CN111500777A - Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method - Google Patents
Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method Download PDFInfo
- Publication number
- CN111500777A CN111500777A CN202010359815.4A CN202010359815A CN111500777A CN 111500777 A CN111500777 A CN 111500777A CN 202010359815 A CN202010359815 A CN 202010359815A CN 111500777 A CN111500777 A CN 111500777A
- Authority
- CN
- China
- Prior art keywords
- pcr
- kit
- fluorescent
- novel coronavirus
- nucleic acid
- 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.)
- Pending
Links
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 61
- 238000003757 reverse transcription PCR Methods 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 42
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 26
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 26
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 58
- 239000000523 sample Substances 0.000 claims abstract description 44
- 230000035945 sensitivity Effects 0.000 claims abstract description 16
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 238000002474 experimental method Methods 0.000 claims description 18
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000013641 positive control Substances 0.000 claims description 13
- 238000010839 reverse transcription Methods 0.000 claims description 13
- 125000006853 reporter group Chemical group 0.000 claims description 9
- 239000013642 negative control Substances 0.000 claims description 7
- 238000010791 quenching Methods 0.000 claims description 7
- 230000000171 quenching effect Effects 0.000 claims description 7
- 108091032955 Bacterial small RNA Proteins 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 6
- 238000000338 in vitro Methods 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 4
- -1 BHQ1 Chemical compound 0.000 claims description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000013612 plasmid Substances 0.000 claims description 4
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 101710116435 Outer membrane protein Proteins 0.000 claims description 3
- 102000006382 Ribonucleases Human genes 0.000 claims description 3
- 108010083644 Ribonucleases Proteins 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000013518 transcription Methods 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 241000700605 Viruses Species 0.000 description 7
- 101150013191 E gene Proteins 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 102100034349 Integrase Human genes 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000012268 genome sequencing Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 210000002345 respiratory system Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 241000008904 Betacoronavirus Species 0.000 description 1
- 208000001528 Coronaviridae Infections Diseases 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 206010047924 Wheezing Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for detecting novel coronavirus nucleic acid based on a fluorescent RT-PCR method, which comprises a microcarrier, a primer pair and a probe; the microcarrier has coding information corresponding to coronavirus one by one, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, and the probe sequence is shown as SEQ ID NO:3, respectively. The invention also discloses a method for detecting the novel coronavirus. The kit has high sensitivity and strong specificity; the detection method is rapid and high in accuracy.
Description
Technical Field
The invention relates to virus detection, in particular to a kit for detecting novel coronavirus nucleic acid based on a fluorescence RT-PCR method.
Background
The novel coronavirus belongs to the family of coronaviridae and the genus beta coronavirus, and is a novel coronavirus which is epidemic and identified and found in 12 months in 2019 to 1 month in 2020. The new coronavirus can cause human diseases and can be transmitted between human and animals, and symptoms of the new coronavirus infection include fever, wheeze, pneumonia and the like.
Because the novel coronavirus is a brand new type coronavirus identified and discovered in 2020, no specific detection method exists at present, the virus causes severe respiratory infection of human respiratory tract, and has certain infectivity, and patients who are diagnosed need isolation treatment, a molecular method capable of rapidly and accurately identifying the novel coronavirus is urgently needed to be developed, so that the aims of effectively controlling epidemic spread of epidemic situation and rapidly diagnosing isolation treatment of patients are fulfilled.
RT-PCR (Reverse Transcription-Polymerase Chain Reaction) is a technique that combines Reverse Transcription (RT) of RNA with Polymerase Chain amplification (PCR) of cDNA. Firstly, cDNA is synthesized from RNA under the action of reverse transcriptase, and then the target fragment is amplified and synthesized under the action of DNA polymerase by taking the cDNA as a template. The RT-PCR technology is sensitive and has wide application, and can be used for detecting the gene expression level in cells, the content of RNA viruses in the cells and directly cloning cDNA sequences of specific genes.
Therefore, a kit and a method for detecting novel coronavirus based on fluorescent RT-PCR detection technology are expected to effectively solve the defects in the prior art.
Disclosure of Invention
The invention mainly aims to overcome the defects of slow detection rate and low detection accuracy in the existing virus detection, and provides a kit for detecting novel coronavirus nucleic acid based on a fluorescent RT-PCR method. The kit disclosed by the invention is high in detection speed and accuracy and has a wide application prospect.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.
