CN117344062A - Kit for rapidly detecting novel coronavirus nucleic acid and application method - Google Patents
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Classifications
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- 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
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- 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
Abstract
The invention relates to a kit for rapidly detecting novel coronavirus nucleic acid and a use method thereof, belongs to the technical field of nucleic acid detection, and particularly relates to a multiplex fluorescence quantitative PCR method based on a silicon-based chip for novel coronavirus detection. The invention judges whether the novel coronavirus is detected by the negative positive of the ORF1ab gene and the N gene of the novel coronavirus, and meanwhile, the invention sets an internal standard, namely human ribonuclease P (RNaseP), so that the false negative of the detection result can be effectively avoided, and the acquisition, transportation and extraction processes of the detection sample can be monitored.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit for rapidly detecting novel coronavirus nucleic acid and a use method thereof.
Background
The novel coronavirus is a novel coronavirus of the genus coronavirus beta (named SARS-CoV-2 by the International Commission on the classification of viruses), has a coating, and has a circular or oval particle shape with a diameter of 60-140nm. Is sensitive to ultraviolet rays and heat, and can effectively inactivate viruses by 30 minutes at 56 ℃, diethyl ether, 75% alcohol, chlorine-containing disinfectants, peracetic acid, chloroform and other lipid solvents. The main transmission path is transmission through respiratory tract spray and close contact, and in the case of long-time exposure to high-concentration aerosol in a relatively closed environment, the transmission through aerosol is possible; faeces and urine may also be transmitted. SARS-CoV-2 is a single-stranded positive strand RNA virus, which is easy to mutate and has gene characteristics which are obviously different from those of SARS and MERS. The variants published by WHO at present are mainly: alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron these variant mutation sites occur mainly in the spike protein receptor binding domain, making it easier to bind to the human ACE2 receptor, having a stronger infectivity and being susceptible to large-scale epidemics. There is an urgent need for a sensitive and accurate method that does not leak detection of variants for rapid detection of SARS-CoV-2.
The amplification time of the existing novel coronavirus nucleic acid detection reagent is not less than 30min, and the risk of missed detection of variant strains possibly exists, so that the development of a kit capable of rapidly and accurately detecting SARS-CoV-2 has very important significance for prevention and control and treatment measures of pneumonia.
Disclosure of Invention
The invention aims at solving the problems in the prior art, and provides a kit and a detection method for rapidly detecting novel coronavirus nucleic acid, in particular to a kit and a method for detecting novel coronavirus based on a multiplex fluorescence quantitative PCR method, wherein the detection amplification time is within 12min, and the kit and the method can rapidly and accurately detect novel coronavirus containing main variant strain (Alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron).
According to the invention, specific amplification primers and Taqman probes are respectively designed according to human genome internal standard fragments, a novel coronavirus ORF1ab gene conserved region and an N gene conserved region. The invention judges whether the novel coronavirus is detected by the negative positive of the ORF1ab gene and the N gene of the novel coronavirus, and meanwhile, the invention sets an internal standard, namely human ribonuclease P (RNaseP), so that the false negative of the detection result can be effectively avoided, and the acquisition, transportation and extraction processes of the detection sample can be monitored. The invention can adopt a rapid amplification program to shorten the conventional amplification time from 1 to 2 hours to within 30 minutes; with a specific instrument, the time can be shortened to within 12 minutes. The ORF1ab and the N genes of the two target genes have the same detection limit, are not easy to generate single positive, and reduce the occurrence of confusing results.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a kit for rapidly detecting novel coronavirus nucleic acid, which comprises novel coronavirus (2019-nCoV) reaction liquid, enzyme mixed liquid, positive control and blank control.
As one embodiment of the present invention, the novel coronavirus (2019-nCoV) reaction solution includes: PCR buffer, mgCl 2 dNTPs, ORF1ab primer probe combinations, N primer probe combinations, RNaseP primer probe combinations, and RNase water.
