WO2021196498A1 - Primer, probe and kit for detecting novel coronavirus - Google Patents

Primer, probe and kit for detecting novel coronavirus Download PDF

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WO2021196498A1
WO2021196498A1 PCT/CN2020/109072 CN2020109072W WO2021196498A1 WO 2021196498 A1 WO2021196498 A1 WO 2021196498A1 CN 2020109072 W CN2020109072 W CN 2020109072W WO 2021196498 A1 WO2021196498 A1 WO 2021196498A1
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gene
seq
sequence
probe
primer
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PCT/CN2020/109072
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French (fr)
Chinese (zh)
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陈绍宇
何瑰
李楚明
刘慧�
李灵鸽
黄颖
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广州安必平医药科技股份有限公司
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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
    • 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]
    • 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

Definitions

  • the invention relates to the technical field of virus detection, in particular to a primer, a probe and a kit for detecting a novel coronavirus.
  • Nucleic acid test is the "gold standard" for the diagnosis of new coronary pneumonia.
  • the National Medical Products Administration urgently approved the first batch of 7 companies' new coronavirus 2019-nCoV nucleic acid detection kits 2020-2-11 No. 002], the approved products are based on the open reading frame 1a/b (Open reading frame 1ab, ORF1ab), Envelope protein (E) and Nucleocapsid (Nucleocapsid) of the novel coronavirus genome.
  • protein, N Three gene design.
  • RT-PCR Optimizing diagnostic strategy for novel coronavirus pneumonia, a m ⁇ lti-center study in Eastern China[J].medRxiv, 2020.] It is believed that RT-PCR has 15% false negatives When designing RT-PCR, it is recommended to amplify more than one gene target for analysis.
  • the present invention adopts the following technical solutions:
  • An RT-PCR kit for detecting novel coronavirus contains specific primers for ORF1ab gene, N gene, E gene, S gene and M gene;
  • the specific primer sequence of the ORF1ab gene is shown in SEQ ID NO: 1 to 2
  • the specific primer sequence of the N gene is shown in SEQ ID NO: 4 to 5
  • the specific primer sequence of the E gene is shown in SEQ ID NO: 7 to 8
  • the specific primer sequence of the S gene is shown in SEQ ID NO: 10 to 11
  • the specific primer sequence of the M gene is shown in SEQ ID NO: 13 to 14.
  • the kit further contains probes for ORF1ab gene, N gene, E gene, S gene and M gene; the probe sequence of ORF1ab gene is shown in SEQ ID NO: 3, and the probe for N gene
  • the needle sequence is shown in SEQ ID NO: 6
  • the probe sequence of the E gene is shown in SEQ ID NO: 9
  • the probe sequence of the S gene is shown in SEQ ID NO: 12
  • the probe sequence of the M gene The probe sequence is shown in SEQ ID NO: 15.
  • reaction system of the kit is:
  • the reaction conditions of the kit are: 55°C 15min, 1 cycle; 95°C 30s, 1 cycle; 95°C 10s, 55°C 30s, 10 cycles; 95°C 10s, 55°C 30s, 35 cycles cycle.
  • a RT-PCR specific primer for detecting the ORF1ab gene of the novel coronavirus Its sequence is shown in SEQ ID NO: 1 ⁇ 2.
  • An RT-PCR probe for detecting the ORF1ab gene of the novel coronavirus Its sequence is shown in SEQ ID NO: 3.
  • a specific RT-PCR primer for detecting the N gene of the novel coronavirus whose sequence is shown in SEQ ID NO: 4 to 5.
  • An RT-PCR probe for detecting the N gene of the novel coronavirus Its sequence is shown in SEQ ID NO: 6.
  • a RT-PCR specific primer for detecting the E gene of the novel coronavirus and its sequence is shown in SEQ ID NO: 7-8.
  • An RT-PCR probe for detecting the E gene of the novel coronavirus the sequence of which is shown in SEQ ID NO: 9.
  • a specific RT-PCR primer for detecting the S gene of the novel coronavirus whose sequence is shown in SEQ ID NO: 10-11.
  • An RT-PCR probe for detecting the S gene of the novel coronavirus Its sequence is shown in SEQ ID NO: 12.
  • a specific RT-PCR primer for detecting the M gene of the novel coronavirus, and its sequence is shown in SEQ ID NO: 13-14.
  • An RT-PCR probe for detecting the M gene of the novel coronavirus Its sequence is shown in SEQ ID NO: 15.
  • the present invention has the following beneficial effects:
  • the present invention found that the ORF1ab gene, E gene, N gene, S gene and M gene of the new coronavirus 2019-nCoV are highly conserved, and designed primers and probes specific to the 5 genes for use in the new coronavirus Identification test.
  • the detection kit of the present invention adds the detection of surface glycoprotein (S) and membrane glycoprotein (M) genes. The combined detection of five targets and comprehensive judgment can improve the detection of RT-PCR methods. The accuracy of the new coronavirus.
  • the specific primer probe and kit for detecting the novel coronavirus 2019-nCoV of the present invention have high sensitivity, precision, accuracy, good specificity, good stability, small amount of template required for detection, and objective and accurate detection results.
  • the design of five targets can better overcome the false negatives caused by the variation of the RNA virus passaging process, can effectively increase the positive detection rate, and avoid missed detection as much as possible. High clinical application value.
  • Figure 1 is a computer comparison diagram of different primer probe combinations for ORF1ab gene detection of low-value pseudoviruses. Among them, A is primer probe set 1 and B is primer probe set 2.
  • Figure 2 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the N gene. Among them, A is primer probe set 1 and B is primer probe set 2.
  • Figure 3 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the E gene. Among them, A is primer probe set 1 and Figure B is primer probe set 2.
  • Figure 4 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the S gene. Among them, A is primer probe set 1 and Figure B is primer probe set 2.
  • Figure 5 is a computer comparison diagram of detecting low-value pseudoviruses with different primer probe combinations of the M gene. Among them, A is primer probe set 1 and B is primer probe set 2.
  • Fig. 6 is the comparison result of the primers and probes of the present invention and the primers and probes recommended by CDC.
  • A is the amplification curve of ORF1ab gene primers and probe recommended by China CDC
  • B is the amplification curve of primers and probes of the present invention
  • C is the amplification curve of N gene primers and probes recommended by China CDC
  • D Amplification curve of ORF1ab primers and probes recommended by CDC in the United States.
  • Curves 1 to 5 respectively correspond to the results of the computer-based detection of pseudoviral RNA containing 5 related genes of the novel coronavirus (ORF1ab, N, E, S, and M).
  • Figure 7 is a linear relationship diagram of ORF1ab gene, N gene and E gene of the one-step mixed system.
  • Figure 8 shows the linear relationship between the M gene and the S gene of the one-step mixed system.
  • Figure 9 shows the amplification curves of the one-step method under different reaction conditions.
  • A is the amplification curve of reaction condition 1
  • B is the amplification curve of reaction condition 2.
  • Figure 10 shows the test results of the positive reference product. Among them, A is the detection result of mixed system 1, and B is the detection result of mixed system 2.
  • Figure 11 shows the detection results of ORF1ab, E and N gene detection limit reference products.
  • A is the results of the computer-based testing of the 1 ⁇ 10 4 copies/ml sample in the hybrid system 1
  • B is the computer-based testing results of the 1 ⁇ 10 3 copies/ml sample in the hybrid system 1
  • C is the copy of the hybrid system 1.
  • Count the test results of 5 ⁇ 10 2 copies/ml samples on the machine.
  • Figure 12 shows the detection results of the reference samples for the detection limit of S and M genes.
  • A is the computer-based test result of the 1 ⁇ 10 4 copies/ml sample in the hybrid system 2
  • B is the computer-based test result of the 1 ⁇ 10 3 copies/ml sample in the hybrid system 2
  • C is the copy in the hybrid system 2.
  • Count the test results of 5 ⁇ 10 2 copies/ml samples on the machine.
  • FIG 13 shows the results of the analysis of specific reference products. Wherein A is the detection result of mixed system 1, and B is the detection result of mixed system 2.
  • Figure 14 is the test result of the specificity of the human coronavirus HCoV detected by the kit of the present invention.
  • A is the on-machine test result of the OC43 sample
  • B is the on-board test result of the HKU1 sample
  • C is the on-board test result of the 229E sample
  • D is the on-board test result of the NL63 sample.
  • Figure 15 shows the specific test results of SARS, MERS, influenza A and influenza B viruses detected by the kit of the present invention.
  • A is the on-board testing result of SARS samples
  • B is the on-board testing results of MERS samples
  • C is the on-board testing results of H1N1 samples
  • D is the on-board testing results of INFB samples.
  • Figure 16 shows the specific test results of the kit of the present invention for detecting respiratory syncytial virus, human parainfluenza virus, and adenovirus.
  • A is the on-board testing result of RSV samples
  • B is the on-board testing results of PV samples
  • C is the on-board testing results of ADV samples.
  • Figure 17 is a test result of the kit of the present invention for detecting human genomic DNA and the specificity of 2019-nCoVRNA negative samples. Among them, A is human genomic DNA, and B is a negative sample.
  • Figure 18 shows the test results of system 1 and system 2 precision reference products. Among them, A is the low precision test result of mixed system 1, B is the low precision test result of mixed system 2, C is the median precision test result of mixed system 1, and D is the median precision test result of mixed system 2.
  • Figure 19 shows the test results of the control group and the I1 interfering substance reference. Among them, A is the control positive RNA detection result, B is the detection result of the interfering substance I1 in the mixed system 1, and C is the detection result of the interfering substance I1 in the mixed system 2.
  • Figure 20 shows the detection results of I2 and I3 interfering substance reference products.
  • A is the detection result of interfering substance I2 in mixed system 1
  • B is the detection result of interfering substance I2 in mixed system 2
  • C is the detection result of interfering substance I3 in mixed system 1
  • D is the detection result of interfering substance I3 in mixed system 2. Test results.
  • Fig. 21 is the test result of the accuracy of detecting ORF1ab, E, and N genes by the kit of the present invention.
  • A is the ORF1ab gene detection result in mixed system 1
  • B is the E gene detection result in mixed system 1
  • C is the N gene detection result in mixed system 1.
  • Figure 22 is the test result of the accuracy of detecting M and S genes by the kit of the present invention. Among them, A is the detection result of the M gene in the mixed system 2, and B is the detection result of the S gene in the mixed system 2.
  • test methods in the following examples are all conventional methods.
  • the experimental materials and reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
  • primer probes In the design of primer probes, it is necessary to select conserved and specific regions within the group for design, so as to avoid false negatives caused by missed detection, or false positives caused by poor specificity.
  • the present invention finds through sequence comparison and literature search that the 2019-nCoV structural genes S, M, ORF1ab, E and N genes are highly conserved and can be used as target regions for identification and detection of new coronaviruses.
  • sequences of endemic human coronaviruses HKU1, OC43, 229E, and NL63
  • SARS coronavirus SARS coronavirus
  • MERS coronavirus MERS coronavirus
  • bat coronavirus MG772933_Bat_bat_SL_CoVZC45
  • SARS-like virus MG772934_Bat_SARS_like_bat_SL_CoVZXC21
  • Open reading frame 1ab (ORF1ab), nucleocapsid protein (N), envelope protein (E), surface glycoprotein (S) and membrane glycoprotein (M) genes are specific in different viruses and can be used for design specificity Sex primer and probe sequences.
  • ORF-F (SEQ ID NO: 16): 5’-CCCTGTGGGTTTTACACTTAA-3’;
  • ORF-R (SEQ ID NO: 17): 5’-ACGATTGTGCATCAGCTGA-3’;
  • ORF-P (SEQ ID NO: 18): 5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’;
  • ORF-R (SEQ ID NO: 2): 5’-TTCTAATAGCATTCTTTGCATTTT-3’;
  • ORF-P (SEQ ID NO: 3): 5'-TTACAATCTAGTCAAGCGTGGCAACCG-3'.
  • the 5'end of the ORF1ab gene probe is labeled with FAM group, and the 3'end is labeled with BHQ1 group.
  • N-F (SEQ ID NO: 4): 5’-TGCCACTAAAGCATACAATGTAA-3’;
  • N-R (SEQ ID NO: 5): 5’-GTTCCTTGTCTGATTAGTTCCTG-3’;
  • N-P (SEQ ID NO: 6): 5'-AGCTTTYGGCAGACGTGGTCCAG-3'; where Y is a merging base and refers to C or T. In the following, Y refers to the same as this one, and will not be repeated here.
  • N-F (SEQ ID NO: 19): 5’-CGCAAATTGCACAATTT-3’;
  • N-R (SEQ ID NO: 20): 5’-TTCTTTTTGTCCTTTTTAGG-3’;
  • N-P (SEQ ID NO: 21): 5’-CGTGGTTGACCTACACAGGTGCCATC-3’;
  • the 5'end of the N gene probe is labeled with CY5 group, and the 3'end is labeled with BHQ2 group.
