CN113151590A - Novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof - Google Patents
Novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof Download PDFInfo
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Abstract
The invention discloses a novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and a preparation method and a detection method thereof, wherein the preparation method comprises the following steps: specific primers 2019-nCoV-1ab-F, nCoV-1ab-R and a probe CoV-1 ab-P; a specific primer 2019-nCoV-N-F, nCoV-N-R and a probe nCoV-N-P; specific primers 2019-nCoV-E-F, nCoV-E-R and a probe nCoV-E-P; the invention designs a multi-target fluorescent PCR detection primer aiming at three conserved genes (1 ab, N and E genes) of 2019-nCoV, is a more accurate, simple, convenient and rapid diagnostic reagent, has great advantages in epidemiology investigation and application, and is beneficial to epidemic disease prevention and control, so that early treatment is carried out to avoid causing larger economic loss.
Description
Technical Field
The invention relates to a novel coronavirus fluorescent PCR detection method, in particular to a novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof, and belongs to the technical field of bioengineering.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is an infectious Disease of people caused by coronavirus 2019-nCoV, and the main symptoms are fever, hypodynamia, cough and breath obstruction, and in more serious cases, severe acute respiratory syndrome, renal failure and even death can be caused. The main transmission modes are droplet transmission and contact transmission, and are susceptible to any age group. Because no effective vaccine prevention and treatment exists, it is important to establish a rapid and accurate detection method.
The TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of the TaqMan fluorescent probe carries a fluorescent group, such as FAM, TET, VIC, HEX and the like, and the 3' end of the TaqMan fluorescent probe carries a quenching group, such as TAMRA, BHQ and the like. During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, when the probe is complete, a fluorescent signal emitted by a reporter group is absorbed by a quenching group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter 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 TaqMan method has the advantages of high specificity, good repeatability and high price, and is only suitable for a specific target. The TaqMan probe method fluorescent quantitative PCR is suitable for pathogen detection, disease drug resistance gene research, drug efficacy assessment and genetic disease diagnosis.
Currently, many units at home and abroad develop fluorescent PCR kits aiming at 2019-nCoV, but most of the kits only aim at one or two genes for detection, false negative may exist, and epidemic situation monitoring is not facilitated.
Based on the method, according to a novel pneumonia diagnosis and treatment scheme infected by coronavirus, the conserved ORF1ab, N and E genes of 2019-nCoV are selected as target genes, specific primers and probes are designed, and a 2019-nCoV specific fluorescent PCR detection method is established. The novel coronavirus (2019-nCoV) nucleic acid constant-temperature fluorescence detection kit only needs about 5H from DNA extraction to detection result in the whole detection process, so that the manual operation is greatly reduced, and the time is shortened. And the invention can detect a plurality of samples at one time, and is particularly suitable for detecting a large number of samples.
Disclosure of Invention
The invention aims to provide a novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and a preparation method and a detection method thereof, so that the rapid detection of 2019-nCoV is realized.
In order to realize the aim, the invention provides a novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and a preparation method and a detection method thereof,
the technical scheme is as follows:
the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises: specific primers 2019-nCoV-1ab-F, 2019-nCoV-1ab-R and probes 2019-nCoV-1 ab-P; specific primers 2019-nCoV-N-F, 2019-nCoV-N-R and probes 2019-nCoV-N-P; specific primers 2019-nCoV-E-F, 2019-nCoV-E-R and probes 2019-nCoV-E-P; the reporter fluorescent dyes marked by the probe are respectively FAM, VIC and NED, and the marked fluorescence quenching groups are BHQ; wherein the specific primer and probe sequences are as follows:
①2019-nCoV-1ab-F:5′- CCACATATATCACGTCAAC-3′；
2019-nCoV-1ab-R:5′- GTCACAATTACCTTCATCA-3′；
2019-nCoV-1ab-P:5- FAM-AATACACAATGGCAGACCTCGT-BHQ-3′；
②2019-nCoV-N-F:5′- GCAACAGTTCAAGAAATTC-3′；
2019-nCoV-N-R:5′- CTGGTTCAATCTGTCAAG-3′；
2019-nCoV-N-P:5- VIC-AAGCAAGAGCAGCATCACCG-BHQ-3′；
③2019-nCoV-E-F:5′- TCGTGGTATTCTTGCTAG-3′；
2019-nCoV-E-R:5′- CCAGAAGATCAGGAACTC-3′；
2019-nCoV-E-P:5- NED-ACACTAGCCATCCTTACTGCG-BHQ-3′。
the detection kit further comprises: fluorescent PCR reaction solution, ROX, primers, probes, sterilized water, negative control and positive control;
the fluorescent PCR reaction solution contains reverse transcriptase, exonuclease, dNTP and magnesium ions; the primers comprise 6 primers as described in claim 1; the probes comprise 3 probes as described in claim 1; the positive control is a plasmid containing the target gene sequences of ORF1ab, N and E of the novel coronavirus; the negative control was an empty vector plasmid.
