CN114686618A - Primer probe composition, kit and detection method for detecting SARS-COV-2 - Google Patents
Primer probe composition, kit and detection method for detecting SARS-COV-2 Download PDFInfo
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Abstract
The invention discloses a primer probe composition, a kit and a detection method for detecting SARS-COV-2. The primer probe composition comprises a first specific primer and a first specific probe for specific amplification detection of ORF1ab gene; a second specific primer and a second specific probe for specific amplification detection of the N gene; a third specific primer and a third specific probe for specific amplification and detection of the reference gene beta-actin. In the invention, the defects of low nucleic acid positive detection rate and inaccurate detection result in the process of detecting SARS-COV-2 by adopting real-time fluorescence quantitative PCR based on the kit and the kit in the prior art are solved by specifically amplifying and detecting ORF1ab gene and N gene in SARS-COV-2.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a primer probe composition, a kit and a detection method for detecting SARS-COV-2.
Background
The novel coronavirus (SARS-COV-2) is a newly discovered coronavirus, and can cause serious respiratory diseases. The common signs include respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. Like many classical respiratory viruses, the novel coronavirus (SARS-COV-2) is rapidly transmitted among people, and some infected people do not have obvious infection symptoms, which brings great difficulty to the control of the virus. Currently, the number of infected people and dead people worldwide is rapidly rising, and before no effective treatment method appears, the screening and control of viruses are the best means for preventing and controlling further propagation.
The nucleic acid detection of SARS-COV-2 is the only accurate and effective detection means at present. Nucleic acid detection is usually performed by fluorescent quantitative PCR or gene sequencing. Although the gene sequencing method is accurate, the detection time is long, the detection cost is high, and the detection method is difficult to be widely used in clinic. The real-time fluorescent quantitative PCR technology is a method widely applied in the nucleic acid detection process, and the whole reaction process is monitored by adding a fluorescent label in the PCR reaction and monitoring the accumulation of a fluorescent signal in real time. Therefore, compared with the gene sequencing method, the real-time fluorescence quantitative PCR (namely the real-time fluorescence qPCR) SARS-COV-2 pair has the advantages of high sensitivity, high specificity, high flux, rapidness and the like, thereby being capable of being used for rapidly screening and detecting SARS-COV-2.
However, the conventional real-time fluorescent quantitative PCR has the defect of low nucleic acid positive detection rate when detecting SARS-COV-2, and the essential reason is that the specificity detection of SARS-COV-2 in the kit used for the real-time fluorescent quantitative PCR detection is poor.
In view of the above, there is a need for improvement of the related art kit and method for rapid specific detection of SARS-COV-2, so as to solve the above problems.
Disclosure of Invention
The invention aims to disclose a primer probe composition, a kit and a detection method for detecting SARS-COV-2, which are used for solving the defects of low nucleic acid positive detection rate and inaccurate detection result in the prior art of the kit and the process of detecting SARS-COV-2 by adopting real-time fluorescence quantitative PCR based on the kit.
To achieve the above first object, the present invention provides a primer probe composition for detecting SARS-COV-2, comprising:
a first specific primer and a first specific probe for specific amplification detection of ORF1ab gene in SARS-COV-2;
a second specific primer and a second specific probe for specific amplification detection of the N gene in the SARS-COV-2;
a third specific primer and a third specific probe for specific amplification detection of the reference gene beta-actin;
the forward primer of the first specific primer has a nucleotide sequence shown as SEQ NO.1, the reverse primer of the first specific primer has a nucleotide sequence shown as SEQ NO.2, and the first specific probe has a nucleotide sequence shown as SEQ NO. 3;
the forward primer of the second specific primer has a nucleotide sequence shown as SEQ NO.4, the reverse primer of the second specific primer has a nucleotide sequence shown as SEQ NO.5, and the second specific probe has a nucleotide sequence shown as SEQ NO. 6;
the forward primer of the third specific primer has a nucleotide sequence shown as SEQ NO.7, the reverse primer of the third specific primer has a nucleotide sequence shown as SEQ NO.8, and the third specific probe has a nucleotide sequence shown as SEQ NO. 9.