  • E-F (SEQ ID NO: 7): 5’-TTGCTTTCGTGGTATTCTTGC-3’;
  • E-R (SEQ ID NO: 8): 5’-ATATTGCAGCAGTACGCACACA-3’;
  • E-P (SEQ ID NO: 9): 5’-ACACTAGCCATCCTTACTGCGCTTCG-3’;
  • E-F (SEQ ID NO: 22): 5’-TACTGCTGCAATATTGTTAACG-3’;
  • E-R (SEQ ID NO: 23): 5’-ATCAGGAACTCTAGAAGAATTCAG-3’;
  • E-P (SEQ ID NO: 24): 5’-TCTTGTAAAACCTTCTTTTTACGTTTACTCT-3’;
  • the 5'end of the E gene probe is labeled with ROX group, and the 3'end is labeled with BHQ2 group.
  • S-F (SEQ ID NO: 10): 5’-TTGTGCCCTTTTGGTGAAGT-3’;
  • S-R (SEQ ID NO: 11): 5’-TAGGACAGAATAATCAGCAACACA-3’;
  • S-P (SEQ ID NO: 12): 5’-CCACCAGATTTGCATCTGTTTATGCTTG-3’;
  • S-F (SEQ ID NO: 25): 5’-TTTGTAATTAGAGGTGATGAAGTCA-3’;
  • S-R (SEQ ID NO: 26): 5’-TAACGCAGCCTGTAAAATCAT-3’;
  • S-P (SEQ ID NO: 27): 5’-ACAAATCGCTCCAGGGCAAACTGG-3’;
  • the 5'end of the S gene probe is labeled with FAM group, and the 3'end is labeled with BHQ1 group.
  • M-F (SEQ ID NO: 28): 5’-TATTCTGACCAGACCGCTTCT-3’;
  • M-R (SEQ ID NO: 29): 5’-TTTAGGCAGGTCCTTGATGTC-3’;
  • M-P (SEQ ID NO: 30): 5’-CGGAGCTGTGATCCTTCGTGGACAT-3’;
  • M-F (SEQ ID NO: 13): 5’-TGCYGTTTACAGAATAAATTGGA-3’;
  • M-P (SEQ ID NO: 15): 5'-CGGTGGAATTGCTATCGCAATGG-3'.
  • the 5'end of the M gene probe is labeled with ROX group, and the 3'end is labeled with BHQ2 group.
  • Example 1 In order to evaluate the preferred primers and probes of Example 1, the CDC (Center for Disease Control and Prevention) recommended sequence was used as a comparison sequence for detection and evaluation.
  • ORF1ab-F (SEQ ID NO: 16): 5’-CCCTGTGGGTTTTACACTTAA-3’;
  • ORF1ab-R (SEQ ID NO: 17): 5’-ACGATTGTGCATCAGCTGA-3’;
  • ORF1ab-P (SEQ ID NO: 18): 5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’;
  • N-F (SEQ ID NO: 31): 5’-GGGGAACTTCTCCTGCTAGAAT-3’;
  • N-R (SEQ ID NO: 32): 5’-CAGACATTTTGCTCTCAAGCTG-3’;
  • N-P (SEQ ID NO: 33): 5'-TTGCTGCTGCTTGACAGATT-3'.
  • the US CDC does not recommend primer and probe sequences for N gene, E gene, S gene and M gene. It only recommends primer and probe sequences for ORF1ab gene.
  • the recommended primer and probe sequences are as follows:
  • ORF1ab-F (SEQ ID NO: 34): 5’-GGGAGCCTTGAATACACCAAAA-3’;
  • ORF1ab-R (SEQ ID NO: 35): 5’-TGTAGCACGATTGCAGCATTG-3’;
  • ORF1ab-P (SEQ ID NO: 36): 5'-AYCACATTGGCACCCGCAATCCTG-3'.
  • the reaction system used for detection and evaluation is: 2 ⁇ One Step RT-PCR Buffer III 10 ⁇ l, 5U/ ⁇ l Takara Ex Taq HS 0.6 ⁇ l, PrimeScript RT Enzyme Mix II 0.4 ⁇ l, 10 ⁇ M Primer 0.4 ⁇ l each, 10 ⁇ M Probe 0.8 ⁇ l, Total RNA 2 ⁇ l , RNase Free dH 2 O 5.4 ⁇ l.
  • reaction conditions used for detection and evaluation are: 55°C for 15 minutes, 1 cycle; 95°C for 30s, 1 cycle; 95°C for 10s; 55°C for 30s, 45 cycles.
  • the present invention compares the detection effects of the two reaction systems, first evaluates the detection effects of a single pair of primer probes, and then performs a combined test on the synthetic sequence according to the reaction tube.
  • the two-step method uses Takara RNA PCR Kit (RR019A) and Takara Ex TAQ HS (RR390L), and operates according to the product manual.
  • the two-step reverse transcription reaction system is shown in Table 2.
  • Reverse transcription program 142°C 30min, 1 cycle; 295°C 5min, 1 cycle; 35°C 5min, 1 cycle.
  • qPCR program 195°C for 30s, 1 cycle; 295°C for 10s; 55°C for 30s, 45 cycles [Signal collection: Collect FAM, Cy5, ROX and HEX (or VIC) signals at 55°C].
  • the one-step method uses Takara Primer Script one step RT-PCR Kit (RR064A) and operates according to the product manual.
  • the one-step reverse transcription system is shown in Table 4.
  • One-step computer program 155°C 15min, 1 cycle; 295°C 30s, 1 cycle; 395°C 10s; 55°C 30s, 45 cycles [Signal collection: 55°C collect FAM, Cy5, ROX and HEX (or VIC )signal of ⁇ .
  • Table 5 compares the detection effects of the two detection systems.
  • the one-step system has higher sensitivity than the two-step system, and the detection limit is twice that of the two-step method; however, the two-step system has twice the sample in the reverse transcription step After conversion, the amplification efficiency of the two systems is the same.
  • the amplification efficiency of the one-step method and the two-step method system are reduced, and the detection limit of the two-step method is reduced to 10 3 copies /ml, and the detection limit of the one-step system is reduced to 500copies/ml. After conversion, the amplification efficiency of the two is still the same.
  • a one-step detection system is preferred.
  • the detection limit of the kit is 500copies/ml, and the detection sensitivity is high; the linear range is 0.983 ⁇ 0.999; the variability of precision is less than 5%.
  • This example further optimized the reaction conditions of the one-step method of the five-target joint inspection system.
  • Computer program (reaction conditions) 1 (45 cycles): 155°C 15min, 1 cycle; 295°C 30s, 1 cycle; 395°C 10s; 55°C 30s, 45 cycles [Signal collection: 55°C collect FAM, Cy5 , ROX and HEX (or VIC) signals].
  • Computer program 2 (10+35 cycles): 155°C 15min, 1 cycle; 295°C 30s, 1 cycle; 395°C 10s; 55°C 30s, 10 cycles; 495°C 10s; 55°C 30s, 35 cycles [Signal collection: Collect FAM, Cy5, ROX and HEX (or VIC) signals at 55°C].
  • the five-target joint test kit for the detection of new coronavirus (2019-nCoV) of the present invention adopts multiple Taqman fluorescent probe technology to select 2019 new coronavirus (2019-nCoV) open reading frame 1ab (ORF1ab), nucleocapsid protein (N), envelope protein (E), surface glycoprotein (S) and membrane glycoprotein (M) genes are used as amplification target regions, design specific primers and fluorescent probes; select ribonuclease P (RNase P) genes As an internal standard, specific primers and fluorescent probes are designed as follows.
  • Rnase P-F (SEQ ID NO: 37): 5’-AGATTTGGACCTGCGAGC-3’;
  • Rnase P-PSEQ ID NO39: 5'-TTCTGACCTGAAGGCTCTGCGCG-3'.
  • the probe has a HEX group at 5'and a BHQ1 group at 3'.
  • the kit of the present invention includes: 2019-nCoV reaction solution A, 2019-nCoV reaction solution B, 2019-nCoV reaction solution C, 2019-nCoV negative control substance, and 2019-nCoV positive quality control substance.
  • ORF1ab, N and E gene-specific primers and probes are in reaction solution A
  • S and M gene-specific primers and probes are in reaction solution B.
  • the concentration of each probe and primer is 10-15 pmol, preferably 12 pmol.
  • reaction solution A and reaction solution B also contain buffer and dNTPs.
  • the reaction solution C is an enzyme solution, such as mMLV enzyme, HS-Taq enzyme, RNasin, UDG enzyme and so on.
  • the above-mentioned four fluorescent signals are detected simultaneously through the four channels of the automatic fluorescent PCR detector (see Table 6).
  • the detection performance is evaluated through the dilution gradient of artificial plasmids and high-concentration pseudoviruses, and the sequence/system specificity is evaluated through the performance evaluation standard plate (from Guangzhou Bang Desheng).
  • the new coronavirus ribonucleic acid (COVID-19 RNA) liquid series performance evaluation standard plate contains positive reference products, detection limit reference products, analysis specific reference products, precision reference products and interference reference products.
  • the positive reference P1 ⁇ P10 are composed of pseudoviruses containing 5 related genes (ORF1ab, N, E, S and M) of the new coronavirus with different copy numbers, and the copy number is 1.0 ⁇ 10 3 copies/mL ⁇ 1.0 ⁇ 10 6 copies/mL.
  • the detection limit reference materials L1 ⁇ L3 are composed of pseudoviruses containing 5 related genes (ORF1ab, N, E, S, and M) of the new coronavirus with different copy numbers.
  • the copy numbers are 1.0 ⁇ 10 5 copies/mL, 1.0 ⁇ 10 4 copies/mL and 5.0 ⁇ 10 2 copies/mL.
  • Reference interferer article I1 ⁇ I3 hemoglobin containing 2.0 ⁇ 10 3 copies / mL positive samples (I1), albumin-containing 2.0 ⁇ 10 3 copies / mL positive samples (I2) and ribavirin containing azithromycin + 2.0 ⁇ 10 3 Copies/mL positive samples (I3) are composed, and the positive control group is 2.0 ⁇ 10 3 copies/ml/ml positive samples.
  • the evaluation result requirements of the performance evaluation standard plate the test result of the positive reference product should be positive, the test result of the detection limit reference product should be positive, the test result of the analysis-specific reference product should be negative, and the test result of the precision reference product should be positive. And the coefficient of variation (CV, %) of the CT value is not more than 5.0%.
  • the test result of the interference reference substance should be positive.
  • test results of the positive reference product are shown in Figure 10.
  • the test results of the positive reference product ORF1ab, N, E, S, and M genes of different copy numbers are all positive.
  • the detection results of the detection limit reference product are shown in Figure 11 and Figure 12.
  • the detection of ORF1ab, E, N, S and M genes using 1.0 ⁇ 10 3 , 1.0 ⁇ 10 3 and 5.0 ⁇ 10 3 are all positive, and Good repeatability.
  • the minimum detection limit of the kit is 500copies/ml.
  • the test results of the analysis-specific reference product are shown in Figure 13.
  • the analysis-specific reference product is only positive for the internal standard in System 1 and System 2, and the target genes ORF1ab, N, E, S and M genes to be detected are all negative.
  • the precision test result is shown in Figure 18.
  • the test shows that the low-precision reference product (2019-nCoV RNA 2.0 ⁇ 10 3 copies/ml positive sample) and the median precision reference product (2019-nCoV RNA 2.0 ⁇ 10 5 copies/ml positive sample) were used to repeat the test.
  • Machine using system 1 (ORF1ab, N and E genes) and system 2 (S and M genes) were tested separately, the results were all positive, the kit has good repeatability, and the CV value is not more than 5.0%.
  • the detection results of the interference reference product are shown in Figure 19 and Figure 20.
  • the control sample and the three interference sample simulation samples are used as the test objects, and the system 1 and system 2 are used for the detection.
  • the results are all positive.
  • the three interference substances are in the system. 1. It can be detected in system 2. Interfering substances such as hemoglobin, albumin, ribavirin/azithromycin, etc. will not interfere with the test results.
  • the new coronavirus nucleic acid national standard material (Chinese Academy of Metrology) (NCRM)-GBW(E)091090 (low concentration) is in vitro transcribed RNA, and the absolute quantitative method-digital PCR is used to determine the gene copy number concentration.
  • the target gene range covers the three main genes of the new coronavirus that have been announced: the full length of the nucleocapsid protein N gene, the full length of the envelope protein E gene, and the open reading frame 1ab (ORF1ab) gene fragment (genomic coordinates: 14911-15910); (Table 7) is the copy number concentration of N, E and ORF1ab genes.
  • N gene-1, N gene-2, N gene-3, and N gene-4 are 9.8 ⁇ 10 4 copies/ml, 9.8 ⁇ 10 3 copies/ml, 9.8 ⁇ 10 2 copies/ml, respectively. 4.9 ⁇ 0 2 copies/ml.
  • E gene-1, E gene-2, E gene-3, and E gene-4 are 7.63 ⁇ 10 4 copies/ml, 7.63 ⁇ 10 3 copies/ml, 7.63 ⁇ 10 2 copies/ml, 3.82 ⁇ 10 2 copies/ml.
  • the kit of the present invention has reasonable design and feasible technology; the quality control system is stable and reliable; the stability of the product is good, the amount of template required for detection is small, and the detection result is objective and accurate.
  • the design of five targets can better overcome the false negatives caused by the variation of the RNA virus passaging process, can effectively increase the positive detection rate, and avoid missed detection as much as possible. High clinical application value.