The preparation method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps:
(1) artificially synthesizing target gene sequences of open reading frames 1a/b, N and E in a 2019-nCoV novel coronavirus genome, cloning the target gene sequences onto a pUC57 vector, and extracting plasmids as positive control after pure culture of escherichia coli engineering bacteria;
(2) preparation of primer group and probe:
according to ORF1ab, N and E gene target sequences, respectively designing specific primers 2019-nCoV-1ab-F and 2019-nCoV-1ab-R and a probe 2019-nCoV-1 ab-P; 2019-nCoV-N-F and 2019-nCoV-N-R, and probe 2019-nCoV-N-P; 2019-nCoV-E-F and 2019-nCoV-E-R, and probe 2019-nCoV-E-P;
labeling the 5 'ends of the probes with FAM, VIC and NED fluorophores, respectively, and labeling the 3' ends of all the probes with BHQ1-dT quencher; when the probe is kept complete, a fluorescence signal excited by the 5 'end fluorescence reporter group is absorbed by the 3' end quenching group, and no fluorescence signal change is emitted; when a target gene exists in a reaction system, a specific nucleic acid fragment can be amplified, and the fluorescent probe can be hybridized according to the base pairing principle; when PCR enters an extension period, Taq enzyme starts from the 3' end of a primer and moves along a DNA template along with the extension of a new strand, and when the Taq enzyme moves to a probe combination position, exonuclease activity in a reagent is used for cracking the probe, so that a fluorescent group and a quenching group are separated, and a fluorescent signal is generated;
(3) the fluorescent PCR reaction solution is commercially prepared.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps:
1) and (3) optimizing the reaction system:
| composition (I) | Dosage of |
| 2019-nCoV fluorescent | 5 μL |
| Primer | 0.5. mu.L each |
| Probe | 0.25. mu.L each |
| ROX（40 μM） | 0.05 μL |
| Sample | 2 μL |
| Sterilized water | Make up to 20. mu.L |
2) And (3) optimizing the reaction conditions:
reaction conditions are as follows: (Instrument: ABI7500 Fast)
3) And (3) judging rules:
the instrument directly reads the detection result, and the baseline and threshold settings are adjusted according to the noise condition of the instrument;
the effective standard of the experiment is as follows:
positive control ORF1ab, N and E genes generate an amplification curve (Ct value is less than 30), and negative control has no amplification curve;
the above conditions should be met at the same time, otherwise, the test is regarded as invalid.
4) And (4) judging a result:
under the condition that the test is established, ORF1ab and E genes are preferentially analyzed and judged according to the following rule:
the results are illustrated in detail in the description of FIG. 1.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit is characterized in that 3-target fluorescence PCR information of the detection method is summarized as follows:
the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following table after a primer probe sequence is optimized:
| name (R) | Sequence (5 '-3') | Size and breadth |
| ORF1ab-F | CCACATATATCACGTCAAC | 87bp |
| ORF1ab-R | GTCACAATTACCTTCATCA | |
| ORF1ab-probe | FAM-AATACACAATGGCAGACCTCGT-BHQ | |
| N-F | GCAACAGTTCAAGAAATTC | 116bp |
| N-R | CTGGTTCAATCTGTCAAG | |
| N-probe | VIC-AAGCAAGAGCAGCATCACCG-BHQ | |
| E-F | TCGTGGTATTCTTGCTAG | 156bp |
| E-R | CCAGAAGATCAGGAACTC | |
| E-probe | NED-ACACTAGCCATCCTTACTGCG-BHQ |
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, the dosage of ROX is properly adjusted according to a fluorescence PCR instrument which is actually used; 0.05 μ L of ROX (40 μm) added here was the recommended amount for ABI7500/7500Fast fluorescent PCR instrument; if the instrument is ABI StepOne series, 0.5 μ L ROX (40 μm) is added; the RotorQ series instrument does not require the addition of ROX.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps of when a fluorescence signal is collected, detecting fluorescence selection: ORF1ab gene (FAM); e gene (NED); n gene (VIC); if the quantitative PCR instrument is ABI series, ROX is selected as reference fluorescence; if the instrument is of the Rotor series, ROX is not required as a reference.
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, if amplification occurs in the negative control, pollution is likely to exist in the operation process, and the experiment is carried out after the experiment environment is changed.
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, the suspected sample is recommended to be rechecked; if the Ct values of the ORF1ab and the E gene in the retest are both less than or equal to 38, the result is judged to be positive; otherwise, the result is judged to be negative.
