CN111733295A - Primer group and kit for detecting novel coronavirus - Google Patents

Primer group and kit for detecting novel coronavirus Download PDF

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Publication number
CN111733295A
CN111733295A CN202010759566.8A CN202010759566A CN111733295A CN 111733295 A CN111733295 A CN 111733295A CN 202010759566 A CN202010759566 A CN 202010759566A CN 111733295 A CN111733295 A CN 111733295A
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novel coronavirus
gene
detecting
kit
primer
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CN111733295B (en
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张秀杰
曹艳
高秀洁
梁建芳
张晓刚
陈廷友
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Beijing Innotech Biotechnology Co.,Ltd.
Guangzhou lingshangyuan Biotechnology Co., Ltd;
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/166Oligonucleotides used as internal standards, controls or normalisation probes
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a primer group and a kit for detecting novel coronavirus, belonging to the technical field of gene detection. The invention establishes a set of novel coronavirus (COVID-19) constant-temperature rapid nucleic acid amplification detection method based on recombinase polymerase amplification technology, constructs a primer probe combination and a kit for constant-temperature rapid detection of novel coronavirus ORF genes and N genes based on the detection method, has the reaction duration of only 18-20min, has the characteristics of constant-temperature rapid and simple and convenient operation of POCT products, and is more time-saving than the conventional RT-qPCR method.

Description

Primer group and kit for detecting novel coronavirus
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer group and a kit for detecting novel coronavirus.
Background
The novel coronavirus pneumonia is acute infectious pneumonia, and the pathogen of the novel coronavirus is a new emerging coronavirus which is named as '2019-nCoV' by the world health organization. The novel coronavirus belongs to the genus coronavirus beta, is an nonsegmented single-stranded positive-strand RNA virus, has an envelope, is mainly transmitted through droplets, has strong infectious capacity, is mainly characterized by fever, hypodynamia and dry cough after infecting people, gradually shows severe manifestations such as dyspnea and the like, has good prognosis for most patients, and can cause pneumonia, severe acute respiratory syndrome, renal failure and even death for some severe cases. Currently, there is still a lack of effective antiviral drugs against new coronaviruses.
Since the discovery of new coronavirus pneumonia cases, this acute respiratory infectious disease spread rapidly. Since the WHO announced that new coronavirus pneumonia epidemic was listed as an emergent public health incident of international concern, new coronavirus pneumonia has become a global problem. In view of this, the demand for detection of novel coronas has been sharply rising.
However, the common novel coronavirus nucleic acid detection products in the market at present are common fluorescence PCR detection products, the time consumption is long, the amplification detection process basically needs 1.5-2 hours, and the requirements on instruments and laboratory environments are high.
Therefore, a rapid, highly sensitive and highly specific nucleic acid detection kit is continuously developed, and can be applied to rapid field screening in hospitals, laboratories, entry and exit ports and the like.
Disclosure of Invention
Accordingly, there is a need to provide a primer set for detecting a novel coronavirus, which can be amplified at a constant temperature based on a recombinase polymerase amplification technique depending on three enzymes, namely a recombinase capable of binding single-stranded nucleic acid, a single-stranded DNA binding protein (SSB) and a strand displacement DNA polymerase, and can terminate the reaction within 20min, and which is applicable to fluorescence detection equipment such as a fluorescence PCR instrument and a constant-temperature fluorescence amplification instrument of a common brand, and has the characteristics of high efficiency and high adaptability.
A primer set for detecting a novel coronavirus, comprising: the primer pair is characterized by comprising a novel coronavirus ORF gene specific primer pair and a novel coronavirus N gene specific primer pair, wherein the ORF gene specific primer pair is as follows:
a forward primer: 5'-GTATGTGGAAAGGTTATGGCTGTAGTTGTG-3' (SEQ. ID. No: 1);
reverse primer: 5'-GTGCCGCACGGTGTAAGACGGGCTGCACTTAC-3' (SEQ. ID. No: 2);
the N gene specific primer pair is as follows:
a forward primer: 5'-AACATTGCCAAAAGGCTTCTACGCAGAAGG-3' (SEQ. ID. No: 3);
reverse primer: 5'-GGAGAAGTTCCCCTACTGCTGCCTGGAGTT-3' (SEQ. ID. No: 4).
