CN111394511A - 2019 novel coronavirus detection primer group, probe group and detection kit - Google Patents
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Abstract
The invention provides a 2019 novel coronavirus nucleic acid detection primer group, a probe group, a detection kit and a detection method. The 2019 novel coronavirus detection primer group comprises the following primer pairs: a specific primer pair for an S gene fragment of the 2019 novel coronavirus; a specific primer pair aiming at ORF1ab gene segment of the 2019 novel coronavirus; a pair of specific primers for the N gene segment of 2019 novel coronavirus. Through the design of the three pairs of specific primers, the accuracy and precision of the 2019 novel coronavirus detection are improved.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a 2019 novel coronavirus detection primer and a detection kit.
Background
In 12 days 1 month 2020, the national Weijian Commission shares the sequence information of the found novel coronavirus gene with the world health organization. In addition, the world health organization was announced in geneva, switzerland at total labor Tanshi 2, 11 d, and the formal name of the disease caused by 2019-nCoV infection was COVID-19, in which most infections caused pneumonia, and thus were called novel coronavirus pneumonia/new corona pneumonia.
According to the diagnosis and treatment scheme for pneumonia infected by novel coronavirus (trial fifth revision), infection sources are mainly patients with pneumonia caused by novel coronavirus, and asymptomatic patients can also be called as infection sources. From the current evidence, it was determined that 2019-nCoV could continue to be a biographer. The transmission via respiratory droplets is the main transmission route, and can be transmitted by contact and aerosol, but whether the transmission can be carried out through feces and mouth is not determined.
In order to solve the outbreak of epidemic situation, a nucleic acid detection reagent which can be used for 2019-nCoV is needed to provide technical support for the control of virus epidemic.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a detection primer group, a probe group, a detection kit and a detection method for the 2019 novel coronavirus.
In a first aspect, an embodiment of the present invention provides a primer set for detecting a 2019 novel coronavirus, which includes three pairs of specific primer pairs with the following nucleotide sequences:
specific primer pair for S gene fragment of 2019 novel coronavirus:
2019-nCov S-F：5′-TGCAGGTATATGCGCTAGT-3′，
2019-nCov S-R：5′-CACCAAGTGACATAGTGTAGG-3′；
specific primer pair aiming at ORF1ab gene fragment of 2019 novel coronavirus:
2019-nCov ORF1ab-F：5′-CCCTGTGGGTTTTACACTTAA-3′，
2019-nCov ORF1ab-R：5′-ACGATTGTGCATCAGCTGA-3′；
specific primer pair for N gene fragment of 2019 novel coronavirus:
2019-nCov N-F：5′-ATTGGCATGGAAGTCACACC-3′，
2019-nCov N-R：5′-TTATTCAGCAAAATGACTTGAT-3′。
the 2019 novel coronavirus detection primer group provided by the embodiment of the invention at least has the following beneficial effects:
the three pairs of specific primer pairs are designed aiming at highly conserved regions of an S gene, an ORF1ab gene and an N gene of the 2019 novel coronavirus respectively, wherein the homology of ORF1ab is low, and the primers can be used as important targets for distinguishing the 2019 novel coronavirus from other coronaviruses; and in the structural proteins of the 2019 novel coronavirus, the N protein is most conservative and stable, and the detection of the sequence-specific N gene fragment can further assist in diagnosing the infection of the 2019 novel coronavirus. Meanwhile, because the false negatives of the two genes are higher, the design of primers for the S gene with stronger specificity is increased, and the false negative rate is reduced. Through the design of the three pairs of specific primers, the accuracy and precision of the 2019 novel coronavirus detection are improved.
In a second aspect, an embodiment of the present invention provides a probe set used in cooperation with the detection primer set of the 2019 novel coronavirus, including the following probes:
probe for S gene fragment of 2019 novel coronavirus:
2019-nCov S-P：5′-CGACTAATTCTCCTCGGCGGG-3′；
probe for ORF1ab gene fragment of 2019 novel coronavirus:
2019-nCov ORF1ab-P：5′-CCGTCTGCGGTATGTGGAAAGGTTA-3′；
probe for N gene fragment of 2019 novel coronavirus:
2019-nCov N-P：5′-AGTTGACCTACACAGGTGCCATC-3′；
wherein, the 5 'ends of the three probes are all marked with fluorescence reporter groups, and the 3' ends are all marked with fluorescence quenching groups.