The invention has the beneficial effects that:
compared with the prior art, the invention designs the multi-target fluorescent PCR detection primer aiming at the three conserved genes (1 ab, N and E genes) of 2019-nCoV, is a more accurate, simple and rapid diagnostic reagent, has great advantages in epidemiology investigation and application, and is beneficial to epidemic disease prevention and control, so that early treatment is carried out to avoid causing larger economic loss.
Drawings
FIG. 1 is a schematic diagram illustrating the determination of the detection result according to the present invention.
FIG. 2 is a schematic diagram showing the validity of fluorescent PCR detection of ORF1ab probe 1 in example 1.
FIG. 3 is a schematic diagram showing the validity of fluorescent PCR detection of ORF1ab probe 2 in example 1.
FIG. 4 is a schematic diagram showing the validity of fluorescent PCR detection of ORF1ab probe 3 in example 1.
FIG. 5 is a schematic diagram showing the validation of the fluorescent PCR detection of the N gene probe 1 in example 2.
FIG. 6 is a schematic diagram showing the validation of the fluorescent PCR detection of the N gene probe 2 in example 2.
FIG. 7 is a schematic diagram showing the validity verification of the fluorescent PCR detection of the E gene probe 1CY3 in example 3.
FIG. 8 is a schematic diagram showing the validation of the E gene probe 1NED fluorescence PCR detection in example 3.
FIG. 9 is a schematic diagram of the sensitivity experiment of multiplex fluorescence PCR of ORF1ab, N and E genes.
FIG. 10 is a schematic diagram of the technical route of the present invention.
FIG. 11 is a schematic diagram showing the validation of multiplex fluorescent PCR for ORF1ab, N and E genes.
FIG. 12 is a schematic diagram of the N gene-duplication experiment of the present invention.
FIG. 13 is a schematic diagram of the E gene-duplication experiment of the present invention.
FIG. 14 is a schematic diagram of ORF1ab gene-repeat experiments according to the present invention.
FIG. 15 is a reference diagram of the sequence alignment of the present invention.
Detailed Description
The present invention is described in further detail below, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
Referring to the 77 genotype reference strain sequences of the published 2019-nCoV, screening conserved regions of ORF1ab, N and E genes, and designing specific primers and probes.
New corona strain reference sequence number: NC _045512.2
Source of sequence data: NCBI Genbank (International Biogene database, Website: https:// www.ncbi.nlm.nih.gov /).
Sequence integrity: the standard used in this project is a plasmid, a primer pair is designed according to conserved fragments of ORF1ab gene, N gene and E gene of sequence NC _045512, and a corresponding sequence fragment (PUC 57 plasmid) is synthesized by Biotechnology engineering (Shanghai) GmbH, and the synthesized sequence fragment information is as follows:
ORF1ab gene:
CCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGT。
gene:
GCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTC。
gene:
CTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTA
a sequence table: the sequence alignment is described in the specification and shown in figure 15.
The invention (II) is implemented as follows:
the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises: specific primers 2019-nCoV-1ab-F, 2019-nCoV-1ab-R and probes 2019-nCoV-1 ab-P; specific primers 2019-nCoV-N-F, 2019-nCoV-N-R and probes 2019-nCoV-N-P; specific primers 2019-nCoV-E-F, 2019-nCoV-E-R and probes 2019-nCoV-E-P; the reporter fluorescent dyes marked by the probe are respectively FAM, VIC and NED, and the marked fluorescence quenching groups are BHQ; wherein the specific primer and probe sequences are as follows:
①2019-nCoV-1ab-F:5′- CCACATATATCACGTCAAC-3′；
2019-nCoV-1ab-R:5′- GTCACAATTACCTTCATCA-3′；
2019-nCoV-1ab-P:5- FAM-AATACACAATGGCAGACCTCGT-BHQ-3′；
②2019-nCoV-N-F:5′- GCAACAGTTCAAGAAATTC-3′；
2019-nCoV-N-R:5′- CTGGTTCAATCTGTCAAG-3′；
2019-nCoV-N-P:5- VIC-AAGCAAGAGCAGCATCACCG-BHQ-3′；
③2019-nCoV-E-F:5′- TCGTGGTATTCTTGCTAG-3′；
2019-nCoV-E-R:5′- CCAGAAGATCAGGAACTC-3′；
2019-nCoV-E-P:5- NED-ACACTAGCCATCCTTACTGCG-BHQ-3′。
the detection kit further comprises: fluorescent PCR reaction solution, ROX, primers, probes, sterilized water, negative control and positive control;
the fluorescent PCR reaction solution contains reverse transcriptase, exonuclease, dNTP and magnesium ions; the primers comprise 6 primers as described in claim 1; the probes comprise 3 probes as described in claim 1; the positive control is a plasmid containing the target gene sequences of ORF1ab, N and E of the novel coronavirus; the negative control was an empty vector plasmid.
