established reaction system and reaction conditions of example 2 using DNA or RNA of SVA, FMDV, PCV2, CSFV, PRRSV, PCV3, TGEV, PEDV, RV, PRV, JEV, and SADS-CoV as templates, and the amplification was found to be positive only for SADS-CoV (FIG. 1), indicating that SADS-CoV can be specifically amplified using the above primers, probes, and amplification method.
Example 4 sensitivity assay for real-time fluorescent RT-RPA
RT-RPA (10 times) was performed using 10-fold serial dilutions of SADS-CoV positive standards as templates6～100copies/. mu.L for a total of 7 gradients to determine their lower detection limit. As a result, the lower limit of RT-RPA detection using the standard as a template was 101copies/. mu.L (FIG. 2).
Example 5 reproducibility of real-time fluorescent RT-RPA
Positive standard quality pellets of SADS-CoV were diluted to 106、104、1023 total gradients of copies/. mu.L were used as templates for the in-group and inter-group reproducibility tests, each gradient was repeated 5 times for 3 reactions. Standard quality grain repeatability tests were performed on the established method of SADS-CoV. Results are shown in Table 1: the Coefficient of Variation (CV) was less than 5% both within and between groups.
TABLE 1 repeatability analysis of SADS-CoV real-time fluorescent RT-RPA amplification of TSAS-RNA
EXAMPLE 6 kit composition
A kit for detecting SADS-CoV was formulated as follows: 10 mu M of upstream primer F, 10 mu M of downstream primer R, 10 mu M of Probe Probe, fluorescent RT-RPA reagent and a positive standard substance.
One reaction system of the kit may be: according to the instructions of the fluorescent RT-RPA reagent (Hangzhou Zhongzhiyu Biotech Co., Ltd.), 2. mu.L of the forward primer F, 2. mu.L of the reverse primer R, 0.6. mu.L of the Probe Probe and 2.0. mu.L of the RNA template were added to the recommended 50. mu.L system.
The reaction conditions for carrying out real-time fluorescence RT-RPA by using the kit are as follows: the reaction was carried out at 39 ℃ for 20 min.
While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> southern China university of agriculture
<120> primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology
<141>2019-12-04
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>35
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>1
ccaacaattg ctgagttgct tccgacacaa gctgc 35
<210>2
<211>30
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>2
catcagcatt taggtcttgt tgagattgag 30
<210>3
<211>45
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>3
agtgaaatca caaccaaaga gtctggtgaa gtagaagtca cctat 45
<210>4
<211>19
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>4
atggccactg ttaattggg 19
<210>5
<211>25
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>5
ctaattaata atctcatcca ccatc 25
Claims (7)
1. A primer and a probe for detecting SADS-CoV based on a real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology are characterized in that the primer comprises an upstream primer F and a downstream primer R, and the sequences of the upstream primer F and the downstream primer R are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the probe is shown as SEQ ID NO: 3, respectively.
2. The use of the primers and probes for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology as claimed in claim 1 for preparing a detection reagent for specifically detecting SADS-CoV fluorescent RT-RPA, wherein RNA of a sample to be detected is extracted as a template, and the primers and probes as claimed in claim 1 are used for amplification and real-time fluorescent detection.
3. A fluorescent RT-RPA kit for detecting SADS-CoV, comprising the primers and probes of claim 1.
4. The kit according to claim 3, wherein the concentrations of the forward primer F, the reverse primer R and the probe are 10 μ M, respectively.
5. The kit of claim 4, further comprising a positive standard and a fluorescent RT-RPA reagent.
6. The kit of claim 5, wherein the kit performs the fluorescent RT-RPA detection reaction system as follows: fluorescent RT-RPA reagent, 2. mu.L of upstream primer F, 2. mu.L of downstream primer R, 0.6. mu.L of Probe Probe, and 2.0. mu.L of RNA template, with a total volume of 50. mu.L.