The invention provides a kit for detecting novel coronavirus nucleic acid based on a fluorescent RT-PCR method, which comprises a microcarrier, a primer pair and a probe; the microcarrier has coding information corresponding to coronavirus one by one, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, and the probe sequence is shown as SEQ ID NO:3, respectively.
In some embodiments of the invention, the nucleic acid of the probe is linked at the 5 'end to a fluorescent reporter group, and the nucleic acid is linked at the 3' end to a fluorescent quencher group, and the fluorescent reporter groups of the two probes are different.
In some embodiments of the invention, the fluorescent reporter is selected from any one of FAM, VIC, ROX, CY3, and CY 5.
In some embodiments of the invention, the fluorescence quenching group is selected from any one of TAMRA, BHQ1, BHQ2, and NFQ.
In some embodiments of the invention, the kit further comprises a PCR buffer and a Taq enzyme.
In some embodiments of the invention, the kit further comprises a positive control and a negative control.
In some embodiments of the invention, the positive control fluid is an amplified sequence comprising the novel coronavirus.
In some embodiments of the invention, the negative control is RNase Free H2O。
The object of the present invention and the technical problems solved thereby can be further achieved by the following technical measures.
The invention provides a novel RT-PCR detection method of coronavirus, which comprises the following steps:
first, RT-PCR primer and probe design
Based on the obtained gene sequence of the novel coronavirus outer membrane protein, 2 primers and 1 probe for establishing a detection method were designed using software Primer Premier 5, and the sequences of the primers and the probes are shown in SEQ ID NO: 1-3:
secondly, collecting samples, constructing recombinant plasmids containing detection target segments and preparing small RNA segments serving as positive control in vitro, and then carrying out in vitro transcription to obtain the small RNA segments;
third, specificity experiment and sensitivity detection experiment of primer
1) Specificity detection
Performing RT-PCR experiment by using RNA of coronavirus as a template; RT-PCR and PCR reaction conditions and procedures were as follows:
after being mixed uniformly, the mixture is reacted for 1h at 42 ℃, and then quickly placed on ice after being reacted for 15min at 70 ℃, and then the obtained reverse transcription product is used as a template for PCR amplification, wherein the reaction system and the reaction program are as follows:
the PCR procedure was: 15min at 60 ℃; at 95 ℃ for 10s, at 92 ℃ for 30s, at 60 ℃ for 60s, for 45 cycles; 5min at 72 ℃;
2) sensitivity detection experiment
The sensitivity of the method is detected, 2 mul of RNA with different concentration gradients as positive control is taken as a template to carry out RT-PCR, reverse transcription and PCR conditions and procedures with the same specificity experiment, and then 1.5 percent agarose gel electrophoresis is used for detecting the result.
Compared with the prior art, the invention has the obvious advantages and beneficial effects that: the invention utilizes the fluorescent RT-PCR technology to detect the novel coronavirus, and based on the primer pair and the probe sequence designed by the invention, the existence of the novel coronavirus can be rapidly detected, and under the condition of multiple test verifications, the accuracy is high, and the time and the efficiency are saved. The detection method can be used for directly detecting the RNA sample, is simple to operate, can realize the purpose of rapid detection, and shortens the detection time. Meanwhile, the effective sample amount can be increased, and the detection sensitivity is improved.
In conclusion, the special kit for detecting the novel coronavirus nucleic acid based on the fluorescent RT-PCR method has high detection accuracy and is rapid. The detection device has the advantages and practical values, does not have similar design publication or use in similar products, is innovative, has great improvement on the product or function, has great technical progress, produces good and practical effects, has multiple enhanced effects compared with the existing detection products, is more suitable for practical use, has industrial wide utilization value, and is a novel, advanced and practical new design.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given of preferred embodiments of the present invention with reference to the accompanying drawings.
The specific kit and the detection method of the present invention are given in detail by the following examples and the accompanying drawings.
