CN111518951A - Primer, probe, kit and detection method for detecting SARS-CoV-2 by RT-RAA fluorescence method - Google Patents
Primer, probe, kit and detection method for detecting SARS-CoV-2 by RT-RAA fluorescence method Download PDFInfo
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- CN111518951A CN111518951A CN202010374163.1A CN202010374163A CN111518951A CN 111518951 A CN111518951 A CN 111518951A CN 202010374163 A CN202010374163 A CN 202010374163A CN 111518951 A CN111518951 A CN 111518951A
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- 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
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Abstract
The invention provides a primer, a probe, a kit and a detection method for detecting SARS-CoV-2 by an RT-RAA fluorescence method. The primer for detecting SARS-CoV-2 by RT-RAA fluorescence method of the invention comprises the primer of the sequence shown in SEQ ID NO.2 and the primer of the sequence shown in SEQ ID NO.3, and the probe comprises the probe of the sequence shown in SEQ ID NO.4 or the probe modified on the basis of the sequence shown in SEQ ID NO. 4. The detection method has the characteristics of short detection time, high sensitivity, strong specificity, simple operation and the like, and can analyze the result only by reacting for 5-15 min at 37-42 ℃; has very important significance for rapidly detecting SARS-CoV-2 and has larger application prospect.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a primer, a probe, a kit and a detection method for detecting SARS-CoV-2 by an RT-RAA fluorescence method.
Background
Coronaviruses are a large family of viruses known to cause influenza in general and more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). 2019 the novel coronavirus (SARS-CoV-2) is a novel RNA coronavirus, and can cause novel coronavirus pneumonia disease (COVID-19).
2019 the novel coronavirus has strong infectivity. The world health organization announces in 30 days 1 month in 2020, and ranks the new coronavirus epidemic situation as an international emergent public health incident.
After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, no specific treatment method exists for diseases caused by the novel coronavirus, and symptomatic treatment is required according to the clinical condition of a patient.
The present etiological SARS-CoV-2 detecting method is mainly virus nucleic acid detection. For the problems of increasing the number of cases, limited detection conditions in laboratories of primary hospitals and the like, the development of a simple, rapid and high-specificity diagnosis method is very necessary, a patient can be detected as soon as possible, and the method has very important significance for the next step of treating and preventing the spread of diseases.
Disclosure of Invention
An object of the present invention is to provide a primer for detecting a novel coronavirus SARS-CoV-2.
Another object of the present invention is to provide a probe for detecting SARS-CoV-2, a novel coronavirus.
Another object of the present invention is to provide a kit for detecting the novel coronavirus SARS-CoV-2.
Another object of the present invention is to provide the related application of the primer, probe or kit for detecting the novel coronavirus SARS-CoV-2.
In one aspect, the invention provides a primer for detecting SARS-CoV-2 by RT-RAA fluorescence method, which comprises a primer with a sequence shown as SEQ ID NO.2 and a primer with a sequence shown as SEQ ID NO. 3:
5’-CTTCAACCTAGGACTTTTCTATTAAAATATAATG-3’(SEQ ID NO.2)；
5’-GTTGGTTGGACTCTAAAGTTAGAAGTTTGATAG-3’(SEQ ID NO.3)。
on the other hand, the invention also provides a probe for detecting SARS-CoV-2 by RT-RAA fluorescence method, which comprises a probe with a sequence shown in SEQ ID NO.4, or a probe modified on the basis of the sequence shown in SEQ ID NO. 4:
5’-CCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACG-3’(SEQ IDNO.4)。
according to a specific embodiment of the invention, the RT-RAA fluorescence method of the invention is used for detecting the probe of SARS-CoV-2, wherein the modification comprises the modification of a fluorescence reporter group and a fluorescence quencher group. Specifically, the modifications include:
the base T at the position 35bp away from the 5 ' end base of the probe sequence is modified by a fluorescence reporter group FAM, the base T at the position 39bp away from the 5 ' end base of the probe sequence is modified by a fluorescence quenching group BHQ, and the base T at the position 37bp away from the 5 ' end base of the probe sequence is replaced by a tetrahydrofuran residue (THF).