As a further improvement of the invention, the 5 'ends of the first specific probe and the second specific probe are marked with fluorescent reporter groups, and the 3' ends are marked with fluorescent quenching groups; the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophanate.
In order to achieve the second object, the present invention also discloses a kit for detecting SARS-COV-2 for non-diagnostic purposes, comprising:
adopting a primer probe premix solution, an amplification premix solution, a negative quality control product and a positive quality control product which are composed of the primer probe composition for detecting SARS-COV-2, which is created by any one of the above inventions;
the amplification premix solution consists of PCR buffer solution, PCR protective agent, Taq enzyme, dNTPs and MgCl2Composition is carried out;
the positive quality control product is SARS-COV-2 virus;
the negative quality control product is influenza A virus and/or influenza B virus.
Finally, the application also discloses an assay for detecting SARS-COV-2 for non-diagnostic purposes comprising the steps of:
step (1): extracting an RNA sample from a biological detection material sample;
step (2): performing a fluorescent quantitative PCR assay on the RNA sample using the kit for detecting SARS-COV-2 of non-diagnostic interest according to claim 3;
and (3): and (6) analyzing the detection result.
As a further improvement of the invention, the biological sample in the step (1) is a fresh pharyngeal swab.
As a further improvement of the invention, the amplification reaction system used in the fluorescent quantitative PCR detection in the step (2) is 20 μ l:
10 XPCR buffer, MgCl250mM, dNTPs 250. mu.M, 10 XPCR protectant, forward primer 100. mu.M of the first specific primer, reverse primer 100. mu.M of the first specific primer, first specific probe 100. mu.M, forward primer 100. mu.M of the second specific primer, reverse primer 100. mu.M of the second specific primer, second specific probe 100. mu.M, sterilized ultrapure water to 20. mu.l;
the reaction system of the amplification reaction contains a positive quality control substance with the concentration of 10-1000 copies/mu l, and the concentration of a negative quality control substance with the concentration of 10-1000 copies/mu l.
Compared with the prior art, the invention has the beneficial effects that:
by the primer probe composition, the kit and the detection method for detecting SARS-COV-2 disclosed by the invention, the defects of low nucleic acid positive detection rate and inaccurate detection result in the process of detecting SARS-COV-2 by using the kit in the prior art and the kit based on the prior art and adopting real-time fluorescence quantitative PCR are overcome by specifically amplifying and detecting ORF1ab gene and N gene in SARS-COV-2.
Drawings
FIG. 1 is a comparison graph showing the detection effect of ORF1ab gene specificity detection when positive quality control substances are 10 copies, 100 copies and 1000copies respectively by using the kit disclosed in the present invention and the detection method disclosed in the present invention for non-diagnostic purpose of detecting SARS-COV-2;
FIG. 2 is a comparison graph of the specific detection effects of the N gene when the positive quality control substance is 10 copies, 100 copies and 1000copies respectively, using the kit disclosed in the present invention and the detection method disclosed in the present invention for detecting SARS-COV-2 for non-diagnostic purposes;
FIG. 3 is a comparison of the detection effect of the specific detection of the negative quality control material, which is influenza A virus, using the kit of the present invention and the detection method for detecting SARS-COV-2 of the present invention for non-diagnostic purposes.
Detailed Description
The present invention is described in detail with reference to the embodiments shown in the drawings, but it should be understood that these embodiments are not intended to limit the present invention, and those skilled in the art should understand that functional, methodological, or structural equivalents or substitutions made by these embodiments are within the scope of the present invention.
In the examples of the present application, the term "For non-diagnostic purposes"refers to information that is not intended to obtain whether an individual is infected with SARS-CoV-2 (a novel coronavirus) and has suffered pneumonia. The detection method can detect whether SARS-CoV-2 exists in the detection culture in experiments for scientific research.