In the PCR amplification process, when the probe is complete, the fluorescence quenching group is close to the fluorescence reporter group, so that the fluorescence emitted by the fluorescence reporter gene is absorbed by the quenching group and does not emit a fluorescence signal. And when the primer is extended, the probe combined with the template is cut off by Taq enzyme (5'→ 3' exonuclease activity), the fluorescent reporter group is separated from the fluorescent quenching group to generate a fluorescent signal, and the fluorescent quantitative PCR instrument automatically draws a real-time amplification curve according to the detected fluorescent signal, so that the detection of the 2019 novel coronavirus (2019-nCov ORF1ab and N, S fragments) on the nucleic acid level is realized. The detection result can be selected qualitatively or quantitatively, and the qualitative detection can be realized by directly judging whether the corresponding amplification curve exists or not. Drawing a corresponding standard curve according to the Ct values of the standard substances with different concentration gradients, and calculating according to the Ct values of the samples to obtain a quantitative result. In addition, based on the general understanding in the art, the fluorescent reporter groups of different probes should be different so that the amplification of different gene fragments in a single-tube reaction produces fluorescent signals of different wavelength bands for convenient detection.
A probe set according to some embodiments of the invention, the 5 'end of 2019-nCov S-P is labeled with a fluorescence reporter JOE, and the 3' end is labeled with a fluorescence quencher BHQ 1; the 5 'end of the 2019-nCov ORF1ab-P is marked with a fluorescence reporter CY5, and the 3' end is marked with a fluorescence quenching group BHQ 2; the 5 'end of 2019-nCov N-P is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
In a third aspect, an embodiment of the present invention provides a 2019 novel coronavirus detection kit, which includes the primer set and the probe set.
The detection kit according to some embodiments of the invention further comprises an internal standard primer pair and an internal standard probe;
wherein, the nucleotide sequence of the internal standard primer pair is as follows:
RNP-F：5′-AGATTTGGACCTGCGAGC-3′，
RNP-R：5′-GCGGCTGTCTCCACAAGT-3′；
the nucleotide sequence of the internal standard probe RNP-P is as follows: 5'-CTGACCTGAAGGCTCTGCGC-3', respectively;
the 5 'end of the internal standard probe RNP-P is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.
The internal standard used by the kit is human ribonuclease P (RNase P), whether qualified patient tissues or secretion samples are taken is judged through the internal standard, the whole experimental process of collection, transportation and extraction of samples to be detected is monitored, and false negative of detection results is avoided.
The fluorescence reporter group of the internal standard probe should also be a group different from the fluorescence reporter group of the detection probe, for example, ROX is selected, and the fluorescence quencher group is BHQ1, so that fluorescence can be detected in the ROX channel.
The detection kit according to some embodiments of the invention further comprises an RT-PCR enzyme mixture and an RT-PCR buffer.
The detection kit according to some embodiments of the present invention further comprises a positive control and a negative control. The positive control should be capable of completely amplifying the target gene segment corresponding to the primer during the amplification process, for example, it can be inactivated standard strain, plasmid containing the target gene segment (S gene segment, ORFlab gene segment, N gene segment) corresponding to the viral primer probe, pseudovirus, etc. The negative control is water for dissolving the template RNA, and may be DEPC water, for example.
The detection kit according to some embodiments of the present invention, further comprising a nucleic acid extraction reagent. The extraction of the virus nucleic acid in the sample is completed through the corresponding nucleic acid extraction reagent so as to facilitate the subsequent amplification detection procedure.
In a fourth aspect, an embodiment of the present invention provides a 2019 method for detecting a novel coronavirus, including the following steps:
extracting total RNA of a sample to be detected;
taking total RNA of a sample as a template, and amplifying by using the detection primer group and the probe group;
analyzing the amplification product, and judging whether the sample to be detected contains 2019 novel coronavirus according to whether an amplification curve appears;
the detection method is not suitable for diagnosis and treatment of diseases.
According to the detection method of some embodiments of the present invention, the procedure of the PCR amplification reaction is 50 ℃, 20 min; 95 ℃ for 15 min; 95 ℃, 10s, 55 ℃, 40s, 40 cycles.