According to a preferred embodiment of the invention, the probes of the invention modified on the basis of the sequence indicated in SEQ ID No.4 are as follows:
5’-CCATTACAGATGCTGTAGACTGTGCACTTGACCC[FAM-dT]C[THF]C[BHQ-dT]CAGAAACAAAGTGTACG-3’。
in another aspect, the present invention also provides a kit for detecting SARS-CoV-2 by RT-RAA fluorescence method, comprising: the primer of the invention and/or the probe of the invention.
According to a specific embodiment of the invention, the kit further comprises RT-RAA basic fluorescence universal reaction reagent and reaction buffer, wherein the buffer comprises Tris-HCl buffer, MgAc and PEG 10000.
According to a specific embodiment of the present invention, the kit of the present invention further comprises a positive quality control material and a negative quality control material; preferably, the positive quality control material is a specific gene sequence containing SARS-CoV-2, and the negative quality control material is an RT-RAA amplification system without the specific sequence of SARS-CoV-2.
On the other hand, the invention also provides the application of the primer or the probe or the kit in-vitro detection of whether the novel coronavirus SARS-CoV-2 exists in a sample. Namely, the present invention also provides a method for detecting the presence of the novel coronavirus SARS-CoV-2 in a sample in vitro.
According to a specific embodiment of the present invention, in the use of the present invention, the final concentration of the primer in the reaction system is independently 0.42 pM/. mu.l, and the final concentration of the probe in the reaction system is 0.24 pM/. mu.l.
According to a specific embodiment of the invention, in the application of the invention, the reaction system further comprises an RT-RAA-based fluorescence universal reaction reagent and a reaction buffer solution, wherein the reaction buffer solution comprises the following components in parts by weight: Tris-HCl buffer at a concentration of 450mM, pH 7.6, MgAc at a concentration of 260mM and PEG10000 at 10% W/V.
According to a particular embodiment of the invention, the detection method of the invention comprises the following steps:
s1: extracting RNA of a sample to be detected;
s2: preparing a reaction system: adding the reaction buffer solution into the RT-RAA basic fluorescence universal reaction reagent, fully dissolving and uniformly mixing; adding the extracted RNA of the sample to be detected as a template; preferably, the reaction buffer comprises Tris-HCl buffer (pH 7.6) at a concentration of 450 mM; 260mM of MgAc; 10% W/V PEG10000, a probe with a final concentration of 0.24 pM/mul, an upstream primer and a downstream primer with a final concentration of 0.42 pM/mul respectively, and the balance of water as a solvent;
s3: uniformly mixing the reaction system, and putting the mixture into a fluorescence detection instrument for RAA fluorescence reaction in real time; preferably, the real-time RT-RAA fluorescence method reaction conditions are: the reaction temperature is 37-42 ℃, the preferable temperature is 39 ℃, and the reaction time is 5-15 min, the preferable time is 10 min;
s4: and analyzing the result according to the signal detected by the fluorescence detector. And judging the signal to be positive by the fluorescence detector within the reaction time, otherwise, judging the signal to be negative.
The invention has the following beneficial effects:
(1) the invention solves the problems of time and labor waste, long time period, low efficiency, requirement of professional and expensive instruments, high false positive rate and the like in the SARS-CoV-2 detection method in the prior art; the invention adopts RT-RAA technology, overcomes the defects, has the characteristics of short detection time, high sensitivity, strong specificity, simple operation and the like, and can analyze the result only by reacting for 5-15 min at 37-42 ℃ (preferably 39 ℃); has very important significance for rapid detection, prevention and control and clinical isolation treatment of SARS-CoV-2, and has larger application prospect.