The template quality generally affects the amplification reaction, and the invention improves the compatibility of the template by optimizing a buffer system, greatly improves the input volume of the nucleic acid template and reaches 12.5 mu L. The input amount is improved by 2-6 times compared with the conventional input amount of 2-5 mu L.
The PCR buffer is 5 Xbuffer buffer.
The MgCl 2 Is 0-3.3mM; dNTPs are present at a concentration of 0.1-0.4mM, including dATP: dTTP: dCTP: dGTP: dutp=1: 1:1:1:1.
the ORF1ab primer probe combination comprises a primer pair and a specific probe:
ORF1ab specific primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.1;
ORF1ab primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.2;
the ORF1ab probe has a concentration of 0.1-1.0. Mu.M and a sequence of SEQ ID No.7.
The sequence is as follows:
SEQ ID No.1：CTAATGACCCTGTGGGTTTTAC；
SEQ ID No.2：CAACTACAGCCATAACCTTTCC；
SEQ ID No.7：CGCAGACGGTACAGACTGTGTTTTTAAG。
the N primer probe combination comprises a primer pair and a specific probe:
n specific primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.3;
n primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.4;
n probe with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.8.
The sequence is as follows:
SEQ ID No.3：GAGGACAAGGCGTTCCAA；
SEQ ID No.4：CATTTTACCGTCACCACCAC；
SEQ ID No.8：TGGTAGCTCTTCGGTAGTAGCCAATT。
the RNaseP primer probe combination comprises a primer pair and a specific probe:
RNaseP specific primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.5;
RNaseP primer with concentration of 0.1-1.0 mu M and sequence of SEQ ID No.6;
RNaseP probe with concentration of 0.1-1.0 μm and sequence of SEQ ID No.9.
The sequence is as follows:
SEQ ID No.5：ACAGGGAAAATCAAGACCAAT；
SEQ ID No.6：TCAAAACATTGCAGTGAGATGGA；
SEQ ID No.9：CCGAGACAATAATTGTTAATCTAGTTAAAAT。
the two ends of the specific probe are respectively provided with a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or more of FAM, HEX, VIC, TET, TAMRA, ROX, CY 3.5.5 or CY 5; the quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, DABCYL and MGB.
The de-RNase water is used for supplementing the volume of the reaction solution and is based on MgCl 2 The concentration and the addition amount of dNTPs and the like.
As one embodiment of the present invention, the enzyme mixture comprises an RNase inhibitor, a DNA polymerase, a reverse transcriptase, a UDG enzyme. The content of each component is 1-2U/Test. The invention introduces a UDG enzyme anti-pollution system, and can effectively avoid false positive.
As one embodiment of the present invention, the positive control is a synthetic gene fragment containing RNaseP gene fragment, ORF1ab gene conserved region and N gene conserved region. The sequence of the RNaseP gene fragment is SEQ ID No.10, the sequence of the conserved region of the ORF1ab gene is SEQ ID No.11, and the sequence of the conserved region of the N gene is SEQ ID No.12.
As one embodiment of the invention, the novel coronavirus comprises Alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron.
As one embodiment of the invention, the blank is water with RNase.
The invention also provides a using method of the kit, which comprises the following steps:
s1, extracting DNA of sample to be detected
Extracting nucleic acid from the sample to obtain sample nucleic acid;
s2, preparing reagent reaction system
The reaction system comprises the following components in parts by volume
7.5 of a novel coronavirus reaction solution;
enzyme mixed solution 5;
sample nucleic acid 12.5;
preferably as shown in Table 1
TABLE 1 reaction system
S3, setting fluorescent PCR amplification program
S3-1, reverse transcription reaction: the temperature is 50 ℃ and the time is 2min;
s3-2, pre-denaturation: the temperature is 95 ℃ and the time is 10s;
s3-3, denaturation: the temperature is 95 ℃ and the time is 1s;
s3-4, annealing, extension and fluorescence detection: the temperature is 55-60 ℃ and the total time is 5s;
the fluorescence detection is performed during annealing extension, and detection channels are FAM, VIC, ROX respectively; s3-3 and S3-4 are carried out for 40-45 cycles in total;
the setting of the RT-PCR amplification procedure is preferably: 50 ℃ for 2min;95 ℃ for 10s; FAM, VIC, ROX is set at 95℃for 1s,55-60℃for 5s,42-45 cycles.