The first embodiment is as follows:
this example discloses a primer probe composition (hereinafter referred to as "primer probe composition") for detecting SARS-COV-2, which can perform accurate and rapid specific detection on whether a sample to be detected is marked to be SARS-COV-2 (novel coronavirus) positive or not during fluorescence quantitative PCR detection, and can perform specific detection on a gene (ORF 1ab gene) in a target region I and/or a gene (N gene) in a target region II, and can perform specific detection on other currently known influenza viruses or other viruses with similar proteins and genetic structures, thereby improving accurate detection on a sample to be detected carrying SARS-COV-2 and avoiding the phenomenon of SARS-COV-2 nucleic acid detection turning positive.
Specifically, in this embodiment, the primer probe composition comprises: a first specific primer and a first specific probe for specific amplification detection of ORF1ab gene in SARS-COV-2; a second specific primer and a second specific probe for specific amplification detection of the N gene in the SARS-COV-2; a third specific primer and a third specific probe for specific amplification detection of the reference gene beta-actin.
The nucleotide sequence of ORF1ab gene is shown below:
GCTTCATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAATACTGAGTCCTCTTTATGCATTTGC, and designing a first specific primer and a first specific probe for specific amplification detection of ORF1ab gene in SARS-COV-2. The forward primer of the first specific primer has a nucleotide sequence shown as SEQ NO.1, the reverse primer of the first specific primer has a nucleotide sequence shown as SEQ NO.2, and the first specific probe has a nucleotide sequence shown as SEQ NO. 3. Forward primer of the first specific primer: 5'-cggCTGGCTTGAAAACCATTCTTCGT-3', respectively; reverse primer of the first specific primer: 5'-cccGGCACACTTGTTATGGCAACC-3', respectively; first specific probe: FAM-CCTCCAAAGGCAATAGTGCGACCACC-MGB.
The nucleotide sequence of the N gene is shown as follows:
ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAA, and designing a second specific primer and a second specific probe for specific amplification and detection of the N gene in SARS-COV-2. The forward primer of the second specific primer has a nucleotide sequence shown as SEQ NO.4, the reverse primer of the second specific primer has a nucleotide sequence shown as SEQ NO.5, and the second specific probe has a nucleotide sequence shown as SEQ NO. 6. Forward primer of the second specific primer: 5'-ggCCAAGGTTTACCCAATAATACTGCGTCT-3', reverse primer of the second specific primer: 5'-gcGTGTTAATTGGAACGCCTTGTCCTC-3', second specific probe: FAM-TGGTTCACCGCTCTCACTCAACATGGC-MGB.
The forward primer of the third specific primer has a nucleotide sequence shown as SEQ NO.7, the reverse primer of the third specific primer has a nucleotide sequence shown as SEQ NO.8, and the third specific probe has a nucleotide sequence shown as SEQ NO. 9. Forward primer of the third specific primer: 5'-TGAACCCCAAGGCCAACCGC-3', reverse primer of the third specific primer: 5'-gcGTCCTACGGAAAACGGCAGA-3', third specific probe: CY 5-AGGTAGCGGGCCACTCACCT-MGB.
The 5 'ends of the first specific probe and the second specific probe are marked with fluorescent reporter groups, and the 3' ends of the first specific probe and the second specific probe are marked with fluorescent quenching groups; the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophanate.
During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, wherein the probe is an oligonucleotide, and two ends (namely 3 'end and 5' end) are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter fluorophore is absorbed by the quenching fluorophore; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the report fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.
The reference Gene β -actin, as a housekeeping Gene (Home-eating Gene, HKG), generally refers to a protein expressed by a housekeeping Gene code for mammalian cell expression. The expression of the internal reference gene beta-actin is relatively constant in various tissues and cells, and the internal reference gene beta-actin is commonly used as a reference substance when detecting the change of the expression level of protein. Thereby providing accurate reference basis for the specificity detection of the first target region (i.e. ORF1ab gene) and the second target region (i.e. N gene) contained in SARS-COV-2.
