CN114107574B - Kit and method for detecting novel coronavirus and Omicron mutant strain thereof - Google Patents
Kit and method for detecting novel coronavirus and Omicron mutant strain thereof Download PDFInfo
- Publication number
- CN114107574B CN114107574B CN202210098504.6A CN202210098504A CN114107574B CN 114107574 B CN114107574 B CN 114107574B CN 202210098504 A CN202210098504 A CN 202210098504A CN 114107574 B CN114107574 B CN 114107574B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- probe
- primer pair
- kit
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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 discloses a kit and a method for detecting novel coronaviruses and Omicron mutant strains thereof. The invention carries out qualitative detection on the novel coronavirus by carrying out joint detection on three target genes of the novel coronavirus ORF1ab, N and S, and meanwhile, the detection target region of the S gene covers the specific mutation site of the Omicron variant strain, if the novel coronavirus is the Omicron variant strain, the S gene is lost without a signal due to the occurrence of the mutation site, so that the Omicron variant strain and the non-Omicron variant strain are identified. The detection reagent provided by the invention can be used for detecting patients infected by the novel coronavirus and the omacron mutant thereof with high efficiency, high specificity, high stability and low cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for detecting novel coronaviruses and Omicron mutant strains thereof.
Background
The novel coronavirus (SARS-CoV-2) is a single-stranded positive strand RNA virus that is susceptible to mutation. Clinically, the symptoms of new coronavirus infected persons vary greatly, and can occur from asymptomatic to critically ill. In addition to differences in individual factors, viral variation may also be an important factor that leads to large differences in symptoms in infected patients.
Studies have shown that novel coronaviruses mutate at rates of about 2 to 4 mutations per month, and that currently, over 25 mutant variants are found in known strains. Alpha new crown variant, beta new crown variant, gamma new crown variant, delta new crown variant and Omicron new crown variant are discovered in a plurality of countries successively, and are mutated in the Receptor Binding Domain (RBD) of spike protein on the surface of virus, so that the Alpha new crown variant, beta new crown variant, gamma new crown variant and Omicron new crown variant are easier to bind with angiotensin converting enzyme 2 (ACE 2) receptor on the surface of human cells, and the transmission capacity and pathogenicity of the virus are obviously affected by the mutation of the virus.
The novel coronavirus Omikovia variant has more mutation in virus Spike (Spike) protein, and has important amino acid mutation sites of Alpha (Alpha), beta (Beta), gamma (Gamma) and Delta (Delta) Spike proteins of the first 4 variants, including mutation sites for enhancing cell receptor affinity and virus replication capacity, so that the novel coronavirus Omikovia variant has extremely strong transmission capacity.
Therefore, the development of a combined screening kit capable of simultaneously detecting novel coronaviruses and omacron variants has very positive significance for epidemic prevention and control and treatment of definite patients.
Disclosure of Invention
The invention develops a composition, a kit, a method and application for rapidly detecting novel coronaviruses and Omicron mutant strains aiming at the novel coronaviruses and the Omicron mutant strains thereof.
In a first aspect of the invention, there is provided a primer pair set for detecting a novel coronavirus and Omicron variant thereof, the primer pair set comprising a first primer pair:
the first primer pair comprises: a forward primer as shown in SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
In another preferred embodiment, the primer pair set further comprises other primer pairs for detecting novel coronaviruses.
In another preferred embodiment, the primer set further comprises primer pairs for detecting the novel coronavirus ORF1ab gene, and/or the N gene.
In another preferred embodiment, the primer set further comprises a second primer pair that specifically amplifies the novel coronavirus ORF1ab gene, the second primer pair comprising:
a forward primer as shown in SEQ ID NO. 1; and, a reverse primer as shown in SEQ ID NO. 2.
In another preferred embodiment, the primer set further comprises a third primer pair that specifically amplifies a novel coronavirus N gene, the third primer pair comprising:
a forward primer as shown in SEQ ID NO. 4; and, a reverse primer as shown in SEQ ID NO. 5.
In another preferred embodiment, the primer set further comprises a fourth primer pair, which specifically amplifies a reference RNase P gene, the fourth primer pair comprising:
a forward primer as shown in SEQ ID NO. 10; and, a reverse primer as shown in SEQ ID NO. 11.
In a second aspect of the present invention, there is provided a probe set for detecting a novel coronavirus and Omicron mutant thereof, the probe set comprising:
a first probe with a nucleotide sequence shown as SEQ ID NO. 9.
In another preferred embodiment, the probe set further comprises a second probe having a nucleotide sequence shown in SEQ ID NO. 3.
In another preferred embodiment, the probe set further comprises a third probe having a nucleotide sequence shown in SEQ ID NO. 6.
In another preferred embodiment, the probe set further comprises a fourth probe having a nucleotide sequence shown in SEQ ID NO. 12.
In another preferred embodiment, the 5' end of each probe comprises a fluorescent reporter group; and/or, the 3' end of each probe comprises a fluorescence quenching group.
In another preferred embodiment, the fluorescent reporter groups labeled with the respective probes are different from each other.
In a third aspect of the present invention, there is provided a kit for detecting a novel coronavirus and its Omicron mutant, the kit comprising a primer pair for detecting a novel coronavirus and its Omicron mutant, the primer pair comprising: a forward primer as shown in SEQ ID NO. 7; and, a reverse primer as shown in SEQ ID NO. 8.