The preparation method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps:
(1) artificially synthesizing target gene sequences of open reading frames 1a/b, N and E in a 2019-nCoV novel coronavirus genome, cloning the target gene sequences onto a pUC57 vector, and extracting plasmids as positive control after pure culture of escherichia coli engineering bacteria;
(2) preparation of primer group and probe:
according to ORF1ab, N and E gene target sequences, respectively designing specific primers 2019-nCoV-1ab-F and 2019-nCoV-1ab-R and a probe 2019-nCoV-1 ab-P; 2019-nCoV-N-F and 2019-nCoV-N-R, and probe 2019-nCoV-N-P; 2019-nCoV-E-F and 2019-nCoV-E-R, and probe 2019-nCoV-E-P;
labeling the 5 'ends of the probes with FAM, VIC and NED fluorophores, respectively, and labeling the 3' ends of all the probes with BHQ1-dT quencher; when the probe is kept complete, a fluorescence signal excited by the 5 'end fluorescence reporter group is absorbed by the 3' end quenching group, and no fluorescence signal change is emitted; when a target gene exists in a reaction system, a specific nucleic acid fragment can be amplified, and the fluorescent probe can be hybridized according to the base pairing principle; when PCR enters an extension period, Taq enzyme starts from the 3' end of a primer and moves along a DNA template along with the extension of a new strand, and when the Taq enzyme moves to a probe combination position, exonuclease activity in a reagent is used for cracking the probe, so that a fluorescent group and a quenching group are separated, and a fluorescent signal is generated;
(3) the fluorescent PCR reaction solution is commercially prepared.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps:
1) and (3) optimizing the reaction system:
| composition (I) | Dosage of |
| 2019-nCoV fluorescent | 5 μL |
| Primer | 0.5. mu.L each |
| Probe | 0.25. mu.L each |
| ROX（40 μM） | 0.05 μL |
| Sample | 2 μL |
| Sterilized water | Make up to 20. mu.L |
2) And (3) optimizing the reaction conditions:
reaction conditions are as follows: (Instrument: ABI7500 Fast)
3) And (3) judging rules:
the instrument directly reads the detection result, and the baseline and threshold settings are adjusted according to the noise condition of the instrument;
the effective standard of the experiment is as follows:
positive control ORF1ab, N and E genes generate an amplification curve (Ct value is less than 30), and negative control has no amplification curve;
the above conditions should be met at the same time, otherwise, the test is regarded as invalid.
4) And (4) judging a result:
under the condition that the test is established, ORF1ab and E genes are preferentially analyzed and judged according to the following rule:
the results are illustrated in detail in the description of FIG. 1.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit is characterized in that 3-target fluorescence PCR information of the detection method is summarized as follows:
the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following table after a primer probe sequence is optimized:
| name (R) | Sequence (5 '-3') | Size and breadth |
| ORF1ab-F | CCACATATATCACGTCAAC | 87bp |
| ORF1ab-R | GTCACAATTACCTTCATCA | |
| ORF1ab-probe | FAM-AATACACAATGGCAGACCTCGT-BHQ | |
| N-F | GCAACAGTTCAAGAAATTC | 116bp |
| N-R | CTGGTTCAATCTGTCAAG | |
| N-probe | VIC-AAGCAAGAGCAGCATCACCG-BHQ | |
| E-F | TCGTGGTATTCTTGCTAG | 156bp |
| E-R | CCAGAAGATCAGGAACTC | |
| E-probe | NED-ACACTAGCCATCCTTACTGCG-BHQ |
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, the dosage of ROX is properly adjusted according to a fluorescence PCR instrument which is actually used; 0.05 μ L of ROX (40 μm) added here was the recommended amount for ABI7500/7500Fast fluorescent PCR instrument; if the instrument is ABI StepOne series, 0.5 μ L ROX (40 μm) is added; the RotorQ series instrument does not require the addition of ROX.
The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit comprises the following steps of when a fluorescence signal is collected, detecting fluorescence selection: ORF1ab gene (FAM); e gene (NED); n gene (VIC); if the quantitative PCR instrument is ABI series, ROX is selected as reference fluorescence; if the instrument is of the Rotor series, ROX is not required as a reference.
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, if amplification occurs in the negative control, pollution is likely to exist in the operation process, and the experiment is carried out after the experiment environment is changed.
According to the detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit, the suspected sample is recommended to be rechecked; if the Ct values of the ORF1ab and the E gene in the retest are both less than or equal to 38, the result is judged to be positive; otherwise, the result is judged to be negative.
The kit provided by the invention can be used for detecting novel coronavirus nucleic acid (2019-nCoV), and can be used for detecting the novel coronavirus (2019-nCoV) nucleic acid in samples such as throat swabs, blood or serum of suspected infected persons.
(III) sensitivity verification: diluting the positive control plasmid by a multiple ratio of 10 < -1 > to 10 < -6 >, detecting according to the method in the step (2), and verifying the sensitivity of the kit.