S4, judging a result:
yin-yang determination:
observing the Ct value and an amplification curve, wherein the amplification curve is S-shaped, the Ct value is less than or equal to 38.0, and judging that the amplification curve is positive; ct > 40.0 or undetected, and negative.
The method comprises the following steps:
(1) Positive: the single-channel or double-channel detection result Ct of the sample to be detected is less than or equal to 38 (when the Ct of FAM or both FAM and VIC is less than or equal to 40), the curve is S-shaped and has obvious index increment, and the curve is judged to be positive;
(2) Negative: all channels Ct > 40 or are not detected, and the result is judged to be negative;
step S4 further includes a rechecking step:
when any channel and the double-channel detection result are 38 < Ct less than or equal to 40 (when either channel of FAM or VIC or both channels of FAM and VIC are 38 < Ct less than or equal to 40), the sample is repeatedly detected, if the single-channel Ct value of the recheck experiment result is less than or equal to 38 or the Ct value of both channels is in the range of 38-40, the curve is in the standard S shape and has obvious index increment, the curve is positive, otherwise, the curve is negative.
And the Ct of the detection result of the sample internal standard CY5 channel is less than or equal to 40, otherwise, the repeated measurement is carried out. When the sample result is judged to be positive, if the internal standard Ct is more than 40 or is not detected, the result is still reliable.
When the blank control detection result is negative and the positive control detection result is positive, the analysis of the sample detection result is as follows in table 2:
TABLE 2 interpretation of results
As an embodiment of the present invention, the sample comprises one of a pharyngeal swab, a nasopharyngeal swab, and sputum.
Compared with the prior art, the invention has the following beneficial effects:
(1) The kit and the method for rapidly detecting the novel coronavirus nucleic acid can detect the novel coronavirus containing the main variant Alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron by optimizing specific primers and probes of the ORF1ab gene and the N gene of the novel coronavirus.
(2) The amplification procedure is optimized, so that the amplification time is shortened to be within 12min, and the detection sensitivity and the specificity are ensured while the detection efficiency is improved. The invention sets the human genome internal standard fragment, can be used for monitoring the processes of sample collection, preservation and transportation and nucleic acid extraction, and avoids false negative results.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of a novel coronavirus nucleic acid-positive amplification curve (micro-nano chip nucleic acid amplification analyzer);
FIG. 2 is a schematic diagram of a novel coronavirus nucleic acid-positive amplification curve (Shang Weigao family chip nucleic acid amplification analyzer);
FIG. 3 is a schematic representation of the amplification curve of novel coronavirus nucleic acid positivity (BioRad CFX96 fluorescent quantitative PCR instrument);
FIG. 4 is a schematic representation of the amplification curve of novel coronavirus nucleic acid positivity (ABI quantsudio 5Q 5 fluorescent quantitative PCR instrument).
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The following examples, which are presented to provide those of ordinary skill in the art with a detailed description of the invention and to provide a further understanding of the invention, are presented in terms of implementation and operation. It should be noted that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made on the premise of the inventive concept are all within the protection scope of the present invention.
Example 1
Design of specific primers and probes for conserved regions of ORF1ab gene and N gene of novel coronavirus.
First, the sequence of the main variant Alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron of 2022, 4 th to 2022 6 th months was downloaded from the GISAID database, and bioinformatics alignment analysis was performed with the original new coronavirus strain sequence (NC_ 045512.2) to find out the ORF1ab gene conserved region and N gene conserved region.
Table 3 shows the number of sequences of each variant
The sequence of the conserved region of the ORF1ab gene is SEQ ID No.11 (ctaatgaccctgtgggttttacacttaaaaacacagtc tgtaccgtctgcggtatgtggaaaggttatggctgtagttg).
The sequence of the N gene conserved region is SEQ ID No.12 (gaggacaaggcgttccaattaacaccaatagcagtcca gatgaccaaattggctactaccgaagagctaccagacgaattcgtggtggtgacggtaaaatg).