In another preferred embodiment, the kit further comprises a probe that specifically targets the PCR amplification product of the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO. 8.
In another preferred embodiment, the kit comprises a set of primer pairs according to the first aspect of the invention.
In another preferred embodiment, the kit further comprises a probe set according to the second aspect of the invention.
In another preferred embodiment, the kit further comprises one or more components selected from the group consisting of: hot start Taq enzyme, reverse transcriptase, UDG enzyme (uracil-N-glycosylase), dNTPs, mgCl 2 。
In another preferred embodiment, the kit further comprises a negative quality control.
In another preferred embodiment, the kit further comprises a positive quality control.
In another preferred embodiment, the kit includes a first container, and the first container contains a primer-probe mixed solution, where the primer-probe mixed solution includes: nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 12.
In another preferred embodiment, the first container further comprises: mgCl 2 And dNTPs.
In another preferred embodiment, the kit further comprises a second container comprising an enzyme cocktail comprising a hot start Taq enzyme, a reverse transcriptase, and a uracil glycosylase (UNG)).
In another preferred embodiment, the kit comprises a third container comprising a negative control within the third container.
In another preferred embodiment, the kit comprises a fourth container comprising a cationic control within the fourth container.
In a fourth aspect of the invention, there is provided a method of detecting novel coronaviruses and omacron mutants thereof, the method comprising the steps of:
(1) Providing a sample of an object to be detected;
(2) Preparing a fluorescent quantitative PCR reaction system and performing fluorescent quantitative PCR detection:
wherein, the fluorescence quantitative PCR reaction system comprises: the sample provided in step (1), the set of primer pairs according to the first aspect of the invention, and the set of probes according to the second aspect of the invention.
In another preferred embodiment, the sample is a nucleic acid sample.
In another preferred embodiment, the sample may be from a pharyngeal swab sample, an alveolar lavage sample, a blood sample, a sputum sample, a fecal sample, or an environmental sample.
In another preferred embodiment, the method is a detection method for non-diagnostic purposes.
In another preferred embodiment, the fluorescent quantitative PCR reaction system further comprises a positive quality control, and/or a negative quality control.
In another preferred embodiment, the PCR reaction system further comprises a PCR reaction enzyme system.
In a fifth aspect of the invention there is provided the use of a primer set according to the first aspect of the invention and/or a probe set according to the second aspect of the invention for the preparation of a PCR detection kit for the detection of novel coronaviruses and Omicron mutants thereof.
In another preferred embodiment, the PCR is fluorescent quantitative PCR.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIGS. 1 and 2 show the amplification results of detection of novel coronaviruses and Omicron mutants using a multiplex primer probe combination 1 (comprising SEQ ID NO. 1-SEQ ID NO. 12), respectively.
FIGS. 3 and 4 show the results of amplification using the multiplex primer probe combination 2 (comprising SEQ ID NO. 13-15, SEQ ID NO. 28-30, SEQ ID NO. 43-45, SEQ ID NO.52, SEQ ID NO.51, SEQ ID NO. 42) for detecting novel coronaviruses and Omicron mutants, respectively.
FIGS. 5 and 6 show the results of amplification using the multiplex primer probe combinations 3 (including SEQ ID NO.16 to SEQ ID NO.18, SEQ ID NO.31 to SEQ ID NO.33, SEQ ID NO.46 to SEQ ID NO.48, SEQ ID NO.24, SEQ ID NO.23, SEQ ID NO. 12) for detecting novel coronaviruses and Omicron mutants, respectively.
FIGS. 7 and 8 show the results of amplification using the multiplex primer probe combinations 4 (including SEQ ID NO.19 to SEQ ID NO.21, SEQ ID NO.34 to SEQ ID NO.36, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.9, SEQ ID NO.10 to SEQ ID NO. 12), respectively, for detecting novel coronaviruses and Omicron mutants.
FIG. 9 shows the results of concentration gradient amplification of novel coronavirus (2019-nCoV) ORF1ab target gene.
FIG. 10 shows the results of concentration gradient amplification of novel coronavirus (2019-nCoV) N target gene.
FIG. 11 shows the results of novel coronavirus (2019-nCoV) S gene concentration gradient (wild-type) amplification.
FIG. 12 shows the results of a specific assay for pseudoviruses of human coronavirus (HKU 1).
FIG. 13 shows the detection result of the negative sample (-);
FIG. 14 shows the results of detection of Omicron variant samples;
FIG. 15 shows the results of testing samples of the novel crown Delta variant.