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Abstract

A primer, probe and kit for detecting a novel coronavirus. A specific primer and a probe are designed according to an ORF1ab gene, an E gene, an N gene, an S gene and an M gene of novel coronavirus 2019-nCoV, for identifying and detecting the novel coronavirus. The specific primer/the probe and a kit for detecting 2019-nCoV are high in sensitivity, precision and accuracy, good in specificity and stability, small in template quantity required for detection, and objective and accurate in detection result. The five-target design can better overcome the false negative caused by variation in an RNA virus passage process, and can effectively improve the positive detection rate, and avoid missed detection as much as possible. The present invention has a high clinical application value.

Description

一种检测新型冠状病毒的引物、探针及试剂盒A primer, probe and kit for detecting novel coronavirus
本申请要求于2020年04月02日提交中国专利局、申请号为202010253120.8、发明名称为“一种检测新型冠状病毒的引物、探针及试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on April 02, 2020, the application number is 202010253120.8, and the invention title is "A primer, probe and kit for detecting a novel coronavirus", and its entire contents Incorporated in this application by reference.
技术领域Technical field
本发明涉及病毒检测技术领域，具体涉及一种检测新型冠状病毒的引物、探针及试剂盒。The invention relates to the technical field of virus detection, in particular to a primer, a probe and a kit for detecting a novel coronavirus.
背景技术Background technique
核酸检验(RT-PCR)是新冠肺炎诊断的“金标准”。国家药品监督管理局应急审批首批7家企业的新型冠状病毒2019-nCoV核酸检测试剂盒【一文读懂新型冠状病毒的核酸检测,国家药品监督管理局医疗器械技术审评中心,中国医药报,2020-2-11第002版】，批准产品均基于新型冠状病毒基因组中开放读码框1a/b(Openreading frame 1ab，ORF1ab)、包膜蛋白(Envelope protein，E)和核衣壳蛋白(Nucleocapsid protein，N)三个基因设计。Nucleic acid test (RT-PCR) is the "gold standard" for the diagnosis of new coronary pneumonia. The National Medical Products Administration urgently approved the first batch of 7 companies' new coronavirus 2019-nCoV nucleic acid detection kits 2020-2-11 No. 002], the approved products are based on the open reading frame 1a/b (Open reading frame 1ab, ORF1ab), Envelope protein (E) and Nucleocapsid (Nucleocapsid) of the novel coronavirus genome. protein, N) Three gene design.
但病毒的进化非常迅速【Bartoszewicz J M,Seidel A,Renard B Y.Interpretable detection of novel human viruses from genome sequencing data[J].BioRxiv,2020.】，通过对序列的比对和文献检索，可以发现，ORF1ab基因已经发现相关SNP位点突变，编码结构蛋白的基因(如，S、E、M和N)在β冠状病毒属中表现高度保守性，但E基因与蝙蝠物种高度同源，针对E基因的引物探针很难或不能与这两个物种进行区分。But the evolution of the virus is very rapid [Bartoszewicz J M, Seidel A, Renard B Y. Interpretable detection of novel human viruses from gene sequencing data [J]. BioRxiv, 2020.], it can be found through sequence alignment and literature search The ORF1ab gene has found related SNP mutations. The genes encoding structural proteins (such as S, E, M, and N) are highly conserved in the β-coronavirus genus, but the E gene is highly homologous to the bat species and targets E The primer probes of genes are difficult or impossible to distinguish from these two species.
中国科学院主办的《国家科学评论》(National Science Review)于3月3日发表论文《关于SARS-CoV-2的起源和持续进化》(On the origin and continuing evolution of SARS-CoV-2)，中国科研团队最新发现显示：新冠病毒已于近期产生了149个突变点。一份发表在病毒学国际交流平台，由巴西Adolfo Lutz国家研究所、国家参考实验室，以及圣保罗大学、牛津大学热带医学研究所共同完成的题为《Firstreport of COVID-19 in South America》的研究首次分析了南美洲新冠病毒的基因序列，初步的遗传分析表明，这株巴西“Brazil/SPBR1/2020”病毒的基因组，跟中国此前公布 的“Hu-1参考株”有3处不同，表示病毒在传播的过程中已经开始突变。The National Science Review sponsored by the Chinese Academy of Sciences published a paper "On the origin and continuing evolution of SARS-CoV-2" (On the origin and continuing evolution of SARS-CoV-2) on March 3, China The latest findings of the scientific research team show that the new coronavirus has recently produced 149 mutation points. A study titled "First report of COVID-19 in South America", published on the International Exchange Platform of Virology, jointly completed by Brazil's Adolfo Lutz National Institute, National Reference Laboratory, University of Sao Paulo and Institute of Tropical Medicine, University of Oxford The gene sequence of the South American new coronavirus was analyzed for the first time. Preliminary genetic analysis showed that the genome of this Brazilian "Brazil/SPBR1/2020" virus is different from the previously announced "Hu-1 Reference Strain" in China in three places, indicating the virus In the process of spreading, mutations have begun.
因此，目前核酸检测在应用中准确性遭到质疑。一些流行病学史、临床症状吻合，甚至CT显示肺部病毒感染的病患未能得到核酸“阳性”确诊。有学者认为现有核酸检测对于阳性病人，最高30％～50％的阳性率。新型冠状病毒感染的肺炎实验室检测技术指南中推荐检测ORF1ab和N基因，检测阳性判断要求两个基因均为阳性；在检测方法的局限性中强调不能排除因病毒变异导致的假阴性可能。Ai JW【Ai JW,Zhang H C,Xu T,et al.Optimizing diagnostic strategy for novel coronavirus pneumonia,a mμlti-center study in Eastern China[J].medRxiv,2020.】认为RT-PCR有15％假阴性率，建议设计RT-PCR时建议扩增超过1个基因靶标分析。Therefore, the accuracy of nucleic acid detection in applications is currently being questioned. Some patients with epidemiological history, clinical symptoms coincided, and even CT showed that patients with pulmonary virus infection failed to get a “positive” diagnosis of nucleic acid. Some scholars believe that the current nucleic acid test has a maximum positive rate of 30% to 50% for positive patients. The technical guidelines for laboratory testing of pneumonia caused by new coronavirus infection recommend the detection of ORF1ab and N genes. A positive test requires both genes to be positive; the limitations of the detection method emphasize that the possibility of false negatives due to virus mutation cannot be ruled out. Ai JW [Ai JW, Zhang H C, Xu T, et al. Optimizing diagnostic strategy for novel coronavirus pneumonia, a mμlti-center study in Eastern China[J].medRxiv, 2020.] It is believed that RT-PCR has 15% false negatives When designing RT-PCR, it is recommended to amplify more than one gene target for analysis.
综上，在新冠疫情全面爆发的当下，亟需一种特异性强、准确率高的用于检测新型冠状病毒的寡核苷酸组合物及其检测试剂盒和应用。To sum up, in the current full-scale outbreak of the new crown epidemic, there is an urgent need for an oligonucleotide composition with strong specificity and high accuracy for detecting the new coronavirus and its detection kit and application.
发明内容Summary of the invention
为了解决上述技术问题，本发明采用如下技术方案：In order to solve the above technical problems, the present invention adopts the following technical solutions:
一种检测新型冠状病毒的RT-PCR试剂盒，该试剂盒包含ORF1ab基因、N基因、E基因、S基因和M基因的特异性引物；An RT-PCR kit for detecting novel coronavirus, the kit contains specific primers for ORF1ab gene, N gene, E gene, S gene and M gene;
所述ORF1ab基因的特异性引物序列如SEQ ID NO:1～2所示，所述N基因的特异性引物序列如SEQ ID NO:4～5所示，所述E基因的特异性引物序列如SEQ ID NO:7～8所示，所述S基因的特异性引物序列如SEQ ID NO:10～11所示，所述M基因的特异性引物序列如SEQ ID NO:13～14所示。The specific primer sequence of the ORF1ab gene is shown in SEQ ID NO: 1 to 2, the specific primer sequence of the N gene is shown in SEQ ID NO: 4 to 5, and the specific primer sequence of the E gene is shown in SEQ ID NO: 7 to 8, the specific primer sequence of the S gene is shown in SEQ ID NO: 10 to 11, and the specific primer sequence of the M gene is shown in SEQ ID NO: 13 to 14.
优选地，所述试剂盒还包含ORF1ab基因、N基因、E基因、S基因和M基因的探针；所述ORF1ab基因的探针序列如SEQ ID NO:3所示，所述N基因的探针序列如SEQ ID NO:6所示，所述E基因的探针序列如SEQ ID NO:9所示，所述S基因的探针序列如SEQ ID NO:12所示，所述M基因的探针序列如SEQ ID NO:15所示。Preferably, the kit further contains probes for ORF1ab gene, N gene, E gene, S gene and M gene; the probe sequence of ORF1ab gene is shown in SEQ ID NO: 3, and the probe for N gene The needle sequence is shown in SEQ ID NO: 6, the probe sequence of the E gene is shown in SEQ ID NO: 9, the probe sequence of the S gene is shown in SEQ ID NO: 12, and the probe sequence of the M gene The probe sequence is shown in SEQ ID NO: 15.
优选地，所述试剂盒的反应体系为：Preferably, the reaction system of the kit is:
2×One Step RT-PCR Buffer III10μl，2×One Step RT-PCR Buffer III 10μl,
5U/μlTakara Ex Taq HS0.4μl，5U/μlTakara ExTaq HS0.4μl,
PrimeScript RT Enzyme Mix II0.4μl，PrimeScript RT Enzyme Mix II 0.4μl,
10μMForward Primer0.4μl，10μMForward Primer0.4μl,
10μM Reverse Primer0.4μl，10μM Reverse Primer 0.4μl,
Probe0.8μl，Probe0.8μl,
Total RNA2μl，Total RNA2μl,
RNase Free dH 2O5.6μl。 RNase Free dH 2 O5.6μl.
优选地，所述试剂盒的反应条件为：55℃ 15min，1个循环；95℃ 30s，1个循环；95℃ 10s，55℃ 30s，10个循环；95℃ 10s，55℃ 30s，35个循环。Preferably, the reaction conditions of the kit are: 55°C 15min, 1 cycle; 95°C 30s, 1 cycle; 95°C 10s, 55°C 30s, 10 cycles; 95°C 10s, 55°C 30s, 35 cycles cycle.
一种检测新型冠状病毒ORF1ab基因的RT-PCR特异性引物，其序列如SEQ ID NO:1～2所示。A RT-PCR specific primer for detecting the ORF1ab gene of the novel coronavirus. Its sequence is shown in SEQ ID NO: 1~2.
一种检测新型冠状病毒ORF1ab基因的RT-PCR探针，其序列如SEQ ID NO:3所示。An RT-PCR probe for detecting the ORF1ab gene of the novel coronavirus. Its sequence is shown in SEQ ID NO: 3.
一种检测新型冠状病毒N基因的RT-PCR特异性引物，其序列如SEQ ID NO:4～5所示。A specific RT-PCR primer for detecting the N gene of the novel coronavirus, whose sequence is shown in SEQ ID NO: 4 to 5.
一种检测新型冠状病毒N基因的RT-PCR探针，其序列如SEQ ID NO:6所示。An RT-PCR probe for detecting the N gene of the novel coronavirus. Its sequence is shown in SEQ ID NO: 6.
一种检测新型冠状病毒E基因的RT-PCR特异性引物，其序列如SEQ ID NO:7～8所示。A RT-PCR specific primer for detecting the E gene of the novel coronavirus, and its sequence is shown in SEQ ID NO: 7-8.
一种检测新型冠状病毒E基因的RT-PCR探针，其序列如SEQ ID NO:9所示。An RT-PCR probe for detecting the E gene of the novel coronavirus, the sequence of which is shown in SEQ ID NO: 9.
一种检测新型冠状病毒S基因的RT-PCR特异性引物，其序列如SEQ ID NO:10～11所示。A specific RT-PCR primer for detecting the S gene of the novel coronavirus, whose sequence is shown in SEQ ID NO: 10-11.
一种检测新型冠状病毒S基因的RT-PCR探针，其序列如SEQ ID NO:12所示。An RT-PCR probe for detecting the S gene of the novel coronavirus. Its sequence is shown in SEQ ID NO: 12.
一种检测新型冠状病毒M基因的RT-PCR特异性引物，其序列如SEQ ID NO:13～14所示。A specific RT-PCR primer for detecting the M gene of the novel coronavirus, and its sequence is shown in SEQ ID NO: 13-14.
一种检测新型冠状病毒M基因的RT-PCR探针，其序列如SEQ ID NO:15所示。An RT-PCR probe for detecting the M gene of the novel coronavirus. Its sequence is shown in SEQ ID NO: 15.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明发现新型冠状病毒2019-nCoV的ORF1ab基因、E基因、N基因、S基因和M基因具较强保守性，并根据该5基因特异性的设计引物和探针，用于新型冠状病毒的鉴别检测。本发明检测试剂盒除检测ORF1ab、E和N基因外，增加了表面糖蛋白(S)和膜糖蛋白(M)基因的检测，通过五靶标联合检测，综合判断，能够提高RT-PCR方法检测新型冠状病毒的准确性。The present invention found that the ORF1ab gene, E gene, N gene, S gene and M gene of the new coronavirus 2019-nCoV are highly conserved, and designed primers and probes specific to the 5 genes for use in the new coronavirus Identification test. In addition to detecting ORF1ab, E and N genes, the detection kit of the present invention adds the detection of surface glycoprotein (S) and membrane glycoprotein (M) genes. The combined detection of five targets and comprehensive judgment can improve the detection of RT-PCR methods. The accuracy of the new coronavirus.