The primer group for detecting the novel coronavirus is designed according to the characteristics of isothermal amplification reaction, and the amplification primer pair can be used for efficiently amplifying the target gene fragment under the conditions of constant temperature and high speed without losing the detection precision. The kit has the advantages of rapidness, high sensitivity and strong specificity, and can be widely applied to the fields of hospitals, inspection centers, entry and exit, ports and the like which need on-site rapid screening.
In one embodiment, the kit further comprises a novel coronavirus ORF gene specific probe and a novel coronavirus N gene specific probe; the ORF gene specific probe is: 5'-TAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGC-3' (SEQ. ID. No: 5); the N gene specific probe is as follows: 5'-AGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCA CGTA GTCGC-3' (SEQ. ID. No: 6).
The probe is used for detection, and has the advantage of high detection precision.
In one example, the ORF gene specific probe labels tetrahydrofuran at 34bp from the 5 'end, T bases upstream of the tetrahydrofuran site label FAM fluorophore, T bases downstream of the tetrahydrofuran site label BHQ1 fluorophore, and a C3-spacer modifier group at the 3' end;
the N gene specific probe is used for marking tetrahydrofuran at a position 34bp away from a 5 'end, marking FAM fluorescent group at an upstream T base of a tetrahydrofuran site, marking BHQ1 fluorescent group at a downstream T base of the tetrahydrofuran site, and marking a C3-spacer modifying group at a 3' end.
By optimizing the probe, the detection performance of the probe is improved.
The invention also discloses application of the primer group for detecting the novel coronavirus as a constant-temperature amplification detection reagent in novel coronavirus detection.
In one embodiment, the conditions for the isothermal amplification assay are: reacting at a constant temperature of 39-42 ℃ for 18-20 min.
It can be understood that the isothermal amplification detection reagent of the invention can also be used in a conventional fluorescence PCR instrument, parameter conditions can be set to 39-42 ℃ for 30sec, 36-40 cycles, and fluorescence is collected.
The invention also discloses a kit for detecting the novel coronavirus, which comprises the following components in part by weight: a recombinase that binds to a single-stranded nucleic acid, a single-stranded DNA binding protein, a strand displacement DNA polymerase, dNTPs, and the primer set described above.
The kit provided by the invention utilizes the principle of recombinase polymerase amplification technology, utilizes specific conserved regions of ORF genes and N genes of novel coronavirus, and designs target nucleotide primers and probes aiming at the conserved regions. The specific primers and probes of the ORF gene and the N gene of the novel coronavirus are respectively put in a system for recombinase polymerase amplification, so that whether the novel coronavirus is infected or not can be identified.
It is understood that the recombinase of the single-stranded nucleic acid, the single-stranded DNA binding protein, the strand displacement DNA polymerase, dNTPs, and the like may be used in accordance with conventional commercial reagents.
In one embodiment, the concentration of the ORF gene specific primer pair and the concentration of the N gene specific primer pair are both 0.4-0.6 mu mol/L, the concentration of the ORF gene specific probe and the concentration of the N gene specific probe are both 0.12-0.15 mu mol/L, and the dosage of the dNTP is 6-10 mmol.
In one embodiment, the concentration of the single-stranded nucleic acid-binding recombinase is 100-120 ng/. mu.L, the concentration of the single-stranded DNA-binding protein is 80-100 ng/. mu.L, and the concentration of the strand displacement DNA polymerase is 30-35 ng/. mu.L.
In one embodiment, the kit further comprises: reagent I: buffer solution of reaction solution; and (2) reagent II: 280mM (mmol/L) magnesium acetate.
in one embodiment, the kit further comprises a negative quality control product and a positive quality control product, wherein the negative quality control product is physiological saline, the positive quality control product is a pseudovirus comprising a target fragment of ORF gene of the novel coronavirus and a pseudovirus comprising a target fragment of N gene of the novel coronavirus, and the concentration of the pseudoviruses is 1.0 × 103~1.0×105copies/μl。
During detection, two quality control products need to be detected simultaneously, and only when the ORF gene and the N gene positive quality control products are detected, the fluorescence signals of the corresponding channels are positive, and when the negative quality control products are detected, the fluorescence signals are negative, the detection result of the sample to be detected is effective.
it is understood that the pseudovirus can be prepared by conventional methods, such as artificially synthesizing a target fragment of ORF gene of the novel coronavirus and a target fragment of N gene of the novel coronavirus respectively, cloning the synthesized fragments to an expression vector containing a phage gene, generating an RNA fragment coated by a capsid protein of the phage by induction expression, measuring the concentration by titer, and diluting to 1.0 × 103~1.0×105And (5) copies/mu l to obtain the product.
