WO2021238087A1 - 基于热对流pcr的新型冠状病毒快速检测试剂盒 - Google Patents

基于热对流pcr的新型冠状病毒快速检测试剂盒 Download PDF

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WO2021238087A1
WO2021238087A1 PCT/CN2020/127752 CN2020127752W WO2021238087A1 WO 2021238087 A1 WO2021238087 A1 WO 2021238087A1 CN 2020127752 W CN2020127752 W CN 2020127752W WO 2021238087 A1 WO2021238087 A1 WO 2021238087A1
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seq
primer
probe
kit
primer pair
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French (fr)
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蒋析文
黄桃生
卢卓健
陈绮娴
林卫萍
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广州达安基因股份有限公司
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Priority to EP20937608.6A priority Critical patent/EP4159875A4/en
Priority to US18/000,044 priority patent/US20230203575A1/en
Publication of WO2021238087A1 publication Critical patent/WO2021238087A1/zh

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    • 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]
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • 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 present invention belongs to the field of biotechnology and molecular diagnosis. Specifically, the present invention relates to a novel coronavirus rapid detection kit based on thermal convection PCR.
  • the novel coronavirus (2019 novel coronavirus, 2019-nCoV, SARS-CoV-2) belongs to the beta coronavirus, has an envelope, and has round or elliptical particles, often pleomorphic, with a diameter of 60-140nm.
  • S protein is one of the main proteins of the virus. Its coding gene is used for virus typing.
  • the molecular mechanism of interaction between S-protein and human ACE2 is used to infect human respiratory epithelial cells.
  • the common sign of human being infected with the coronavirus is fever. , Cough, shortness of breath and difficulty breathing. Severe infections can lead to pneumonia, severe malformed respiratory syndrome, kidney failure, and even death.
  • This new type of coronavirus has a strong infectivity to humans, and the lung infection caused by it is named "new type of coronavirus pneumonia" (COVID-19).
  • Nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • RNA ribonucleic acid
  • the new coronavirus is a virus that only contains RNA. The specific RNA sequence in the virus distinguishes the virus from other pathogens. Markers. After the emergence of the new coronavirus, scientists completed the analysis of the entire genome sequence of the new coronavirus in a very short time, and found the specific nucleic acid sequence in the new coronavirus by comparing it with the genome sequences of other species.
  • the specific nucleic acid sequence of the new coronavirus is detected in the patient's sample, it should indicate that the patient may be infected by the new coronavirus.
  • the most common method for detecting the specific sequence of the new coronavirus is fluorescent quantitative PCR (polymerase chain reaction).
  • the PCR reaction template is only DNA. Therefore, the new coronavirus RNA should be reverse transcribed into DNA before the PCR reaction.
  • a pair of specific primers and a Taqman probe are included.
  • the probe is a specific oligonucleotide sequence with a reporter fluorophore and a quencher fluorophore labeled at both ends.
  • the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the enzyme's exonuclease activity along the template to detect the target sequence.
  • the needle is digested and degraded, the reporter group is separated from the quenching group, and it emits fluorescence. Every time a DNA strand is amplified, a fluorescent molecule is produced.
  • the fluorescence quantitative PCR instrument can detect that the number of cycles (Ct value) at which the fluorescence reaches a preset threshold is related to the concentration of viral nucleic acid. The higher the concentration of viral nucleic acid, the smaller the Ct value. The products of different manufacturers will determine the positive judgment value of this product based on the performance of their own products.
  • the currently used fluorescent quantitative PCR equipment has problems such as high instrument cost and large laboratory operation space; and it needs to be submitted for inspection and tested in a dedicated PCR laboratory, which cannot be quickly tested on site; the experiment time is long, Excluding the nucleic acid processing process, the amplification detection time is generally about 2 hours; the above-mentioned various reasons cause the current nucleic acid detection kit to be unable to conduct large-scale cell investigations, which limits the scope of application of existing nucleic acid detection products. Unable to push the diagnosis forward and downward.
  • the present invention develops a rapid diagnosis system for a new coronavirus based on the N gene and ORF1 ab gene of the new coronavirus genome. In order to be able to detect patients with new coronavirus infection with high efficiency, high specificity and low cost, and prevent possible variability.
  • a primer pair set for detecting nucleic acid of a novel coronavirus includes:
  • a first primer pair comprising:
  • the primer pair set further includes:
  • a second primer pair comprising:
  • the primer pair set further includes:
  • a pair of internal standard primers includes:
  • the second aspect of the present invention provides a probe set for multiple detection of nucleic acid of a novel coronavirus, the probe set including a first probe whose nucleotide sequence is shown in SEQ ID NO.3.
  • the probe set further includes a second probe whose nucleotide sequence is shown in SEQ ID NO.6.
  • the probe set further includes an internal standard probe whose nucleotide sequence is shown in SEQ ID NO.9.
  • the 5'end of each probe contains a fluorescent reporter group; and/or the 3'end of each probe contains a fluorescence quenching group.
  • the fluorescent reporter groups labeled with the probes are different from each other.
  • the third aspect of the present invention provides a kit for multiple detection of novel coronavirus nucleic acid, which includes the primer pair set described in the first aspect of the present invention.
  • the kit further includes the probe set according to the second aspect of the present invention.
  • the kit includes a first container, and the first container contains a primer-probe mixture, and the primer-probe mixture contains a sequence as shown in SEQ ID NO.1-6 Polynucleotides; preferably also dNTPs.
  • the primer-probe mixture also contains a polynucleotide whose sequence is shown in SEQ ID NO. 7-9.
  • the kit further includes a second container containing a PCR reaction enzyme system, and the PCR reaction enzyme system includes a hot-start Taq enzyme and a reverse transcriptase C-MMLV.
  • the kit further includes a third container, and the third container contains a negative control substance.
  • the kit further includes a fourth container, and the fourth container contains a positive quality control product.
  • the fourth aspect of the present invention provides a method for multiple detection of novel coronavirus nucleic acid, the method comprising the steps:
  • the PCR reaction system includes: the nucleic acid sample provided in step (1), the primer pair set described in the first aspect of the present invention, and the probe set described in the second aspect of the present invention.
  • the nucleic acid sample may be from a throat swab sample, an alveolar lavage fluid sample, a blood sample, a sputum sample, a stool sample, or an environmental sample.
  • the method is a detection method for non-diagnostic purposes.
  • the PCR reaction system further includes a positive quality control product and/or a negative quality control product.
  • the PCR reaction system further includes a PCR reaction enzyme system.
  • the PCR is fluorescent quantitative PCR or thermal convection PCR.
  • the fifth aspect of the present invention provides the use of the primer pair set according to the first aspect of the present invention and/or the probe set according to the second aspect of the present invention for preparing a PCR detection kit, the PCR detection The kit is used to detect the nucleic acid of the new coronavirus.
  • the PCR is fluorescent quantitative PCR or thermal convection PCR.