本发明检测新型冠状病毒2019-nCoV的特异性引物探针和试剂盒，灵敏度、精密度、准确度高，特异性好，稳定性好，检测所需模板量少，检测结果客观准确。五靶标(包括ORF1ab、N、E、S和M基因)设计能够更好的克服RNA病毒传代过程变异导致的假阴性，可有效提升阳性检出率，尽可能避免漏检的情况出现，具有较高的临床应用价值。The specific primer probe and kit for detecting the novel coronavirus 2019-nCoV of the present invention have high sensitivity, precision, accuracy, good specificity, good stability, small amount of template required for detection, and objective and accurate detection results. The design of five targets (including ORF1ab, N, E, S and M genes) can better overcome the false negatives caused by the variation of the RNA virus passaging process, can effectively increase the positive detection rate, and avoid missed detection as much as possible. High clinical application value.
附图说明Description of the drawings
图1为ORF1ab基因不同引物探针组合检测低值假病毒上机对比图。其中，A为引物探针组1，B为引物探针组2。Figure 1 is a computer comparison diagram of different primer probe combinations for ORF1ab gene detection of low-value pseudoviruses. Among them, A is primer probe set 1 and B is primer probe set 2.
图2为N基因不同引物探针组合检测低值假病毒上机对比图。其中，A为引物探针组1，B为引物探针组2。Figure 2 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the N gene. Among them, A is primer probe set 1 and B is primer probe set 2.
图3为E基因不同引物探针组合检测低值假病毒上机对比图。其中，A为引物探针组1图B为引物探针组2。Figure 3 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the E gene. Among them, A is primer probe set 1 and Figure B is primer probe set 2.
图4为S基因不同引物探针组合检测低值假病毒上机对比图。其中，A为引物探针组1图B为引物探针组2。Figure 4 is a comparison diagram of the computer-based detection of low-value pseudoviruses with different primer probe combinations of the S gene. Among them, A is primer probe set 1 and Figure B is primer probe set 2.
图5为M基因不同引物探针组合检测低值假病毒上机对比图。其中，A为引物探针组1，B为引物探针组2。Figure 5 is a computer comparison diagram of detecting low-value pseudoviruses with different primer probe combinations of the M gene. Among them, A is primer probe set 1 and B is primer probe set 2.
图6为本发明引物和探针与CDC推荐的引物和探针对比结果。其中，A为中国CDC推荐的ORF1ab基因引物和探针的扩增曲线，B为本发明引物和探针的扩增曲线，C为中国CDC推荐的N基因引物和探针的扩增 曲线，D为美国CDC推荐的ORF1ab引物和探针的扩增曲线。1～5曲线分别依次对应包含新型冠状病毒5个相关基因(ORF1ab、N、E、S和M)的假病毒RNA的上机检测结果。Fig. 6 is the comparison result of the primers and probes of the present invention and the primers and probes recommended by CDC. Among them, A is the amplification curve of ORF1ab gene primers and probe recommended by China CDC, B is the amplification curve of primers and probes of the present invention, C is the amplification curve of N gene primers and probes recommended by China CDC, D Amplification curve of ORF1ab primers and probes recommended by CDC in the United States. Curves 1 to 5 respectively correspond to the results of the computer-based detection of pseudoviral RNA containing 5 related genes of the novel coronavirus (ORF1ab, N, E, S, and M).
图7为一步法混合体系的ORF1ab基因、N基因和E基因线性关系图。Figure 7 is a linear relationship diagram of ORF1ab gene, N gene and E gene of the one-step mixed system.
图8为一步法混合体系的M基因和S基因线性关系图。Figure 8 shows the linear relationship between the M gene and the S gene of the one-step mixed system.
图9为一步法不同反应条件的扩增曲线。其中A为反应条件1的扩增曲线，B为反应条件2的扩增曲线。Figure 9 shows the amplification curves of the one-step method under different reaction conditions. A is the amplification curve of reaction condition 1, and B is the amplification curve of reaction condition 2.
图10为阳性参考品检测结果。其中A为混合体系1检测结果，B为混合体系2检测结果。Figure 10 shows the test results of the positive reference product. Among them, A is the detection result of mixed system 1, and B is the detection result of mixed system 2.
图11为ORF1ab、E和N基因的检测限参考品的检测结果。其中A为混合体系1中拷贝数1×10 4copies/ml样品上机检测结果，B为混合体系1中拷贝数1×10 3copies/ml样品上机检测结果，C为混合体系1中拷贝数5×10 2copies/ml样品上机检测结果。 Figure 11 shows the detection results of ORF1ab, E and N gene detection limit reference products. Among them, A is the results of the computer-based testing of the 1×10 4 copies/ml sample in the hybrid system 1, B is the computer-based testing results of the 1×10 3 copies/ml sample in the hybrid system 1, and C is the copy of the hybrid system 1. Count the test results of 5×10 2 copies/ml samples on the machine.
图12为S和M基因的检测限参考品的检测结果。其中A为混合体系2中拷贝数1×10 4copies/ml样品上机检测结果，B为混合体系2中拷贝数1×10 3copies/ml样品上机检测结果，C为混合体系2中拷贝数5×10 2copies/ml样品上机检测结果。 Figure 12 shows the detection results of the reference samples for the detection limit of S and M genes. Among them, A is the computer-based test result of the 1×10 4 copies/ml sample in the hybrid system 2, B is the computer-based test result of the 1×10 3 copies/ml sample in the hybrid system 2, and C is the copy in the hybrid system 2. Count the test results of 5×10 2 copies/ml samples on the machine.
图13为分析特异性参考品的检测结果。其中A为混合体系1的检测结果，B为混合体系2的检测结果。Figure 13 shows the results of the analysis of specific reference products. Wherein A is the detection result of mixed system 1, and B is the detection result of mixed system 2.
图14为本发明试剂盒检测人冠状病毒HCoV特异性测试结果。其中A为OC43样品上机检测结果，B为HKU1样品上机检测结果，C为229E样品上机检测结果，D为NL63样品上机检测结果。Figure 14 is the test result of the specificity of the human coronavirus HCoV detected by the kit of the present invention. Among them, A is the on-machine test result of the OC43 sample, B is the on-board test result of the HKU1 sample, C is the on-board test result of the 229E sample, and D is the on-board test result of the NL63 sample.
图15为本发明试剂盒检测SARS、MERS、甲型流感和乙型流感病毒特异性测试结果。其中A为SARS样品上机检测结果，B为MERS样品上机检测结果，C为H1N1样品上机检测结果，D为INFB样品上机检测结果。Figure 15 shows the specific test results of SARS, MERS, influenza A and influenza B viruses detected by the kit of the present invention. Among them, A is the on-board testing result of SARS samples, B is the on-board testing results of MERS samples, C is the on-board testing results of H1N1 samples, and D is the on-board testing results of INFB samples.
图16为本发明试剂盒检测呼吸道合胞病毒、人副流感病毒、腺病毒特异性测试结果。其中A为RSV样品上机检测结果，B为PV样品上机 检测结果，C为ADV样品上机检测结果。Figure 16 shows the specific test results of the kit of the present invention for detecting respiratory syncytial virus, human parainfluenza virus, and adenovirus. Among them, A is the on-board testing result of RSV samples, B is the on-board testing results of PV samples, and C is the on-board testing results of ADV samples.
图17为本发明试剂盒检测人类基因组DNA、2019-nCoVRNA阴性样本特异性测试结果。其中A为人类基因组DNA，B为阴性样本。Figure 17 is a test result of the kit of the present invention for detecting human genomic DNA and the specificity of 2019-nCoVRNA negative samples. Among them, A is human genomic DNA, and B is a negative sample.
图18为体系1、体系2精密度参考品的检测结果。其中A为混合体系1低值精密度检测结果，B为混合体系2低值精密度检测结果，C为混合体系1中值精密度检测结果，D为混合体系2中值精密度检测结果。Figure 18 shows the test results of system 1 and system 2 precision reference products. Among them, A is the low precision test result of mixed system 1, B is the low precision test result of mixed system 2, C is the median precision test result of mixed system 1, and D is the median precision test result of mixed system 2.
图19为对照组、I1干扰物参考品的检测结果。其中A为对照阳性RNA检测结果，B为干扰物I1在混合体系1的检测结果，C为干扰物I1在混合体系2的检测结果。Figure 19 shows the test results of the control group and the I1 interfering substance reference. Among them, A is the control positive RNA detection result, B is the detection result of the interfering substance I1 in the mixed system 1, and C is the detection result of the interfering substance I1 in the mixed system 2.
图20为I2和I3干扰物参考品的检测结果。其中A为干扰物I2在混合体系1的检测结果，B为干扰物I2在混合体系2的检测结果，C为干扰物I3在混合体系1的检测结果，D为干扰物I3在混合体系2的检测结果。Figure 20 shows the detection results of I2 and I3 interfering substance reference products. Where A is the detection result of interfering substance I2 in mixed system 1, B is the detection result of interfering substance I2 in mixed system 2, C is the detection result of interfering substance I3 in mixed system 1, and D is the detection result of interfering substance I3 in mixed system 2. Test results.
图21为本发明试剂盒检测ORF1ab、E、N基因准确度测试结果。其中A为混合体系1中ORF1ab基因检测结果，B为混合体系1中E基因检测结果，C为混合体系1中N基因检测结果。Fig. 21 is the test result of the accuracy of detecting ORF1ab, E, and N genes by the kit of the present invention. Among them, A is the ORF1ab gene detection result in mixed system 1, B is the E gene detection result in mixed system 1, and C is the N gene detection result in mixed system 1.
图22为本发明试剂盒检测M、S基因准确度测试结果。其中A为混合体系2中M基因检测结果，B为混合体系2中S基因检测结果。Figure 22 is the test result of the accuracy of detecting M and S genes by the kit of the present invention. Among them, A is the detection result of the M gene in the mixed system 2, and B is the detection result of the S gene in the mixed system 2.
图23(NCRM)-GBW(E)091090(低浓度)梯度样本上机结果。Figure 23 (NCRM)-GBW(E)091090 (low concentration) gradient sample computer results.
具体实施方式Detailed ways
下面结合具体实施方式对本发明作进一步的说明。下述实施例中的试验方法，如无特别说明，均为常规方法。下述实施例中所用的实验材料及试剂，如无特别说明，均购自常规生化试剂商店。The present invention will be further described below in conjunction with specific embodiments. The test methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials and reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
实施例1、特异性引物和探针的设计Example 1. Design of specific primers and probes
引物探针的设计中，需要选择组内保守和组间特异的区域进行设计，从而避免漏检造成假阴性，或特异性不佳导致的假阳性。In the design of primer probes, it is necessary to select conserved and specific regions within the group for design, so as to avoid false negatives caused by missed detection, or false positives caused by poor specificity.
本发明通过序列比对、文献检索发现，2019-nCoV结构基因S、M、ORF1ab、E和N基因具较强保守性，可作为用于新冠病毒的鉴别检测的 靶区。The present invention finds through sequence comparison and literature search that the 2019-nCoV structural genes S, M, ORF1ab, E and N genes are highly conserved and can be used as target regions for identification and detection of new coronaviruses.
本实施例选择地方性人类冠状病毒(HKU1、OC43、229E和NL63)、SARS冠状病毒、MERS冠状病毒、蝙蝠冠状病毒(MG772933_Bat_bat_SL_CoVZC45)、SARS样病毒(MG772934_Bat_SARS_like_bat_SL_CoVZXC21)序列进行比对，结果如表1所示，其中，框内标示为引物、探针的结合位点。开放读码框1ab(ORF1ab)、核壳蛋白(N)、包膜蛋白(E)、表面糖蛋白(S)和膜糖蛋白(M)基因在不同病毒中具有特异性，可以用于设计特异性引物和探针序列。In this example, the sequences of endemic human coronaviruses (HKU1, OC43, 229E, and NL63), SARS coronavirus, MERS coronavirus, bat coronavirus (MG772933_Bat_bat_SL_CoVZC45), and SARS-like virus (MG772934_Bat_SARS_like_bat_SL_CoVZXC21) were selected for alignment, and the results are shown in Table 1. In the box, the binding sites of primers and probes are indicated in the box. Open reading frame 1ab (ORF1ab), nucleocapsid protein (N), envelope protein (E), surface glycoprotein (S) and membrane glycoprotein (M) genes are specific in different viruses and can be used for design specificity Sex primer and probe sequences.