Compared with the defects of low detection precision and negative to positive conversion existing in the specific detection of part of bases in the target region I or the target region II in the prior art, the primer probe composition disclosed by the embodiment can realize high-flux, rapid and accurate detection on a sample to be detected which is positive for representing SARS-COV-2.
Meanwhile, in the embodiment, through the special optimization design of the forward primer and the reverse primer of the first specific primer, the first specific probe, the forward primer and the reverse primer of the second specific primer, the second specific probe, the forward primer and the reverse primer of the third specific primer and the third specific probe, the detection time of the kit containing the primer probe composition by adopting fluorescence quantitative PCR amplification detection is shortened by 5-10min compared with the detection time of the conventional primer probe composition, and the rapid detection and screening of the SARS-COV-2 virus can be efficiently carried out.
Meanwhile, the kit containing the primer probe composition can be applied to a qPCR platform, quantitative PCR amplification is carried out on ORF1ab gene and N gene contained in SARS-COV-2 by designing a group of specific primers, and the amplified product is detected by using the specific fluorescence labeled probe, so that a band detection sample carrying SARS-COV-2 virus can be effectively detected, and the kit has the characteristics of stability, rapidness, high specificity, low detection cost and the like.
Example two:
based on the primer probe composition disclosed in the first embodiment, the applicant further proposes a kit for detecting SARS-COV-2 for non-diagnostic purposes (hereinafter referred to as "kit"), the kit comprising: amplification Using primer Probe premix solution comprising primer Probe composition for detecting SARS-COV-2 as disclosed in example oneMixing liquid, negative quality control product and positive quality control product; wherein the amplification premix solution comprises PCR buffer solution, PCR protective agent, Taq enzyme, dNTPs and MgCl2Composition is carried out; the positive quality control product is SARS-COV-2 virus; the negative quality control product is influenza A virus and/or influenza B virus.
Example three:
based on the kit disclosed in example two, the applicant also proposed a method for detecting SARS-COV-2 for non-diagnostic purposes using the kit, comprising the steps of:
step (1): extracting an RNA sample from a biological detection material sample; the biological test material sample in the step (1) is a fresh pharyngeal swab.
Step (2): performing a fluorescent quantitative PCR assay on the RNA sample using the kit for detecting SARS-COV-2 of non-diagnostic interest according to claim 3;
and (3): and (6) analyzing the detection result.
In this embodiment, the amplification reaction system used in the fluorescent quantitative PCR detection in step (2) is 20 μ l:
10 XPCR buffer, MgCl250mM, dNTPs 250. mu.M, 10 XPCR protectant, 100. mu.M forward primer of the first specific primer, 100. mu.M reverse primer of the first specific primer, 100. mu.M first specific probe, 100. mu.M forward primer of the second specific primer, 100. mu.M reverse primer of the second specific primer, 100. mu.M second specific probe, sterilized ultrapure water to 20. mu.l;
the reaction system of the amplification reaction contains a positive quality control substance with the concentration of 10-1000 copies/mu l, and the negative quality control substance with the concentration of 10-1000 copies/mu l.
The kit comprises a primer probe premix (PPmix), an amplification premix (APmix), a negative quality control (NC) and a positive quality control (PC), and the system preparation of an amplification reaction system is shown in the following table I:
| components | Volume (μ L) |
| APmix (amplification premix) | 8.0-10.0 |
| PPmix (primer probe premix) | 1.5-2.0 |
| Enzyme mix (Enzyme mixture such as Taq, reverse transcription, UDG, etc.) | 0.5-1.0 |
| RNA extraction,or PC/NC* | 8.0-10.0 |
| |
20 |
Watch 1
In order to ensure the success of the test operation and the subsequent analysis of the test results, a PC control group and an NC control group are required in each detection of ORF1ab gene and N gene. And (3) sequentially putting the PCR tube or the PCR96 pore plate into a PCR instrument, performing real-time fluorescent quantitative PCR amplification according to the amplification program in the step (3), and adopting a fluorescent probe method to utilize a fluorescent reporter group (such as FAM or VIC) marked at the 5 'end, and a fluorescent quencher group (such as MGB or BHQ) marked at the 3' end. The BHQ as the fluorescence quenching group may be BHQ-1, BHQ-2 or BHQ-3.
Setting parameters of a qPCR amplification instrument (model ABI 7500) real-time fluorescence quantitative PCR instrument: at 95 ℃ for 5min, (at 95 ℃ for 15sec, at 70 ℃ for 30sec)40cycles, the system is 20ul, ORF1ab gene locus FAM channel and VIC channel, N gene locus VIC channel, the sample is put into a PCR amplification instrument, the position where the sample is put is selected, the sample is stored in a designated folder, and then the program can be started to run for PCR amplification reaction. The qPCR operating parameters are detailed in table two below.
Watch two
Quality control:
blank control: the channel detection of FAM, VIC and CY5 has no Ct value or the Ct value is 0;
positive control: both FAM and VIC channel detection show S-type amplification curves, and Ct value is less than or equal to 33.5;
controlling internal parameters: the CY5 channel detection amplification curve is S-shaped, and the Ct value is less than or equal to 32.
And (4) interpretation of results:
the FAM channel of the sample to be detected is an S-shaped amplification curve, and if the Ct value is less than or equal to 36, the detection result is positive for SARS-CoV-2;
the FAM channel of the sample to be detected detects a non-S-type amplification curve, or the Ct value is 0/no Ct value, and the Ct value of the CY5 channel is less than or equal to 32, which is negative in SARS-CoV-2 detection;
if the CY5 channel of the sample to be tested is S-shaped amplification curve and Ct value is more than 32: the weak FAM channel is in an S-shaped amplification curve, and the Ct value is less than or equal to 36, the detection result is positive for SARS-CoV-2; if the FAM channel detects a non-S-type amplification curve or the Ct value is 0/no Ct value, resampling is needed for detection.
Referring to FIG. 1, the detection method disclosed in this example showed that ORF1ab gene takes off at 33.2 min, 30.8 min and 27.2 min when PCR amplification reactions were carried out at 10 copies, 100 copies and 1000copies of positive quality control, respectively, and typical amplification curves were obtained, thus demonstrating that ORF1ab gene can be specifically detected by the detection method using the kit.
Referring to fig. 2, when the PCR amplification reactions were performed on N gene at the concentrations of positive quality control 10 copies, 100 copies, and 1000copies, respectively, by the detection method disclosed in this example, the N gene took off at 33 minutes, 30.4 minutes, and 27.1 minutes, respectively, and had typical amplification curves, thereby demonstrating that the detection method using the kit can specifically detect N gene.
Referring to FIG. 3, when the detection method disclosed in this example is used to detect a negative sample, the sample to be detected has no obvious S-type amplification curve, thus demonstrating that the detection method using the kit can not detect a sample to be detected other than SARS-CoV-2, such as influenza A virus or influenza B virus, and thus demonstrating that the kit and the detection method can rapidly and specifically detect SARS-CoV-2 and can not detect a sample to be detected other than SARS-CoV-2. Compared with the conventional primer probe composition, the primer probe composition-based kit and the detection method based on the kit, the detection time of the detection method is shortened by 5-10min, so that the sensitive detection of SARS-CoV-2 (novel coronavirus) can be efficiently carried out, and an effective screening means is provided for prevention and treatment of SARS-CoV-2.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
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Claims (6)
1. A primer probe composition for detecting SARS-COV-2, comprising:
a first specific primer and a first specific probe for specific amplification detection of ORF1ab gene in SARS-COV-2;
a second specific primer and a second specific probe for specific amplification detection of the N gene in the SARS-COV-2;
a third specific primer and a third specific probe for specific amplification detection of the reference gene beta-actin;
the forward primer of the first specific primer has a nucleotide sequence shown as SEQ NO.1, the reverse primer of the first specific primer has a nucleotide sequence shown as SEQ NO.2, and the first specific probe has a nucleotide sequence shown as SEQ NO. 3;
the forward primer of the second specific primer has a nucleotide sequence shown as SEQ NO.4, the reverse primer of the second specific primer has a nucleotide sequence shown as SEQ NO.5, and the second specific probe has a nucleotide sequence shown as SEQ NO. 6;
the forward primer of the third specific primer has a nucleotide sequence shown as SEQ NO.7, the reverse primer of the third specific primer has a nucleotide sequence shown as SEQ NO.8, and the third specific probe has a nucleotide sequence shown as SEQ NO. 9.
2. The primer-probe composition of claim 1, wherein the first specific probe and the second specific probe are labeled with a fluorescent reporter at their 5 'end and a fluorescent quencher at their 3' end; the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophanate.
3. A kit for detecting SARS-COV-2 for non-diagnostic purposes, comprising:
the primer probe premix, the amplification premix, the negative quality control product and the positive quality control product which are composed of the primer probe composition for detecting SARS-COV-2 according to claim 1 or 2;
the amplification premix solution consists of PCR buffer solution, PCR protective agent, Taq enzyme, dNTPs and MgCl2Composition is carried out;
the positive quality control product is SARS-COV-2 virus;
the negative quality control product is influenza A virus and/or influenza B virus.
4. A method for detecting SARS-COV-2 for non-diagnostic purposes, comprising the steps of:
step (1): extracting an RNA sample from a biological detection material sample;
step (2): performing a fluorescent quantitative PCR assay on the RNA sample using the kit for detecting SARS-COV-2 of claim 3;
and (3): and (6) analyzing the detection result.
5. The detection method according to claim 4, wherein the biological sample in step (1) is a fresh throat swab.
6. The detection method according to claim 4, wherein the amplification reaction system used in the fluorescent quantitative PCR detection in step (2) is 20 μ l:
10 XPCR buffer, MgCl250mM, dNTPs 250. mu.M, 10 XPCR protectant, forward primer 100. mu.M of the first specific primer, reverse primer 100. mu.M of the first specific primer, first specific probe 100. mu.M, forward primer 100. mu.M of the second specific primer, reverse primer 100. mu.M of the second specific primer, second specific probe 100. mu.M, sterilized ultrapure water to 20. mu.l;
the reaction system of the amplification reaction contains a positive quality control substance with the concentration of 10-1000 copies/mu l, and the concentration of a negative quality control substance with the concentration of 10-1000 copies/mu l.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349720A (en) * | 2020-03-19 | 2020-06-30 | 中国医学科学院病原生物学研究所 | Nucleic acid reagent, kit, system and method for detecting respiratory tract infection virus |
| CN111471800A (en) * | 2020-05-26 | 2020-07-31 | 海关总署(北京)国际旅行卫生保健中心 | Kit for detecting novel coronavirus and amplification primer composition thereof |
| CN111621604A (en) * | 2020-06-24 | 2020-09-04 | 上海五色石医学研究股份有限公司 | Novel primer probe composition, kit and method for coronavirus nucleic acid detection |
| CN111808990A (en) * | 2020-06-24 | 2020-10-23 | 阿吉安(福州)基因医学检验实验室有限公司 | 2019-nCoV nucleic acid detection kit |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349720A (en) * | 2020-03-19 | 2020-06-30 | 中国医学科学院病原生物学研究所 | Nucleic acid reagent, kit, system and method for detecting respiratory tract infection virus |
| CN111471800A (en) * | 2020-05-26 | 2020-07-31 | 海关总署(北京)国际旅行卫生保健中心 | Kit for detecting novel coronavirus and amplification primer composition thereof |
| CN111621604A (en) * | 2020-06-24 | 2020-09-04 | 上海五色石医学研究股份有限公司 | Novel primer probe composition, kit and method for coronavirus nucleic acid detection |
| CN111808990A (en) * | 2020-06-24 | 2020-10-23 | 阿吉安(福州)基因医学检验实验室有限公司 | 2019-nCoV nucleic acid detection kit |
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Application publication date: 20220701 |