(2) The invention can rapidly detect SARS-CoV-2 by utilizing RAA technology, especially can be used for detecting samples with lower DNA content and has low requirement on the quality of detection materials; the operation is simple and convenient, the used time is greatly shortened, large-scale instruments and equipment are not needed, and the method is suitable for large-scale screening; and can be used for screening and detecting SARS-CoV-2 in the primary medical and health institution, can rapidly perform clinical early warning, isolation and treatment, has wide market prospect and great economic and social benefits, and is suitable for large-scale popularization and application.
Drawings
FIG. 1 shows the sensitivity results of RT-RAA fluorescence detection of SARS-CoV-2 of the present invention, wherein line 1, line 2, line 3, line 4, line 5, line 6 and line 7 represent 1.0 × 10 respectively7copies/mL、1.0×106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、1.0×102copies/mL、1.0×101A gene sequence containing SARS-CoV-2 of copies/mL; line 8 is ddH2O；
FIG. 2 shows the specificity results of the RT-RAA fluorescence detection of SARS-CoV-2 of the present invention. Line 1, line 2, line 3, line 4, line 5, line 6, line 7, line 8, line 9, line 10, line 11, line 12, line 13, line 14, line 15, line 16, line 17 represent SARS-CoV-2 virus, SARS virus, human coronavirus, rhinovirus, human bocavirus, adenovirus, influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, and ddH, respectively2O。
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.
Example 1
In this example, primers for RT-RAA detection of SARS-CoV-2 were designed based on the target sequence of specific gene in SARS-CoV-2 genome (GenBank accession No. MN908947.3) to qualitatively and quantitatively detect SARS-CoV-2 gene in human body specimen or other samples to be detected. The conserved target sequence specific to the SARS-CoV-2 gene is shown as follows:
CTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAAC(SEQ ID NO.1)。
the primer sequences obtained by the design of this example are as follows:
SARS-CoV-2-F：5’-CTTCAACCTAGGACTTTTCTATTAAAATATAATG-3’(SEQ ID NO.2)；
SARS-CoV-2-R：5’-GTTGGTTGGACTCTAAAGTTAGAAGTTTGATAG-3’(SEQ ID NO.3)。
modifying the probe by adopting a fluorescence reporter group (FAM) and a fluorescence quenching group (BHQ); the modified probe is as follows:
5’-CCATTACAGATGCTGTAGACTGTGCACTTGACCC[FAM-dT]C[THF]C[BHQ-dT]CAGAAACAAAGTGTACG-3’-block。
modifying a fluorescent reporter group FAM at a position 35bp away from the 5' end base number of a probe sequence; the fluorescence quenching group BHQ is modified on the position 39bp away from the 5 'end base number of the probe sequence, wherein the base on the position 37bp away from the 5' end base number is replaced by a tetrahydrofuran residue.
Primers and probes were synthesized by Competition Biotechnology (Shanghai) Ltd.
Example 2 RNA extraction of SARS-CoV-2 Virus-containing specimen
The virus genome RNA is extracted by using a QIAamp Viral RNA mini Kit according to the reference instruction, and the specific extraction method comprises the following steps:
(1) 560. mu.l of AVL buffer containing carrier RNA was taken into a 1.5mL centrifuge tube, followed by addition of 140. mu.l of tissue fluid, vortexing for 15s, and mixing. Standing at room temperature for 10min, and centrifuging briefly to collect liquid attached to the tube wall and the tube cover;
(2) adding 560 μ l of anhydrous ethanol into 1.5mL of the centrifuge tube in the step (1), and fully and uniformly mixing by swirling for 15 s; then, the liquid drops on the cover are thrown back to the bottom of the pipe by instantaneous centrifugation;
(3) adding 630. mu.l (including precipitate) of the mixture into QIAamp Mini spinogum, and placing the column into a 2mL collection tube; covering the centrifuge tube cover tightly, and centrifuging at 6000 Xg (8000rpm) for 1 min;