Compared with the prior art, the invention has the following beneficial effects:
according to the primer group for detecting the novel coronavirus, the specific conserved regions of the ORF gene and the N gene of the novel coronavirus are utilized, the target nucleotide primer and the probe are designed aiming at the conserved regions, the amplification primer pair is designed according to the characteristics of constant-temperature amplification reaction, a target gene fragment can be efficiently amplified under the conditions of constant temperature and high speed, and the detection precision is not lost.
On the basis of recombinase polymerase amplification technology, the kit for detecting the novel coronavirus disclosed by the invention applies recombinase capable of combining single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB), strand displacement DNA polymerase and optimized reaction Buffer, and compared with common real-time fluorescence PCR, the time for completing constant-temperature rapid detection can be shortened to 20min from 100-120 min, so that the detection efficiency is greatly improved.
Moreover, the kit for detecting the novel coronavirus is applicable to detection equipment with a fluorescence capture function, such as fluorescent quantitative PCR instruments and constant-temperature fluorescent amplification instruments of various brands, the result is easy to judge, real-time observation can be realized, the reaction time is short, the reaction time is only 20min, the kit has the characteristics of quick constant temperature and simple and convenient POCT product operation, is more time-saving than the conventional RT-qPCR method, can judge suspected cases in a short time, is convenient for taking necessary prevention and control and treatment measures in time in clinic, improves the cure rate of severe cases, and has great significance for entry and exit inspection, quarantine, epidemic disease monitoring, prevention and control and the like.
Drawings
FIG. 1 is a graph showing the amplification results of a primer probe combination test for ORF gene portions, the concentration of the novel coronavirus nucleic acid being 1.0 × 102copies/μL;
FIG. 2 is a graph showing the amplification results of the primer probe combination test for N gene portion, the concentration of the novel coronavirus nucleic acid is 1.0 × 102copies/μL;
FIG. 3 shows that the 10-fold dilution concentrations of the novel coronavirus nucleic acid standard substance were 1.0X 103、1.0×102、1.0×101、1.0×100The amplification result chart of the ORF gene of copies/. mu.L;
FIG. 4 shows that the 10-fold dilution concentrations of the novel coronavirus nucleic acid standard substance were 1.0X 103、1.0×102、1.0×101、1.0×100A graph showing the result of N gene amplification of copies/. mu.L;
FIG. 5 shows the samples were all diluted to 1.0 × 103copies/μ L endemic human coronavirus (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus (H1N1), influenza A virus (H3N2), influenza B virus, respiratory syncytial virus, human adenovirus (ADV3), human adenovirus (ADV7), human adenovirus (ADV55), human parainfluenza virus, Coxsackie virus B, EB virus, mumps virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Diplococcus meningitidis, rubella virus, etcA diagram of the detection result of ORF genes of the original culture or the pseudovirus (the positive amplification curve in the diagram is a positive control curve);
FIG. 6 shows the samples were all diluted to 1.0 × 103A test result graph of N genes of pathogen cultures or pseudoviruses such as copies/μ L endemic human coronaviruses (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus (H1N1), influenza A virus (H3N2), influenza B virus, respiratory syncytial virus, human adenovirus (ADV3), human adenovirus (ADV7), human adenovirus (ADV55), human parainfluenza virus, Coxsackie virus B, EB virus, mumps virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Diplococcus meningitidis, rubella virus and the like (a positive amplification curve in the graph is a positive control curve);
FIG. 7 shows the dilution of the novel coronavirus standard substance to 1.0 × 103ORF gene amplification result chart of 10 times of repeated detection of copies/mu L template;
FIG. 8 shows the dilution of the novel coronavirus standard substance to 5.0 × 101ORF gene amplification result chart of 10 times of repeated detection of copies/mu L template;
FIG. 9 shows the dilution of the novel coronavirus standard substance to 1.0 × 103A graph of the result of N gene amplification of 10 repeated detections of copies/mu L template;
FIG. 10 shows the dilution of the novel coronavirus standard substance to 5.0 × 101A graph of the result of N gene amplification of 10 repeated detections of copies/mu L template;
FIG. 11 is a graph showing the results of ORF gene amplification in 3 cases of clinical specimen detection.