  • Figures 6 and 7 show the detection results of nCoV-N-F3 and nCoV-N-R3 (Figure 6N gene single weight; Figure 7 multiple system);
  • Figures 8 and 9 show the detection results of nCoV-N-F4 and nCoV-N-R4 in a common fluorescent system ( Figure 8) and a thermal convection fast system ( Figure 9), respectively.
  • the present invention has obtained high sensitivity, strong specificity, and repeatability from a large number of primer probe combinations after multiple rounds of screening and verification. Good performance, suitable for thermal convection PCR and ordinary fluorescence quantitative PCR, and can be multiplexed detection primer probe combination.
  • kit and detection method of the present invention based on thermal convection PCR, ultra-fast PCR amplification and detection of 50 cycles can be completed in 18 minutes, and the detection sensitivity is equivalent to that of ordinary fluorescent quantitative PCR.
  • Thermal convection PCR is to maintain the temperature of the upper and lower surfaces unchanged in a closed space, which produces the regular rolling pattern phenomenon of the hot fluid rising and the cold fluid falling, and the thermal convection caused by the change of the density of the fluid in the space caused by the temperature difference between the upper and lower temperature Applied to DNA amplification.
  • the lower part of the capillary is affected by continuous heating to cause denaturation of the sample at the bottom of the capillary. At the same time, its density decreases and the buoyancy gradually increases. When it is greater than the viscous force and gravity, it floats upwards and rises to the lower temperature area near the top of the tube. Annealing and extension reactions occur again, and at the same time it is affected by cooling, the density increases again, and it starts to sink slowly and repeats the heating and cooling process. In this way, the PCR reaction is achieved by thermal convection.
  • thermal convection PCR has many difficulties. For example, it is difficult to ensure that the thermal convection fluid path is regular, which makes it impossible to ensure that the reaction reagents can fully complete the three major steps of deformation, annealing and extension. Problems such as low amplification efficiency, poor amplification specificity, and large differences between tubes. Therefore, thermal convection PCR has higher requirements for the design of reaction reagents, especially primers, probes and other specific reagents.
  • the thermal convection PCR equipment currently on the market includes the POCKIT TM nucleic acid analyzer developed by Taiwan Ruiji Marine Biotechnology Co., Ltd., and the thermal convection PCR detector developed by Achem Biosystems (Korea).
  • Multiplex PCR also known as multiple primer PCR or multiple PCR
  • multiple PCR is a PCR reaction in which more than two pairs of primers are added to the same PCR reaction system to amplify multiple nucleic acid fragments at the same time.
  • the reaction principle, reagents and operation The process is the same as general PCR.
  • the reaction system is unbalanced.
  • the unbalance of the reaction system leads to rapid amplification of certain advantageous primers and their templates in the previous rounds of reactions, and a large amount of amplification products are obtained, and these amplification products are also DNA polymerases.
  • a good inhibitor Therefore, with the emergence of a large number of amplified products, the polymerase’s ability to polymerize is more and more strongly inhibited. Therefore, primers and templates that are inferior in the early stage are more difficult to react at this time, resulting in a very large amount of amplified products. It is too small to detect.
  • the optimal annealing temperature is inconsistent. Put multiple pairs of primers into one system for amplification. Since the annealing temperature of the PCR reaction is the same, the optimal annealing temperature of each pair of primers is required to be close.
  • Primer dimers including the dimers between primers and the hairpin structure formed by the primers themselves, and a third-party DNA-mediated polymer. These dimers and non-specific primers will interfere with the primers and Competition for target binding sites affects amplification efficiency.
  • the present invention provides a kit for specifically detecting nucleic acid of a novel coronavirus in a sample, comprising a combination of primers and probes for detecting N gene, wherein the sequence of the primers for detecting N gene is:
  • SEQ ID NO. 1 GGGGAACTTCTCCTGCTAGAAT and SEQ ID NO. 2: CAGACATTTTGCTCTCAAGCTG,
  • the sequence of the corresponding detection probe is SEQ ID NO. 3: TTGCTGCTGCTTGACAGATT.
  • the kit further includes a primer and probe combination for detecting ORF1ab gene, wherein the primer sequence for detecting ORF1ab gene is:
  • SEQ ID NO. 4 CCCTGTGGGTTTTACACTTAA and SEQ ID NO. 5: ACGATTGTGCATCAGCTGA,
  • the sequence of the corresponding detection probe is SEQ ID NO. 6: CCGTCTGCGGTATGTGGAAAGGTTATGG.
  • the kit further includes internal standard quality control amplification primers and detection probes; the internal standard amplification primer sequences are:
  • SEQ ID NO. 7 CTCCGTACACTCTCCTACCCTC and SEQ ID NO. 8: ACACCTCTGGATGCCACT,
  • the sequence of the corresponding detection probe is SEQ ID NO. 9: CCATTGCCAGTCCGCCGTC.
  • the 5'end of each probe contains a fluorescent reporter group; and/or the 3'end of each probe contains a fluorescence quenching group.
  • the fluorescent reporter groups labeled with the probes are different from each other.
  • the fluorescent group is selected from the following group: FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5.
  • the quenching gene is selected from the group consisting of MGB, BHQ-1, BHQ-2, and BHQ-3.
  • the kit further includes a positive quality control product (including a pseudovirus containing a target fragment and a pseudovirus containing an internal standard fragment), and a negative control product (a pseudovirus containing an internal standard fragment) .
  • the kit includes PCR reaction solution A and PCR reaction solution B, wherein PCR reaction solution A includes specific primers and probes (SEQ ID NO.: 1-9), tris Methane-hydrochloric acid buffer, Mg 2+ and dNTPs; PCR reaction solution B includes hot-start Taq enzyme and c-MMLV enzyme.
  • PCR reaction solution A includes specific primers and probes (SEQ ID NO.: 1-9), tris Methane-hydrochloric acid buffer, Mg 2+ and dNTPs
  • PCR reaction solution B includes hot-start Taq enzyme and c-MMLV enzyme.
  • GISAID BetaCov/Wuhan/WH01/2019
  • oligonucleotide sequence information of its E gene please refer to the literature (Roujian Lu, Xiang Zhao, Juan Li, et.al, Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020 Jan 30).
  • the nucleic acid extraction or purification kit (magnetic bead method) Yue Sui Equipment No. 20170583 and Yue Sui Equipment No. 20150302 produced by Sun Yat-Sen University Daan Gene Co., Ltd. can be used; other commercial products can also be used. Please refer to the specific operation steps. Follow the instructions of the kit instructions.
  • NC(ORF1ab/N) PCR reaction solution A and NC(ORF1ab/N) PCR reaction solution B from the kit, thawed at room temperature, shake and mix well, centrifuge at 8000rpm for a few seconds before use
  • NC single-part amplification system is prepared as follows:
  • the program is set as follows: first perform reverse transcription at 50°C for 4 minutes, and then perform 50 cycles of thermal convective PCR at 95°C.