The real-time fluorescence RT-PCR method only needs 3 hours in the whole process from extraction of nucleic acid to detection completion, is convenient to operate, strong in specificity and high in sensitivity, greatly shortens the detection time of 2019 novel coronavirus, and provides a quick and effective laboratory detection method for laboratory emergency diagnosis of epidemic outbreaks.
By adopting the fluorescence PCR method, the RNA of 2019-nCoV is detected from a throat swab or other respiratory tract samples under the condition of eliminating sample quality, sample collection time, pollution and technical problems, and especially the RNA positivity of 2019-nCoV of a plurality of times, a plurality of samples and a plurality of detection kits has important supporting significance for etiology diagnosis.
The three pairs of specific primer pairs are designed aiming at highly conserved regions of an S gene, an ORF1ab gene and an N gene of the 2019 novel coronavirus respectively, wherein the homology of ORF1ab is low, and the primers can be used as important targets for distinguishing the 2019 novel coronavirus from other coronaviruses; and in the structural proteins of the 2019 novel coronavirus, the N protein is most conservative and stable, and the detection of the sequence-specific N gene fragment can further assist in diagnosing the infection of the 2019 novel coronavirus. Meanwhile, because the false negatives of the two genes are higher, the design of primers for the S gene with stronger specificity is increased, and the false negative rate is reduced. Through the design of the three pairs of specific primers, the accuracy and precision of the 2019 novel coronavirus detection are improved.
The detection method utilizing the kit can be used for auxiliary diagnosis of the 2019 novel coronavirus, epidemiological information about 2019 novel coronavirus outbreak is quickly obtained, reliable technical support is provided for prevention and control of 2019 novel coronavirus outbreak, and the kit has important significance in dealing with the unknown 2019 novel coronavirus outbreak.
Drawings
Fig. 1 shows the amplification result of the 2019 novel coronavirus multiplex PCR detection kit of example 3 of the present invention.
FIG. 2 shows the results of the sensitivity detection of the 2019 novel coronavirus multiplex PCR detection kit of example 4 of the present invention.
Fig. 3 shows the result of the repetitive detection of the 2019 novel coronavirus multiplex PCR detection kit of example 5 of the present invention.
Fig. 4 is a gradient detection result of the 2019 novel coronavirus multiplex PCR detection kit of example 6 of the present invention.
Fig. 5 shows the gradient detection results of the three 2019 novel coronavirus single-plex PCR detection kits of the control group in example 6 of the present invention.
Fig. 6 shows the gradient detection result of the 2019 novel coronavirus multiplex PCR detection kit of example 7 of the present invention.
FIG. 7 shows the results of gradient detection in the control group of example 7 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1
This example provides primers and probes for a 2019 novel coronavirus. Based on a Taqman fluorescent probe technology, corresponding specificity detection primers and Taqman probes are designed by highly conserved regions of an S gene, an ORF1ab gene and an N gene of a 2019 novel coronavirus, and meanwhile, a corresponding internal standard primer pair and an internal standard probe are designed by taking human ribonuclease P (RNase P) as an internal standard. The details are shown in table 1 below:
TABLE 1 primer and Probe information
Wherein, the 5 'end of 2019-nCov S-P is marked with a fluorescence reporter JOE, and the 3' end is marked with a fluorescence quenching group BHQ 1; the 5 'end of the 2019-nCov ORF1ab-P is marked with a fluorescence reporter CY5, and the 3' end is marked with a fluorescence quenching group BHQ 2; the 5 'end of 2019-nCov N-P is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1; the 5 'end of RNP-P is marked with a fluorescence reporter group ROX, and the 3' end is marked with a fluorescence quencher group BHQ 1.
Example 2
The embodiment provides a 2019 novel coronavirus detection kit, which comprises an RT-PCR enzyme mixed solution, an RT-PCR buffer solution, an RT-PCR primer mixed solution, an RT-PCR probe, an RT-PCR positive control substance and an RT-PCR negative control substance.