(IV) specificity verification: and (3) detecting nucleic acids of the bovine coronavirus, the porcine epidemic diarrhea virus, the transmissible gastroenteritis virus, the avian infectious bronchitis virus and the canine respiratory coronavirus according to the method in the step (2), and verifying the specificity of the kit.
The name of the technical scheme is as follows: novel coronavirus (2019-nCoV) ORF1ab, N and E gene detection kit (fluorescence PCR method)
2. The technical scheme comprises the following contents: the novel coronavirus pneumonia has the characteristics of strong outbreak, strong infectivity, high concealment, strong pathogenicity and the like. A great deal of resources are invested in clinical and basic research on the detection and treatment of 2019-nCoV at home and abroad. The project designs a multi-target fluorescent PCR detection primer aiming at three conserved genes (1 ab, N and E genes) of 2019-nCoV, and aims to develop a more accurate, simple, convenient and rapid diagnostic reagent.
Intended target
3, targeting: ORF1ab/N/E, obtaining stable primer pair and probe
Sample collection to detection time: about 3h
Minimum detection limit: 500 copies
Accuracy: domestic leading level
4. The technical scheme is briefly introduced:
the technical scheme is that a time axis is planned:
5. the technical route of the technical scheme
See the description of fig. 10 in detail
2019-nCoV ORF1ab, N and E gene fluorescence PCR single gene detection
6. Example 1: 2019-nCoV ORF1ab fluorescence PCR detection:
the experimental method comprises the following steps: primer design → plasmid standard construction → preliminary validation. (Instrument: ABI7500 fast)
Designing a primer: 3 pairs of primer probes are designed according to the sequence conserved region of 2019-nCoV ORF1ab published on NCBI, and the probes are all FAM markers.
Reaction system:
| reagent | Dosage of |
| 2×One Step RT-PCR buffer | 10μL |
| Ex Taq HS | 0.5μL |
| PrimeScript RT Enzyme Mix | 0.5μL |
| Primer-F/R (10μM) | Each 1 mu L |
| Probe (10μM) | 0.5μL |
| Rox Dye (4μM) | 0.5μL |
| Nucleic acids | 2μL |
| Sterilized water | Make up to 20. mu.L |
Reaction conditions are as follows: 10min at 42 ℃ and 10s at 95 ℃, 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
2019-nCoV ORF1ab gene fluorescence PCR validity verification
See figures 2-4 of the specification for details.
Validation results of Probe 1, Probe 2, and Probe 3
2019-nCoV ORF1ab, the probes 1 and 2 pass the validity verification (both the primer probe and the standard product are valid), the probe 3 does not pass the validity verification, and the ORF1ab, the probe 1 and the probe 2 are adopted for the next step of experiments.
Example 2: 2019-nCoV N gene fluorescence PCR detection
The experimental method comprises the following steps: primer design → plasmid standard construction → preliminary validation. (Instrument: ABI7500 fast)
Designing a primer: 2 pairs of primer probes are designed according to a 2019-nCoV N gene sequence conserved region published on NCBI, and the probes are all VIC markers.
Reaction system:
| reagent | Dosage of |
| 2×One Step RT-PCR buffer | 10μL |
| Ex Taq HS | 0.5μL |
| PrimeScript RT Enzyme Mix | 0.5μL |
| Primer-F/R (10μM) | Each 1 mu L |
| Probe (10μM) | 0.5μL |
| Rox Dye (4μM) | 0.5μL |
| Nucleic acids | 2μL |
| Sterilized water | Make up to 20. mu.L |
Reaction conditions are as follows: 10min at 42 ℃ and 10s at 95 ℃, 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
2019-nCoV N gene fluorescence PCR detection validity verification:
see figures 5-6 of the specification for details.
2019-nCoV N gene probe 1 and probe 2 pass validity verification (both the primer probe and the standard substance are valid), and the N gene probe 1 and probe 2 are adopted to carry out the next step of experiment.
Example 3: 2019-nCoV E gene fluorescence PCR detection
The experimental method comprises the following steps: primer design → plasmid standard construction → preliminary validation. (Instrument: ABI7500 fast)
Designing a primer: 1 pair of primer probes are designed according to 2019-nCoV E gene sequence conserved region published on NCBI
Reaction system:
| reagent | Dosage of |
| 2×One Step RT-PCR buffer | 10μL |
| Ex Taq HS | 0.5μL |
| PrimeScript RT Enzyme Mix | 0.5μL |
| Primer-F/R (10μM) | Each 1 mu L |
| Probe (10μM) | 0.5μL |
| Rox Dye (4μM) | 0.5μL |
| Nucleic acids | 2μL |
| Sterilized water | Make up to 20. mu.L |
Reaction conditions are as follows: 10min at 42 ℃ and 10s at 95 ℃, 1 cycle; 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles.