表1多种冠状病毒靶基因序列比对结果Table 1 Sequence comparison results of multiple coronavirus target genes
Figure PCTCN2020109072-appb-000001
Figure PCTCN2020109072-appb-000001
应用Mega对Genbank中检索到的2019-nCoV病毒序列进行同源性比较，分别对应5个基因的序列确认保守区段设计两组用于靶基因区检测的引物和探针。将设计的引物探针组1和引物探针组2，分别结合其他组分配制成PCR反应体系，以低值假病毒(1.49×103copies/ml)为样本，进行检测，通过对检测CT值和扩增曲线线型进行对比，确认优选地的引物和探针组，作为检测新冠病毒2019-nCoV靶基因检测的特异性引物和探针序列。Use Mega to compare the homology of the 2019-nCoV virus sequences retrieved in Genbank, and design two sets of primers and probes for the detection of target gene regions corresponding to the sequences of 5 genes to confirm the conserved segments. The designed primer probe set 1 and primer probe set 2 were combined with other groups to form a PCR reaction system. The low-value pseudovirus (1.49×103copies/ml) was used as the sample for detection, and the CT value and The amplification curve line type is compared to confirm the preferred primer and probe set as the specific primer and probe sequence for the detection of the new coronavirus 2019-nCoV target gene.
ORF1ab基因的引物探针组1：Primer probe set of ORF1ab gene 1:
ORF-F(SEQ ID NO:16)：5’-CCCTGTGGGTTTTACACTTAA-3’；ORF-F (SEQ ID NO: 16): 5’-CCCTGTGGGTTTTACACTTAA-3’;
ORF-R(SEQ ID NO:17)：5’-ACGATTGTGCATCAGCTGA-3’；ORF-R (SEQ ID NO: 17): 5’-ACGATTGTGCATCAGCTGA-3’;
ORF-P(SEQ ID NO:18)：5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’；ORF-P (SEQ ID NO: 18): 5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’;
RF1ab基因的引物探针组2：Primer probe set of RF1ab gene 2:
ORF-F(SEQ ID NO:1)：5’-CCATGTAGAAACATTTTACCCA-3’；ORF-F(SEQ ID NO:1): 5’-CCATGTAGAAACATTTTACCCA-3’;
ORF-R(SEQ ID NO:2)：5’-TTCTAATAGCATTCTTTGCATTTT-3’；ORF-R (SEQ ID NO: 2): 5’-TTCTAATAGCATTCTTTGCATTTT-3’;
ORF-P(SEQ ID NO:3)：5’-TTACAATCTAGTCAAGCGTGGCAACCG-3’。ORF-P (SEQ ID NO: 3): 5'-TTACAATCTAGTCAAGCGTGGCAACCG-3'.
ORF1ab基因探针的5’端标记FAM基团，3’端标记BHQ1基团。The 5'end of the ORF1ab gene probe is labeled with FAM group, and the 3'end is labeled with BHQ1 group.
检测结果如图1所示，ORF1ab基因两组引物探针组的CT值相似-21，但引物探针组2表现进入平台期更早。故选择引物探针组2进行后续试验。The detection result is shown in Figure 1. The CT value of the two primer probe groups of ORF1ab gene is similar to -21, but the primer probe group 2 appears to enter the plateau earlier. Therefore, primer probe set 2 was selected for subsequent experiments.
N基因的引物探针组1：N gene primer probe set 1:
N-F(SEQ ID NO:4)：5’-TGCCACTAAAGCATACAATGTAA-3’；N-F (SEQ ID NO: 4): 5’-TGCCACTAAAGCATACAATGTAA-3’;
N-R(SEQ ID NO:5)：5’-GTTCCTTGTCTGATTAGTTCCTG-3’；N-R (SEQ ID NO: 5): 5’-GTTCCTTGTCTGATTAGTTCCTG-3’;
N-P(SEQ ID NO:6)：5’-AGCTTTYGGCAGACGTGGTCCAG-3’；其中Y为兼并碱基，指C或T，下文中Y指代与本处相同，不再赘述。N-P (SEQ ID NO: 6): 5'-AGCTTTYGGCAGACGTGGTCCAG-3'; where Y is a merging base and refers to C or T. In the following, Y refers to the same as this one, and will not be repeated here.
N基因的引物探针组2：N gene primer probe set 2:
N-F(SEQ ID NO:19)：5’-CGCAAATTGCACAATTT-3’；N-F (SEQ ID NO: 19): 5’-CGCAAATTGCACAATTT-3’;
N-R(SEQ ID NO:20)：5’-TTCTTTTTGTCCTTTTTAGG-3’；N-R (SEQ ID NO: 20): 5’-TTCTTTTTGTCCTTTTTAGG-3’;
N-P(SEQ ID NO:21)：5’-CGTGGTTGACCTACACAGGTGCCATC-3’；N-P (SEQ ID NO: 21): 5’-CGTGGTTGACCTACACAGGTGCCATC-3’;
N基因探针的5’端标记CY5基团，3’端标记BHQ2基团。The 5'end of the N gene probe is labeled with CY5 group, and the 3'end is labeled with BHQ2 group.
检测结果如图2所示，N基因两组引物探针组的CT值相似-35，但引物探针组1表现进入平台期更早。故选择引物探针组1进行后续试验。The detection result is shown in Figure 2. The CT value of the two sets of primer probes for the N gene is similar to -35, but the primer probe set 1 appears to enter the plateau earlier. Therefore, primer probe set 1 was selected for subsequent experiments.
E基因的引物探针组1：E gene primer probe set 1:
E-F(SEQ ID NO:7)：5’-TTGCTTTCGTGGTATTCTTGC-3’；E-F (SEQ ID NO: 7): 5’-TTGCTTTCGTGGTATTCTTGC-3’;
E-R(SEQ ID NO:8)：5’-ATATTGCAGCAGTACGCACACA-3’；E-R (SEQ ID NO: 8): 5’-ATATTGCAGCAGTACGCACACA-3’;
E-P(SEQ ID NO:9)：5’-ACACTAGCCATCCTTACTGCGCTTCG-3’；E-P (SEQ ID NO: 9): 5’-ACACTAGCCATCCTTACTGCGCTTCG-3’;
E基因的引物探针组2：E gene primer probe set 2:
E-F(SEQ ID NO:22)：5’-TACTGCTGCAATATTGTTAACG-3’；E-F (SEQ ID NO: 22): 5’-TACTGCTGCAATATTGTTAACG-3’;
E-R(SEQ ID NO:23)：5’-ATCAGGAACTCTAGAAGAATTCAG-3’；E-R (SEQ ID NO: 23): 5’-ATCAGGAACTCTAGAAGAATTCAG-3’;
E-P(SEQ ID NO:24)：5’-TCTTGTAAAACCTTCTTTTTACGTTTACTCT-3’；E-P (SEQ ID NO: 24): 5’-TCTTGTAAAACCTTCTTTTTACGTTTACTCT-3’;
E基因探针的5’端标记ROX基团，3’端标记BHQ2基团。The 5'end of the E gene probe is labeled with ROX group, and the 3'end is labeled with BHQ2 group.
检测结果如图3所示，E基因两组引物探针组的CT值相似-19，但引物探针组1表现进入平台期更早。故选择引物探针组1进行后续试验。The detection result is shown in Figure 3. The CT value of the two primer probe groups of the E gene is similar to -19, but the primer probe group 1 enters the plateau earlier. Therefore, primer probe set 1 was selected for subsequent experiments.
S基因的引物探针组1：S gene primer probe set 1:
S-F(SEQ ID NO:10)：5’-TTGTGCCCTTTTGGTGAAGT-3’；S-F (SEQ ID NO: 10): 5’-TTGTGCCCTTTTGGTGAAGT-3’;
S-R(SEQ ID NO:11)：5’-TAGGACAGAATAATCAGCAACACA-3’；S-R (SEQ ID NO: 11): 5’-TAGGACAGAATAATCAGCAACACA-3’;
S-P(SEQ ID NO:12)：5’-CCACCAGATTTGCATCTGTTTATGCTTG-3’；S-P (SEQ ID NO: 12): 5’-CCACCAGATTTGCATCTGTTTATGCTTG-3’;
S基因的引物探针组2：S gene primer probe set 2:
S-F(SEQ ID NO:25)：5’-TTTGTAATTAGAGGTGATGAAGTCA-3’；S-F (SEQ ID NO: 25): 5’-TTTGTAATTAGAGGTGATGAAGTCA-3’;
S-R(SEQ ID NO:26)：5’-TAACGCAGCCTGTAAAATCAT-3’；S-R (SEQ ID NO: 26): 5’-TAACGCAGCCTGTAAAATCAT-3’;
S-P(SEQ ID NO:27)：5’-ACAAATCGCTCCAGGGCAAACTGG-3’；S-P (SEQ ID NO: 27): 5’-ACAAATCGCTCCAGGGCAAACTGG-3’;
S基因探针的5’端标记FAM基团，3’端标记BHQ1基团。The 5'end of the S gene probe is labeled with FAM group, and the 3'end is labeled with BHQ1 group.
检测结果如图4所示，S基因引物探针组1的CT值为-20，引物探针 组2的CT值为-22。故选择引物探针组1进行后续试验。The detection result is shown in Fig. 4, the CT value of S gene primer probe set 1 is -20, and the CT value of primer probe set 2 is -22. Therefore, primer probe set 1 was selected for subsequent experiments.
M基因的引物探针组1：M gene primer probe set 1:
M-F(SEQ ID NO:28)：5’-TATTCTGACCAGACCGCTTCT-3’；M-F (SEQ ID NO: 28): 5’-TATTCTGACCAGACCGCTTCT-3’;
M-R(SEQ ID NO:29)：5’-TTTAGGCAGGTCCTTGATGTC-3’；M-R (SEQ ID NO: 29): 5’-TTTAGGCAGGTCCTTGATGTC-3’;
M-P(SEQ ID NO:30)：5’-CGGAGCTGTGATCCTTCGTGGACAT-3’；M-P (SEQ ID NO: 30): 5’-CGGAGCTGTGATCCTTCGTGGACAT-3’;
M基因的引物探针组2：Primer probe set of M gene 2:
M-F(SEQ ID NO:13)：5’-TGCYGTTTACAGAATAAATTGGA-3’；M-F (SEQ ID NO: 13): 5’-TGCYGTTTACAGAATAAATTGGA-3’;
M-R(SEQ ID NO:14)：5’-GAAGTAGCTGAGCCACATCAA-3’；M-R (SEQ ID NO: 14): 5’-GAAGTAGCTGAGCCACATCAA-3’;
M-P(SEQ ID NO:15)：5’-CGGTGGAATTGCTATCGCAATGG-3’。M-P (SEQ ID NO: 15): 5'-CGGTGGAATTGCTATCGCAATGG-3'.
M基因探针的5’端标记ROX基团，3’端标记BHQ2基团。The 5'end of the M gene probe is labeled with ROX group, and the 3'end is labeled with BHQ2 group.
检测结果如图5所示，M基因引物探针组1的CT值为-20，引物探针组2的CT值为-19。故选择引物探针组2进行后续试验。The detection result is shown in Fig. 5, the CT value of M gene primer probe set 1 is -20, and the CT value of primer probe set 2 is -19. Therefore, primer probe set 2 was selected for subsequent experiments.
实施例2、本发明特异性引物和探针与CDC推荐序列对比分析Example 2. Comparative analysis of specific primers and probes of the present invention and CDC recommended sequences
为评价实施例1优选的引物和探针，使用CDC(疾病预防控制中心)推荐序列作为对比序列，进行检测评价。In order to evaluate the preferred primers and probes of Example 1, the CDC (Center for Disease Control and Prevention) recommended sequence was used as a comparison sequence for detection and evaluation.
中国CDC针对E基因、S基因和M基因没有推荐引物和探针序列，仅针对ORF1ab和N基因推荐了引物和探针序列，其推荐的针对ORF1ab和N区的引物和探针序列如下：China CDC does not recommend primer and probe sequences for E gene, S gene and M gene. It only recommends primer and probe sequences for ORF1ab and N genes. The recommended primer and probe sequences for ORF1ab and N region are as follows:
ORF1ab-F(SEQ ID NO:16)：5’-CCCTGTGGGTTTTACACTTAA-3’；ORF1ab-F (SEQ ID NO: 16): 5’-CCCTGTGGGTTTTACACTTAA-3’;
ORF1ab-R(SEQ ID NO:17)：5’-ACGATTGTGCATCAGCTGA-3’；ORF1ab-R (SEQ ID NO: 17): 5’-ACGATTGTGCATCAGCTGA-3’;
ORF1ab-P(SEQ ID NO:18)：5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’；ORF1ab-P (SEQ ID NO: 18): 5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’;
N-F(SEQ ID NO:31)：5’-GGGGAACTTCTCCTGCTAGAAT-3’；N-F (SEQ ID NO: 31): 5’-GGGGAACTTCTCCTGCTAGAAT-3’;
N-R(SEQ ID NO:32)：5’-CAGACATTTTGCTCTCAAGCTG-3’；N-R (SEQ ID NO: 32): 5’-CAGACATTTTGCTCTCAAGCTG-3’;
N-P(SEQ ID NO:33)：5’-TTGCTGCTGCTTGACAGATT-3’。N-P (SEQ ID NO: 33): 5'-TTGCTGCTGCTTGACAGATT-3'.