Drawings
FIG. 1 is a fluorescent amplification curve of one embodiment of the detection kit of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
One embodiment of the invention provides a kit for detecting novel coronavirus nucleic acid based on a fluorescent RT-PCR method, which comprises a microcarrier, a primer pair and a probe; the microcarrier has coding information corresponding to coronavirus one by one, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, and the probe sequence is shown as SEQ ID NO:3, respectively. The nucleic acid of the probe is connected with a fluorescence reporter group at the 5 'end, the nucleic acid of the probe is connected with a fluorescence quenching group at the 3' end, and the fluorescence reporter groups of the two probes are different. Preferably, the fluorescent reporter group is selected from any one of FAM, VIC, ROX, CY3 and CY 5. The fluorescence quenching group is selected from any one of TAMRA, BHQ1, BHQ2 and NFQ.
In some embodiments, the above-mentioned components may constitute an RT-PCR reaction system with other components, or may themselves comprise components necessary for an RT-PCR reaction for performing fluorescent RT-PCR detection. When the components are independently used as an RT-PCR reaction system, the kit comprises a PCR buffer solution and Taq enzyme besides a primer pair SEQ ID NO. 1 and SEQ ID NO. 2 and a probe SEQ ID NO. 3, and the volume can be supplemented by adding water, wherein the PCR buffer solution contains magnesium ions and dNTP.
In some embodiments of the invention, the kit further comprises components required for reverse transcription, including dNTPMix, 5 × Primescript buffer, RNase inhibitor and Primescript reverse transcriptase, and when detection is performed, the RNA of the sample to be detected can be added into the reverse transcription component system and the PCR system respectively to perform RT-PCR detection, and whether the detection result is positive or not is determined according to the detection result.
In some embodiments, the primer and probe sequences are:
upstream primer SEQIDNO 15 '-ATCTAGTAGCAACTATAGTGCATTC-3'
Downstream primer SEQIDNO 25 '-GCAGTGCCTATGCTGAGCAA-3'
The probe SEQIDNO is 35 '-FAM-TGTATCAGCTCAGATGCTGAC-3' -NFQ,
in some embodiments, the fluorescent group attached to the 5' end of the probe sequence may be FAM or VIC fluorescent group, and other suitable fluorescent groups may be selected, such as ROX, CY3 and CY 5. The quenching group NFQ connected to the 3' end of the probe sequence may be a quenching group known to those skilled in the art, or other suitable groups may be selected, including but not limited to TAMRA, BHQ1, BHQ 2. When the fluorescent group at the 5 ' end of the probe and the quenching group at the 3 ' end are close to each other, the fluorescent reporter group can not emit fluorescence, but the fluorescent group at the 5 ' end falls off along with the hydrolysis of the probe along with the progress of PCR amplification reaction, so that the fluorescent group can emit fluorescence, and the unknown template can be quantitatively analyzed by detecting the accumulation of fluorescent signals.
In some embodiments, the kit further comprisesPositive and negative controls were included. The positive control is a standard substance of the novel coronavirus; negative control was RNase Free H2O。
The detection kit provided by the invention is simple and convenient to operate, has a short detection period, and realizes rapid detection of a sample.
An embodiment of the present invention also provides a detection method for a novel coronavirus, which is performed by using the detection kit provided by the present invention, and the detection method comprises the following steps:
first, RT-PCR primer and probe design
Based on the obtained gene sequence of the novel coronavirus outer membrane protein, 2 primers and 1 probe for establishing a detection method were designed using software Primer Premier 5, and the sequences of the primers and the probes are shown in SEQ ID NO:1 to 3.
Secondly, collecting samples, constructing recombinant plasmids containing detection target segments and preparing small RNA segments serving as positive control in vitro, and then carrying out in vitro transcription to obtain the small RNA segments;
third, specificity experiment and sensitivity detection experiment of primer
1) Specificity detection
Performing RT-PCR experiment by using RNA of coronavirus as a template; RT-PCR and PCR reaction conditions and procedures were as follows:
after being mixed uniformly, the mixture is reacted for 1h at 42 ℃, and then quickly placed on ice after being reacted for 15min at 70 ℃, and then the obtained reverse transcription product is used as a template for PCR amplification, wherein the reaction system and the reaction program are as follows:
the PCR procedure was: 15min at 60 ℃; at 95 ℃ for 10s, at 92 ℃ for 30s, at 60 ℃ for 60s, for 45 cycles; 5min at 72 ℃;
2) sensitivity detection experiment
The sensitivity of the method is detected, 2 mul of RNA with different concentration gradients as positive control is taken as a template to carry out RT-PCR, reverse transcription and PCR conditions and procedures with the same specificity experiment, and then 1.5 percent agarose gel electrophoresis is used for detecting the result.