2019-nCoV E gene fluorescence PCR detection validity verification
See figures 7-8 of the specification for details.
Gene probe 1-Cy3 labeling (left) and NED labeling (right) validity verification results
The effectiveness of the 2019-nCoV E gene probe 1 (marked by Cy3 and marked by NED) is verified to pass (both the primer probe and the standard product are effective), and the amplification effect of the NED marked probe is better than that of the Cy3 marked probe. The next experiment was performed using NED-labeled E gene probe 1.
Small knot
Successfully screening 2019-nCoV ORF1ab gene, N gene and E gene detection primers and probes, and successfully preparing a standard substance.
The effectiveness test is detected through a single target.
The experimental results provide technical support for the next step of establishing multi-target detection.
Information summarization after target fluorescence PCR optimization:
10. and (3) optimizing the reaction system:
| composition (I) | Dosage of |
| 2019-nCoV fluorescent | 5 μL |
| Primer & Probe Mix | 5.25μL |
| ROX（40 μM） | 0.05 μL* |
| Sample | 2 μL |
| Sterilized water | Make up to 20. mu.L |
The dose of ROX was adjusted appropriately according to the actual fluorescence PCR instrument used. 0.05 μ L of ROX (40 μm) was added here as recommended by ABI7500/7500Fast fluorescent PCR instrument.
If the instrument is ABI StepOne series, 0.5 μ L ROX (40 μm) is added; the RotorQ series instrument does not require the addition of ROX.
And reaction conditions after optimization are as follows:
reaction conditions are as follows: (Instrument: ABI7500 Fast)
Detection of fluorescence selection: ORF1ab gene (FAM); e gene (NED); n gene (VIC)
If the quantitative PCR instrument is ABI series, ROX is selected as reference fluorescence; if the instrument is of the Rotor series, ROX is not required as a reference.
And (3) judging rules:
the instrument directly reads the detection result, and the baseline and threshold settings are adjusted according to the noise condition of the instrument.
Experimental validation criteria:
the positive control ORF1ab, N and E genes produced amplification curves (Ct values < 30), and the negative control had no amplification curve.
The above conditions should be met at the same time, otherwise, the test is regarded as invalid.
If the negative control is amplified, there may be contamination during the operation, please replace the experimental environment and then perform the experiment.
And judging the result:
under the condition that the test was established, ORF1ab and E genes were preferentially analyzed and judged according to the following table rule
For suspicious samples, a review is recommended. If the Ct values of the ORF1ab and the E gene in the retest are both less than or equal to 38, the result is judged to be positive; otherwise, the result is judged to be negative.
Illustration of the results
See the attached figure 1 of the specification for details.
2019-nCoV ORF1ab, N and E gene multiplex fluorescence PCR detection
The experimental method comprises the following steps: and respectively premixing the screened primer probes into a reaction system for primary verification and effectiveness verification. (Instrument: ABI7500 fast)
Reaction system:
| reagent | Dosage of |
| 2019-nCoV fluorescent PCR reaction solution (One-step Taq Mix) | 5μL |
| Primer-F/R | Each 1 mu L |
| Probe | 0.1. mu.L each |
| Rox Dye (4μM) | 0.5μL |
| Nucleic acids | 2μL |
| Sterilized water | Make up to 20. mu.L |
Reaction conditions are as follows: 2min at 25 ℃; 10min at 53 ℃; 2mi at 95 ℃; 95 ℃ for 3s, 60 ℃ for 30s, 40 cycles.
2019-nCoV ORF1ab, and N and E gene multiplex fluorescence PCR validity verification:
3 the effectiveness of the target fluorescence PCR experiment was passed (see FIG. 11). By comparison, the combination effect of the E-P1-NED, N-P2-VIC and ORF1ab-P1-FAM primer probe is better than that of the other three groups.
2019-nCoV ORF1ab, and N and E gene multiplex fluorescence PCR annealing temperature optimization:
reaction reagents: one-step multiplex fluorescence PCRmix;
reaction conditions are as follows: according to the instruction, 20 mul of reaction system, 1 mul of primer and 0.1 mul of probe are respectively carried out; the annealing temperatures were 60 ℃, 64 ℃ and 68 ℃, respectively.
Sample preparation: a positive plasmid standard; negative control was water
As a result: 60 ℃ is the optimal annealing extension reaction temperature.
2019-nCoV ORF1ab, N and E gene multiplex fluorescence PCR sensitivity experiment:
and carrying out sensitivity experiments by using the optimized experimental conditions.
Sample preparation: ORF1ab, N, E plasmid standards (mixed), and the standards were diluted in gradient.
Reagent: 2019-nCoV fluorescent PCR reaction solution.
The instrument comprises the following steps: ABI7500 fast.