7. The kit according to claim 6, wherein the positive standard is prepared by a method comprising:
s1, extracting RNA from a SADS-CoV positive disease material, and performing RNA extraction by using a nucleic acid sequence shown as SEQ ID NO: 4 and SEQ ID NO: 5, specifically amplifying the SADS-CoV N gene by the primer of the RT-PCR to obtain an amplification product;
s2, recovering the target fragment, connecting the target fragment with a vector, and obtaining an in vitro transcription positive standard TSADS-RNA through purification and in vitro transcription.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228922.7A CN110951916A (en) | 2019-12-04 | 2019-12-04 | Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228922.7A CN110951916A (en) | 2019-12-04 | 2019-12-04 | Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110951916A true CN110951916A (en) | 2020-04-03 |
Family
ID=69979752
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911228922.7A Pending CN110951916A (en) | 2019-12-04 | 2019-12-04 | Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110951916A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154923A (en) * | 2020-04-07 | 2020-05-15 | 中国医学科学院医学实验动物研究所 | Primer for amplifying nucleotide fragment of novel coronavirus (SARS-CoV-2) gene and application thereof |
| CN111621597A (en) * | 2020-05-09 | 2020-09-04 | 清华大学 | Virus recombinase-polymerase amplification detection method |
| CN111621606A (en) * | 2020-05-25 | 2020-09-04 | 成都海之元生物科技有限公司 | Novel real-time fluorescent RPA detection kit for coronavirus |
| CN112522443A (en) * | 2020-12-18 | 2021-03-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Primer composition and kit for detecting porcine acute diarrhea syndrome coronavirus and application |
| CN113528704A (en) * | 2020-04-14 | 2021-10-22 | 华南农业大学 | Primer group, probe, kit and detection method for rapidly identifying novel coronavirus |
| CN113930547A (en) * | 2021-10-25 | 2022-01-14 | 华南农业大学 | RT-RAA fluorescence method detection primer pair, kit and detection method for porcine epidemic diarrhea virus N gene |
| CN114134252A (en) * | 2021-10-19 | 2022-03-04 | 中国检验检疫科学研究院 | Primer and kit for detecting coronavirus |
| CN116287460A (en) * | 2023-03-07 | 2023-06-23 | 中国农业大学 | RAA-CRISPR/Cas12a detection composition of PEAV, reaction device, test strip and kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299156A (en) * | 2017-08-22 | 2017-10-27 | 河北省检验检疫科学技术研究院 | Detect nucleic acid, real-time fluorescence RT RPA kits and the method for Porcine epidemic diarrhea virus |
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108411040A (en) * | 2018-05-21 | 2018-08-17 | 浙江大学 | Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method |
| CN109439799A (en) * | 2018-11-06 | 2019-03-08 | 江西农业大学 | It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus |
-
2019
- 2019-12-04 CN CN201911228922.7A patent/CN110951916A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299156A (en) * | 2017-08-22 | 2017-10-27 | 河北省检验检疫科学技术研究院 | Detect nucleic acid, real-time fluorescence RT RPA kits and the method for Porcine epidemic diarrhea virus |
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108411040A (en) * | 2018-05-21 | 2018-08-17 | 浙江大学 | Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method |
| CN109439799A (en) * | 2018-11-06 | 2019-03-08 | 江西农业大学 | It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus |
Non-Patent Citations (5)
| Title |
|---|
| HUANAN WANG等: "Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV)", 《J VIROL METHODS》 * |
| LING ZHOU等: "Development of a TaqMan-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs", 《J VIROL METHODS》 * |
| LING ZHOU等: "Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China", 《TRANSBOUNDARY AND EMERGING DISEASES》 * |
| MATTHEW D MORRE等: "Recombinase polymerase amplification: a promising point of care detection methods for enteric viruses", 《FUTURE VIROLOGY》 * |
| 景志刚等: "重组酶聚合酶扩增技术研究进展", 《生物技术通报》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154923A (en) * | 2020-04-07 | 2020-05-15 | 中国医学科学院医学实验动物研究所 | Primer for amplifying nucleotide fragment of novel coronavirus (SARS-CoV-2) gene and application thereof |
| CN113528704A (en) * | 2020-04-14 | 2021-10-22 | 华南农业大学 | Primer group, probe, kit and detection method for rapidly identifying novel coronavirus |