(4) repeating the step (3);
(5) the centrifuge tube was removed and placed in a clean 2mL collection tube (provided by the kit); discarding the collection tube containing liquid; add 500. mu.l of buffer AW1 carefully to QIAamp Mini spin column; covering the centrifuge tube cover tightly, and centrifuging at 6000 Xg (8000rpm) for 1 min;
(6) the centrifuge tube was removed and placed in a clean 2mL collection tube (provided by the kit); discarding the collection tube containing liquid; add 500. mu.l of buffer AW2 carefully to QIAamp Mini spin column; the centrifuge tube lid was closed and centrifuged at 20000 Xg (14000rpm) for 3 min; the centrifuge tube was removed and placed in a clean 2mL collection tube (provided by the kit); discarding the collection tube containing liquid;
(7) recommending: the QIAamp Mini spin column was placed into a new 2mL collection tube (self-contained) and the liquid containing tube was discarded; centrifuging at full speed for 1 min; the buffer AW2 is removed as far as possible, and the residue of the buffer AW2 can possibly influence the subsequent experiment;
(8) the QIAamp Mini spin column was placed into a clean 1.5mL collection tube (self-contained) and the liquid containing tube was discarded; carefully add 60. mu.l of buffer AVE to the QIAamp Mini spin column; standing at room temperature for 1min, and centrifuging at 6000 Xg (8000rpm) for 1 min;
(9) repeating the step (8); the collected liquid is SARS-CoV-2 genome-containing RNA; detecting the concentration and purity of the extracted RNA by adopting an ultraviolet spectroscopy method, covering a collecting pipe, and storing at-80 ℃ for later use.
Example 3 sensitivity assay for RT-RAA fluorescence detection of SARS-CoV-2
The sensitivity was carried out using the following detection method:
(1) SARS-CoV-2 genome RNA extracted by the extraction method of example 2 was used as a stock solution to prepare different gradient working standards, which were:
working standard 1, containing 1.0 × 107copies/. mu.L of SARS-CoV-2 genome;
working Standard 2, containing 1.0 × 106copies/μ l of SARS-CoV-2 genome;
working Standard 3, containing 1.0 × 105copies/μ l of SARS-CoV-2 genome;
working standard 4, containing 1.0 × 104copies/μ l of SARS-CoV-2 genome;
working standard 5, containing 1.0 × 103copies/μ l of SARS-CoV-2 genome;
working standard 6, containing 1.0 × 102copies/μ l of SARS-CoV-2 genome;
working standard 7, containing 1.0 × 101copies/μ l of SARS-CoV-2 genome;
(2) preparing a reaction system: adding 48 μ l reaction buffer solution into lyophilized RT-RAA basic fluorescence universal reaction reagent lyophilized powder (F00R 01 provided by Jiangsu Qitian gene biotechnology limited, 2 μ g lyophilized powder per tube) to dissolve completely and mix well; then adding 2 mul of extracted RNA of a sample to be detected as a template, wherein the total volume is 50 mul; wherein the reaction buffer comprises Tris-HCl buffer (pH 7.6) at a concentration of 450 mM; 260mM of MgAc; 10% W/V PEG10000, 0.24 pM/. mu.l of the designed probe, 0.42 pM/. mu.l of the designed upstream primer and downstream primer, respectively, and the balance of water as a solvent.
(3) Placing the reaction system into a matched instrument B6100 (provided by Jiangsu Qitianjing gene biotechnology limited) which is started up in advance and preheated to 39 ℃, and oscillating and mixing uniformly at constant temperature for 3 s;
(4) placing into FAM fluorescence detection instrument (QT-RAA-F1620 fluorescence detector provided by Jiangsu Qitian Genencor Biotech Co., Ltd.) which is started up in advance and preheated to 39 deg.C for real-time RAA fluorescence reaction; the reaction conditions of the real-time RT-RAA fluorescence method are as follows: the reaction temperature is 37-42 ℃, preferably 39 ℃, and the reaction time is 5-15 min, preferably 10 min.