The kit and the detection method are as follows:
EXAMPLE 1 preparation of fluorescent RT-PCR kit
1. In the embodiment, an obtained E gene sequence of the specific coding envelope protein of the novel coronavirus is selected to design a pair of specific primers and a specific fluorescent probe, and a fluorescent RT-PCR technology is constructed to detect the novel coronavirus. The E gene is an important structural gene of the coronavirus, and the primer and the probe designed by the invention are only 100% similar to the E gene of the novel coronavirus and are not matched with other coronaviruses, so that the primer and the probe can be specifically combined with the E gene of the novel coronavirus and initiate amplification, and other types of coronaviruses are not amplified.
The primer sequences and probe sequences designed in this embodiment of the present invention are as follows:
upstream primer SEQIDNO 15 '-ATCTAGTAGCAACTATAGTGCATTC-3'
Downstream primer SEQIDNO 25 '-GCAGTGCCTATGCTGAGCAA-3'
The probe SEQIDNO is 35 '-FAM-TGTATCAGCTCAGATGCTGAC-3' -NFQ,
the kit also comprises a positive control: a novel coronavirus standard; negative control: RNaseFree H2O；
2. Fluorescent RT-PCR detection method for novel coronavirus
(1) Extraction of viral nucleic acids
The extraction of viral nucleic acid was carried out using the RNA extraction Kit EZ-press RNA Purification Kit (from EZBioscience) according to the Kit instructions. 200 parts of nucleic acid of a throat swab specimen of a suspected respiratory virus infected patient is extracted by a method of an instruction.
(2) Reverse transcription of RNA
Performing RT-PCR experiment by using RNA of coronavirus as a template; RT-PCR and PCR reaction conditions and procedures were as follows:
after mixing evenly, the mixture reacts for 1h at 42 ℃, and then quickly placed on ice after the reaction is stopped for 15min at 70 ℃.
(3) Fluorescent PCR
The obtained reverse transcription product is used as a template for PCR amplification, and the reaction system and the reaction program are as follows:
the PCR procedure was: 15min at 60 ℃; at 95 ℃ for 10s, at 92 ℃ for 30s, at 60 ℃ for 60s, for 45 cycles; 5min at 72 ℃;
2) sensitivity detection experiment
The sensitivity of the method is detected, 2 mul of RNA with different concentration gradients as positive control is taken as a template to carry out RT-PCR, reverse transcription and PCR conditions and procedures with the same specificity experiment, and then 1.5 percent agarose gel electrophoresis is used for detecting the result.
The method constructed above is used for detecting 200 parts of throat swab specimens of suspected respiratory virus infected patients, and 6 positive cases of novel coronavirus are detected.
EXAMPLE 2 Performance determination of fluorescent RT-PCR kits for novel coronaviruses
1. Accuracy verification
The gold standard for virus nucleic acid detection is genome sequencing, and the detection result of the kit is compared with the virus genome sequencing to analyze the accuracy of the detection result. In this example, 6 specimens determined to be novel coronaviruses by genome sequencing were selected, and the results of detection using the kit provided by the present invention are shown in table 1 below. As can be seen from the results, 6 positive samples were detected, indicating that the accuracy of the fluorescent RT-PCR of the novel coronavirus provided by the present invention is 100%.
TABLE 1 accuracy analysis of the kits of the invention
| Viral types | Number of examples | Positive in sequencing | The method is positive | Accuracy (%) |
| Novel coronavirus | 6 | 6 | 4 | 100 |
2. Specificity verification
The specificity of the kit was evaluated by detecting other pathogens, and 36 positive specimens of respiratory tract and other common pathogens or plasmid-mimic positive specimens were selected in this example, with the results shown in table 2 below. Through detection, the invention has no amplification on 36 respiratory tract and other common pathogen positive specimens, and shows that the specificity of the fluorescence RT-PCR of the novel coronavirus provided by the invention is 100%.