| CN111621597A (en) * | 2020-05-09 | 2020-09-04 | 清华大学 | Virus recombinase-polymerase amplification detection method |
| CN111621606A (en) * | 2020-05-25 | 2020-09-04 | 成都海之元生物科技有限公司 | Novel real-time fluorescent RPA detection kit for coronavirus |
| CN112522443A (en) * | 2020-12-18 | 2021-03-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Primer composition and kit for detecting porcine acute diarrhea syndrome coronavirus and application |
| CN114134252A (en) * | 2021-10-19 | 2022-03-04 | 中国检验检疫科学研究院 | Primer and kit for detecting coronavirus |
| CN114134252B (en) * | 2021-10-19 | 2024-03-08 | 中国检验检疫科学研究院 | Primers and kit for detecting coronavirus |
| CN113930547A (en) * | 2021-10-25 | 2022-01-14 | 华南农业大学 | RT-RAA fluorescence method detection primer pair, kit and detection method for porcine epidemic diarrhea virus N gene |
| CN113930547B (en) * | 2021-10-25 | 2022-07-26 | 华南农业大学 | RT-RAA fluorescence detection primer pair, kit and detection method for porcine epidemic diarrhea virus N gene |
| CN116287460A (en) * | 2023-03-07 | 2023-06-23 | 中国农业大学 | RAA-CRISPR/Cas12a detection composition of PEAV, reaction device, test strip and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110951916A (en) | Primer and kit for detecting SADS-CoV based on real-time fluorescent reverse transcription recombinase polymerase nucleic acid amplification technology | |
| CN110777220B (en) | Primer group, probe, RPA test strip kit and identification method | |
| CN108676920B (en) | Primer and kit for rapidly detecting mouse norovirus and RT-RPA method thereof | |
| CN110004240B (en) | Real-time fluorescence detection kit and test strip detection kit for mycoplasma gallisepticum based on RPA and application of kit and test strip detection kit | |
| CN107988340B (en) | PCR amplification primer for rapidly detecting mycoplasma ovipneumoniae and application thereof | |
| CN112176112A (en) | Triple fluorescent quantitative RT-PCR detection kit for avian influenza virus H5, H7 and H9 subtypes and application thereof | |
| CN110904270A (en) | Multiplex RT-PCR detection method for porcine delta coronavirus, porcine epidemic diarrhea virus and porcine sapelovirus and application | |
| CN106947834B (en) | Multiplex PCR method for detecting six duck susceptibility viruses | |
| CN108588276B (en) | Fluorescent quantitative PCR (polymerase chain reaction) primer pair and kit for D-type influenza virus | |
| CN107574261B (en) | Detection primer, detection kit and detection method for detecting hantavirus | |
| CN112662821A (en) | Fluorescent probe primer combination, kit and application of porcine epidemic diarrhea virus M gene | |
| CN111471802A (en) | Porcine delta coronavirus rapid detection primer, kit and application thereof | |
| CN107058630B (en) | Multiple RT-PCR detection method for 3 genotypes of bovine parainfluenza 3 virus | |
| CN107937615B (en) | Primers and probes for distinguishing wild strains and vaccine strains of swine Japanese encephalitis virus | |
| CN112458211A (en) | One-step RT-PCR kit and method for identifying bluetongue virus serotype | |
| CN110735005A (en) | SIV and PRRSV multiple RT-PCR rapid detection kit and primer | |
| CN116515840B (en) | Kit and detection method for detecting bovine viral diarrhea virus type 3 | |
| CN114292963B (en) | Duck tembusu virus nucleic acid CRISPR-Cas13a detection system, RPA primer pair and crRNA | |
| CN109338012B (en) | K subgroup avian leukosis virus RT-PCR detection primer and detection method | |
| CN110387437B (en) | Amplification method for detecting mouse encephalomyelitis virus RT-RPA and kit thereof | |
| CN116064968A (en) | Kit and application of probe group for detecting acarb virus RT-RAA primer | |
| CN117143866A (en) | Primer group, probe, detection kit and application of primer group, probe and detection kit in preparation of products for detecting porcine epidemic diarrhea | |
| CN116769972A (en) | RAA primer pair, composition, detection method and application for detecting TGEV | |
| CN116064961A (en) | Products and methods for fluorescent RT-RAA detection of genotype 2 porcine reproductive and respiratory syndrome virus | |
| CN117568525A (en) | Primer group for detecting Nipah virus, modified primer group and application thereof |
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: 20200403 |
|
| RJ01 | Rejection of invention patent application after publication |