(5) The results of the detection are shown in FIG. 1. The results show that: working standards 1, 2, 3, 4, 5, 6, 7 all had specific gene amplification of SARS-CoV-2 (line 1-line 7 in the figure); sample 8 was a simultaneous negative control with ddH template2O, a straight line (line 8 in the figure), without SARS-CoV-2 specific gene amplification.
Example 4 specificity assay for RAA fluorescence detection of SARS-CoV-2
The specificity detection is carried out by adopting the following detection method:
(1) RNA was extracted from a sample to be tested (containing SARS-CoV-2, SARS virus, human coronavirus, rhinovirus, human bocavirus, adenovirus, influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, each biological material from the institute of the first Coco., Ltd.) according to the method described in example 2.
(2) In each reaction system, 48 μ l of reaction buffer (same as that in example 3) was added to lyophilized RT-RAA basic fluorescence universal reaction reagent lyophilized powder (F00R 01 provided by Jiangsu Qitian gene Biotechnology Co., Ltd., 2 μ g of lyophilized powder per tube) to be fully dissolved and mixed; then adding 2 mul of extracted RNA of the sample to be detected as a template, wherein the total volume is 50 mul.
(3) Placing the reaction system into a matched instrument B6100 (provided by Jiangsu Qitianjing gene biotechnology limited) which is started up in advance and preheated to 39 ℃, and oscillating and mixing uniformly at constant temperature for 3 s;
(4) putting into FAM fluorescence detection instrument (QT-RAA-F1620 fluorescence detector provided by Jiangsu Qitian Genencor Biotechnology Co., Ltd.) which is started and preheated to 39 ℃ in advance for real-time RT-RAA fluorescence reaction; the reaction conditions of the real-time RT-RAA fluorescence method are as follows: the reaction temperature is 37-42 ℃, preferably 39 ℃, and the reaction time is 5-15 min, preferably 10 min.
(5) The results of the detection are shown in FIG. 2. The result shows that the amplification starts within 2min as soon as possible, the standard work containing SARS-CoV-2 has fluorescence signals within 10min, and the rest has no fluorescence signals, thus having good specificity.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> institute of first-capital science
Primer, probe, kit and detection method for detecting SARS-CoV-2 by RT-RAA fluorescence method
<130>GAI20CN2018
<160>4
<170>PatentIn version 3.5
<210>1
<211>161
<212>DNA
<213>SARS-CoV-2
<400>1
cttcaaccta ggacttttct attaaaatat aatgaaaatg gaaccattac agatgctgta 60
gactgtgcac ttgaccctct ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa 120
aaaggaatct atcaaacttc taactttaga gtccaaccaac 161
<210>2
<211>34
<212>DNA
<213>Artificial Sequence
<220>
<223> primer
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cttcaaccta ggacttttct attaaaatat aatg 34
<210>3
<211>33
<212>DNA
<213>Artificial Sequence
<220>
<223> primer
<400>3
gttggttgga ctctaaagtt agaagtttga tag 33
<210>4
<211>56
<212>DNA
<213>Artificial Sequence
<220>
<223> Probe
<400>4
ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag tgtacg 56
Claims (10)
1. A primer for detecting SARS-CoV-2 by RT-RAA fluorescence method comprises a primer with a sequence shown as SEQ ID NO.2 and a primer with a sequence shown as SEQ ID NO. 3:
5’-CTTCAACCTAGGACTTTTCTATTAAAATATAATG-3’(SEQ ID NO.2)；
5’-GTTGGTTGGACTCTAAAGTTAGAAGTTTGATAG-3’(SEQ ID NO.3)。
2. a probe for detecting SARS-CoV-2 by RT-RAA fluorescence method comprises a probe with a sequence shown in SEQ ID NO.4, or a probe modified on the basis of the sequence shown in SEQ ID NO. 4:
5’-CCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACG-3’(SEQ IDNO.4)。
3. the RT-RAA fluorescence method SARS-CoV-2 detection probe according to claim 2, wherein the modification comprises:
the base T at the position 35bp away from the 5 ' end base of the probe sequence is modified by a fluorescence reporter group FAM, the base T at the position 39bp away from the 5 ' end base of the probe sequence is modified by a fluorescence quenching group BHQ, and the base T at the position 37bp away from the 5 ' end base of the probe sequence is replaced by a tetrahydrofuran residue (THF).