Detailed Description
The invention develops a kit or a method for rapidly detecting novel coronaviruses and identifying omacron variant strains, and has very positive significance for epidemic prevention and control and treatment of definite patients. The novel coronavirus is qualitatively detected by simultaneously carrying out joint detection on three target genes of the novel coronavirus ORF1ab, N and S in the 1 tube, and meanwhile, the detection target region of the S gene covers the specific mutation site of the Omicron variant strain, if the novel coronavirus is the Omicron variant strain, the S gene is lost without a signal due to the occurrence of the mutation site, so that the Omicron variant strain and the non-Omicron variant strain can be further identified by judging whether the S gene is lost. The detection reagent provided by the invention can be used for detecting patients infected by the novel coronavirus and the omacron mutant thereof with high efficiency, high specificity, high stability and low cost.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Real-time fluorescent PCR
Is a technique based on the principle of Fluorescence Resonance Energy Transfer (FRET). Wherein, the real-time fluorescent PCR technology based on TaqMan fluorescent labeled probes utilizes the thermostable DNA polymerase Taq enzyme to have polymerase activity in the 5'-3' direction and exonuclease activity in the 5'-3' direction of nucleotide sequences bound with target sequences encountered in the polymerization extension process. TaqMan fluorescent probes label the fluorescent emitting group and the quenching group at the 5' and 3' ends, respectively, and the 3' end of the probe is phosphorylated to prevent the probe from being extended during PCR amplification. The quenching group inhibits fluorescence emission of the emitting group while the probe remains intact. Upon separation of the emitting group from the quenching group, the inhibition is released and the optical density at the emission wavelength of the fluorescent emitting group is increased to be detected by the fluorescent detection system. The renaturation period probe hybridizes with the template DNA, the extension period Taq enzyme moves along the DNA template along with the extension of the primer, when the extension period Taq enzyme moves to the probe position, the 5' exonuclease activity of Taq DNA polymerase can degrade the specific fluorescent labeled probe, the fluorescent reporter group is separated from the quenching group, and fluorescence is emitted.
Multiplex PCR
Multiplex PCR (multiplex PCR), also called multiplex primer PCR or multiplex PCR, is a PCR reaction in which more than two pairs of primers are added in the same PCR reaction system and simultaneously a plurality of nucleic acid fragments are amplified, and the reaction principle, reaction reagents and operation process are the same as those of the general PCR.
There are many factors that affect multiplex PCR reactions, such as:
(1) The imbalance of the reaction system results in rapid amplification of certain advantageous primers and templates thereof in the previous rounds of reaction, resulting in large amounts of amplified products which are also good inhibitors of DNA polymerase. Therefore, with the large amount of amplified products, the polymerization ability of the polymerase is more and more strongly inhibited, and therefore, the primer and its template, which are at a disadvantage in the early stage, are more difficult to react, eventually resulting in an amount of amplified products that is too small to be detected.
(2) Primer specificity, if the primer binds more strongly to other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, resulting in a decrease in amplification efficiency.
(3) The optimal annealing temperatures are not uniform, and a plurality of pairs of primers are placed in a system for amplification, so that the optimal annealing temperature of each pair of primers is required to be close because the annealing temperatures for carrying out PCR reactions are the same.
(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are also third party DNA mediated polymers, which, like non-specific primers, interfere with the competition of primers with the target binding sites, affecting amplification efficiency.
Although several factors affecting amplification efficiency are mentioned above, more factors are not yet clear. To date, there is no effective method by which amplification efficiency can be predicted explicitly.
In a preferred embodiment of the present invention, the present invention provides a primer and probe combination for detecting novel coronaviruses, omicron mutants thereof and reference genes, which have good specificity, high amplification efficiency and are capable of multiplex detection:
in another preferred embodiment of the present invention, the present invention provides a kit comprising the above primer and probe combination, the kit comprising:
an RT-PCR reaction solution comprising: mgCl 2 dNTPs, primers and probe combinations of the present invention;
an enzyme mixed solution comprising: hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG));
negative quality control: DEPC H 2 O；
Controlling the nature of yang: the plasmid contains novel coronavirus (2019-nCoV) ORF1ab target gene, novel coronavirus (2019-nCoV) N target gene, novel coronavirus (2019-nCoV) Omicron specific mutation site and reference gene target fragment.
In a preferred embodiment, the concentration of each primer is 0.3. Mu.M.
In a preferred embodiment, the concentration of each probe is 0.15. Mu.M.
The invention has the beneficial effects that:
(1) The invention establishes a detection method and a nucleic acid detection kit for simultaneously detecting novel coronaviruses and Omicron mutant strains thereof.
(2) The novel coronavirus and Omicron mutant detection kit provided by the invention have extremely high sensitivity.
(3) The detection method and the kit have extremely strong specificity aiming at novel coronavirus Omicron mutant strains, and the detection result is stable and reliable.
(4) The invention designs and screens a large number of specific detection primers and probes for the novel coronavirus and the Omicron mutant thereof, obtains a primer pair capable of specifically identifying the Omicron mutant mutation sites, and unexpectedly discovers that the primer pair has no non-specific amplification for the wild type novel coronavirus and other novel coronavirus mutant strains after repeated verification, thereby not only greatly simplifying the detection flow of the novel coronavirus Omicron mutant, but also obviously improving the accuracy of identifying the novel coronavirus Omicron mutant.
The invention is suitable for detecting novel coronaviruses, provides reliable basis for virus identification and typing, prevention and control, and is worthy of popularization and application. In addition, the method of the invention is also suitable for non-diagnostic purposes, for example, in epidemic prevention and control processes, the detection method of the invention is used for detecting virus nucleic acid in the environment, and the virus nucleic acid information can be used as public health management requirement.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook J et al, america (Huang Peitang et al, beijing: science Press, 2002), or under the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified. For example, the hot start Taq enzyme, UDG enzyme, C-MMLV enzyme referred to in the examples of the present invention are available from Invitrogen corporation.
Example 1 kit for multiplex rapid detection of novel coronaviruses (2019-nCoV) and identification of Omicron mutant strains
In order to obtain a multiple primer probe system combination with better specificity and sensitivity, the inventor designs tens of primers and probes in the research and development process, and respectively combines the primers and the probes to perform multiple tests.
Meanwhile, a human internal reference gene is designed to monitor the sample collection, the nucleic acid extraction process and the PCR amplification process.