Wherein the RT-PCR enzyme mixed solution comprises 2-3U/mu L hot start Taq enzyme, 1-2U/mu L reverse transcriptase, 15-30mM Tris, 10% -30% glycerol and 0.5-1.5mM DTT;
the RT-PCR buffer comprises 250mM Tris, 25mM MgCl250mM KCl, 0.025% Triton X-100 buffer system and 6.5mM dNTPs;
the RT-PCR primer mixture and the RT-PCR probe are the primers and the probes in the embodiment 1;
the RT-PCR positive control is a plasmid solution of primer targeting fragments of 2019 novel coronavirus S gene, ORF1ab gene and N gene which take pUC57 as a plasmid vector and an internal standard gene.
The RT-PCR negative control was DEPC water.
Example 3
The embodiment provides a 2019 novel coronavirus detection method, which comprises the following steps:
1. extraction of nucleic acids
Obtaining a sample to be tested from a throat swab, inactivating the sample (heating at 56 ℃ for 30min), and then extracting the sample according to the method of the extraction kit, wherein a nucleic acid extraction or purification reagent (docket number: 20180040, yushen instrument) produced by Shenzhen catalpan key biotechnologies ltd is adopted in this embodiment.
PCR amplification
An RT-PCR reaction solution system was prepared according to Table 2:
TABLE 2 RT-PCR reaction solution System
Wherein the RT-PCR enzyme mixture and the RT-PCR buffer solution are the RT-PCR enzyme mixture and the RT-PCR buffer solution in the embodiment 2.
And (2) taking the RNA extracted in the step (1) as a template, adding 5 mu L into the reaction solution system, and performing multiple fluorescence PCR amplification reaction on an ABI 7500 instrument, wherein the amplification reaction procedure comprises 40 cycles of 50 ℃, 20min, 95 ℃, 15min, 95 ℃, 10s, 55 ℃, 40 s.
3. Result judgment
Setting a baseline and a threshold line, wherein the baseline is generally 3-15 cycles, and the threshold line is the highest point just exceeding the negative control.
The positive control substance needs to meet the requirement that JOE, FAM, CY5 and ROX channels have obvious S-shaped amplification curves; the negative control needs to satisfy no amplification curve of other channels; and the result of the sample to be detected can be analyzed by meeting the conditions.
If any two channels of the JOE channel, the FAM channel and the CY5 channel detect an obvious S-type amplification curve, the fact that the sample to be detected contains the 2019 novel coronavirus is shown, and only one channel detects the obvious S-type amplification curve and needs to be detected again.
Fig. 1 shows the amplification result of the 2019 novel coronavirus multiplex PCR detection kit of example 3 of the present invention. As can be seen from the figure, JOE, FAM and CY5 channels have obvious S-shaped amplification curves, indicating that the sample contains 2019 novel coronavirus.
Example 4
Sensitivity detection
Will be calibrated to be 1 × 108Artificially synthesized RNA containing 2019 novel coronavirus ORF1ab, N and S fragments of copies/m L is respectively diluted to 1 × 107copies/mL、1×106copies/mL、1×105copies/mL、1×104copies/mL、 1×103The sensitivity test is carried out by using the amplification reaction system in the example 3 according to the copies/m L gradient, the amplification result is shown in figure 2, the figure 2 is the sensitivity detection result of the 2019 novel coronavirus multiplex PCR detection kit in the example 4 of the invention, and as can be seen from figure 2, JOE, FAM, CY5 channel and ROX channel of the detection internal standard all have obvious 5S-shaped amplification curves, which shows that the sensitivity of the kit can reach 1000copies/m L, and the Ct value can reach 37.
Example 5
Repeatability detection
At 1 × 104Of copies/m LThe RNA solution in example 4 is used as a sample, 10 times of repeated amplification experiments are performed, and the result is shown in fig. 3, and fig. 3 is the repeated detection result of the 2019 novel coronavirus multiplex PCR detection kit in example 5 of the present invention. As can be seen from the figure, the repeatability of the kit is high, and the Ct value CV% is calculated to be less than 5%.
Example 6
Multiple to single comparison
A comparison experiment is carried out by using a novel coronavirus (2019-nCov) N gene nucleic acid detection kit (fluorescent PCR method), a novel coronavirus (2019-nCov) S gene nucleic acid detection kit (fluorescent PCR method), a novel coronavirus (2019-nCov) ORF1ab gene nucleic acid detection kit (fluorescent PCR method) and the reaction system in example 3, which are researched by Shenzhen catalpan Biotech Limited, and 5 2019 novel coronavirus nucleic acid samples with different concentrations are detected simultaneously.
The comparative experiment results are shown in fig. 4 and 5, and fig. 4 is the gradient detection result of the 2019 novel coronavirus multiplex PCR detection kit of example 6 of the invention. Fig. 5 shows the gradient detection results of the three 2019 novel coronavirus single-plex PCR detection kits of the control group in example 6 of the present invention. As can be seen by comparing FIG. 4 and FIG. 5, the amplification effect of the multiplex PCR detection kit provided by the embodiment of the present invention on the positive sample is consistent with the amplification effect of the single-fold kit.
Example 7
Comparison with products of other companies
In this example, a new coronavirus (2019-nCov) ORF1ab gene nucleic acid detection kit (fluorescence PCR method), a new coronavirus (2019-nCov) N gene nucleic acid detection kit (fluorescence PCR method), and a new coronavirus (2019-nCov) S gene nucleic acid detection kit (fluorescence PCR method) developed by tianlong biotechnology limited were used as a control group, and the control group was subjected to a detection comparison experiment with the reaction system provided in example 3 to simultaneously detect 5 new coronavirus nucleic acid samples with different concentrations, and sample detection and amplification were performed with an upper computer program according to the kit instructions, and the amplification results are shown in fig. 6 and 7. Fig. 6 shows the gradient detection result of the 2019 novel coronavirus multiplex PCR detection kit of example 7 of the present invention. FIG. 7 shows the results of gradient detection in the control group of example 7 of the present invention. Through comparison of amplification curves of the detected samples, the amplification effect of the low-concentration positive sample detected by the reaction system provided by the embodiment 3 is 2 Ct values of the same sample detected by Tianlong biotechnology company, which shows that the detection kit provided by the embodiment of the invention has higher detection sensitivity than a control group and has good effect of reagent amplification of the sample.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen catalpen Biotechnology Limited
<120>2019 novel coronavirus detection primer group, probe group and detection kit
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Claims (10)
1. The primer group for detecting the 2019 novel coronavirus is characterized by comprising three pairs of specific primer pairs with the nucleotide sequences as follows:
specific primer pair for S gene fragment of 2019 novel coronavirus:
2019-nCov S-F：5′-TGCAGGTATATGCGCTAGT-3′，
2019-nCov S-R：5′-CACCAAGTGACATAGTGTAGG-3′；
specific primer pair aiming at ORF1ab gene fragment of 2019 novel coronavirus:
2019-nCov ORF1ab-F：5′-CCCTGTGGGTTTTACACTTAA-3′，
2019-nCov ORF1ab-R：5′-ACGATTGTGCATCAGCTGA-3′；
specific primer pair for N gene fragment of 2019 novel coronavirus:
2019-nCov N-F：5′-ATTGGCATGGAAGTCACACC-3′，
2019-nCov N-R：5′-TTATTCAGCAAAATGACTTGAT-3′。
2. the probe set used in combination with the 2019 novel coronavirus detection primer set of claim 1, wherein the probe set comprises three probes with the following nucleotide sequences:
probe for S gene fragment of 2019 novel coronavirus:
2019-nCov S-P：5′-CGACTAATTCTCCTCGGCGGG-3′；
probe for ORF1ab gene fragment of 2019 novel coronavirus:
2019-nCov ORF1ab-P：5′-CCGTCTGCGGTATGTGGAAAGGTTA-3′；
probe for N gene fragment of 2019 novel coronavirus:
2019-nCov N-P：5′-AGTTGACCTACACAGGTGCCATC-3′；
wherein, the 5 'ends of the three probes are marked with fluorescence reporter groups, and the 3' ends are marked with fluorescence quenching groups.
3. The probe set of claim 2, wherein the 5 'end of 2019-nCov S-P is labeled with a fluorescence reporter group JOE and the 3' end is labeled with a fluorescence quencher group BHQ 1; the 5 'end of the 2019-nCov ORF1ab-P is marked with a fluorescence reporter CY5, and the 3' end is marked with a fluorescence quenching group BHQ 2; the 5 'end of 2019-nCov N-P is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
4. A detection kit for a 2019 novel coronavirus, which is characterized by comprising a detection primer group and a probe group, wherein the detection primer group is the detection primer group for the 2019 novel coronavirus in claim 1, and the probe group is the probe group in claim 2 or 3.