  • test results of the FAM channel and the VIC channel of the sample to be tested are Ct ⁇ 40, the curve is S-shaped or has a significant exponential growth period, or the FAM channel and the VIC have a Ct ⁇ 40 and the other Ct ⁇ 45, which is judged to be 2019 new Coronavirus (2019-nCoV) positive;
  • the test results of the FAM channel and the VIC channel of the sample to be tested are both at 40 ⁇ Ct ⁇ 45. At this time, the sample should be tested repeatedly. If the period is increased, the 2019 new coronavirus (2019-nCoV) is judged to be positive, otherwise the 2019 new coronavirus (2019-nCoV) is negative;
  • NC (ORF1ab/N) negative control FAM and VIC detection channels have no obvious amplification curves, and Cy5 channels have obvious amplification curves;
  • Cy5 channel internal standard channel
  • the negative result report format is: no 2019 novel coronavirus (2019-nCoV) RNA is detected in the sample, and the concentration is lower than the sensitivity of the kit;
  • the format of the positive result report is: the sample detects the 2019 novel coronavirus (2019-nCoV) RNA.
  • the experimental results show that the kit of the present invention has a good detection effect, and the detection results are basically not much different from ordinary fluorescent PCR.
  • the detection limit concentration is 300copies/mL.
  • ultra-rapid PCR amplification detection can be realized in 18 minutes to complete 50 cycles, and the detection sensitivity is equivalent to ordinary fluorescent quantitative PCR, and the sensitivity can reach 300 copies/ml.
  • the overall detection time does not exceed 30 minutes, which is about 1.5 hours faster than the existing known rapid detection equipment.
  • the present invention can detect patients infected with the new coronavirus with high efficiency, high specificity and low cost, and prevent false results that may occur due to virus variability. Negative, to confirm the missed detection that may be caused by mutation, which significantly improves the accuracy of virus identification.
  • the detection kit of the present invention includes both a new coronavirus N gene and ORF1ab gene nucleic acid target detection system and an endogenous internal standard detection system. It is a multiple fluorescence detection kit that can perform sample collection, nucleic acid extraction process and PCR The amplification process is monitored to reduce the occurrence of false negative results.
  • the present invention has obtained a primer probe with high sensitivity, strong specificity, and good repeatability from a large number of primer probe combinations, suitable for thermal convection PCR and ordinary fluorescent quantitative PCR, and capable of multiple detection. Needle combination.
  • the invention is suitable for the detection of the nucleic acid of the novel coronavirus 2019-nCoV, provides a reliable basis for virus identification and prevention and control, and is worthy of popularization and application.
  • the method of the present invention is also suitable for non-diagnostic purposes. For example, in the process of epidemic prevention and control, the detection method of the present invention is used to detect viral nucleic acids in the environment, and the viral nucleic acid information can be used for public health management.
  • the conserved regions of its genome are analyzed.
  • dozens of sets of specific primers and probe sequences for the N gene and ORF1ab gene of the new coronavirus have been designed.
  • internal standard primers and probes are designed.
  • primers and probes try to avoid the formation of hairpin structures, primer internal dimers, inter-primer dimers, and mismatches.
  • NCBI Blast online database https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • NCBI Blast online database https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • the 5'-end fluorescent group of nCoV-NP is FAM, and the 3'-end quenching group is BHQ1;
  • the 5'-end fluorescent group of nCoV-ORF1ab-P is VIC, and the 3'-end quenching group is BHQ1;
  • the 5'end fluorophore of P7 is CY5, and the 3'end quenching group is BHQ2.
  • sequence of the N gene target fragment is as follows:
  • the sequence of the ORF 1ab gene target fragment is as follows:
  • the internal standard fragment sequence is as follows:
  • the final reaction system is as follows:
  • PCR reaction solution A includes specific primers and probes (SEQ ID NO.: 1-9), tris-hydrochloric acid buffer, MgCl 2 , dNTPs (4 mmol);
  • PCR reaction solution B includes hot-start Taq enzyme and reverse transcriptase (c-MMLV enzyme).
  • Hot-start Taq enzymes, reverse transcriptases, and dNTPs can all be commercially available products, such as products from Qiagen, in which the amount of hot-start Taq enzyme in PCR reaction solution B per person is 24 U, and the amount of reverse transcriptase is 24 U.
  • Construct a virus-like particle containing the fragment of interest Dilute the pseudovirus with the measured concentration to an appropriate concentration and then perform a 10-fold dilution.
  • the concentrations are 3.00E+06, 3.00E+05, 3.00E+04, 3.00E+03, 3.00E+02copies/ml.
  • the above-mentioned pseudoviruses are detected with the above-identified detection system and circulation parameters.
  • the results show that the detection method of the present invention has high sensitivity, and the sensitivity can reach 300 copies/ml.
  • Influenza A/B, Coronavirus 229E, Coronavirus NL63, Coronavirus OC43, Coronavirus HKU1, Respiratory Syncytial Virus, Adenovirus, Mycoplasma Pneumoniae, and Chlamydia pneumoniae were tested as specific samples.
  • test results are shown in Figure 2.
  • the test results show that the test specific reference products (A/B influenza virus, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae), the test results are all negative, and the negative nature is normally detected, indicating that the kit of the present invention has good specificity.
  • test specific reference products A/B influenza virus, coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae
  • test results refer to Figure 3 (1.00E+04copies/ml), and the results show that the kit and the test method of the present invention have good repeatability.
  • the nucleic acid extraction or purification kit (magnetic bead method) Yue Sui Equipment No. 20170583 and Yue Sui Equipment No. 20150302 produced by Sun Yat-Sen University Daan Gene Co., Ltd. can be used; other commercial products can also be used. Please refer to the specific operation steps. Follow the instructions of the kit instructions.
  • NC (ORF1ab/N) PCR reaction solution A and NC (ORF1ab/N) PCR reaction solution B from the kit, thawed at room temperature, shake and mix, and centrifuge at 8000 rpm for a few seconds before use.
  • the single amplification system is prepared as follows:
  • the program is set to: first perform reverse transcription at 50°C for 4 minutes, and then perform 50 cycles of thermal convection PCR at 95°C, about 18 minutes.
  • test results of the FAM channel and the VIC channel of the sample to be tested are Ct ⁇ 40, the curve is S-shaped or has a significant exponential growth period, or the FAM channel and the VIC have a Ct ⁇ 40 and the other Ct ⁇ 45, which is judged to be 2019 new Coronavirus (2019-nCoV) positive;
  • the test results of the FAM channel and the VIC channel of the sample to be tested are both at 40 ⁇ Ct ⁇ 45. At this time, the sample should be tested repeatedly. If the period is increased, the 2019 new coronavirus (2019-nCoV) is judged to be positive, otherwise the 2019 new coronavirus (2019-nCoV) is negative;
  • NC (ORF1ab/N) negative control FAM and VIC detection channels have no obvious amplification curves, and Cy5 channels have obvious amplification curves;
  • Cy5 channel internal standard channel
  • the negative result report format is: no 2019 novel coronavirus (2019-nCoV) RNA is detected in the sample, and the concentration is lower than the sensitivity of the kit;
  • the format of the positive result report is: the sample detects the 2019 novel coronavirus (2019-nCoV) RNA.