Finally, a set of primer and probe sequences with optimal sensitivity and specificity are determined.
Because of excessive content, partial results are selected for presentation, partial primer probes are shown in the following table 1, and partial primer probe combination test results are shown in fig. 1, 2, 3 and 4.
According to the experiment, the novel coronavirus Omicron mutant strain and the novel coronavirus wild type have extremely small nucleic acid differences, and most primer pairs designed for different mutation sites of the Omicron mutant strain can show nonspecific amplification on the wild type, so that the Omicron mutant strain is difficult to accurately identify.
The invention designs and screens a large number of specific detection primers and probes for novel coronavirus Omicron mutant strains to obtain a primer pair (SEQ ID NO.7 and SEQ ID NO. 8) capable of specifically identifying the corresponding mutation sites of the Omicron mutant strains, wherein the primer pair specifically amplifies the nucleic acid fragments which are not present in the Omicron mutant strain nucleic acid and correspond to the Omicron mutant sites, and when the amplification products of the primer pair exist in the detection result, the fact that the Omicron mutant strain nucleic acid does not exist in a sample is indicated, and otherwise, the fact that the Omicron mutant strain nucleic acid exists in the sample is indicated.
Through repeated verification, the primer pair is unexpectedly found to have no non-specific amplification on the Omicron mutant strain, so that the detection flow of the novel coronavirus Omicron mutant strain is greatly simplified, and the accuracy of identifying the novel coronavirus Omicron mutant strain is remarkably improved.
The multiplex primer probe combination 1 comprises SEQ ID NO. 1-SEQ ID NO.12;
the multiplex primer probe combination 2 comprises SEQ ID NO. 13-15, SEQ ID NO. 28-30, SEQ ID NO. 43-45, SEQ ID NO.52, SEQ ID NO.51 and SEQ ID NO.42;
the multiplex primer probe combination 3 comprises SEQ ID NO. 16-18, SEQ ID NO. 31-33, SEQ ID NO. 46-48, SEQ ID NO.24, SEQ ID NO.23 and SEQ ID NO.12;
the multiplex primer probe combination 4 comprises SEQ ID NO. 19-21, SEQ ID NO. 34-36, SEQ ID NO. 49-50, SEQ ID NO.9, and SEQ ID NO. 10-12;
the multiplex primer probe combination 5 comprises SEQ ID NO.22, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.39, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12;
the multiplex primer probe combination 6 comprises SEQ ID NO. 25-27, SEQ ID NO. 4-6 and SEQ ID NO. 7-9,SEQ ID NO.51,SEQ ID NO.42,SEQ ID NO.24.
Multiple detection results show that the amplification efficiency of the primer probe combination 1, the primer probe combination 5 and the primer probe combination 6 is higher, and further performance tests can be carried out. During multiplex amplification, the primer probe combination 2, the primer probe combination 3 and the primer probe combination 4 obviously inhibit the amplification efficiency of partial targets, so that the sensitivity is poor, and a nonspecific signal is generated for omacron variants.
Fig. 1 to 8 show the detection results of a partially representative multiplex primer probe combination.
FIGS. 1 and 2 show the amplification results of detection of novel coronaviruses and Omicron mutants using a multiplex primer probe combination 1 (comprising SEQ ID NO. 1-SEQ ID NO. 12), respectively. The nucleic acid concentration of the novel coronavirus wild type standard substance is 100 copies/mL, the wild type is detected at 100%, and the Omicron variant strain shows no signal due to S gene loss, so that the Omicron variant strain can be accurately identified.
FIGS. 3 and 4 show the results of amplification using the multiplex primer probe combination 2 (comprising SEQ ID NO. 13-15, SEQ ID NO. 28-30, SEQ ID NO. 43-45, SEQ ID NO.52, SEQ ID NO.51, SEQ ID NO. 42) for detecting novel coronaviruses and Omicron mutants, respectively. The nucleic acid concentration of the novel coronavirus wild type standard substance is 100 copies/mL, the combination effect is represented by poor wild type detection sensitivity, and the Omicron variant strain still has nonspecific signals after S genes of the Omicron variant strain are lost, so that the Omicron variant strain cannot be accurately identified.
FIGS. 5 and 6 show the results of amplification using the multiplex primer probe combinations 3 (including SEQ ID NO.16 to SEQ ID NO.18, SEQ ID NO.31 to SEQ ID NO.33, SEQ ID NO.46 to SEQ ID NO.48, SEQ ID NO.24, SEQ ID NO.23, SEQ ID NO. 12) for detecting novel coronaviruses and Omicron mutants, respectively. The nucleic acid concentration of the novel coronavirus wild type standard substance is 100 copies/mL, the combination effect is represented by poor wild type detection sensitivity, and the Omicron variant strain still has nonspecific signals after S genes of the Omicron variant strain are lost, so that the Omicron variant strain cannot be accurately identified.
FIGS. 7 and 8 show the results of amplification using the multiplex primer probe combinations 4 (including SEQ ID NO.19 to SEQ ID NO.21, SEQ ID NO.34 to SEQ ID NO.36, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.9, SEQ ID NO.10 to SEQ ID NO. 12), respectively, for detecting novel coronaviruses and Omicron mutants. The nucleic acid concentration of the novel coronavirus wild type standard substance is 100 copies/mL, the combination effect is represented by poor wild type detection sensitivity, and the Omicron variant strain still has nonspecific signals after S genes of the Omicron variant strain are lost, so that the Omicron variant strain cannot be accurately identified.