美国CDC针对N基因、E基因、S基因和M基因没有推荐引物和探针序列，仅针对ORF1ab基因推荐了引物和探针序列，其推荐的引物和探针序列如下：The US CDC does not recommend primer and probe sequences for N gene, E gene, S gene and M gene. It only recommends primer and probe sequences for ORF1ab gene. The recommended primer and probe sequences are as follows:
ORF1ab-F(SEQ ID NO:34)：5’-GGGAGCCTTGAATACACCAAAA-3’；ORF1ab-F (SEQ ID NO: 34): 5’-GGGAGCCTTGAATACACCAAAA-3’;
ORF1ab-R(SEQ ID NO:35)：5’-TGTAGCACGATTGCAGCATTG-3’；ORF1ab-R (SEQ ID NO: 35): 5’-TGTAGCACGATTGCAGCATTG-3’;
ORF1ab-P(SEQ ID NO:36)：5’-AYCACATTGGCACCCGCAATCCTG-3’。ORF1ab-P (SEQ ID NO: 36): 5'-AYCACATTGGCACCCGCAATCCTG-3'.
检测评价使用的反应体系为：2×One Step RT-PCR Buffer III10μl，5U/μl的Takara Ex Taq HS0.6μl，PrimeScript RT Enzyme Mix II0.4μl，10μM Primer各0.4μl，10μM Probe0.8μl，Total RNA2μl，RNase Free dH 2O 5.4μl。 The reaction system used for detection and evaluation is: 2×One Step RT-PCR Buffer III 10μl, 5U/μl Takara Ex Taq HS 0.6μl, PrimeScript RT Enzyme Mix II 0.4μl, 10μM Primer 0.4μl each, 10μM Probe 0.8μl, Total RNA 2μl , RNase Free dH 2 O 5.4μl.
检测评价使用的反应条件为：55℃ 15min，1个循环；95℃ 30s，1个循环；95℃ 10s；55℃ 30s，45个循环。The reaction conditions used for detection and evaluation are: 55°C for 15 minutes, 1 cycle; 95°C for 30s, 1 cycle; 95°C for 10s; 55°C for 30s, 45 cycles.
结果如图6所示，其中1～5曲线分别依次对应包含新型冠状病毒5个相关基因(ORF1ab、N、E、S和M)的假病毒RNA的上机检测结果。假病毒的拷贝数在1.0×10 3copies/mL～1.0×10 7copies/mL。中国CDC推荐的ORF1ab的引物和探针荧光值低且曲线不佳，中国CDC和美国CDC推荐的N基因的引物和探针灵敏度和扩增曲线不佳。 The results are shown in Figure 6, where curves 1 to 5 respectively correspond to the results of the on-machine detection of pseudoviral RNA containing 5 related genes of the novel coronavirus (ORF1ab, N, E, S, and M). The copy number of the pseudovirus ranges from 1.0×10 3 copies/mL to 1.0×10 7 copies/mL. The primers and probes of ORF1ab recommended by the Chinese CDC have low fluorescence values and poor curves, and the primers and probes of the N gene recommended by the Chinese CDC and the US CDC have poor sensitivity and amplification curves.
实施例3、RT-PCR方法的优化试验Example 3. Optimization test of RT-PCR method
基于荧光RT-PCR的病毒RNA检测有两种方法，即先逆转录成cDNA后，再进行PCR的两步法，和在同一个体系中同时进行RT-PCR的一步法体系。两者各有优缺点。使用两步法体系，逆转录得到的cDNA易于保存；而一步法快速、简便、减少污染机会。对于多重PCR来说，需要考虑引物和探针间的干扰情况。There are two methods for the detection of viral RNA based on fluorescent RT-PCR, namely, a two-step method in which PCR is performed after reverse transcription into cDNA, and a one-step method in which RT-PCR is performed simultaneously in the same system. Both have their advantages and disadvantages. Using a two-step system, the cDNA obtained by reverse transcription is easy to store; while the one-step method is fast, simple and reduces the chance of contamination. For multiplex PCR, the interference between primers and probes needs to be considered.
本发明对比了两种反应体系的检测效果，先评估单对引物探针的检测效果，再将合成序列按反应管进行组合测试。The present invention compares the detection effects of the two reaction systems, first evaluates the detection effects of a single pair of primer probes, and then performs a combined test on the synthetic sequence according to the reaction tube.
本实施例中，两步法使用Takara RNA PCR Kit(RR019A)和Takara Ex TAQ HS(RR390L)，按产品说明书操作。两步法的逆转录反应体系如表2所示。In this embodiment, the two-step method uses Takara RNA PCR Kit (RR019A) and Takara Ex TAQ HS (RR390L), and operates according to the product manual. The two-step reverse transcription reaction system is shown in Table 2.
表2两步法的逆转录反应体系Table 2 Two-step reverse transcription reaction system
Figure PCTCN2020109072-appb-000002
Figure PCTCN2020109072-appb-000002
逆转录上机程序：①42℃ 30min，1循环；②95℃ 5min，1循环；③5℃ 5min，1循环。Reverse transcription program: ①42℃ 30min, 1 cycle; ②95℃ 5min, 1 cycle; ③5℃ 5min, 1 cycle.
两步法的qPCR反应体系如表3所示。The two-step qPCR reaction system is shown in Table 3.
表3两步法的qPCR反应体系Table 3 Two-step qPCR reaction system
试剂Reagent 使用量Usage amount 终浓度Final concentration
Premix Ex Taq(2×)Premix Ex Taq(2×) 12.5μl12.5μl 1×1×
Forward PrimerForward Primer 0.5μl0.5μl 0.5μM0.5μM
Reverse PrimerReverse Primer 0.5μl0.5μl 0.5μM0.5μM
probeprobe 1μl1μl 1μM1μM
模板template 5μl5μl //
水water 5.5μl5.5μl //
TotalTotal 20μl20μl //
qPCR程序：①95℃ 30s，1个循环；②95℃ 10s；55℃ 30s，45个循环【信号收集：55℃收集FAM、Cy5、ROX和HEX(或VIC)的信号】。qPCR program: ①95°C for 30s, 1 cycle; ②95°C for 10s; 55°C for 30s, 45 cycles [Signal collection: Collect FAM, Cy5, ROX and HEX (or VIC) signals at 55°C].
一步法使用Takara Primer Script one step RT-PCR Kit(RR064A)，按产品说明书操作。一步法逆转录体系如表4所示。The one-step method uses Takara Primer Script one step RT-PCR Kit (RR064A) and operates according to the product manual. The one-step reverse transcription system is shown in Table 4.
表4一步法逆转录体系Table 4 One-step reverse transcription system
试剂Reagent 使用量Usage amount 终浓度 Final concentration
2×One Step RT-PCR Buffer III2×One Step RT-PCR Buffer III 10μl10μl 1x1x
Takara Ex Taq HS(5U/μl)Takara Ex Taq HS(5U/μl) 0.4μl0.4μl //
PrimeScript RT Enzyme Mix IIPrimeScript RT Enzyme Mix II 0.4μl0.4μl //
Forward Primer(10μM)Forward Primer(10μM) 0.4μl0.4μl 0.2μM0.2μM
Reverse Primer(10μM)Reverse Primer(10μM) 0.4μl0.4μl 0.2μM0.2μM
ProbeProbe 0.8μl0.8μl //
Total RNATotal RNA 2μl2μl //
RNase Free dH 2O RNase Free dH 2 O 5.6μl5.6μl //
TotalTotal 20μl20μl //
一步法上机程序：①55℃ 15min，1个循环；②95℃ 30s，1个循环；③95℃ 10s；55℃ 30s，45个循环【信号收集：55℃收集FAM、Cy5、ROX和HEX(或VIC)的信号】。One-step computer program: ①55℃ 15min, 1 cycle; ②95℃ 30s, 1 cycle; ③95℃ 10s; 55℃ 30s, 45 cycles [Signal collection: 55℃ collect FAM, Cy5, ROX and HEX (or VIC )signal of】.
一步法的检测线性关系效果如图7和图8所示。其中，ORF1ab基因的荧光基团为FAM，斜率为-3.356，截距为34.722，相关系数为0.998，扩增效率为98.614％，线性关系式为y＝-3.356x+116.527(R2＝0.998)；N基因的荧光基团为CY5，斜率为-3.928，截距为38.636，相关系数为0.986，扩增效率为79.714％，线性关系式为y＝-3.928x+151.762(R2＝0.986)；E基因的荧光基团为ROX，斜率为-2.998，截距为34.439，相关系数为0.984，扩增效率为115.554％，线性关系式为y＝-2.998x+103.248(R2＝0.984)；M基因的荧光基团为ROX，斜率为-3.216，截距为37.082，相关系数为0.996，扩增效率为104.61％，线性关系式为y＝-3.216x+119.256(R2＝0.996)；S基因的荧光基团为FAM，斜率为-2.942，截距为34.832，相关系数为0.997，扩增效率为118.757％，线性关系式为y＝-2.942x+102.476(R2＝0.997)。The linear relationship effect of the one-step method is shown in Figure 7 and Figure 8. Among them, the fluorophore of ORF1ab gene is FAM, the slope is -3.356, the intercept is 34.722, the correlation coefficient is 0.998, the amplification efficiency is 98.614%, and the linear relationship is y=-3.356x+116.527 (R2=0.98); The fluorescent group of the N gene is CY5, the slope is -3.928, the intercept is 38.636, the correlation coefficient is 0.986, the amplification efficiency is 79.714%, and the linear relationship is y=-3.928x+151.762 (R2=0.986); E gene The fluorophore of is ROX, the slope is -2.998, the intercept is 34.439, the correlation coefficient is 0.984, the amplification efficiency is 115.554%, and the linear relationship is y=-2.998x+103.248 (R2=0.984); the fluorescence of the M gene The group is ROX, the slope is -3.216, the intercept is 37.082, the correlation coefficient is 0.996, the amplification efficiency is 104.61%, and the linear relationship is y = -3.216x + 119.256 (R2 = 0.996); the fluorophore of the S gene For FAM, the slope is -2.942, the intercept is 34.832, the correlation coefficient is 0.997, the amplification efficiency is 118.757%, and the linear relationship is y=-2.942x+102.476 (R2=0.997).
表5对比了两种检测体系的检测效果。单体系(针对单一靶标的检测)方面，一步法的体系在灵敏度上较两步法体系高，检测限是两步法体的2倍；但是两步法体系在逆转录步骤有样本的2倍的稀释关系，换算之后，两体系的扩增效率是相同的。Table 5 compares the detection effects of the two detection systems. In terms of single system (detection of a single target), the one-step system has higher sensitivity than the two-step system, and the detection limit is twice that of the two-step method; however, the two-step system has twice the sample in the reverse transcription step After conversion, the amplification efficiency of the two systems is the same.
混合体系(针对五种靶标的检测)方面，由于混合体系中引物之间的相互干扰，一步法和两步法体系的扩增效率均有所下降，两步法的检测限降为10 3copies/ml，而一步法体系的检测限降为500copies/ml，进行换算之后，二者的扩增效率依然相同。 In the mixed system (for the detection of five targets), due to the mutual interference between the primers in the mixed system, the amplification efficiency of the one-step method and the two-step method system are reduced, and the detection limit of the two-step method is reduced to 10 3 copies /ml, and the detection limit of the one-step system is reduced to 500copies/ml. After conversion, the amplification efficiency of the two is still the same.
综合一步法体系的简便性和高灵敏度，优选一步法检测体系。试剂盒的检测限500copies/ml，检测灵敏度高；线性范围0.983～0.999；精密度的变异性数≤5％。Considering the simplicity and high sensitivity of the one-step system, a one-step detection system is preferred. The detection limit of the kit is 500copies/ml, and the detection sensitivity is high; the linear range is 0.983～0.999; the variability of precision is less than 5%.
表5两种体系检测结果对比Table 5 Comparison of test results of two systems
Figure PCTCN2020109072-appb-000003
Figure PCTCN2020109072-appb-000003
实施例4、RT-PCR反应条件优化试验Example 4, RT-PCR reaction conditions optimization test
本实施例进一步优化了五靶标联检体系一步法的反应条件。This example further optimized the reaction conditions of the one-step method of the five-target joint inspection system.
上机程序(反应条件)1(45循环)：①55℃ 15min，1个循环；②95℃ 30s，1个循环；③95℃ 10s；55℃ 30s，45个循环【信号收集：55℃收集FAM、Cy5、ROX和HEX(或VIC)的信号】。Computer program (reaction conditions) 1 (45 cycles): ①55℃ 15min, 1 cycle; ②95℃ 30s, 1 cycle; ③95℃ 10s; 55℃ 30s, 45 cycles [Signal collection: 55℃ collect FAM, Cy5 , ROX and HEX (or VIC) signals].
上机程序2(10+35循环)：①55℃ 15min，1个循环；②95℃ 30s，1个循环；③95℃ 10s；55℃ 30s，10个循环；④95℃ 10s；55℃ 30s，35个循环【信号收集：55℃收集FAM、Cy5、ROX和HEX(或VIC)的信号】。Computer program 2 (10+35 cycles): ①55°C 15min, 1 cycle; ②95°C 30s, 1 cycle; ③95°C 10s; 55°C 30s, 10 cycles; ④95°C 10s; 55°C 30s, 35 cycles [Signal collection: Collect FAM, Cy5, ROX and HEX (or VIC) signals at 55°C].