FIG. 12 is a graph showing the results of amplification of N gene in 3 cases of clinical specimens.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
And (3) screening and preparing primers and probes.
Referring to the diagnosis and treatment guidelines of the novel coronavirus, nucleic acid detection is a gold standard for judging the confirmed diagnosis of infection of the novel coronavirus, and 2 regions of ORF gene and N gene of the novel coronavirus conserved region are selected for detection in order to ensure the detection of the novel coronavirus.
One of the keys of the invention is to design a primer probe based on a recombinase polymerase amplification technology, and the primer and probe design of the technology cannot be carried out by auxiliary software and only can depend on manual design. In order to ensure the amplification speed and the detection sensitivity, the length of the primer should be controlled to be 30-35 bp, the length of the fluorescent probe should be controlled to be 46-52 bp, the design of the primer probe is particularly important, and the success or failure of the whole experiment is concerned.
The base modification in the probe is mainly concentrated at the middle rear part of the sequence, and the Spacer appears in the middle of the probe, so that the probe is more complex than a probe used for real-time fluorescent RT-PCR, and the successful design not only needs to be designed according to experience, but also needs a large amount of experimental verification to obtain a primer and a probe which are more reliable, high in sensitivity and good in specificity. Therefore, in order to obtain primer probes with good specificity and sensitivity and suitable for the recombinase polymerase amplification technology, 12 probes of 60 primers are designed at a plurality of different positions of ORF genes and N genes, and 162 primer-probe combination tests are carried out. As the content is excessive, 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 and fig. 2.
Table 1. partial primers and probes designed in this example
Based on the above-mentioned numerous works, the combinations of primers and probes for ORF gene and N gene which are excellent in specificity and high in amplification efficiency are finally selected and selected as shown in Table 2.
TABLE 2 primers and probes used in the present invention
Specifically, the 31 st base T in the ORF gene probe shown in SEQ ID NO.5 is marked with a fluorescent reporter group selected from the following groups: FAM, HEX, JOE, CY3, CY5, ROX, Texas Red, 34 th base G marks a dSpacer (tetrahydrofuran, THF) as an exonuclease recognition site, and 35 th base T marks a quenching group selected from the following group: TAMARA, BHQ1, BHQ2 and CY5, and a modification group marked at the 3' end can be an amino group, a phosphate group and a C3-spacer.
The 31 st basic group T in the N gene probe shown in SEQ ID NO.6 is marked with a fluorescent reporter group selected from the following groups: FAM, HEX, JOE, CY3, CY5, ROX, Texas Red, 33 th base A marks a dSpacer (tetrahydrofuran, THF) as an exonuclease recognition site, and 34 th base T marks a quenching group selected from the following group: TAMARA, BHQ1, BHQ2 and CY5, and a modification group marked at the 3' end can be an amino group, a phosphate group and a C3-spacer.
Example 2
A kit for detecting a novel coronavirus, comprising:
reagent I (1.8 mL/tube): reaction buffer.
Reagent II (150 μ l/tube): 280mmol/L magnesium acetate.
Reagent iii (lyophilized): single-stranded nucleic acid-binding recombinase (115 ng/. mu.L), single-stranded DNA-binding protein (90 ng/. mu.L), strand-displacement DNA polymerase (30 ng/. mu.L), dNTPs (10mmol), and the primers and probes shown in Table 2 of example 1 (the concentration of the primer pair is 0.4. mu. mol/L, and the concentration of the probe is 0.12. mu. mol/L).
Positive quality control (500. mu.l/tube): physiological saline.
negative quality control material (500. mu.l/tube) including pseudovirus containing target fragment of ORF gene (SEQ ID No:31) of novel coronavirus and pseudovirus containing target fragment of N gene (SEQ ID No:32) of novel coronavirus, wherein the concentration of the pseudoviruses is 1.0 × 103~1.0×105copies/μl。
Example 3
A detection method for a novel coronavirus isothermal nucleic acid rapid detection kit.
Using the kit of example 2, detection was carried out in the following manner.
1. Specimen type: nasopharyngeal swab, alveolar lavage fluid.
2. Nucleic acid extraction:
adopting a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on a silica gel membrane centrifugal column method or a nucleic acid extraction reagent based on a magnetic bead method, operating according to the kit specification, finally collecting 80 mu l of RNA solution, and directly detecting or storing at-80 ℃. The negative quality control product and the positive quality control product are extracted.