Based on the above-mentioned great amount of work, finally, a novel coronavirus primer probe combination (including internal reference) with good specificity and high amplification efficiency is preferable as shown in table 2:
the present example provides a kit for multiplex rapid detection of novel coronaviruses (2019-nCoV) and identification of omacron mutants, comprising:
RT-PCR reaction (925 μl): mgCl 2 (4.5 mM), dNTPs (0.2 mM), novel coronavirus (2019-nCoV) ORF1ab target gene, novel coronavirus (2019-nCoV) N target gene, novel coronavirus (2019-nCoV) Omicron-specific mutation site were each 0.3. Mu.M in primer concentration and 0.15. Mu.M in probe concentration.
Enzyme cocktail (75 μl): including hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG);
negative quality control: DEPC H 2 O。
Controlling the nature of yang: the plasmid contains novel coronavirus (2019-nCoV) ORF1ab target gene, novel coronavirus (2019-nCoV) N target gene, novel coronavirus (2019-nCoV) Omicron specific mutation site and reference gene target fragment.
Example 2 multiplex rapid detection of novel coronaviruses (2019-nCoV) and methods for identifying Omicron mutants
This example provides multiplex rapid detection of novel coronaviruses (2019-nCoV) and identification of mutant omacron using the kit of example 1.
Specimen type: oropharyngeal swab and nasopharyngeal swab.
Nucleic acid extraction:
and (3) adopting a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on a silica gel membrane centrifugal column method or a reagent based on a magnetic bead method nucleic acid extraction method, operating according to a kit instruction, and finally collecting 80 mu L of RNA solution for direct detection.
Or stored at-80 ℃. The negative quality control and the positive quality control are all extracted.
And (3) system configuration:
according to the total reaction number N (including the number of samples to be detected, negative quality control products and positive quality control products) required by detection, 18.5 mu l of RT-PCR amplification solution and 1.5 mu l of enzyme mixed solution are added into each PCR. Calculating the required total amount, mixing uniformly, and then sub-packaging into a special PCR reaction tube.
Sample adding:
respectively adding negative quality control substances, sample RNA solution and positive quality control substances into the PCR reaction tube with the reagents, wherein the sample adding amount is 10 mu L, closing the tube cover, mixing uniformly, centrifuging and collecting the solution, and placing the solution at the bottom of the tube.
And (5) performing amplification detection on the machine:
the fluorescent detection channels were set as shown in table 3:
the settings for the RT-PCR amplification procedure are shown in Table 4:
6. analysis of results:
on the premise of meeting the amplification effectiveness, the positive result judgment of the target gene is shown in the following table 5:
the novel crown Omicron variant was judged according to the following table 6:
example 3 sensitivity detection
The genome RNA standard substance (standard substance number: GBW (E) 091099) of wild type novel coronavirus non-omacron variant purchased from China measuring institute was diluted to a proper concentration and then diluted by 10-fold ratio, wherein the concentrations were respectively 1×10 6 copies/mL、1×10 5 copies/mL、1×10 4 copies/mL、1×10 3 copies/mL、1×10 2 copies/mL、1×10 1 copies/mL。
The 6 gradient concentrations of the novel coronavirus genomic RNA standard are measured using the above-identified detection system and cycling parameters. The amplification result Ct is shown in FIGS. 9 to 11.
The results showed sensitivity data of 1×10 2 copies/mL. The detection method has higher sensitivity, and the CT value of the detection result is shown in the following table 7.
Fig. 9 to 11: the dilution concentration of the novel coronavirus genome RNA standard substance of China measuring institute by 10 times ratio is 1 multiplied by 10 respectively 6 copies/mL、1×10 5 copies/mL、1×10 4 copies/mL、1×10 3 copies/mL、1×10 2 copies/mL、1×10 1 Amplification plot of copies/mL. FIG. 9 shows the results of concentration gradient amplification of novel coronavirus (2019-nCoV) ORF1ab target gene; FIG. 10 shows the results of concentration gradient amplification of novel coronavirus (2019-nCoV) N target gene; FIG. 11 shows the results of novel coronavirus (2019-nCoV) S gene concentration gradient (wild-type) amplification.
Example 4 specific assay
Diluting local human coronavirus (HKU 1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, H1N1 influenza A virus, H3N2 influenza A virus, H5N1 influenza A virus, H7N9 influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, rhinovirus, adenovirus, enterovirus pseudovirus to 1×10 6 The copies/mL was tested as a specific test sample.
The detection results show that 16 pathogens, namely HKU1, OC43, NL63 and 229E, SARS coronavirus, MERS coronavirus, H1N1 influenza A virus, H3N2 influenza A virus, H5N1 influenza A virus, H7N9 influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, rhinovirus, adenovirus and enterovirus, are negative, and the negative and positive and negative quality control products are normally detected, so that the kit has good specificity.
Fig. 12: pseudo-virus specific assay for human coronavirus (HKU 1).