结果如图9所示。上机程序1(图A)和上机程序2(图B)对扩增体系的灵敏度基本上没有影响，只是对低浓度的扩增曲线会有影响，使用上机程序2(10+35循环)的低浓度样本的扩增曲线会更加挺拔，更S型。因此选择上机程序2作后续试验。The result is shown in Figure 9. The computer program 1 (Figure A) and the computer program 2 (Figure B) basically have no effect on the sensitivity of the amplification system, but it will affect the low concentration amplification curve. Use the computer program 2 (10+35 cycles) ) The amplification curve of low-concentration samples will be more upright and more S-shaped. So choose the computer program 2 for follow-up test.
实施例5、五靶标联检试剂盒Example 5. Five-target joint inspection kit
本发明用于新型冠状病毒(2019-nCoV)检测的五靶标联检试剂盒，采用多重Taqman荧光探针技术，选取2019新型冠状病毒(2019-nCoV)开放读码框1ab(ORF1ab)、核壳蛋白(N)、包膜蛋白(E)、表面糖蛋白(S)和膜糖蛋白(M)基因作为扩增靶区域，设计特异性引物及荧光探针；选取核糖核酸酶P(RNase P)基因作为内标，设计特异性的引物和荧光探针如下。The five-target joint test kit for the detection of new coronavirus (2019-nCoV) of the present invention adopts multiple Taqman fluorescent probe technology to select 2019 new coronavirus (2019-nCoV) open reading frame 1ab (ORF1ab), nucleocapsid protein (N), envelope protein (E), surface glycoprotein (S) and membrane glycoprotein (M) genes are used as amplification target regions, design specific primers and fluorescent probes; select ribonuclease P (RNase P) genes As an internal standard, specific primers and fluorescent probes are designed as follows.
Rnase P-F(SEQ ID NO：37)：5’-AGATTTGGACCTGCGAGC-3’；Rnase P-F (SEQ ID NO: 37): 5’-AGATTTGGACCTGCGAGC-3’;
Rnase P-RSEQ ID NO：38)：5’-AACAACTGAATAGCCAAGGTG-3’；Rnase P-RSEQ ID NO: 38): 5’-AACAACTGAATAGCCAAGGTG-3’;
Rnase P-PSEQ ID NO39：)5’-TTCTGACCTGAAGGCTCTGCGCG-3’。该探针5’有HEX基团，3’有BHQ1基团。Rnase P-PSEQ ID NO39:) 5'-TTCTGACCTGAAGGCTCTGCGCG-3'. The probe has a HEX group at 5'and a BHQ1 group at 3'.
本发明试剂盒包括：2019-nCoV反应液A、2019-nCoV反应液B、2019-nCoV反应液C和2019-nCoV阴性质控品、2019-nCoV阳性质控品。其中，ORF1ab、N和E基因特异引物和探针在反应液A中，S和M基因特异引物和探针在反应液B中。各探针和引物的浓度为10-15pmol，优选12pmol。此外，反应液A和反应液B中还含有缓冲液和dNTPs。反应液C为酶液，如mMLV酶、HS-Taq酶、RNasin、UDG酶等。The kit of the present invention includes: 2019-nCoV reaction solution A, 2019-nCoV reaction solution B, 2019-nCoV reaction solution C, 2019-nCoV negative control substance, and 2019-nCoV positive quality control substance. Among them, ORF1ab, N and E gene-specific primers and probes are in reaction solution A, and S and M gene-specific primers and probes are in reaction solution B. The concentration of each probe and primer is 10-15 pmol, preferably 12 pmol. In addition, reaction solution A and reaction solution B also contain buffer and dNTPs. The reaction solution C is an enzyme solution, such as mMLV enzyme, HS-Taq enzyme, RNasin, UDG enzyme and so on.
为了验证本发明试剂盒的灵敏度、特异性、精密度、干扰因素、准确度，通过全自动荧光PCR检测仪的4个通道同时检测上述4种荧光信号(见表6)。In order to verify the sensitivity, specificity, precision, interference factors, and accuracy of the kit of the present invention, the above-mentioned four fluorescent signals are detected simultaneously through the four channels of the automatic fluorescent PCR detector (see Table 6).
表6检测通道及其检测基因对应表Table 6 Correspondence table of detection channels and detection genes
荧光通道Fluorescence channel FAMFAM Cy5Cy5 ROXROX VIC/HEXVIC/HEX
体系1System 1 ORF1ab基因ORF1ab gene N基因N gene E基因E gene 内标基因Internal standard gene
体系2System 2 S基因S gene // M基因M gene 内标基因Internal standard gene
通过人工质粒和高浓度假病毒稀释梯度评估检测性能，通过性能评估标准盘(来源于广州邦德盛)评价序列/体系特异性。新型冠状病毒核糖 核酸(COVID-19 RNA)液体系列性能评估标准盘包含阳性参考品、检测限参考品、分析特异性参考品、精密度参考品和干扰物参考品。The detection performance is evaluated through the dilution gradient of artificial plasmids and high-concentration pseudoviruses, and the sequence/system specificity is evaluated through the performance evaluation standard plate (from Guangzhou Bang Desheng). The new coronavirus ribonucleic acid (COVID-19 RNA) liquid series performance evaluation standard plate contains positive reference products, detection limit reference products, analysis specific reference products, precision reference products and interference reference products.
阳性参考品P1～P10，是由拷贝数不同的包含新型冠状病毒5个相关基因(ORF1ab、N、E、S和M)的假病毒构成，拷贝数在1.0×10 3copies/mL～1.0×10 6copies/mL。 The positive reference P1～P10 are composed of pseudoviruses containing 5 related genes (ORF1ab, N, E, S and M) of the new coronavirus with different copy numbers, and the copy number is 1.0×10 3 copies/mL～1.0× 10 6 copies/mL.
检测限参考品L1～L3，是由拷贝数不同的包含新型冠状病毒5个相关基因(ORF1ab、N、E、S和M)的假病毒构成，拷贝数分别为1.0×10 5copies/mL、1.0×10 4copies/mL和5.0×10 2copies/mL。 The detection limit reference materials L1～L3 are composed of pseudoviruses containing 5 related genes (ORF1ab, N, E, S, and M) of the new coronavirus with different copy numbers. The copy numbers are 1.0×10 5 copies/mL, 1.0×10 4 copies/mL and 5.0×10 2 copies/mL.
分析特异性参考品N1～N20，是由含人冠状病毒HCoV-OC43 RNA阳性、含人冠状病毒HCoV-HKU1 RNA阳性、含人冠状病毒HCoV-229E RNA阳性、含人冠状病毒HCoV-NL63 RNA阳性、含新型冠状病毒SARS RNA阳性、含中东呼吸综合征症状病毒MERS RNA阳性、含甲型HIN1流感病毒HIN1 RNA阳性、含乙型流感病毒INFB RNA阳性、含呼吸道合胞病毒A+B型阳性、含人副流感病毒阳性、含腺病毒阳性、含肠道病毒阳性、含肺炎支原体阳性、含EB病毒阳性、含人巨细胞病毒阳性、含结核分枝杆菌阳性、含人类基因组DNA、含人类基因组DNA、含COVID-19 RNA阴性、含COVID-19 RNA阴性样品构成。Analysis of specific reference products N1～N20, which contain human coronavirus HCoV-OC43 RNA positive, human coronavirus HCoV-HKU1 RNA positive, human coronavirus HCoV-229E RNA positive, and human coronavirus HCoV-NL63 RNA positive , Containing new coronavirus SARS RNA positive, Containing Middle East respiratory syndrome virus MERS RNA positive, Containing influenza A HIN1 influenza virus HIN1 RNA positive, Containing influenza B virus INFB RNA positive, Containing respiratory syncytial virus A+B positive, Positive for human parainfluenza virus, positive for adenovirus, positive for enterovirus, positive for Mycoplasma pneumoniae, positive for Epstein-Barr virus, positive for human cytomegalovirus, positive for Mycobacterium tuberculosis, human genomic DNA, human genome Consists of DNA, COVID-19-containing RNA negative, and COVID-19-containing RNA negative samples.
精密度参考品R1(低值2.0×10 3copies/mL)和R2(中值2.0×10 5copies/mL)。 Precision reference products R1 (low 2.0×10 3 copies/mL) and R2 (median 2.0×10 5 copies/mL).
干扰物参考品I1～I3由含血红蛋白2.0×10 3copies/mL阳性样本(I1)、含白蛋白2.0×10 3copies/mL阳性样本(I2)和含利巴韦林+阿奇霉素2.0×10 3copies/mL阳性样本(I3)组成，阳性对照组为2.0×10 3copies/ml/ml阳性样本。 Reference interferer article I1 ~ I3 hemoglobin containing 2.0 × 10 3 copies / mL positive samples (I1), albumin-containing 2.0 × 10 3 copies / mL positive samples (I2) and ribavirin containing azithromycin + 2.0 × 10 3 Copies/mL positive samples (I3) are composed, and the positive control group is 2.0×10 3 copies/ml/ml positive samples.
性能评估标准盘的评估结果要求：阳性参考品的检测结果应为阳性，检测限参考品检测结果应为阳性，分析特异性参考品检测结果应为阴性，精密度参考品检测结果均为阳性，且CT值的变异系数(CV，％)不大于5.0％。干扰物参考品检测结果应为阳性。The evaluation result requirements of the performance evaluation standard plate: the test result of the positive reference product should be positive, the test result of the detection limit reference product should be positive, the test result of the analysis-specific reference product should be negative, and the test result of the precision reference product should be positive. And the coefficient of variation (CV, %) of the CT value is not more than 5.0%. The test result of the interference reference substance should be positive.
阳性参考品的检测结果如图10所示，不同拷贝数的阳性参考品ORF1ab、N、E、S和M基因的检测结果均为阳性。The test results of the positive reference product are shown in Figure 10. The test results of the positive reference product ORF1ab, N, E, S, and M genes of different copy numbers are all positive.
检测限参考品的检测结果如图11和图12所示，使用1.0×10 3、1.0 ×10 3和5.0×10 3进行ORF1ab、E、N、S和M基因的检测，均为阳性，且重复性好。试剂盒的最低检测限为500copies/ml。 The detection results of the detection limit reference product are shown in Figure 11 and Figure 12. The detection of ORF1ab, E, N, S and M genes using 1.0×10 3 , 1.0×10 3 and 5.0×10 3 are all positive, and Good repeatability. The minimum detection limit of the kit is 500copies/ml.
分析特异性参考品的检测结果如图13所示，分析特异性参考品在体系1、体系2中仅内标为阳性，待检测靶基因ORF1ab、N、E、S和M基因均为阴性。The test results of the analysis-specific reference product are shown in Figure 13. The analysis-specific reference product is only positive for the internal standard in System 1 and System 2, and the target genes ORF1ab, N, E, S and M genes to be detected are all negative.
使用含相关病毒的阳性样本进行分别进行体系1和体系2的特异性测试，结果如图14～图17所示。检测可见，结果除内标体系外，待检测靶标均为阴性(箭头示内标扩增曲线)。结果表明，与地方性人类冠状病毒(HKU1、OC43、229E和NL63)、SARS冠状病毒、MERS冠状病毒无交叉反应，与甲型H1N1流感病毒(H1N1Influenza virus，H1N1)、乙型流感病毒(Influenza B virus，INFB)、呼吸道合胞病毒(Respiratory syncytial virus，RSV)、人副流感病毒(Parainfluenza virus，PV)、腺病毒(Adenovirus，ADV)和人类基因组DNA等无交叉反应。The specificity tests of System 1 and System 2 were performed using positive samples containing related viruses, and the results are shown in Figures 14-17. The results can be seen, except for the internal standard system, the targets to be tested are all negative (the arrow shows the internal standard amplification curve). The results show that it has no cross-reactivity with endemic human coronaviruses (HKU1, OC43, 229E, and NL63), SARS coronavirus, and MERS coronavirus. virus, INFB), respiratory syncytial virus (RSV), human parainfluenza virus (parainfluenza virus, PV), adenovirus (ADV) and human genomic DNA, etc. have no cross-reactivity.
精密度测试结果如图18所示。检测可见，分别使用低值精密度参考品(2019-nCoV RNA 2.0×10 3copies/ml阳性样本)和中值精密度参考品(2019-nCoV RNA 2.0×10 5copies/ml阳性样本)重复上机，使用体系1(ORF1ab、N和E基因)和体系2(S和M基因)分别检测，结果均为阳性，试剂盒重复性好，CV值不大于5.0％。 The precision test result is shown in Figure 18. The test shows that the low-precision reference product (2019-nCoV RNA 2.0×10 3 copies/ml positive sample) and the median precision reference product (2019-nCoV RNA 2.0×10 5 copies/ml positive sample) were used to repeat the test. Machine, using system 1 (ORF1ab, N and E genes) and system 2 (S and M genes) were tested separately, the results were all positive, the kit has good repeatability, and the CV value is not more than 5.0%.