The sequence of the internal standard RNase P gene is SEQ ID No.10 (gaattcggcacgaggtgggacttcagcatggcggtgt ttgcagatttggacctgcgagcgggttctgacctgaaggctctgcgcggacttgtggagacagccgctcaccttggctattcagttgttgctatcaatcatatcgttgactttaaggaaaagaaacaggaaattgaaaaaccagtagctgtttctgaactcttcacaactttgccaattgtacagggaaaatcaagaccaattaaaattttaactagattaacaattattgtctcggatccatctcactgcaatgttttgagagcaacttcttcaagggcccggctctatgatgttgttgcagtttttccaaagacagaaaagctttttcatattgcttgcacacatttagatgtggatttagtctgcataactgtaacagagaaactaccattttacttcaaaagacctcctattaatgtggcgattgaccgaggcctggcttttgaacttgtctatagccctgctatcaaagactccacaatgagaaggtatacaatttccagtgccctcaatttgatgcaaatctgcaaaggaaagaatgtaattatatctagtgctgcagaaaggcctttagaaataagagggccatatgacgtggcaaatctaggcttgctgtttgggctctctgaaagtgacgccaaggctgcggtgtccaccaactgccgagcagcgcttctccatggagaaactagaaaaactgcttttggaattatctctacagtgaagaaacctcggccatcagaaggagatgaagattgtcttccagcttccaagaaagccaagtgtgagggctgaaaagaatgccccagtctctgtcagcactcccttcttcccttttatagttcatcagccacaacaaaaataaaacctttgtgtg).
Specific amplification primers and Taqman probes are respectively designed aiming at the novel coronavirus ORF1ab gene conserved region and N gene conserved region by utilizing Primer 5 software, and the novel coronavirus ORF1ab gene conserved region is synthesized by Shanghai Shuoyang biosciences and technologies. The primers and probes used for detection are shown in Table 4:
TABLE 4 primer and probe sequence information
The fluorescent groups respectively carried at two ends of the ORF1ab, N gene and RNaseP specific probe are FAM, VIC, CY, and the quenching groups are BHQ1, BHQ1 and BHQ2.
Example 2
Sample nucleic acid extraction
(1) Collecting, storing and transporting clinical samples: is suitable for throat swab, nasopharyngeal swab and sputum sample type.
Pharyngeal swab: the swab is passed over the tongue root, the tonsils of the pharynx on both sides of the person to be collected are slightly rubbed back and forth for at least 3 times, then the swab head is rubbed up and down on the back wall of the pharynx for at least 3 times, the swab head is immersed into a tube containing virus preservation solution (isotonic saline solution, tissue culture solution or phosphate buffer solution can be used), the tail is discarded, and the tube cover is screwed.
Nasopharyngeal swab: when the plastic rod swab with 1 polypropylene fiber head is gently inserted into the back wall of the nasal meatus nasopharyngeal cavity, the swab slowly rotates to withdraw after staying for a moment. Another polypropylene fiber head swab is taken to collect the nostril at the other side in the same way. The two swabs were immersed in the same tube containing 3mL of virus preservation solution, the tail was discarded, and the tube cap was screwed.
Sputum: the expectorated sputum was collected in 50 mL screw plastic tubes or sputum cassettes containing virus stock. Adding the sputum sample into an equal volume of sputum digestion liquid (1 g/L phosphate buffer solution containing proteinase K), shaking and mixing uniformly, standing for 5 minutes, and digesting and then extracting nucleic acid. The digested sputum sample can be used as a conventional swab sample for subsequent processing.
The sample for virus separation and nucleic acid detection should be detected as soon as possible, and the sample which can be detected within 24 hours can be stored at 4 ℃; samples that could not be detected within 24 hours should be stored at-70 ℃ or below (if no-70 ℃ storage conditions exist, the samples should be stored in a refrigerator at-20 ℃). The freezing and thawing of the sample can not be performed for more than 5 times, otherwise, the detection result is affected.