Example 5 authentication detection
In order to verify the accuracy and effectiveness of differential diagnosis of the Omicron detection kit and other variants of the novel coronavirus, a pseudovirus sample of the novel coronavirus wild type and other variants Alpha, beta, gamma, delta, lambda, mu, kappa, iota is verified, the result of the Omicron detection kit shows that the Omicron variant has negative S gene loss, the wild type and other variants have positive S gene loss, and the result shows no cross reaction, so that the kit can accurately carry out differential diagnosis on the Omicron variant and the non-Omicron variant. The partial detection results are shown in fig. 13 to 15:
FIG. 13 shows the detection result of the negative sample (-);
FIG. 14 shows the results of detection of Omicron variant samples;
FIG. 15 shows the results of testing samples of the novel crown Delta variant.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Shenzhen Co., ltd
<120> kit and method for detecting novel coronavirus and Omicron mutant thereof
<130> 00004
<160> 52
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
tgacatagcc aagaaaccaa ctga 24
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
caccattacg ggcatttcta aat 23
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
cgatttgtgc accactcact gtcttttttg 30
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
acatggcaag gaagacctta aatt 24
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
tggtcatctg gactgctatt gg 22
<210> 6
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cctcgaggac aaggcgttcc aattaa 26
<210> 7
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ttacttggtt ccatgctata catgtc 26
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
acaataagta gggactgggt cttc 24
<210> 9
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
tttgataacc ctgtcctacc attta 25
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
cttccaagaa agccaagtgt ga 22
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
aggttttatt tttgttgtgg ctgat 25
<210> 12
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
aatgccccag tctctgtcag cactcc 26
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
gcgcacctgt tgtctatgtg a 21
<210> 14
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
aaccccattg ttgaacatca atc 23
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
acgtgccaca tgcttttcca ctgct 25
<210> 16
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
cagggaccac ctggtactgg taa 23
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
ggcatgagag caagctgtat aca 23
<210> 18
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tggcctagct ctctactacc cttctgctcg 30
<210> 19
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
tggcatacct aaggacatga ccta 24
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
gaagccaatc catgcacgta 20
<210> 21
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
accctaacat gtttatcacc cgcgaaga 28
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
agggaccacc tggtactggt aa 22
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
ggctgatgaa ctataaaagg gaaga 25
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
agtgttgtca atgccagatt acgt 24
<210> 26
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
ggttctagtg tgcccttagt tagca 25
<210> 27
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
cattggcgac cctgctcaat tacctg 26
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
gcacgattgc agcattgtta g 21
<210> 30
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
aaagatcaca ttggcacccg caatcc 26
<210> 31
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
caactgaggg agccttgaat acac 24
<210> 32
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
cgaaggtgtg acttccatg 19
<210> 33
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
agatcacatt ggcacccgca atcc 24
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
<210> 35
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
tctggttact gccagttgaa tctg 24
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
agttgtagca cgattgcagc at 22
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
gtcttggttc accgctctca ct 22
<210> 38
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
ttggtcatct ggactgctat tgg 23
<210> 39
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
taaattccct cgaggacaag gcgttcc 27
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
ctctcactca acatggcaag ga 22
<210> 41
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
tcggtagtag ccaatttggt cat 23
<210> 42
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
caagctccgc ctcctgggtt ca 22
<210> 43
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
gttacttggt tccatgctat acatg 25
<210> 44
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
ccagcctctt attatgttag acttctc 27
<210> 45
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
ttgataaccc tgtcctacca ttta 24
<210> 46
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
gttacttggt tccatgctat acatgt 26
<210> 47
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
ctcartggaa gcaaaataaa cacc 24
<210> 48
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
tttgataacc ctgtcctacc att 23
<210> 49
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
ttacttggtt ccatgctata catgtct 27
<210> 50
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
ctcartggaa gcaaaataaa cac 23
<210> 51
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
accccgtctc tgctaaatac atg 23
<210> 52
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
gagtgcagtg gctcgatctc t 21
Claims (9)
1. A kit for detecting a novel coronavirus, characterized in that the kit consists of a primer set and a probe set; the primer pair set consists of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair,
the first primer pair consists of a forward primer shown as SEQ ID NO.7 and a reverse primer shown as SEQ ID NO. 8;
the second primer pair consists of a forward primer shown as SEQ ID NO.1 and a reverse primer shown as SEQ ID NO. 2;
the third primer pair consists of a forward primer shown as SEQ ID NO.4 and a reverse primer shown as SEQ ID NO. 5;
the fourth primer pair consists of a forward primer shown as SEQ ID NO.10 and a reverse primer shown as SEQ ID NO. 11;
the probe set consists of a first probe with a nucleotide sequence shown as SEQ ID NO.9, a second probe with a nucleotide sequence shown as SEQ ID NO.3, a third probe with a nucleotide sequence shown as SEQ ID NO.6 and a fourth probe with a nucleotide sequence shown as SEQ ID NO.12;
when the kit is used for detecting a sample of an object to be detected, a reaction system comprising the sample, the primer pair set and the fluorescent quantitative PCR detection of the probe set is configured for fluorescent quantitative PCR detection, wherein the reaction program of the fluorescent quantitative PCR detection is as follows: step 1: 10min at 50 ℃, step 2:95 ℃ for 3min, step 3:95℃for 5s and 60℃for 20s, the step 3 was cycled 45 times in total and fluorescence signals were collected at 60 ℃.
2. The kit of claim 1, wherein the kit comprises a first container comprising a primer probe mixture therein, the primer probe mixture comprising: nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 12.
3. The kit of claim 2, wherein the first container further comprises: mgCl 2 And dNTPs.
4. The kit of claim 3, further comprising a second container comprising an enzyme cocktail comprising a hot start Taq enzyme, a reverse transcriptase, and a uracil glycosylase (UNG).