3. And (3) amplification reaction:
the complete detection system of each tube should include 37.5. mu.l of reagent I, 2.5. mu.l of reagent II, reagent III (lyophilized), and 10. mu.l of quality control substance or nucleic acid of the sample to be tested.
The detection can be specifically carried out according to the following operation steps:
3.1 reagent preparation: adding 37.5 mul of reagent I and 2.5 mul of reagent II into each tube of reagent III (freeze-drying), or calculating the total amount of the required reagent I and reagent II according to the total reaction number N (including the number of samples to be detected, 1 part of negative quality control product and 1 part of positive quality control product) required by detection, mixing uniformly and then packaging into PCR reagent III (freeze-drying).
3.2 sample adding: adding a negative quality control product into the PCR reagent III (freeze-drying) which is subpackaged with the reagents, adding a positive quality control product, adding a sample RNA solution, wherein the sample adding amount is 10 mu l, tightly covering a tube cover, uniformly mixing, centrifuging, collecting the solution, and placing the solution at the bottom of the tube.
3.3 detection by amplification on machine: the ABI 7500 fluorescence quantitative PCR instrument and the software version V2.4 are adopted for detection, the operation module of the selection instrument is 7500(96Wells), the experiment type is quantitization-Standard Curve, and the reagent type is TaqMan Reagents. Four New Targets are set under the Define Targets and Samples sub-interface of the "Plate Setup" interface, and the Target Name, Reporter, Quencher and Colour are respectively: N/ORF, FAM/HEX, None, default. Under the sub-interface of 'Assigne Targets and Samples', selecting a hole site with sample adding as a positive quality control product, setting Task as 'S' (Standard), setting a hole site of the Negative quality control product as 'N' (Negative), and setting a sample adding hole site of a sample to be detected as 'U' (unknown). Continuing to set the "Graphical View" to "cycling stage" under the "Run Method" interface, 42 ℃ for 30sec (default collected fluorescence); the Number of Cycles is 40. Default settings are used at the "Reaction Setup" and "Materials List" interfaces. And saving the file after all the settings are finished, and clicking 'START' to RUN under the 'RUN' main interface.
3.4 analysis of results:
and after the reaction is finished, storing the detection data file, checking the curve condition of each sample, and checking the corresponding detection result in the sample hole. Reaction wells with obvious amplification curves are positive results, and reaction wells without obvious amplification curves are negative results.
Example 4
This example performed sensitivity detection.
The RNA transcripts of the novel coronavirus important characteristic gene nucleocapsid protein N gene (full length) and the gene segment of open reading frame 1ab (ORF1ab) (genome coordinates: 132011-15910, GenBank No. NC-045512) are obtained by in vitro transcription by carrying out sensitivity detection by using the novel coronavirus nucleic acid standard substance (GBW (E)091089) to transcribe the RNA transcripts in vitro. And (3) determining copy number concentration of ORF1ab gene and N gene by an absolute quantitative method-digital PCR method to obtain a standard value of the standard substance.
Diluting the novel coronavirus nucleic acid standard substancediluting to appropriate concentration by 10 times, the concentrations are 1.0 × 103、1.0×102、1.0×101、1.0×100copies/μL。
ORF1ab gene and N gene detection were carried out on 4 gradients of standard dilution using the reaction system and amplification conditions determined in example 3, respectively, and the results showed sensitivity data of 1.0 × 100copies/. mu.L, which shows that the detection method has higher sensitivity, and the detection results are shown in FIG. 3 and FIG. 4.
Example 5
This example carried out a specific assay.
respectively detecting and diluting to 1.0 × 103copies/μ L endemic human coronavirus (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza a virus (H1N1), influenza a virus (H3N2), influenza b virus, respiratory syncytial virus, human adenovirus (ADV3), human adenovirus (ADV7), human adenovirus (ADV55), human parainfluenza virus, coxsackie virus B, EB virus, mumps virus, mycoplasma pneumoniae, chlamydia pneumoniae, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, diplococcus meningitidis, rubella virus and other pathogen cultures or pseudoviruses, and the detection results are shown in fig. 5 and 6. ORF and N gene detection results show that endemic human coronavirus (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus (H1N1), influenza A virus (H3N2), influenza B virus, respiratory syncytial virus, human adenovirus (ADV3), human adenovirus (ADV7), human adenovirus (ADV55), human parainfluenza virus, coxsackie virus B, EB virus, mumps virus, mycoplasma pneumoniae, chlamydia pneumoniae, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, diplococcus meningitidis and rubella virus are all negative without amplification, which indicates that the kit has good specificity.
Example 6
This example was conducted for repetitive detection.
diluting the new coronavirus standard substance to 1.0 × 103copies/μL、5.0×101Two concentrations of copies/. mu.L, using the reagent of example 2The kit and the experimental conditions of example 3, the ORF1ab gene and the N gene were subjected to the above-mentioned 10 repeated detections using 2 dilution templates, and the detection results are shown in table 3 and fig. 7, fig. 8, fig. 9, and fig. 10, indicating that the kit of the present invention has good reproducibility.
TABLE 3 results of the reproducibility measurements
1.0×103copies/μL 5.0×101copies/μL
ORF1ab gene 10 tests, 10 positive results 10 tests, 10 positive results
N gene 10 tests, 10 positive results 10 tests, 10 positive results
Example 7
This example performed clinical specimen testing.
Selecting 3 nasopharyngeal swab samples which are confirmed to be novel coronavirus by clinical and virus culture methods, extracting nucleic acid from the samples, performing on-machine amplification detection and result analysis according to the step of the example 2, and simultaneously performing negative and positive quality control product detection.
The detection result shows that the negative quality control product has no amplification curve, and the corresponding channels of the positive quality control product ORF1ab gene and the N gene both present obvious amplification curves, which indicates that the detection result is effective. The detection results of the clinical samples of the 3 cases are shown in FIGS. 11 and 12, and are all positive, and the results are consistent with the isolated culture identification results, which shows that the constant temperature rapid kit can be used for detecting the novel coronavirus ORF1ab gene and N gene.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<213> Artificial Sequence (Artificial Sequence)
<400>8
tagaggtatg gtacttggta gtttagctgc 30
<210>9
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
catcttaaca caattagtga ttggttgtcc 30
<210>10
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gctagataat ctttgtaagc tttagcagca tc 32
<210>11
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
cgtctacaag ctggtaatgc aacagaagtg cctgccaatt caactgt 47
<210>12
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
aacacagtct gtaccgtctg cggtatgtgg 30
<210>13
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
cacaggcact agtactgatg tcgtatacag 30
<210>14
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gattgtccag ctgttgctaa acatgacttc 30
<210>15
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
atagacggtg acatggtacc acatatatca c 31
<210>16
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gtatgtgaca agtatttctt ttaatgtgtc ac 32
<210>17
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
ctaagttggc gtatacgcgt aatatatctg gg 32
<210>18
<211>49
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caacgtctta ctaaatacac aatggcagac ctcgtctatg ctttaaggc 49
<210>19
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
gtgctacaac ttcctcaagg aacaacattg 30
<210>20
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
ccattctagc aggagaagtt cccctactgc 30
<210>21
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
ctgctgctga ggcttctaag aagcctcggc 30
<210>22
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
gaaatctgct gctgaggctt ctaagaagcc 30
<210>23
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
tcagttcctt gtctgattag ttcctggtcc 30
<210>24
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
gcggccaatg tttgtaatca gttccttgtc 30
<210>25
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
catacaatgt aacacaagct ttcggcagac gtggtccaga acaaaccc 48
<210>26
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
taccgcagag acagaagaaa cagcaaactg 30
<210>27
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
agcaaactgt gactcttctt cctgctgcag 30
<210>28
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
agcccatctg ccttgtgtgg tctgcatgag 30
<210>29
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
acgtttatat agcccatctg ccttgtgtgg 30
<210>30
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
ggatgatttc tccaaacaat tgcaacaatc catgagcagt gctgactc 48
<210>31
<211>501
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
atcgtgttgt ctgtactgcc gttgccacat agatcatcca aatcctaaag gattttgtga 60
cttaaaaggt aagtatgtac aaatacctac aacttgtgct aatgaccctg tgggttttac 120
acttaaaaac acagtctgta ccgtctgcgg tatgtggaaa ggttatggct gtagttgtga 180
tcaactccgc gaacccatgc ttcagtcagc tgatgcacaa tcgtttttaa acgggtttgc 240
ggtgtaagtg cagcccgtct tacaccgtgc ggcacaggca ctagtactga tgtcgtatac 300
agggcttttg acatctacaa tgataaagta gctggttttg ctaaattcct aaaaactaat 360
tgttgtcgct tccaagaaaa ggacgaagat gacaatttaa ttgattctta ctttgtagtt 420
aagagacaca ctttctctaa ctaccaacat gaagaaacaa tttataattt acttaaggat 480
tgtccagctg ttgctaaaca t 501
<210>32
<211>1260
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260

Claims (10)

1. A primer set for detecting a novel coronavirus, comprising: the primer pair is characterized by comprising a novel coronavirus ORF gene specific primer pair and a novel coronavirus N gene specific primer pair, wherein the ORF gene specific primer pair is as follows:
a forward primer: 5'-GTATGTGGAAAGGTTATGGCTGTAGTTGTG-3' (SEQ. ID. No: 1);
reverse primer: 5'-GTGCCGCACGGTGTAAGACGGGCTGCACTTAC-3' (SEQ. ID. No: 2);
the N gene specific primer pair is as follows:
a forward primer: 5'-AACATTGCCAAAAGGCTTCTACGCAGAAGG-3' (SEQ. ID. No: 3);
reverse primer: 5'-GGAGAAGTTCCCCTACTGCTGCCTGGAGTT-3' (SEQ. ID. No: 4).
2. The primer set for detecting a novel coronavirus according to claim 1, further comprising a novel coronavirus ORF gene-specific probe and a novel coronavirus N gene-specific probe; the ORF gene specific probe is: 5'-TAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGC-3' (SEQ. ID. No: 5); the N gene specific probe is as follows: 5'-AGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTA GTCGC-3' (SEQ. ID. No: 6).
3. The primer set for detecting a novel coronavirus according to claim 2, wherein the ORF gene-specific probe labels tetrahydrofuran at 34bp from the 5 'end, T bases upstream of the tetrahydrofuran site label FAM fluorophore, T bases downstream of the tetrahydrofuran site label BHQ1 fluorophore, and C3-spacer modifier at the 3' end;
the N gene specific probe is used for marking tetrahydrofuran at a position 34bp away from a 5 'end, marking FAM fluorescent group at an upstream T base of a tetrahydrofuran site, marking BHQ1 fluorescent group at a downstream T base of the tetrahydrofuran site, and marking a C3-spacer modifying group at a 3' end.
4. Use of the primer set for detecting a novel coronavirus according to any one of claims 1 to 3 as an isothermal amplification detection reagent in the detection of a novel coronavirus.
5. The use of claim 4, wherein the conditions for the isothermal amplification assay are: reacting at a constant temperature of 39-42 ℃ for 18-20 min.
6. A kit for detecting a novel coronavirus, comprising: a recombinase that binds to a single-stranded nucleic acid, a single-stranded DNA binding protein, a strand displacement DNA polymerase, dNTPs, and the primer set according to any one of claims 1 to 3.
7. The kit for detecting the novel coronavirus according to claim 6, wherein the concentration of the ORF gene specific primer pair and the concentration of the N gene specific primer pair are both 0.4-0.6 μmol/L, the concentration of the ORF gene specific probe and the concentration of the N gene specific probe are both 0.12-0.15 μmol/L, and the dosage of the dNTP is 6-10 mmol.
8. The kit for detecting a novel coronavirus according to claim 6, wherein the concentration of the single-stranded nucleic acid-binding recombinase is 100 to 120ng/μ L, the concentration of the single-stranded DNA-binding protein is 80 to 100ng/μ L, and the concentration of the strand displacement DNA polymerase is 30 to 35ng/μ L.
9. The kit for detecting a novel coronavirus according to claim 6, further comprising: reagent I: a reaction buffer; and (2) reagent II: 280mM magnesium acetate.
10. the kit for detecting a novel coronavirus according to claim 6, further comprising a negative quality control substance and a positive quality control substance, wherein the negative quality control substance is physiological saline, the positive quality control substance is pseudovirus comprising a target fragment of ORF gene of the novel coronavirus and pseudovirus comprising a target fragment of N gene of the novel coronavirus, and the concentration of the pseudoviruses is 1.0 × 103~1.0×105copies/μl。
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