  • the experimental results show that the kit of the present invention has a good detection effect, and the detection results are basically not much different from ordinary fluorescent PCR.
  • the detection limit concentration is 1000copies/mL.
  • the sequencing verification result showed that the detection accuracy of the detection system of the present invention reached 100%, which further proved the clinical detection accuracy of the detection of the system of the present invention.
  • the kit of the present invention has a good detection effect, and the detection result is not much different from ordinary fluorescent PCR.
  • the inventors screened dozens of PCR primers and probes against the target nucleic acid sequence of the new coronavirus. After a large number of tests, they finally obtained sensitivity and specificity that can meet the needs of clinical detection, suitable for thermal convection PCR, and A combination of primers and probes capable of multiplex detection.
  • the inventors For the N gene detection target of the new coronavirus, the inventors have gone through a large number of screenings and combinations.
  • Control upstream primer nCoV-N-F2 CTAAGAAGCCTCGGCAAAA (SEQ ID NO.10)
  • Control downstream primer nCoV-N-R2 CGTCTGCCGAAAGCTTG (SEQ ID NO.11)
  • Control probe nCoV-N-P2 TTACATTGTATGCTTTAGTGGCAGT (SEQ ID NO.12)
  • Control upstream primer nCoV-N-F3 GACCCCAAAATCAGCGAAAT (SEQ ID NO.13)
  • Control downstream primer nCoV-N-R3 TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO.14)
  • Control probe nCoV-N-P3 ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO.15)
  • Control upstream primer nCoV-N-F4 TTACAAACATTGGCCGCAAA (SEQ ID NO.16)
  • Control downstream primer nCoV-N-R4 GCGCGACATTCCGAAGAA (SEQ ID NO.17)
  • Control probe nCoV-N-P4 ACAATTTGCCCCCAGCGCTTCAG (SEQ ID NO.18)
  • nCoV-N-F2 and nCoV-N-R2 are shown in Figure 5.
  • the detection results indicate that the primer pair has poor specificity.
  • nCoV-N-F3 and nCoV-N-R3 show that the primer pair has better specificity and sensitivity to N gene target nucleic acid in a single detection system, but in a multiple detection system, N gene target low-concentration nucleic acid amplification Obviously inhibited, the single-plex and multiple-system detection results are shown in Figure 6 and Figure 7, respectively. It shows that the control primer pair nCoV-N-F3 and nCoV-N-R3 cannot be used in a multiplex detection system.
  • nCoV-N-F4 and nCoV-N-R4 show that the primer pair can perform multiple detection in a conventional fluorescent quantitative PCR detection system, and has better specificity and sensitivity. However, in thermal convection PCR, the sensitivity is significantly reduced. It shows that the control primer pair nCoV-N-F4 and nCoV-N-R4 cannot be applied to the thermal convection PCR detection system.
  • Figures 8 and 9 show the detection results of nCoV-N-F4 and nCoV-N-R4 in a common fluorescent system ( Figure 8) and a thermal convection fast system ( Figure 9), respectively.

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Abstract

本发明提供基于热对流PCR的新型冠状病毒快速检测试剂盒及多重检测新型冠状病毒核酸的方法，所述试剂盒包括用于检测新型冠状病毒基因组N基因和ORF1 ab基因的引物和探针组合。

Description

基于热对流PCR的新型冠状病毒快速检测试剂盒 技术领域
本发明属于生物技术和分子诊断领域，具体地说，本发明涉及一种基于热对流PCR的新型冠状病毒快速检测试剂盒。