干扰物参考品的检测结果如图19和图20所示，以对照样品和三种干扰物模拟样本为检测对象，使用体系1和体系2分别检测，结果均为阳性，三个干扰物质在体系1、体系2中均可检出，干扰物质血红蛋白、白蛋白、利巴韦林/阿奇霉素等对检测结果不产生干扰。The detection results of the interference reference product are shown in Figure 19 and Figure 20. The control sample and the three interference sample simulation samples are used as the test objects, and the system 1 and system 2 are used for the detection. The results are all positive. The three interference substances are in the system. 1. It can be detected in system 2. Interfering substances such as hemoglobin, albumin, ribavirin/azithromycin, etc. will not interfere with the test results.
准确度测试结果如图21和图22所示。使用各基因的人工质粒(1×10 4copies/ml)进行检测，结果为各型别对应阳性，其他型别质粒未见阳性扩增。 The accuracy test results are shown in Figure 21 and Figure 22. The artificial plasmids (1×10 4 copies/ml) of each gene were used for testing, and the results were positive for each type, and no positive amplification was seen for other types of plasmids.
实施例6、五靶标联检试剂盒国家标准物质评估结果Example 6. Evaluation result of national standard substance of five-target joint inspection kit
新型冠状病毒核酸国家标准物质(中国计量科学研究院)(NCRM)-GBW(E)091090(低浓度)为体外转录RNA，采用绝对定量方法-数字PCR对基因拷贝数浓度进行测定。目标基因范围涵盖已公布的新冠病毒3个主要基因：核壳蛋白N基因全长、包膜蛋白E基因全长、开放 阅读框1ab(ORF1ab)基因片段(基因组坐标：14911-15910)；量值(表7)为N、E和ORF1ab基因的拷贝数浓度。The new coronavirus nucleic acid national standard material (Chinese Academy of Metrology) (NCRM)-GBW(E)091090 (low concentration) is in vitro transcribed RNA, and the absolute quantitative method-digital PCR is used to determine the gene copy number concentration. The target gene range covers the three main genes of the new coronavirus that have been announced: the full length of the nucleocapsid protein N gene, the full length of the envelope protein E gene, and the open reading frame 1ab (ORF1ab) gene fragment (genomic coordinates: 14911-15910); (Table 7) is the copy number concentration of N, E and ORF1ab genes.
表7(NCRM)-GBW(E)091090(低浓度)特性量值及不确定度Table 7 (NCRM)-GBW(E)091090 (low concentration) characteristic value and uncertainty
Figure PCTCN2020109072-appb-000004
Figure PCTCN2020109072-appb-000004
经检索，除了ORF1ab因区段不同，国家标准物质可用于本发明的新型冠状病毒(2019-nCoV)五靶标联检试剂盒的N和E基因性能评价。将定值标准物质(批次编号2001)使用DEPC处理水十倍梯度稀释，得到梯度样本。使用实施例5中的检测试剂盒，将定值标准物质梯度样本加入到反应体系中，使用实施例4上机程序2(10+35循环)进行RT-PCR扩增和荧光采集。检测结果见图23。After searching, in addition to ORF1ab due to different segments, national standard materials can be used for the N and E gene performance evaluation of the novel coronavirus (2019-nCoV) five-target joint test kit of the present invention. The definite value standard substance (batch number 2001) was diluted ten-fold with DEPC treated water to obtain a gradient sample. Using the detection kit in Example 5, add a gradient sample of a fixed value standard substance to the reaction system, and use the computer program 2 (10+35 cycle) of Example 4 for RT-PCR amplification and fluorescence collection. The test results are shown in Figure 23.
N基因-1、N基因-2、N基因-3、N基因-4对应的理论拷贝数分别为9.8×10 4copies/ml、9.8×10 3copies/ml、9.8×10 2copies/ml、4.9×0 2copies/ml。 The theoretical copy numbers corresponding to N gene-1, N gene-2, N gene-3, and N gene-4 are 9.8×10 4 copies/ml, 9.8×10 3 copies/ml, 9.8×10 2 copies/ml, respectively. 4.9×0 2 copies/ml.
E基因-1、E基因-2、E基因-3、E基因-4对应的理论拷贝数分别为7.63×10 4copies/ml、7.63×10 3copies/ml、7.63×10 2copies/ml、3.82×10 2copies/ml。 The theoretical copy numbers corresponding to E gene-1, E gene-2, E gene-3, and E gene-4 are 7.63×10 4 copies/ml, 7.63×10 3 copies/ml, 7.63×10 2 copies/ml, 3.82×10 2 copies/ml.
结果表明，试剂盒能够检测出低值定值标准品梯度样本，对于N基因490copies/ml和E基因382copies/ml样本检出效果好。The results show that the kit can detect low-value fixed-value standard gradient samples, and the detection effect is good for N gene 490copies/ml and E gene 382copies/ml samples.
综上，本发明试剂盒设计合理，技术可行；质量控制体系稳定可靠；产品稳定性能良好，检测所需模板量少，检测结果客观准确。五靶标(包括ORF1ab、N、E、S和M基因)设计能够更好的克服RNA病毒传代过程变异导致的假阴性，可有效提升阳性检出率，尽可能避免漏检的情况出现，具有较高的临床应用价值。In summary, the kit of the present invention has reasonable design and feasible technology; the quality control system is stable and reliable; the stability of the product is good, the amount of template required for detection is small, and the detection result is objective and accurate. The design of five targets (including ORF1ab, N, E, S and M genes) can better overcome the false negatives caused by the variation of the RNA virus passaging process, can effectively increase the positive detection rate, and avoid missed detection as much as possible. High clinical application value.
以上所述实施例仅表达了本发明的诸多实施方式中的少数，虽然其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应 当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。The above-mentioned embodiments only express a few of the many embodiments of the present invention. Although the descriptions are more specific and detailed, they should not be understood as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention.

Claims (16)

  1. 一种检测新型冠状病毒的RT-PCR试剂盒，其特征在于，所述试剂盒用于ORF1ab基因、N基因、E基因、S基因和M基因的检测。An RT-PCR kit for detecting a novel coronavirus is characterized in that the kit is used for the detection of ORF1ab gene, N gene, E gene, S gene and M gene.
  2. 根据权利要求1所述的试剂盒，其特征在于，所述试剂盒包含ORF1ab基因、N基因、E基因、S基因和M基因的特异性引物；The kit according to claim 1, wherein the kit contains specific primers for ORF1ab gene, N gene, E gene, S gene and M gene;
    所述ORF1ab基因的特异性引物序列如SEQ ID NO:1～2所示，所述N基因的特异性引物序列如SEQ ID NO:4～5所示，所述E基因的特异性引物序列如SEQ ID NO:7～8所示，所述S基因的特异性引物序列如SEQ ID NO:10～11所示，所述M基因的特异性引物序列如SEQ ID NO:13～14所示。The specific primer sequence of the ORF1ab gene is shown in SEQ ID NO: 1 to 2, the specific primer sequence of the N gene is shown in SEQ ID NO: 4 to 5, and the specific primer sequence of the E gene is shown in SEQ ID NO: 7 to 8, the specific primer sequence of the S gene is shown in SEQ ID NO: 10 to 11, and the specific primer sequence of the M gene is shown in SEQ ID NO: 13 to 14.
  3. 根据权利要求1所述的试剂盒，其特征在于，所述试剂盒还包含ORF1ab基因、N基因、E基因、S基因和M基因的探针；所述ORF1ab基因的探针序列如SEQ ID NO:3所示，所述N基因的探针序列如SEQ ID NO:6所示，所述E基因的探针序列如SEQ ID NO:9所示，所述S基因的探针序列如SEQ ID NO:12所示，所述M基因的探针序列如SEQ ID NO:15所示。The kit according to claim 1, wherein the kit further comprises probes for ORF1ab gene, N gene, E gene, S gene, and M gene; the probe sequence of ORF1ab gene is as SEQ ID NO : 3, the probe sequence of the N gene is shown in SEQ ID NO: 6, the probe sequence of the E gene is shown in SEQ ID NO: 9, and the probe sequence of the S gene is shown in SEQ ID As shown in NO: 12, the probe sequence of the M gene is as shown in SEQ ID NO: 15.
  4. 根据权利要求1所述的试剂盒，其特征在于，所述试剂盒包含反应液A、反应液B、反应液C和阴性质控品、2019-nCoV阳性质控品，所述反应液A含有ORF1ab、N和E基因的特异引物和探针，所述反应液B含有S和M基因特异引物和探针，反应液C为酶溶液。The kit according to claim 1, wherein the kit comprises a reaction solution A, a reaction solution B, a reaction solution C and a negative control substance, a 2019-nCoV positive quality control substance, and the reaction solution A contains Specific primers and probes for ORF1ab, N and E genes, the reaction solution B contains specific primers and probes for the S and M genes, and the reaction solution C is an enzyme solution.
  5. 根据权利要求4所述的试剂盒，其特征在于，所述试剂盒的反应体系为：The kit according to claim 4, wherein the reaction system of the kit is:
    2×One Step RT-PCR Buffer III 10μl，2×One Step RT-PCR Buffer III 10μl,
    5U/μl Takara Ex Taq HS 0.4μl，5U/μl Takara Ex Taq HS 0.4μl,
    PrimeScript RT Enzyme Mix II 0.4μl，PrimeScript RT Enzyme Mix II 0.4μl,
    10μM Forward Primer 0.4μl，10μM Forward Primer 0.4μl,
    Probe 0.8μl，Probe 0.8μl,
    Total RNA 2μl，Total RNA 2μl,
    RNase Free dH 2O 5.6μl。 RNase Free dH 2 O 5.6μl.
  6. 根据权利要求1～5任一项所述的试剂盒，其特征在于，所述试剂盒的反应条件为：55℃ 15min，1个循环；95℃ 30s，1个循环；95℃ 10s，55℃ 30s，10个循环；95℃ 10s，55℃ 30s，35个循环。The kit according to any one of claims 1 to 5, characterized in that the reaction conditions of the kit are: 55°C 15min, 1 cycle; 95°C 30s, 1 cycle; 95°C 10s, 55°C 30s, 10 cycles; 95°C for 10s, 55°C for 30s, 35 cycles.
  7. 一种检测新型冠状病毒2019-nCoV的ORF1ab基因RT-PCR特异性引物，其特征在于，其序列如SEQ ID NO:1～2所示。A RT-PCR specific primer for the ORF1ab gene for detecting the novel coronavirus 2019-nCoV, which is characterized in that its sequence is shown in SEQ ID NO:1~2.
  8. 一种检测新型冠状病毒ORF1ab基因的RT-PCR探针，其特征在于，其序列如SEQ ID NO:3所示。An RT-PCR probe for detecting the ORF1ab gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 3.
  9. 一种检测新型冠状病毒N基因的RT-PCR特异性引物，其特征在于，其序列如SEQ ID NO:4～5所示。A specific RT-PCR primer for detecting the N gene of the novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 4 to 5.
  10. 一种检测新型冠状病毒N基因的RT-PCR探针，其特征在于，其序列如SEQ ID NO:6所示。An RT-PCR probe for detecting the N gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 6.
  11. 一种检测新型冠状病毒E基因的RT-PCR特异性引物，其特征在于，其序列如SEQ ID NO:7～8所示。A specific RT-PCR primer for detecting the E gene of the novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 7-8.
  12. 一种检测新型冠状病毒E基因的RT-PCR探针，其特征在于，其序列如SEQ ID NO:9所示。An RT-PCR probe for detecting the E gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 9.
  13. 一种检测新型冠状病毒S基因的RT-PCR特异性引物，其特征在于，其序列如SEQ ID NO:10～11所示。A specific RT-PCR primer for detecting the S gene of the novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 10-11.
  14. 一种检测新型冠状病毒S基因的RT-PCR探针，其特征在于，其序列如SEQ ID NO:12所示。An RT-PCR probe for detecting the S gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 12.
  15. 一种检测新型冠状病毒M基因的RT-PCR特异性引物，其特征在于，其序列如SEQ ID NO:13～14所示。A specific RT-PCR primer for detecting the M gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 13-14.
  16. 一种检测新型冠状病毒M基因的RT-PCR探针，其特征在于，其序列如SEQ ID NO:15所示。An RT-PCR probe for detecting the M gene of a novel coronavirus, characterized in that its sequence is shown in SEQ ID NO: 15.
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CN113999938A (en) * 2021-11-29 2022-02-01 中国计量科学研究院 Digital PCR detection reagent for detecting infectious live new coronavirus and application thereof
CN114410840A (en) * 2022-01-10 2022-04-29 广州达安基因股份有限公司 Kit for detecting novel coronavirus and N501Y mutation site thereof and detection method
CN115725801A (en) * 2022-12-02 2023-03-03 广州呼吸健康研究院 Primer pair, probe and kit for detecting new coronavirus variant
CN115961099A (en) * 2022-12-02 2023-04-14 珠海贝索基因技术有限公司 Primer pair, probe and kit for detecting new coronavirus mutant strain
CN115725801B (en) * 2022-12-02 2023-06-13 广州呼吸健康研究院 Primer pair, probe and kit for detecting novel coronavirus variant
CN115961099B (en) * 2022-12-02 2023-08-18 珠海贝索基因技术有限公司 Primer pair, probe and kit for detecting novel coronavirus mutant strain

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