5. The detection kit according to claim 4, further comprising an internal standard primer pair and an internal standard probe;
wherein, the nucleotide sequence of the internal standard primer pair is as follows:
RNP-F：5′-AGATTTGGACCTGCGAGC-3′，
RNP-R：5′-GCGGCTGTCTCCACAAGT-3′；
the nucleotide sequence of the internal standard probe is as follows:
RNP-P：5′-CTGACCTGAAGGCTCTGCGC-3′；
the 5 'end of RNP-P is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quencher group.
6. The detection kit according to claim 5, wherein RNP-P is labeled at the 5 'end with a fluorescence reporter ROX and at the 3' end with a fluorescence quencher BHQ 1.
7. The detection kit of claim 4, further comprising RT-PCR enzyme mixture and RT-PCR buffer.
8. The test kit of claim 4, further comprising a positive control and a negative control.
9. A2019 novel coronavirus detection method is characterized by comprising the following steps:
extracting total RNA of a sample to be detected;
amplifying by using total RNA of a sample to be detected as a template by using the detection primer group of claim 1 and the probe group of claim 2 or 3;
analyzing the amplification product, and judging whether the sample to be detected contains 2019 novel coronavirus according to whether an amplification curve appears;
the detection method is not suitable for diagnosis and treatment of diseases.
10. The detection method according to claim 9, wherein the procedure of the PCR amplification reaction is 50 ℃, 20 min; 95 ℃ for 15 min; 95 ℃, 10s, 55 ℃, 40s, 40 cycles.
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304368A (en) * | 2020-03-19 | 2020-06-19 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting novel coronavirus |
| CN111676315A (en) * | 2020-04-21 | 2020-09-18 | 中国贵州茅台酒厂(集团)有限责任公司职工医院 | Primer and probe for detecting novel coronavirus ORF1ab gene, kit and method thereof |
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| IT202000006754A1 (en) * | 2020-03-31 | 2021-10-01 | Diasorin S P A | Assays for the detection of SARS-CoV-2 |
| WO2021198325A1 (en) * | 2020-03-31 | 2021-10-07 | Diasorin S.P.A. | Assays for the detection of sars-cov-2 |
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| CN113930481A (en) * | 2020-07-14 | 2022-01-14 | 中国科学院大连化学物理研究所 | Inhibitor screening kit for papain-like protease and application thereof |
| WO2022095731A1 (en) * | 2020-11-03 | 2022-05-12 | 厦门大学 | Kit and method for detecting sars-cov-2 |
| CN114622036A (en) * | 2020-12-10 | 2022-06-14 | 中国科学院大连化学物理研究所 | Method for identifying novel coronavirus COVID-19 through specific primer probe |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1906306A (en) * | 2003-08-22 | 2007-01-31 | 博驰生物医疗研究有限公司 | Multi-allelic molecular detection of SARS-associated coronavirus |
| CN101415844A (en) * | 2003-09-12 | 2009-04-22 | 贝克顿·迪金森公司 | Assay for SARS coronavirus by amplification and detection of nucleocapsid RNA sequence |
-
2020
- 2020-03-09 CN CN202010156972.5A patent/CN111394511B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1906306A (en) * | 2003-08-22 | 2007-01-31 | 博驰生物医疗研究有限公司 | Multi-allelic molecular detection of SARS-associated coronavirus |
| CN101415844A (en) * | 2003-09-12 | 2009-04-22 | 贝克顿·迪金森公司 | Assay for SARS coronavirus by amplification and detection of nucleocapsid RNA sequence |
Non-Patent Citations (4)
| Title |
|---|
| CHENGFENG QIU等: ""Transmission and clinical characteristics of coronavirus disease 2019 in 104 outside-Wuhan patients, China"", 《MEDRXIV》 * |
| YU JIN JUNG等: ""Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2"", 《BIORXIV》 * |
| ZHEN ZHAO等: ""A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2"", 《BIORXIV》 * |
| 王雪宁等: ""新型冠状病毒(SARS-CoV-2)的检测技术"", 《生命的化学》 * |
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