5. The kit of claim 4, further comprising a third container comprising a negative control within the third container.
6. The kit of claim 5, further comprising a fourth container comprising a cationic control therein.
7. The kit of any one of claims 1-6, wherein each probe comprises a fluorescent reporter group at the 5 'end, wherein each probe comprises a fluorescent quenching group at the 3' end, and wherein the fluorescent reporter groups labeled with each probe are different from each other.
8. A method for detecting a novel coronavirus for non-diagnostic purposes, said method comprising the steps of:
(1) Providing a sample of an object to be detected;
(2) Preparing a fluorescent quantitative PCR reaction system and performing fluorescent quantitative PCR detection:
wherein, the reaction program of the fluorescent quantitative PCR detection is as follows: step 1: 10min at 50 ℃, step 2:95 ℃ for 3min, step 3: cycling the step 3 for 45 times at 95 ℃ for 5s and 60 ℃ for 20s, and collecting fluorescent signals at 60 ℃;
the fluorescent quantitative PCR reaction system comprises: the sample, primer pair set and probe set provided in step (1);
the primer pair set consists of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair,
the first primer pair consists of a forward primer shown as SEQ ID NO.7 and a reverse primer shown as SEQ ID NO. 8;
the second primer pair consists of a forward primer shown as SEQ ID NO.1 and a reverse primer shown as SEQ ID NO. 2;
the third primer pair consists of a forward primer shown as SEQ ID NO.4 and a reverse primer shown as SEQ ID NO. 5;
the fourth primer pair consists of a forward primer shown as SEQ ID NO.10 and a reverse primer shown as SEQ ID NO. 11;
the probe set consists of a first probe with a nucleotide sequence shown as SEQ ID NO.9, a second probe with a nucleotide sequence shown as SEQ ID NO.3, a third probe with a nucleotide sequence shown as SEQ ID NO.6 and a fourth probe with a nucleotide sequence shown as SEQ ID NO. 12.
9. The use of a primer set and a probe set in the preparation of a PCR detection kit for detecting a novel coronavirus, characterized in that,
the primer pair set consists of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair,
the first primer pair consists of a forward primer shown as SEQ ID NO.7 and a reverse primer shown as SEQ ID NO. 8;
the second primer pair consists of a forward primer shown as SEQ ID NO.1 and a reverse primer shown as SEQ ID NO. 2;
the third primer pair consists of a forward primer shown as SEQ ID NO.4 and a reverse primer shown as SEQ ID NO. 5;
the fourth primer pair consists of a forward primer shown as SEQ ID NO.10 and a reverse primer shown as SEQ ID NO. 11;
the probe set consists of a first probe with a nucleotide sequence shown as SEQ ID NO.9, a second probe with a nucleotide sequence shown as SEQ ID NO.3, a third probe with a nucleotide sequence shown as SEQ ID NO.6 and a fourth probe with a nucleotide sequence shown as SEQ ID NO.12;
when the PCR detection kit is used for detecting a sample of an object to be detected, a reaction system comprising the sample, the primer pair set and the fluorescent quantitative PCR detection of the probe set is configured for carrying out fluorescent quantitative PCR detection, wherein the reaction program of the fluorescent quantitative PCR detection is as follows: step 1: 10min at 50 ℃, step 2:95 ℃ for 3min, step 3:95℃for 5s and 60℃for 20s, the step 3 was cycled 45 times in total and fluorescence signals were collected at 60 ℃.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310662965.6A CN116555497B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronaviruses |
| CN202210098504.6A CN114107574B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronavirus and Omicron mutant strain thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210098504.6A CN114107574B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronavirus and Omicron mutant strain thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310662965.6A Division CN116555497B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronaviruses |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114107574A CN114107574A (en) | 2022-03-01 |
| CN114107574B true CN114107574B (en) | 2023-05-12 |
Family
ID=80361320
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310662965.6A Active CN116555497B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronaviruses |
| CN202210098504.6A Active CN114107574B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronavirus and Omicron mutant strain thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310662965.6A Active CN116555497B (en) | 2022-01-27 | 2022-01-27 | Kit and method for detecting novel coronaviruses |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN116555497B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116555497B (en) * | 2022-01-27 | 2024-03-19 | 深圳联合医学科技有限公司 | Kit and method for detecting novel coronaviruses |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023170297A1 (en) * | 2022-03-10 | 2023-09-14 | Genxpro Gmbh | Methods for detecting genomic variants of sars-cov-2 in multiplex assays |
| CN114369688B (en) * | 2022-03-22 | 2022-06-03 | 深圳联合医学科技有限公司 | Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte |
| CN114592097B (en) * | 2022-05-07 | 2022-07-29 | 北京生物制品研究所有限责任公司 | Primer and probe for identifying novel coronavirus Omicron strain BA.1 and/or BA.3 sublines and application thereof |
| CN115948609B (en) * | 2022-08-05 | 2023-10-20 | 深圳市儿童医院 | Composition for detecting SARS-CoV-2, kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025748A (en) * | 2021-01-14 | 2021-06-25 | 复旦大学附属华山医院北院 | Primer composition and kit for rapidly detecting 69-70del mutation of novel coronavirus |