(2) Nucleic acid extraction: the sample nucleic acid was extracted using QIAamp Viral RNA Mini Kit kit from Qiagen, and the nucleic acid was extracted according to the instructions.
Samples used in the examples were extracted with pharyngeal swabs, specifically:
1. mu.L of prepared buffer AVL (containing carrier RNA) was pipetted into a 1.5ml centrifuge tube.
2. mu.L of the sample was added to a centrifuge tube containing buffer AVL-carrier RNA. Vortex for 15 seconds and mix well.
3. The mixture was left at room temperature for 10min.
4. And (5) carrying out instantaneous centrifugation to throw the liquid drops on the cover back to the bottom of the pipe.
5. 560 mu L absolute ethyl alcohol (96% -100%) is added into the sample, and vortex 15s is fully mixed. Then, the liquid drops on the cover are thrown back to the bottom of the tube by instantaneous centrifugation.
6. The 630. Mu.L of solution from the previous step was carefully added to column (loaded into a 2ml centrifuge tube) taking care not to hit the edges of the column. The lid was closed and centrifuged at 6000 Xg (8000 rpm) for 1min. Column was placed in a new 2ml centrifuge tube and the old collection tube was discarded.
7. Carefully open the column lid and repeat step 6.
8. Carefully open the column lid and add 500 μl buffer AW1. The lid was closed and centrifuged at 8000rpm for 1min. Column was placed in a new 2ml collection tube (Kit provided) and the old collection tube was discarded.
9. Carefully open the column lid and add 500 μl buffer AW2. The lid was closed and centrifuged at full speed (14000 rpm) for 3min. Then, step 11 is performed, or step 10 is performed first to avoid buffer AW2 residues, and then step 11 is performed.
10. Recommendation: column was placed in a new 2ml collection tube (not provided in Kit), the old collection tube was discarded, and centrifuged at full speed for 1min.11. Column was placed in a 1.5ml centrifuge tube (not provided in kit). The old collection tube was discarded. The column was carefully opened and 60. Mu.L of room temperature buffer AVE was added. Cover the lid and leave at room temperature for 1min. Centrifugal at 8000rpm for 1min.
Specific samples of example 5: all kinds of pathogens and human genome
Coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, SARS coronavirus, MERS coronavirus, influenza a virus H1N1, influenza a virus H3N2, influenza b virus Victoria, parainfluenza virus (type i), adenovirus (type 7), respiratory syncytial virus (type a), human genome.
Detection limit sample of example 6: novel coronavirus samples.
Inclusion sample of example 7: alpha, beta, gamma, delta, lambda, eta, iota, mu, omicron.
Example 3
Detection of nucleic acid from the extracted sample (Omicon strain)
Preparation of the amplification reaction system was performed according to Table 5:
TABLE 5 preparation of amplification reaction System
The novel coronavirus (2019-nCoV) reaction solution comprises PCR buffer solution and 3.3mM MgCl 2 0.2mM dNTPs (dATP: dTTP: dCTP: dGTP: dUTP=1:1:1:1:1), primer probes (SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 at a concentration of 0.4. Mu.M; SEQ ID No.2, SEQ ID No.4, SEQ ID No.6 at a concentration of 0.4. Mu.M; SEQ ID No.7-9 at a concentration of 0.2. Mu.M) and RNase water.
The enzyme mixture comprises an RNase inhibitor, a DNA polymerase, a reverse transcriptase and a UDG enzyme. The content of each component is 1-2U/Test.
RT-PCR amplifications were performed on different nucleic acid amplification analyzers according to the following procedure:
1) Micro-nano chip nucleic acid amplification analyzer for milk lattice organism
Program 1:50 ℃ for 2min;95 ℃ for 10s; a total of 45cycles were carried out at 95℃for 1s and at 60℃for 5 s.
2) Shang Weigao family
Program 2:50 ℃ for 2min;95 ℃ for 10s; cycling at 95 ℃ for 1s,60 ℃ for 10s and 10s; cycling at 95 ℃ for 1s,60 ℃ for 10s and 32;
3）Cfx96
program 3:50 ℃ for 10min;95 ℃ for 60s; a total of 43 cycles were carried out at 95℃for 5s and 60℃for 30 s.