TABLE 2 specificity analysis of the kit of the invention
3. Sensitivity detection
The sensitivity, i.e., the lowest detection limit, is the probability statistically > 95% that a target nucleic acid will be detected in the same sample at the lowest dilution gradient after the positive sample is diluted with the gradient. The number of detections of the sample for sensitivity assessment at each concentration level to be assessed should be not less than 20, and at least 19 positive amplification signals are qualified.
After the positive standard plasmid of the novel coronavirus is diluted according to a certain copy number multiple ratio, each dilution is averagely divided into 20 samples, the detection is carried out by the method, the copy number of the positive samples which are 19 times or more is the lowest detection limit, and the result is shown in table 3.
TABLE 3 sensitivity analysis of the kit of the invention
The invention provides a fluorescent RT-PCR detection primer pair, a probe, a kit and a detection method of the novel coronavirus, which not only shorten the operation time, reduce the pollution, but also reduce the cost of sample diagnosis and have potential application value.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A kit for detecting novel coronavirus nucleic acid based on a fluorescent RT-PCR method comprises a microcarrier, a primer pair and a probe; the microcarrier has coding information corresponding to coronavirus one by one, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, and the probe sequence is shown as SEQ ID NO:3, respectively.
2. The kit for detecting the novel coronavirus nucleic acid based on the fluorescent RT-PCR method as claimed in claim 1, wherein the nucleic acid of the probe is connected with a fluorescent reporter group at the 5 'end, and the fluorescent quencher group at the 3' end, and the fluorescent reporter groups of the two probes are different.
3. The kit for detecting novel coronavirus nucleic acid based on the fluorescent RT-PCR method according to claim 1, wherein the fluorescent reporter group is selected from any one of FAM, VIC, ROX, CY3 and CY 5.
4. The kit for detecting novel coronavirus nucleic acid based on the fluorescent RT-PCR method according to claim 1, wherein the fluorescence quenching group is selected from any one of TAMRA, BHQ1, BHQ2 and NFQ.
5. The kit for detecting novel coronavirus nucleic acid based on the fluorescent RT-PCR method according to claim 1, wherein the kit further comprises a PCR buffer and Taq enzyme.
6. The kit for detecting novel coronavirus nucleic acid based on the fluorescent RT-PCR method according to claim 1, wherein the kit further comprises a positive control and a negative control.
7. The kit for detecting novel coronavirus nucleic acid based on the fluorescent RT-PCR method according to claim 6, wherein the positive control solution is an amplification sequence containing the novel coronavirus.
8. The kit for detecting novel coronavirus nucleic acid based on fluorescent RT-PCR method according to claim 6, wherein the negative control is RNase Free H2O。
9. A method for detecting a novel coronavirus through RT-PCR, which comprises the following steps:
first, RT-PCR primer and probe design
Based on the obtained gene sequence of the novel coronavirus outer membrane protein, 2 primers and 1 probe for establishing a detection method were designed using software Primer Premier 5, and the sequences of the primers and the probes are shown in SEQ ID NO: 1-3:
secondly, collecting samples, constructing recombinant plasmids containing detection target segments and preparing small RNA segments serving as positive control in vitro, and then carrying out in vitro transcription to obtain the small RNA segments;
third, specificity experiment and sensitivity detection experiment of primer
1) Specificity detection
Performing RT-PCR experiment by using RNA of coronavirus as a template; RT-PCR and PCR reaction conditions and procedures were as follows:
after being mixed uniformly, the mixture is reacted for 1h at 42 ℃, and then quickly placed on ice after being reacted for 15min at 70 ℃, and then the obtained reverse transcription product is used as a template for PCR amplification, wherein the reaction system and the reaction program are as follows:
the PCR procedure was: 15min at 60 ℃; at 95 ℃ for 10s, at 92 ℃ for 30s, at 60 ℃ for 60s, for 45 cycles; 5min at 72 ℃;
2) sensitivity detection experiment
The sensitivity of the method is detected, 2 mul of RNA with different concentration gradients as positive control is taken as a template to carry out RT-PCR, reverse transcription and PCR conditions and procedures with the same specificity experiment, and then 1.5 percent agarose gel electrophoresis is used for detecting the result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010359815.4A CN111500777A (en) | 2020-04-30 | 2020-04-30 | Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010359815.4A CN111500777A (en) | 2020-04-30 | 2020-04-30 | Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111500777A true CN111500777A (en) | 2020-08-07 |
Family
ID=71867932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010359815.4A Pending CN111500777A (en) | 2020-04-30 | 2020-04-30 | Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111500777A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982943A (en) * | 2020-03-03 | 2020-04-10 | 中山大学达安基因股份有限公司 | Novel coronavirus RT-PCR detection method and kit |
| CN111074005A (en) * | 2020-01-17 | 2020-04-28 | 西安博睿康宁生物医学中心有限公司 | Double-target-site reverse transcription fluorescence PCR primer, probe and kit for detecting 2019 novel coronavirus |
-
2020
- 2020-04-30 CN CN202010359815.4A patent/CN111500777A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111074005A (en) * | 2020-01-17 | 2020-04-28 | 西安博睿康宁生物医学中心有限公司 | Double-target-site reverse transcription fluorescence PCR primer, probe and kit for detecting 2019 novel coronavirus |
| CN110982943A (en) * | 2020-03-03 | 2020-04-10 | 中山大学达安基因股份有限公司 | Novel coronavirus RT-PCR detection method and kit |
Non-Patent Citations (4)
| Title |
|---|
| CORMAN VM等: "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR" * |
| HUANG C等: "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China" * |
| WANG H等: "The genetic sequence, origin, and diagnosis of SARS-CoV-2" * |
| 吴建国等: "重型/危重型新型冠状病毒肺炎患者多种生物样本核酸检测结果分析" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| WO2021174984A1 (en) | Rt-pcr detection method and kit for novel coronavirus | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| CN112063756B (en) | Method and kit for multiple detection of respiratory virus nucleic acid | |
| CN111500776A (en) | Novel coronavirus 2019-nCoV fluorescent RPA detection primer, probe, kit and method | |
| CN113462820A (en) | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof | |
| CN113930547B (en) | RT-RAA fluorescence detection primer pair, kit and detection method for porcine epidemic diarrhea virus N gene | |
| CN111471800B (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| CN110527747B (en) | Kit for detecting wild strains of classical swine fever viruses | |
| CN111500777A (en) | Kit for detecting novel coronavirus nucleic acid based on fluorescence RT-PCR method | |
| CN116814859A (en) | Primer probe composition, kit and method for identifying African swine fever virus genes I and II | |
| CN111676316A (en) | Primer, probe and detection method for rapidly distinguishing African swine fever virus gene type II from other genotypes | |
| CN107937615B (en) | Primers and probes for distinguishing wild strains and vaccine strains of swine Japanese encephalitis virus | |
| CN110643740A (en) | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for Palimam serogroup virus | |
| CN111549175B (en) | Novel multi-fluorescence RT-PCR detection reagent for detecting port input 2019 coronavirus | |
| CN111647683A (en) | New coronavirus 2019-nCoV nucleic acid detection kit and application thereof | |
| CN114807432B (en) | Kit and method for rapidly detecting novel coronavirus and Delta mutant strain thereof | |
| CN114736990B (en) | Primer probe set and kit for detecting Alapplication virus and enteritis virus of minks based on double microdroplet digital PCR | |
| CN115323076B (en) | Primer probe composition, kit and detection method for detecting monkey D-type retrovirus | |
| CN114317835B (en) | Multiplex PCR detection primer set, kit and detection method for waterfowl parvovirus, duck enteritis virus and goose astrovirus | |
| CN114752709A (en) | Universal fluorescent quantitative PCR (polymerase chain reaction) detection primer and kit for porcine epidemic diarrhea virus and application | |
| CN116814852A (en) | Multiple nucleic acid composition for detecting canine parvovirus, kit development method and application thereof | |
| CN116287477A (en) | Universal fluorescent quantitative PCR detection primer probe set for detecting human circovirus in typing mode, kit and application | |
| CN113151603A (en) | Porcine encephalomyocarditis virus fluorescent quantitative PCR detection kit and detection method thereof | |
| CN115747376A (en) | RPA-based rapid combined detection kit for multiple influenza viruses |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200807 |
|
| WD01 | Invention patent application deemed withdrawn after publication |