4. A kit for detecting SARS-CoV-2 by RT-RAA fluorescence, comprising: the primer of claim 1, and/or the probe of claim 2 or 3.
5. The kit of claim 4, further comprising RT-RAA based fluorescent universal reagents and reaction buffers, including Tris-HCl buffer, MgAc, and PEG 10000.
6. The kit according to claim 4 or 5, further comprising a positive quality control substance and a negative quality control substance; preferably, the positive quality control material is a specific gene sequence containing SARS-CoV-2, and the negative quality control material is an RT-RAA amplification system without the specific sequence of SARS-CoV-2.
7. Use of the primer of claim 1 or the probe of claim 2 or 3 or the kit of any one of claims 4 to 6 for the in vitro detection of the presence of the novel coronavirus SARS-CoV-2 in a sample.
8. The use according to claim 7, wherein the final concentration of the primers in the reaction system is independently 0.42pM/μ l, and the final concentration of the probe in the reaction system is 0.24pM/μ l.
9. The application of claim 8, wherein the reaction system further comprises an RT-RAA basic fluorescence universal reaction reagent and a reaction buffer solution, and the reaction buffer solution comprises the following components in percentage by weight: Tris-HCl buffer at a concentration of 450mM, pH 7.6, MgAc at a concentration of 260mM and PEG10000 at 10% W/V.
10. The use of claim 9, wherein the detecting comprises the steps of:
s1: extracting RNA of a sample to be detected;
s2: preparing a reaction system: adding the reaction buffer solution into the RT-RAA basic fluorescence universal reaction reagent, fully dissolving and uniformly mixing; adding the extracted RNA of the sample to be detected as a template; preferably, the reaction buffer comprises Tris-HCl buffer (pH 7.6) at a concentration of 450 mM; 260mM of MgAc; 10% W/V PEG10000, a probe with a final concentration of 0.24 pM/mul, an upstream primer and a downstream primer with a final concentration of 0.42 pM/mul respectively, and the balance of water as a solvent;
s3: uniformly mixing the reaction system, and putting the mixture into a fluorescence detection instrument for RAA fluorescence reaction in real time; preferably, the real-time RT-RAA fluorescence method reaction conditions are: the reaction temperature is 37-42 ℃, the preferable temperature is 39 ℃, and the reaction time is 5-15 min, the preferable time is 10 min;
s4: and analyzing the result according to the signal detected by the fluorescence detector.
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| CN112391493A (en) * | 2020-12-10 | 2021-02-23 | 浙江省检验检疫科学技术研究院 | RAA fluorescence detection method, primer probe and kit for citrus greening disease Asian species |
| CN112458201A (en) * | 2020-10-20 | 2021-03-09 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | Fluorescent RT-RPA primer, probe and detection method for detecting novel coronavirus |
| CN113046483A (en) * | 2021-03-31 | 2021-06-29 | 山东师范大学 | Novel real-time fluorescent RT-RAA primer, probe and detection kit for coronavirus |
| CN114561495A (en) * | 2022-04-28 | 2022-05-31 | 广东国盛医学科技有限公司 | Nucleic acid composition, application thereof, virus detection kit and virus detection method |
| CN114703322A (en) * | 2022-03-23 | 2022-07-05 | 华南农业大学 | Primer pair, kit and detection method for RT-RAA fluorescence detection of avian leukosis virus P12 gene |
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| CN114703322A (en) * | 2022-03-23 | 2022-07-05 | 华南农业大学 | Primer pair, kit and detection method for RT-RAA fluorescence detection of avian leukosis virus P12 gene |
| CN114561495A (en) * | 2022-04-28 | 2022-05-31 | 广东国盛医学科技有限公司 | Nucleic acid composition, application thereof, virus detection kit and virus detection method |
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