4）ABI Q5
Program 4:50 ℃ for 2min;95 ℃ for 10s; a total of 42 cycles were carried out at 95℃for 1s and 60℃for 10 s.
Example 4
Analysis of results
When the blank control detection result is negative and the positive control detection result is positive, determining the negative and positive conditions of each gene according to the amplification curve graph and the Ct value condition of each fluorescent channel, wherein the negative and positive interpretation standards of each gene are shown in table 6.
Table 6 interpretation of the results of the genes
Results:
TABLE 7 results for each gene
| Method | Procedure 1 × | Procedure 2 x | Procedure 3 × | Procedure 4 x |
| Amplification map | FIG. 1 (Positive sample) | FIG. 2 (Positive sample) | FIG. 3 (Positive sample) | FIG. 4 (Positive sample) |
| Time | Micro-nano chip nucleic acid amplification analyzer for 12min | Shang Weigao chip nucleic acid amplification analyzer for 17min | BioRad CFX96, amplification duration 47min | ABI Q5, amplification duration 28min |
* The rapid program of the invention has certain requirements on the temperature rise and fall rate, fluorescence acquisition time, fluorescence switching time and the like of the instrument, so that only part of instruments support the rapid program. The kit supports detection under a rapid program, reduces the time required by each amplification and improves the detection flux in unit time.
Example 5
Specificity analysis
Multiplex fluorescence quantitative PCR amplification assays were performed on coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, SARS coronavirus, MERS coronavirus, influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, parainfluenza virus (type I), adenovirus (type 7), respiratory syncytial virus (type A) and human genome using the methods of the invention.
TABLE 8 specificity test results
Example 6
Sensitivity analysis
After the collected sample positive to the novel coronavirus is fixed by a digital PCR method, the sample is diluted by a gradient of 2 times of the sample negative to the novel coronavirus, and the minimum detection limit of the detection of the sample is 1.62 multiplied by 10 by performing 20-well multiplex fluorescence quantitative PCR amplification analysis 2 copies/mL。
Table 9 LoD verification of detection system
Example 7
Inclusion detection
The method of the invention is used for multiplex fluorescence quantitative PCR amplification analysis of throat swabs containing novel coronavirus Alpha variant nucleic acid, throat swabs containing novel coronavirus Beta variant nucleic acid, throat swabs containing novel coronavirus Gamma variant nucleic acid, throat swabs containing novel coronavirus Delta variant nucleic acid, throat swabs containing novel coronavirus Lambda variant nucleic acid, throat swabs containing novel coronavirus Eta variant nucleic acid, throat swabs containing novel coronavirus Iota variant nucleic acid, throat swabs containing novel coronavirus Mu variant nucleic acid, and throat swabs containing novel coronavirus Omicron variant nucleic acid.
Table 10 results of detection of novel coronavirus variants
Example 8
Verification of UDG enzyme anti-pollution System
Two sets of PCR amplification reagents were set up in the manner described in example 3 (with the amplification reaction system configured but without the addition of sample nucleic acid). After closing the tube cap, inactivating UDG enzyme at 65deg.C for 30min in group 1, to give group 1; the other group does not perform this operation, set to group 2.
After completion, positive PCR products in example 4 were added to each of the two sets of PCR amplification reagents. Then allowed to stand at 37℃for half an hour. After the completion, the machine was started up, and example 4 was referred to.
Results: group 2 was negative, indicating that the positive PCR product was degraded by UDG enzyme and could not be used as an amplification template to contaminate the amplification system.
TABLE 11 amplification results
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (9)
1. A kit for rapid detection of novel coronavirus nucleic acid, characterized in that the kit comprises a novel coronavirus reaction solution, an enzyme mixed solution, a positive control and a blank control;
the novel coronavirus reaction solution comprises: PCR buffer solution, mgCl 2 dNTPs, ORF1ab primer probe, N primer probe, RNaseP primer probe, and RNase water.
2. The kit of claim 1, wherein the MgCl 2 Is 0-3.3mM; the concentration of dNTPs is 0.1-0.4mM.
3. The kit according to claim 1, wherein,
the ORF1ab primer probe comprises:
ORF1ab specific primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.1;
ORF1ab primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.2;
ORF1ab probe with concentration of 0.1-1. Mu.M and sequence of SEQ ID No.7;
the N primer probe includes:
n specific primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.3;
n primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.4;
n probe with concentration of 0.1-1 mu M and sequence of SEQ ID No.8;
the RNaseP primer probe comprises:
RNaseP specific primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.5;
RNaseP primer with concentration of 0.1-1 mu M and sequence of SEQ ID No.6;
RNaseP probe with concentration of 0.1-1. Mu.M and sequence of SEQ ID No.9.
4. A kit according to claim 3, wherein the ORF1ab probe, N probe, rnase p probe each bear a fluorescent group and a quenching group at both ends, wherein the fluorescent group is selected from any one of FAM, HEX, VIC, TET, TAMRA, ROX, CY3.5, CY 5; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3, DABCYL and MGB.
5. The kit of claim 1, wherein the enzyme cocktail comprises an rnase inhibitor, a DNA polymerase, a reverse transcriptase, a UDG enzyme.
6. The kit according to claim 1, wherein the positive control is an artificially synthesized gene fragment containing the rnase p gene, the ORF1ab gene conserved region and the N gene conserved region; the sequence of RNase P gene is SEQ ID No.10, the sequence of ORF1ab gene conserved region is SEQ ID No.11, and the sequence of N gene conserved region is SEQ ID No.12.
7. The kit of claim 1, wherein the blank is degranolase water.
8. A method of using the kit of claim 1, comprising the steps of:
s1, extracting nucleic acid of sample to be detected
Extracting nucleic acid from the sample to obtain sample nucleic acid;
s2, preparing reagent reaction system
The reaction system comprises the following components in parts by volume
7.5 of a novel coronavirus reaction solution;
enzyme mixed solution 5;
sample nucleic acid 12.5;
s3, setting fluorescent PCR amplification program
S3-1, reverse transcription reaction: the temperature is 50 ℃ and the time is 2min;
s3-2, pre-denaturation: the temperature is 95 ℃ and the time is 10s;
s3-3, denaturation: the temperature is 95 ℃ and the time is 1s;
s3-4, annealing, extension and fluorescence detection: the temperature is 55-60 ℃ and the time is 5s;
the fluorescence detection is performed during annealing extension, and detection channels are FAM, VIC, ROX respectively;
s3-3 and S3-4 are carried out for 40-45 cycles in total.
9. The method of claim 8, wherein in step S1, the sample comprises one of a pharyngeal swab, a nasopharyngeal swab, and sputum.
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| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| CN114410848A (en) * | 2022-03-30 | 2022-04-29 | 深圳联合医学科技有限公司 | Composition, kit, method and use for detecting SARS-CoV-2 |
| KR20220166458A (en) * | 2021-06-10 | 2022-12-19 | 주식회사 팍스젠바이오 | Kit For Diagnosing SARS-CoV-2 and Method of Detecting SARS-CoV-2 Using the Same |
| CN116716435A (en) * | 2023-03-07 | 2023-09-08 | 北京纳捷诊断试剂有限公司 | Composition for detecting novel coronavirus RNA |
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| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| KR20220166458A (en) * | 2021-06-10 | 2022-12-19 | 주식회사 팍스젠바이오 | Kit For Diagnosing SARS-CoV-2 and Method of Detecting SARS-CoV-2 Using the Same |
| CN114410848A (en) * | 2022-03-30 | 2022-04-29 | 深圳联合医学科技有限公司 | Composition, kit, method and use for detecting SARS-CoV-2 |
| CN116716435A (en) * | 2023-03-07 | 2023-09-08 | 北京纳捷诊断试剂有限公司 | Composition for detecting novel coronavirus RNA |
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