As a result:
the lower detection limit of ORF1ab is 101 copies/. mu.L;
the lower limit of N detection is 101 copies/mu L;
the lower limit of E detection is 100 copies/. mu.L.
The amplification effect of the E gene is better than that of ORF1ab gene and N gene, but when the content of the standard substance in the sample is higher than 101 copies/. mu.L, 3 genes can be detected positively. Therefore, the lower limit of the detection of the method 2019-nCoV is preliminarily determined to be 101 copies/mu L.
2019-nCoV ORF1ab, N and E gene multiplex fluorescence PCR sensitivity experiment:
see figure 9 of the specification for details.
2019-nCoV ORF1ab, and multiple fluorescent PCR repeated experiments of N and E genes:
reaction conditions are as follows: optimized system and program
Sample preparation: ORF1ab, N, E plasmid standards (mix), and the standards were diluted in gradient 107-104 copies/. mu.L.
Experiment design: each sample was replicated 5 times in batch to make 3 batches
Reagent: 2019-nCoV fluorescent PCR reaction solution.
The instrument comprises the following steps: ABI7500 fast.
As a result: PASS
| Index (I) | ORF1ab | N | E |
| Inter-batch difference (CV%) | ＜7.0 | ＜6.0 | ＜2.5 |
| Poor batch (CV%) | ＜12.0 | ＜11.0 | ＜1.5 |
19. 2019-nCoV ORF1ab, multiplex fluorescence PCR repeatability experiment for N and E genes-E gene:
see figure 12 of the specification for details.
2019-nCoV ORF1ab, multiplex fluorescence PCR repeatability experiment for N and E genes-N gene:
see figure 13 of the specification for details.
2019-nCoV ORF1ab, multiplex fluorescence PCR repeatability experiment of N and E genes-ORF 1ab gene:
see figure 14 of the specification for details.
2019-nCoV ORF1ab, N and E gene multiplex fluorescent PCR premixed primer probe experiment
ORF1ab, N and E gene fluorescent PCR primers and probes are premixed according to the optimal proportion, and the mixture is stored at-20 ℃ for a period of time and then effectiveness evaluation is carried out
As a result: PASS
ORF1ab, N and E gene fluorescent PCR primers and probes can be premixed according to the optimal proportion, so that the detection steps are simplified, the time is saved for clinical detection, and the preparation of the kit is facilitated
23. Decision rule
The instrument directly reads the detection result, and the baseline and threshold settings are adjusted according to the noise condition of the instrument.
The effective standard of the experiment is as follows:
the positive control ORF1ab, N and E genes produced amplification curves (Ct values < 30), and the negative control had no amplification curve.
The above conditions should be met at the same time, otherwise, the test is regarded as invalid.
If the negative control is amplified, there may be contamination during the operation, please replace the experimental environment and then perform the experiment.
And judging the result:
under the condition that the test was established, ORF1ab and E genes were preferentially analyzed and judged according to the following table rule
For suspicious samples, a review is recommended. If the Ct value of the retest is less than or equal to 38, judging the result as positive; otherwise, the result is judged to be negative.
Results legend:
see the attached figure 1 of the specification for details.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, which is characterized in that: the method comprises the following steps: specific primers 2019-nCoV-1ab-F, 2019-nCoV-1ab-R and probes 2019-nCoV-1 ab-P; specific primers 2019-nCoV-N-F, 2019-nCoV-N-R and probes 2019-nCoV-N-P; specific primers 2019-nCoV-E-F, 2019-nCoV-E-R and probes 2019-nCoV-E-P; the reporter fluorescent dyes marked by the probe are respectively FAM, VIC and NED, and the marked fluorescence quenching groups are BHQ; wherein the specific primer and probe sequences are as follows:
①2019-nCoV-1ab-F:5′- CCACATATATCACGTCAAC-3′；
2019-nCoV-1ab-R:5′- GTCACAATTACCTTCATCA-3′；
2019-nCoV-1ab-P:5- FAM-AATACACAATGGCAGACCTCGT-BHQ-3′；
②2019-nCoV-N-F:5′- GCAACAGTTCAAGAAATTC-3′；
2019-nCoV-N-R:5′- CTGGTTCAATCTGTCAAG-3′；
2019-nCoV-N-P:5- VIC-AAGCAAGAGCAGCATCACCG-BHQ-3′；
③2019-nCoV-E-F:5′- TCGTGGTATTCTTGCTAG-3′；
2019-nCoV-E-R:5′- CCAGAAGATCAGGAACTC-3′；
2019-nCoV-E-P:5- NED-ACACTAGCCATCCTTACTGCG-BHQ-3′。
2. the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit according to claim 1, wherein the kit comprises: the detection kit further comprises: fluorescent PCR reaction solution, ROX, primers, probes, sterilized water, negative control and positive control;
the fluorescent PCR reaction solution contains reverse transcriptase, exonuclease, dNTP and magnesium ions; the primers comprise 6 primers as described in claim 1; the probes comprise 3 probes as described in claim 1; the positive control is a plasmid containing the target gene sequences of ORF1ab, N and E of the novel coronavirus; the negative control was an empty vector plasmid.
3. The method for preparing the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit according to claim 1, which comprises the following steps:
(1) artificially synthesizing target gene sequences of open reading frames 1a/b, N and E in a 2019-nCoV novel coronavirus genome, cloning the target gene sequences onto a pUC57 vector, and extracting plasmids as positive control after pure culture of escherichia coli engineering bacteria;
(2) preparation of primer group and probe:
according to ORF1ab, N and E gene target sequences, respectively designing specific primers 2019-nCoV-1ab-F and 2019-nCoV-1ab-R and a probe 2019-nCoV-1 ab-P; 2019-nCoV-N-F and 2019-nCoV-N-R, and probe 2019-nCoV-N-P; 2019-nCoV-E-F and 2019-nCoV-E-R, and probe 2019-nCoV-E-P;
labeling the 5 'ends of the probes with FAM, VIC and NED fluorophores, respectively, and labeling the 3' ends of all the probes with BHQ1-dT quencher; when the probe is kept complete, a fluorescence signal excited by the 5 'end fluorescence reporter group is absorbed by the 3' end quenching group, and no fluorescence signal change is emitted; when a target gene exists in a reaction system, a specific nucleic acid fragment can be amplified, and the fluorescent probe can be hybridized according to the base pairing principle; when PCR enters an extension period, Taq enzyme starts from the 3' end of a primer and moves along a DNA template along with the extension of a new strand, and when the Taq enzyme moves to a probe combination position, exonuclease activity in a reagent is used for cracking the probe, so that a fluorescent group and a quenching group are separated, and a fluorescent signal is generated;
(3) the fluorescent PCR reaction solution is commercially prepared.
4. The detection method of the novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit according to claim 1, which is characterized in that: the method comprises the following steps:
1) and (3) optimizing the reaction system:
2) and (3) optimizing the reaction conditions:
reaction conditions are as follows: (Instrument: ABI7500 Fast)
3) And (3) judging rules:
the instrument directly reads the detection result, and the baseline and threshold settings are adjusted according to the noise condition of the instrument;
the effective standard of the experiment is as follows:
positive control ORF1ab, N and E genes generate an amplification curve (Ct value is less than 30), and negative control has no amplification curve;
the above conditions should be satisfied simultaneously, otherwise, the test is regarded as invalid;
4) and (4) judging a result:
under the condition that the test is established, ORF1ab and E genes are preferentially analyzed and judged according to the following rule:
the results are illustrated in detail in the description of FIG. 1.
5. The method of claim 4 for detecting the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, wherein the kit comprises: the 3-target fluorescence PCR information of the detection method is summarized as the following table:
6. the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit according to claim 1, wherein the kit comprises: the primer probe sequences are optimized and then are shown in the following table:
。
7. The method of claim 4 for detecting the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, wherein the kit comprises: the dosage of the ROX is properly adjusted according to the actually used fluorescent PCR instrument; 0.05 μ L of ROX (40 μm) added here was the recommended amount for ABI7500/7500Fast fluorescent PCR instrument; if the instrument is ABI StepOne series, 0.5 μ L ROX (40 μm) is added; the RotorQ series instrument does not require the addition of ROX.
8. The method of claim 4 for detecting the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, wherein the kit comprises: when the fluorescence signal is collected, the fluorescence to be detected is selected as follows: ORF1ab gene (FAM); e gene (NED); n gene (VIC); if the quantitative PCR instrument is ABI series, ROX is selected as reference fluorescence; if the instrument is of the Rotor series, ROX is not required as a reference.
9. The method of claim 4 for detecting the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, wherein the kit comprises: and if the negative control is amplified, the operation process may be polluted, and the experiment is carried out after the experiment environment is changed.
10. The method of claim 4 for detecting the novel coronavirus 2019-nCoVORF1ab, N, E gene detection kit, wherein the kit comprises: the suspicious sample is recommended to be rechecked; if the Ct values of the ORF1ab and the E gene in the retest are both less than or equal to 38, the result is judged to be positive; otherwise, the result is judged to be negative.
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| CN113981147A (en) * | 2021-11-17 | 2022-01-28 | 华南农业大学 | Composition for detecting multiple respiratory viruses, kit and application thereof |
| CN113981147B (en) * | 2021-11-17 | 2023-08-01 | 华南农业大学 | Composition for detecting various respiratory viruses, kit and application thereof |
| CN114317830A (en) * | 2022-01-18 | 2022-04-12 | 甘肃国际旅行卫生保健中心(兰州海关口岸门诊部) | Novel coronavirus digital PCR detection kit and detection method |
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