背景技术
新型冠状病毒(2019 novel corona virus,2019-nCoV,SARS-CoV-2)属于β属冠状病毒，有包膜，颗粒呈圆型或椭圆形，常为多形性，直径60-140nm。S蛋白是病毒的主要蛋白之一，其编码基因用于病毒分型通过S-蛋白与人的ACE2相互作用的分子机制来感染人的呼吸道上皮细胞，人感染了该冠状病毒后常见体征为发热、咳嗽、气促和呼吸困难等。感染严重者可导致肺炎、严重畸形呼吸综合征、肾衰竭，更有甚者可导致死亡。这种新型冠状病毒对人类具有有很强的感染力，所引起的肺部感染被命名为“新型冠状病毒肺炎”(COVID-19)。
除朊病毒外所有生物都含有核酸，核酸包括脱氧核糖核酸(DNA)和核糖核酸(RNA)，新型冠状病毒是一种仅含有RNA的病毒，病毒中特异性RNA序列是区分该病毒与其他病原体的标志物。新型冠状病毒出现后，科学家在极短的时间里完成了对新型冠状病毒全基因组序列的解析，并通过与其他物种的基因组序列对比，发现了新型冠状病毒中的特异核酸序列。在临床实验室检测过程中，如果在患者样本中检测到新型冠状病毒的特异核酸序列，应提示该患者可能被新型冠状病毒感染。检测新型冠状病毒特异序列的方法中最常见的是荧光定量PCR(聚合酶链式反应)。PCR反应模板仅为DNA，因此在进行PCR反应前，应将新型冠状病毒RNA逆转录为DNA。在PCR反应体系中，包含一对特异性引物以及一个Taqman探针，该探针为一段特异性寡核苷酸序列，两端分别标记了报告荧光基团和淬灭荧光基团。探针完整时，报告基团发射的荧光信号被淬灭基团吸收；如反应体系存在靶序列，PCR反应时，探针与模板结合，DNA聚合酶沿模板利用酶的外切酶活性将探针酶切降解，报告基团与淬灭基团分离，发出荧光。每扩增一条DNA链，就有一个荧光分子产生。荧光定量PCR仪能够检测出荧光到达预先设定阈值的循环数(Ct值)与病毒核酸浓度有关，病毒核酸浓度越高，Ct值越小。不同生产企业的产品会依据自身产品的性能确定本产品的阳性判断值。
目前使用的荧光定量PCR设备存在仪器成本高，占用较大的实验室操作空间等问题；并且需要集中送检，并在专用的PCR实验室中进行测试，无法进行现场快速检验；实验时间长，排除核酸处理过程，扩增检测时间一般在2h左右；上述种种原因导致目前核酸检测试剂盒无法进行大规模的小区排查，限制现有核酸检测产品的应用范围。无法推动诊断前移下移。
因此，有必要开发新型冠状病毒的快速检测技术。
发明内容
本发明针对新型冠状病毒基因组N基因和ORF1 ab基因开发了新型冠状病毒 快速诊断体系。以便能够高效率、高特异性、低成本的对新型冠状病毒感染患者进行检测，并预防可能出现的变异性。
在本发明的第一方面，提供了一种用于检测新型冠状病毒核酸的引物对集，所述引物对集包括：
第一引物对，所述第一引物对包括：
如SEQ ID NO.1所示的正向引物；和，如SEQ ID NO.2所示的反向引物。
在另一优选例中，所述引物对集还包括：
第二引物对，所述第二引物对包括：
如SEQ ID NO.4所示的正向引物；和，如SEQ ID NO.5所示的反向引物。
在另一优选例中，所述引物对集还包括：
内标引物对，所述内标引物对包括：
如SEQ ID NO.7所示的正向引物；和，如SEQ ID NO.8所示的反向引物。
本发明的第二方面，提供了一种多重检测新型冠状病毒核酸的探针集，所述探针集包括核苷酸序列如SEQ ID NO.3所示的第一探针。
在另一优选例中，所述探针集还包括核苷酸序列如SEQ ID NO.6所示的第二探针。
在另一优选例中，所述探针集还包括核苷酸序列如SEQ ID NO.9所示的内标探针。
在另一优选例中，各探针的5’端包含有荧光报告基团；和/或，各探针的3’端包含有荧光淬灭基团。
在另一优选例中，各探针标记的荧光报告基团互不相同。
本发明的第三方面，提供了一种用于多重检测新型冠状病毒核酸的试剂盒，所述试剂盒包括本发明第一方面所述的引物对集。
在另一优选例中，所述试剂盒还包括本发明第二方面所述的探针集。
在另一优选例中，所述试剂盒包括第一容器，所述第一容器内包含引物探针混合液，所述引物探针混合液中包含序列如SEQ ID NO.1-6所示的多核苷酸；优选地还包括dNTPs。
在另一优选例中，所述引物探针混合液中还包含序列如SEQ ID NO.7-9所示的多核苷酸。
在另一优选例中，所述试剂盒还包括第二容器，所述第二容器内包含PCR反应酶系，所述PCR反应酶系包括热启动Taq酶、和逆转录酶C-MMLV。
在另一优选例中，所述试剂盒还包括第三容器，所述第三容器内包含阴性质控品。
在另一优选例中，所述试剂盒还包括第四容器，所述第四容器内包含阳性质控品。
本发明的第四方面，提供了一种多重检测新型冠状病毒核酸的方法，所述方法包括步骤：
(1)提供待检测对象的核酸样本；
(2)制备PCR反应体系并进行PCR检测：
其中，所述PCR反应体系包括：步骤(1)提供的所述核酸样本、本发明第一方面所述的引物对集、和本发明第二方面所述的探针组。
在另一优选例中，所述核酸样本可来自咽拭子样本、肺泡灌洗液样本、血液样本、痰液样本、粪便样本或环境样本。
在另一优选例中，所述方法为非诊断目的的检测方法。
在另一优选例中，所述PCR反应体系还包括阳性质控品，和/或阴性质控品。
在另一优选例中，所述PCR反应体系还包括PCR反应酶系。
在另一优选例中，所述PCR为荧光定量PCR或热对流PCR。
本发明的第五方面，提供了本发明第一方面所述的引物对集、和/或本发明第二方面所述的探针组的用途，用于制备PCR检测试剂盒，所述PCR检测试剂盒用于检测新型冠状病毒核酸。
在另一优选例中，所述PCR为荧光定量PCR或热对流PCR。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1：灵敏度检测结果；
图2：特异性测试结果；
图3：重复性测试结果；
图4：典型临床样本检测结果；
图5：nCoV-N-F2和nCoV-N-R2的检测结果；
图6和图7为nCoV-N-F3和nCoV-N-R3的检测结果(图6N基因单重；图7多重体系)；
图8和图9分别为nCoV-N-F4和nCoV-N-R4在普通荧光体系(图8)和热对流快速体系(图9)中的检测结果。
具体实施方式
本发明人通过广泛而深入的研究，获得一种快速检测新型冠状病毒核酸的试剂盒及方法，本发明经过多轮筛选验证，从大量引物探针组合中获得了灵敏度高、特异性强、重复性好，适用于热对流PCR和普通荧光定量PCR，并且能够进行多重检测的引物探针组合。使用本发明的试剂盒及检测方法，基于热对流PCR，可实现18min完成50个循环的超快速PCR扩增检测，而且检测灵敏度与普通荧光定量PCR相当。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
热对流PCR
热对流PCR是在一个封闭的空间中，维持其上下表面温度保持不变，产生了热流体上升冷流体下降的规则滚动的斑图现象，把上下温差引起空间内流体的密度变化引起的热对流应用于DNA扩增。毛细管的下部受到不断加热影响使得其底部样品发生变性反应，同时其密度变小，浮力慢慢变大，当大于粘性力和重力后向上浮动，上升到达靠近管顶部的较低温度区域时，样品又发生退火和延伸反应，同时受到冷却影响，密度又变大，开始慢慢下沉并重复进行加热冷却过程。以这种方式，通过热对流实现PCR反应。
但是，使用热对流PCR实现稳定扩增也存在不小的困难，比如热对流流体路径很难保证使规则的，这就导致无法保证反应试剂能充分完成变形、退火和延伸三大步骤，往往存在扩增效率偏低、扩增特异性差及管间差异大等问题。因此，热对流PCR中对反应试剂特别是引物、探针等特异性试剂的设计要求更高。
目前市场上出现的热对流PCR设备有台湾瑞基海洋生物科技股份有限公司开发的POCKIT TM核酸分析仪、阿赫姆生物系统公司(韩国)开发的热对流PCR检测仪。
多重PCR
多重PCR(multiplex PCR)，又称多重引物PCR或复合PCR，它是在同一PCR反应体系里加上二对以上引物，同时扩增出多个核酸片段的PCR反应，其反应原理，反应试剂和操作过程与一般PCR相同。
影响多重PCR反应的因素有很多，比如：
(1)反应体系不平衡，反应体系的不平衡导致在前期的几轮反应中某些优势引物及其模板迅速扩增，获得大量的扩增产物，而这些扩增产物同时又是DNA聚合酶的良好抑制剂。所以，随着扩增产物的大量出现，聚合酶的聚合能力被越来越强烈的抑制，因此，前期处于劣势的引物及其模板，这时就更加难以反应，最终导致扩增产物量非常之小，以至于无法检测。
(2)引物特异性，如果引物与体系中其他非目的基因片段结合力更强，那么目的基因结合引物的能力就会受到竟争，从而导致扩增效率下降。
(3)最佳退火温度不一致，将多对引物放置入一个体系中扩增，由于进行PCR反应的退火温度相同，所以要求每一对引物的最佳退火温度接近。
(4)引物二聚体，包括引物间的二聚体以及引物自身所形成的发卡结构，还有一类是第三方DNA介导的聚体，这些二聚体和非特异引物一样都会干扰引物与目标结合位点的竟争，影响扩增效率。
虽然上述提及了影响扩增效率的几个因素，但更多的因素尚不清楚。到目前为止，还没有一个可以明确预测扩增效率的有效方法。
试剂盒
本发明提供一种特异性检测样品中新型冠状病毒核酸的试剂盒，包括用于检测N基因的引物和探针组合，其中用于检测N基因的引物序列为：
SEQ ID NO.1：GGGGAACTTCTCCTGCTAGAAT和SEQ ID NO.2：CAGACATTTTGCTCTCAAGCTG，
对应的检测探针的序列为SEQ ID NO.3：TTGCTGCTGCTTGACAGATT。
在一个实施例中，所述试剂盒还包括用于检测ORF1ab基因的引物和探针组合，其中用于检测ORF1ab基因的引物序列为：
SEQ ID NO.4：CCCTGTGGGTTTTACACTTAA和SEQ ID NO.5：ACGATTGTGCATCAGCTGA，
对应的检测探针的序列为SEQ ID NO.6：CCGTCTGCGGTATGTGGAAAGGTTATGG。
在一个实施例中，所述试剂盒还包括内标质控扩增引物和检测探针；内标扩增引物序列分别为：
SEQ ID NO.7:CTCCGTACACTCTCCTACCCTC和SEQ ID NO.8:ACACCTCTGGATGCCACT，
对应的检测探针的序列为SEQ ID NO.9：CCATTGCCAGTCCGCCGTC。
在本发明的一个优选地实施方式中，各探针的5’端包含有荧光报告基团；和/或，各探针的3’端包含有荧光淬灭基团。
在另一优选例中，各探针标记的荧光报告基团互不相同。
优选地，所述荧光基团选自下组：FAM、VIC、HEX、NED、ROX、TET、JOE、TAMRA、CY3、CY5。
优选地，所述淬灭基因选自下组：MGB、BHQ-1、BHQ-2、BHQ-3。
在其中一个实施例中，所述试剂盒还包括含阳性质控品(包括含目的片段的假病毒和含内标片段的假病毒)，以及阴性质控品(含内标片段的假病毒)。
在其中一个实施例中，所述试剂盒包括PCR反应液A和PCR反应液B，其中PCR反应液A包括特异性引物和探针(SEQ ID NO.:1-9)、三羟甲基氨基甲烷-盐酸缓冲液、Mg 2+和dNTPs；PCR反应液B包括热启动Taq酶、c-MMLV酶。
本发明中新型冠状病毒的基因序列参见GISAID：BetaCov/Wuhan/WH01/2019|EPI_ISL_406798；关于其E基因的寡核苷酸序列信息可参考文献(Roujian Lu,Xiang Zhao,Juan Li,et.al,Genomic characterisation and epidemiology of 2019 novel coronavirus:implications for virus origins and receptor binding.Lancet.2020 Jan 30)。
本发明中，对特异性引物及探针经大量试验筛选，并对其进行组合、优化、验证，最终优选出组合后不会互相干扰、扩增效率高、特异性好的双重检测最优引探组合。
检测方法
1.样本处理和核酸提取
建议取200μl液态样本进行核酸提取。可采用中山大学达安基因股份有限公司生产的核酸提取或纯化试剂盒(磁珠法)粤穗械备20170583号和粤穗械备20150302号；也可使用其他的商业化产品，具体操作步骤请按照试剂盒说明书指引进行。
本试剂盒中的阴性质控品和阳性质控品均参与提取。
2.PCR试剂准备
从试剂盒中取出NC(ORF1ab/N)PCR反应液A和NC(ORF1ab/N)PCR反应液B，室温融化后振荡混匀，8000rpm离心数秒后使用
取N+1个(N＝待测样本个数+NC(ORF1ab/N)阴性质控品+NC(ORF1ab/N)阳性质控品)PCR反应管
NC单人份扩增体系配制如下表：
Figure PCTCN2020127752-appb-000001
取N+1个专用PCR反应管，使用窄口枪头对PCR试剂进行分装(每管分装15μl)，分装完成后使用离心机8000rpm下离心1min，使PCR反应液流入PCR反应管底。
3.加样
上述PCR反应管中分别加入提取好的样本DNA和阴性质控品、阳性质控品各5μl，盖紧管盖，8000rpm离心3min，离心完成后注意观察反应管中是否有肉眼可见气泡残留，如有需重复一次离心。如无肉眼可见气泡，将各反应管放入便携快速PCR仪(阿赫姆生物系统公司(韩国))中。
4.PCR扩增
程序设置为：首先执行50℃反转录4min，然后95℃热对流PCR反应50个循环。
选择FAM通道、VIC通道、和CY5通道，进行检测。
5.结果分析与判定
在仪器正常，NC(ORF1ab/N)阳性质控品和NC(ORF1ab/N)阴性质控品均正常的情况下进行结果分析。
FAM通道和VIC通道：
5.1阳性：如果待测样本FAM通道和VIC通道检测结果Ct≤40，曲线呈S型或有明显指数增长期，或者FAM通道和VIC有一个Ct≤40，另一个Ct≤45，判断为2019新型冠状病毒(2019-nCoV)阳性；
5.2可疑：待测样本FAM通道和VIC通道检测结果都处于40≤Ct≤45,此时应对样本进行重复检测，如重读结果Ct值仍然存在相同的范围，曲线呈S型或由明显的指数扩增期，则判断2019新型冠状病毒(2019-nCoV)阳性，否则2019新型冠状病毒(2019-nCoV)为阴性；
5.3阴性：如果待测样本FAM通道和VIC通道检测结果Ct≥45或未检出，此次结果判断2019新型冠状病毒(2019-nCoV)阴性。
6.质量控制
6.1 NC(ORF1ab/N)阴性质控品：FAM和VIC检测通道无明显扩增曲线，Cy5通道有明显扩增曲线；
6.2 NC(ORF1ab/N)阳性质控品：FAM和VIC检测通道有明显扩增曲线，Ct值≤32，Cy5通道有或无扩增曲线；
以上各项同时满足的条件下本次实验有效，否则全部试验应重新进行。
7.检验结果的解释
7.1每次实验均需检测阴性质控品，阳性质控品，质控品结果满足质量控制要求时方可进行检测结果的判定；
7.2FAM和VIC检测通道为阳性时，由于体系的竞争关系，Cy5通道(内标通道)结果可能为阴性；
7.3内标结果为阴性时，若该检测管的FAM和VIC检测通道也为阴性，说明体系受抑制或操作失误，试验无效，需对该样品进行复检；
7.4报告建议采用以下格式：
阴性结果报告格式为：样本未检测到2019新型冠状病毒(2019-nCoV)RNA，浓度低于试剂盒的灵敏度；
阳性结果报告格式为：样本检测到2019新型冠状病毒(2019-nCoV)RNA。
7.5实验结果表明本发明的试剂盒具有很好的检测效果，检测结果基本上与普通荧光PCR没有太大的差异。检测限浓度为300copies/mL。
本发明的有益效果在于：
(1)使用本发明的试剂盒及检测方法，基于热对流PCR，可实现18min完成50个循环的超快速PCR扩增检测，而且检测灵敏度与普通荧光定量PCR相当，灵敏度可达300copies/ml。结合直扩裂解法，整体检测时间不超过30min,比现有已知快速检测设备快1.5小时左右。
(2)本发明通过针对性检测新型冠状病毒的N基因和ORF1ab基因，能够高效率、高特异性、低成本的对新型冠状病毒感染患者进行检测，并预防可能因病毒变异性而出现的假阴性，对可能因突变而引起的漏检进行确认，显著提高了病毒鉴定的准确性。
(3)本发明的检测试剂盒，同时包括新型冠状病毒N基因和ORF1ab基因核酸靶标检测系统和内源性内标检测系统，为多重荧光检测试剂盒，可以对标本采集、核酸提取过程及PCR扩增过程进行监控，减少假阴性结果的出现。
(4)本发明经过多轮筛选验证，从大量引物探针组合中获得了灵敏度高、特异性强、重复性好，适用于热对流PCR和普通荧光定量PCR，并且能够进行多重检测的引物探针组合。
本发明适用于对新型冠状病毒2019-nCoV核酸的检测，为病毒鉴定和防控提供可靠的依据，值得推广应用。另外，本发明的方法同样适用于非诊断的目的， 例如，在疫情防控过程中，使用本发明的检测方法对环境中的病毒核酸进行检测，这些病毒核酸信息可以作为公众健康管理之需。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1引物和探针的设计
根据新型冠状病毒的序列，分析其基因组的保守区域。在这些保守区域中设计出数十组针对新型冠状病毒N基因和ORF1ab基因的特异性引物和探针序列。另外为了对标本采集、核酸提取过程及PCR扩增过程进行监控，设计了内标引物和探针。
在上述引物、探针的设计过程中，尽量避免发卡结构、引物内部二聚体、引物间二聚体以及错配的形成。
此外，通过对NCBI Blast在线数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)对上述设计的新型冠状病毒特异性引物和探针序列进行比对分析避免与其他病毒或人类基因发生非特异结核。
在常规实时荧光定量PCR上进通过多轮筛选和优化后，再通过热对流PCR进行验证，最终确定了一套灵敏度和特异性最优的引物、探针序列。
表1新型冠状病毒位点引物和探针序列
Figure PCTCN2020127752-appb-000002
其中，nCoV-N-P的5’端荧光基团为FAM，3’端猝灭基团为BHQ1；nCoV-ORF1ab-P的5’端荧光基团为VIC，3’端猝灭基团为BHQ1；P7的5’端荧光基团为CY5，3’端猝灭基团为BHQ2。
表2试剂盒组成成份
Figure PCTCN2020127752-appb-000003
其中，N基因目的片段序列如下:
Figure PCTCN2020127752-appb-000004
ORF 1ab基因目的片段序列如下:
Figure PCTCN2020127752-appb-000005
内标片段序列如下:
Figure PCTCN2020127752-appb-000006
实施例2检验方法及体系
为了确定引物和探针的检测体系，分别尝试了不同浓度的引物和探针对荧光PCR反应的影响。
结果显示，当nCoV-N-F，nCoV-N-R引物浓度为20pmol，nCoV-N-P探针的浓度为6pmol；nCoV-ORF1ab-F，nCoV-ORF1ab-R引物浓度为15pmol，nCoV-ORF1ab-P探针的浓度为6pmol；F7，R7引物浓度为5pmol，P7探针的浓度为3pmol，扩增曲线最好。
最终确定的反应体系如下：
PCR反应液A     12μL
PCR反应液B     3μL
待检样本核酸   5μL
其中，PCR反应液A包括特异性引物和探针(SEQ ID NO.:1-9)、三羟甲基氨基甲烷-盐酸缓冲液、MgCl 2，dNTPs(4mmol)；
PCR反应液B包括热启动Taq酶、逆转录酶(c-MMLV酶)。热启动Taq酶、逆转录酶、dNTPs均可采用市售产品，如Qiagen公司的产品,其中每人份PCR反应液B中热启动Taq酶的用量为24U，逆转录酶用量为24U。
实施例3灵敏度检测
构建含有目的片段的病毒样颗粒。将测定浓度后的假病毒稀释到合适浓度后再进行10倍倍比稀释，浓度分别为3.00E+06、3.00E+05、3.00E+04、3.00E+03、3.00E+02copies/ml。
用上述确定的检测体系和循环参数对上述假病毒进行检测。部分检测结果参考图1，结果表明，本发明检测方法具有较高的灵敏度，灵敏度能够达到300copies/ml。
实施例4特异性检测
将甲型/乙型流感病毒、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体作为特异性样本进行检测。
检测结果如图2所示，检测结果表明，检测特异性参考品(甲型/乙型流感病毒、冠状病毒229E、冠状病毒NL63、冠状病毒OC43、冠状病毒HKU1、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体)，检测结果均为阴性，阴性质空正常检出，表明本发明的试剂盒特异性良好。
实施例5重复性检测
选取浓度为1.00E+05copies/ml；1.00E+04copies/ml和1.00E+03copies/ml的假病毒进行检测，采用上述确定的检测体系和循环参数，进行重复测试。
检测结果参考图3(1.00E+04copies/ml)，结果表明本发明的试剂盒和检测方法重复性较好。
实施例6临床样本检测
1.样本处理和核酸提取
建议取200μl液态样本进行核酸提取。可采用中山大学达安基因股份有限公司生产的核酸提取或纯化试剂盒(磁珠法)粤穗械备20170583号和粤穗械备20150302号；也可使用其他的商业化产品，具体操作步骤请按照试剂盒说明书指引进行。
本试剂盒中的阴性质控品和阳性质控品均参与提取。
2.PCR试剂准备
从试剂盒中取出NC(ORF1ab/N)PCR反应液A和NC(ORF1ab/N)PCR反应液B，室温融化后振荡混匀，8000rpm离心数秒后使用。
取N+1个(N＝待测样本个数+NC(ORF1ab/N)阴性质控品+NC(ORF1ab/N)阳性质控品)PCR反应管
单人份扩增体系配制如下表：
Figure PCTCN2020127752-appb-000007
取N+1个专用PCR反应管，使用窄口枪头对PCR试剂进行分装(每管分装15μl)，分装完成后使用离心机8000rpm下离心1min，使PCR反应液流入PCR反应管底。
3.加样
上述PCR反应管中分别加入提取好的样本DNA和阴性质控品、阳性质控品各5μl，盖紧管盖，8000rpm离心3min，离心完成后注意观察反应管中是否有肉眼可见气泡残留，如有需重复一次离心。如无肉眼可见气泡，将各反应管放入便携快速PCR仪(阿赫姆生物系统公司(韩国))中。
4.PCR扩增
程序设置为：首先执行50℃反转录4min，然后95℃热对流PCR反应50个循环，约18min。
选择FAM通道、VIC通道、和CY5通道，进行检测。
5.结果分析与判定
在仪器正常，NC(ORF1ab/N)阳性质控品和NC(ORF1ab/N)阴性质控品均正常的情况下进行结果分析。
FAM通道和VIC通道：
5.1阳性：如果待测样本FAM通道和VIC通道检测结果Ct≤40，曲线呈S型或有明显指数增长期，或者FAM通道和VIC有一个Ct≤40，另一个Ct≤45，判断为2019新型冠状病毒(2019-nCoV)阳性；
5.2可疑：待测样本FAM通道和VIC通道检测结果都处于40≤Ct≤45,此时应对样本进行重复检测，如重读结果Ct值仍然存在相同的范围，曲线呈S型或由明显的指数扩增期，则判断2019新型冠状病毒(2019-nCoV)阳性，否则2019新型冠状病毒(2019-nCoV)为阴性；
5.3阴性：如果待测样本FAM通道和VIC通道检测结果Ct≥45或未检出，此次结果判断2019新型冠状病毒(2019-nCoV)阴性
6.质量控制
6.1 NC(ORF1ab/N)阴性质控品：FAM和VIC检测通道无明显扩增曲线，Cy5通道有明显扩增曲线；
6.2 NC(ORF1ab/N)阳性质控品：FAM和VIC检测通道有明显扩增曲线，Ct值≤32，Cy5通道有或无扩增曲线；
以上各项同时满足的条件下本次实验有效，否则全部试验应重新进行。
7.检验结果的解释
7.1每次实验均需检测阴性质控品，阳性质控品，质控品结果满足质量控制要求时方可进行检测结果的判定；
7.2FAM和VIC检测通道为阳性时，由于体系的竞争关系，Cy5通道(内标通道)结果可能为阴性；
7.3内标结果为阴性时，若该检测管的FAM和VIC检测通道也为阴性，说明体系受抑制或操作失误，试验无效，需对该样品进行复检；
7.4报告建议采用以下格式：
阴性结果报告格式为：样本未检测到2019新型冠状病毒(2019-nCoV)RNA，浓度低于试剂盒的灵敏度；
阳性结果报告格式为：样本检测到2019新型冠状病毒(2019-nCoV)RNA。
7.5实验结果表明本发明的试剂盒具有很好的检测效果，检测结果基本上与普通荧光PCR没有太大的差异。检测限浓度为1000copies/mL。
在所检测的32例疑似临床样本中，共检出24例新型冠状病毒2019-nCoV核酸阳性临床样本。典型检测结果如图5所示。
测序验证结果表明本发明检测体系检测准确率达到了100％，进一步证明了本发明体系检测的临床检测准确性。
本发明的试剂盒具有很好的检测效果，检测结果与普通荧光PCR没有太大的差异。
对比例1
本发明人在研究过程中，针对新型冠状病毒目标核酸序列筛选了数十组PCR引物和探针，经过大量测试，最终获得了灵敏度和特异性能够满足临床检测需求，适用于热对流PCR，且能够进行多重检测的引物、探针组合。
针对新型冠状病毒的N基因检测靶标，本发明人经过了大量的筛选、组合。
设计的部分典型引物序列如下：
对照上游引物nCoV-N-F2：CTAAGAAGCCTCGGCAAAA(SEQ ID NO.10)
对照下游引物nCoV-N-R2：CGTCTGCCGAAAGCTTG(SEQ ID NO.11)
对照探针nCoV-N-P2：TTACATTGTATGCTTTAGTGGCAGT(SEQ ID NO.12)
对照上游引物nCoV-N-F3：GACCCCAAAATCAGCGAAAT(SEQ ID NO.13)
对照下游引物nCoV-N-R3：TCTGGTTACTGCCAGTTGAATCTG(SEQ ID NO.14)
对照探针nCoV-N-P3：ACCCCGCATTACGTTTGGTGGACC(SEQ ID NO.15)
对照上游引物nCoV-N-F4：TTACAAACATTGGCCGCAAA(SEQ ID NO.16)
对照下游引物nCoV-N-R4：GCGCGACATTCCGAAGAA(SEQ ID NO.17)
对照探针nCoV-N-P4：ACAATTTGCCCCCAGCGCTTCAG(SEQ ID NO.18)
首先使用常规荧光定量PCR检测进行初筛。
nCoV-N-F2和nCoV-N-R2的特异性检测结果如图5所示，检测结果表明该引物对特异性较差。
nCoV-N-F3和nCoV-N-R3的检测结果表明该引物对在单一检测体系中对N基因靶核酸的特异性和灵敏度较佳，但是在多重检测体系中N基因靶低浓度核酸扩增受到明显抑制，单重和多重体系检测结果分别如图6和图7所示。表明对照引物对nCoV-N-F3和nCoV-N-R3无法应用于多重检测体系中。
使用nCoV-N-F4和nCoV-N-R4的检测结果表明该引物对在常规荧光定量PCR检测体系中能够进行多重检测，具有较佳的特异性和灵敏度。但是，在热对流PCR中，灵敏度显著降低。表明对照引物对nCoV-N-F4和nCoV-N-R4无法应用于热对流PCR检测体系。图8和图9分别为nCoV-N-F4和nCoV-N-R4在普通荧光体系(图8)和热对流快速体系(图9)中的检测结果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于检测新型冠状病毒核酸的试剂盒，其特征在于，所述试剂盒包括引物对集；
    其中，所述引物对集包括第一引物对和第二引物对，
    所述第一引物对包括：
    如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物，
    所述第二引物对包括：
    如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物。
  2. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒还包括探针集；其中，所述探针集包括核苷酸序列如SEQ ID NO.3所示的第一探针，和核苷酸序列如SEQ ID NO.6所示的第二探针。
  3. 如权利要求1所述的试剂盒，其特征在于，所述引物对集还包括内标引物对，所述内标引物对包括：
    如SEQ ID NO.7所示的正向引物；和，如SEQ ID NO.8所示的反向引物。
  4. 如权利要求2所述的试剂盒，其特征在于，所述探针集还包括核苷酸序列如SEQ ID NO.9所示的内标探针。
  5. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒包括第一容器，所述第一容器内包含引物探针混合液，所述引物探针混合液中包含序列如SEQ ID NO.1-6所示的多核苷酸。
  6. 如权利要求5所述的试剂盒，其特征在于，所述引物探针混合液中还包含序列如SEQ ID NO.7-9所示的多核苷酸。
  7. 如权利要求5所述的试剂盒，其特征在于，所述试剂盒还包括第二容器，所述第二容器内包含PCR反应酶系，所述PCR反应酶系包括热启动Taq酶、和逆转录酶C-MMLV。
  8. 如权利要求7所述的试剂盒，其特征在于，所述第一容器还包含dNTPs。
  9. 一种非诊断目的的多重检测新型冠状病毒核酸的方法，其特征在于，所述方法包括步骤：
    (1)提供待检测对象的核酸样本；
    (2)制备PCR反应体系并进行PCR检测：
    其中，所述PCR反应体系包括：步骤(1)提供的所述核酸样本、引物对集和探针集；
    所述引物对集包括第一引物对和第二引物对，
    所述第一引物对包括：
    如SEQ ID NO.1所示的正向引物和如SEQ ID NO.2所示的反向引物，
    所述第二引物对包括：
    如SEQ ID NO.4所示的正向引物和如SEQ ID NO.5所示的反向引物。
    所述探针集包括核苷酸序列如SEQ ID NO.3所示的第一探针，和核苷酸序列如SEQ ID NO.6所示的第二探针。
  10. 权利要求9所述的方法，其特征在于，所述核酸样本来自环境样本。
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