| CN113481325A (en) * | 2021-07-06 | 2021-10-08 | 广州达安基因股份有限公司 | Method and kit for detecting novel coronavirus B.1.1.7 mutant strain |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2970603T3 (en) * | 2020-03-05 | 2024-05-29 | Inst Pasteur | Methods for the specific and sensitive detection of SARS-CoV-2 |
| CN111270013A (en) * | 2020-03-12 | 2020-06-12 | 宁波海尔施基因科技有限公司 | Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) kit and method for detecting 2019 novel coronavirus and primer probe composition |
| KR102338861B1 (en) * | 2020-04-01 | 2021-12-14 | 주식회사 시선바이오머티리얼스 | PNA Probe and Primer for Detecting SARS-CoV-2 Causing Covid-19 Using RT-LAMP and Method for Detecting SARS-CoV-2 Infection Using Thereof |
| US11634762B2 (en) * | 2020-05-14 | 2023-04-25 | Chemgenes Corporation | RT-qPCR molecular detection and diagnosis |
| CN111719016A (en) * | 2020-05-25 | 2020-09-29 | 深圳市疾病预防控制中心 | Composition for detecting new coronavirus 2019-nCoV and influenza A and B viruses and application |
| CN112695134A (en) * | 2020-12-02 | 2021-04-23 | 湖州市中心医院 | Novel coronavirus COVID-19 nucleic acid detection primer group, probe group, detection kit and detection method |
| CN112662809A (en) * | 2020-12-02 | 2021-04-16 | 湖州市中心医院 | Nucleic acid composition for detecting novel coronavirus COVID-19 and application thereof |
| CN112981011B (en) * | 2021-05-12 | 2021-08-17 | 广东凯普生物科技股份有限公司 | Primer composition for detecting SARS-CoV-2 and its application |
| CN113308574B (en) * | 2021-06-01 | 2022-03-15 | 上海伯杰医疗科技股份有限公司 | Primer probe combination, kit and parting detection method for detecting novel coronavirus mutant strain |
| CN113943838A (en) * | 2021-12-09 | 2022-01-18 | 常州国药医学检验实验室有限公司 | New coronavirus Ormckh mutation sequence detection technology based on multiple fluorescence quantitative ARMS-PCR technology and application thereof |
| CN113913561B (en) * | 2021-12-15 | 2022-03-04 | 中国农业大学 | SARS-CoV-2 delta variant strain detection method based on primer design and copper nano cluster |
| CN116555497B (en) * | 2022-01-27 | 2024-03-19 | 深圳联合医学科技有限公司 | Kit and method for detecting novel coronaviruses |
-
2022
- 2022-01-27 CN CN202310662965.6A patent/CN116555497B/en active Active
- 2022-01-27 CN CN202210098504.6A patent/CN114107574B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025748A (en) * | 2021-01-14 | 2021-06-25 | 复旦大学附属华山医院北院 | Primer composition and kit for rapidly detecting 69-70del mutation of novel coronavirus |
| CN113481325A (en) * | 2021-07-06 | 2021-10-08 | 广州达安基因股份有限公司 | Method and kit for detecting novel coronavirus B.1.1.7 mutant strain |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116555497B (en) * | 2022-01-27 | 2024-03-19 | 深圳联合医学科技有限公司 | Kit and method for detecting novel coronaviruses |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116555497B (en) | 2024-03-19 |
| CN114107574A (en) | 2022-03-01 |
| CN116555497A (en) | 2023-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114107574B (en) | Kit and method for detecting novel coronavirus and Omicron mutant strain thereof | |
| US20230114907A1 (en) | Rt-pcr detection method and kit for novel coronavirus | |
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| JP5795259B2 (en) | Primers and probes for detecting human papillomavirus and human beta globin sequences in test samples | |
| US20230203575A1 (en) | Novel coronavirus rapid detection kit based on thermal convection pcr | |
| CN111304369A (en) | Novel dual detection kit for coronavirus | |
| CN112063756A (en) | Method and kit for multiplex detection of respiratory virus nucleic acid | |
| CN113584232B (en) | Novel coronavirus and delta mutant strain detection kit and detection method thereof | |
| CN113774168A (en) | 2019 novel coronavirus, Deltay and lambda variant strain typing nucleic acid detection kit and detection method thereof | |
| CN111733291A (en) | Method and kit for detecting novel coronavirus nucleic acid by digital PCR (polymerase chain reaction) | |
| CN113652505A (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| CN111471800B (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| CN113718045A (en) | DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application | |
| CN113481325A (en) | Method and kit for detecting novel coronavirus B.1.1.7 mutant strain | |
| CN112981007A (en) | Kit for detecting Han beach type hantavirus and detection method thereof | |
| CN105838826B (en) | Double-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strain and wild strain | |
| CN113981140B (en) | Novel coronavirus delta mutant strain detection method and nucleic acid detection kit | |
| CN116479177A (en) | Primer probe combination for detecting 6 mutation sites of novel coronavirus S gene | |
| CN113481324B (en) | Method and kit for detecting novel coronavirus and D614G mutant thereof | |
| CN115125330A (en) | Detection kit for detecting different variants of novel coronavirus and application thereof | |
| CN114807432B (en) | Kit and method for rapidly detecting novel coronavirus and Delta mutant strain thereof | |
| CN112126713A (en) | Coronavirus and influenza virus combined detection product and application thereof | |
| CN114262758B (en) | Kit for detecting novel coronavirus mutant strain and detection method | |
| CN112662815B (en) | Primer-probe combination, kit and method for detecting Takara virus and Tamiami virus | |
| CN114807432A (en) | Kit and method for rapidly detecting novel coronavirus and Delta mutant strain 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |