WO2024045461A1 - 一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法 - Google Patents

一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法 Download PDF

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WO2024045461A1
WO2024045461A1 PCT/CN2022/144290 CN2022144290W WO2024045461A1 WO 2024045461 A1 WO2024045461 A1 WO 2024045461A1 CN 2022144290 W CN2022144290 W CN 2022144290W WO 2024045461 A1 WO2024045461 A1 WO 2024045461A1
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qpcr
seq
virus
tube
primer
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French (fr)
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顾东东
赵百慧
马彬恒
侯巧利
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上海伯杰医疗科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
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    • 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
    • C12Q1/6851—Quantitative amplification
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines
    • 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 disclosure relates to the technical field of reverse transcription fluorescent PCR, and in particular to a one-tube RT-qPCR fully premixed reaction reagent, application and RT-qPCR method.
  • PCR Polymerase chain reaction
  • RT-qPCR is a method that adds reverse transcriptase to PCR and uses RNA as the original template to amplify DNA.
  • RT-qPCR monitors the amplification products of each cycle in the PCR reaction in real time based on changes in the accumulated fluorescence signal intensity of the fluorescent groups added to the reaction system.
  • the currently commonly used RT-qPCR reaction system consists of nucleic acid amplification buffer, enzyme mixture, primer and probe reaction solution, etc. and needs to be stored separately.
  • each component needs to be dissolved at room temperature and mixed thoroughly before use. Add each component in a fixed proportion and mix well before dispensing it into PCR tubes. The entire process requires calculation and careful operation. Mismatching often occurs, and the reaction system needs to be prepared and used immediately.
  • the nucleic acid amplification system needs to be prepared in advance. Stability problems may occur when the buffer, enzyme mixture, and primer-probe reaction solution are premixed and stored frozen or refrigerated, specifically manifested as a decrease in sensitivity and non-specific amplification in negative samples.
  • the amplification efficiency of primers and probes in the RT-qPCR reaction system under the fast program is directly related to the nucleic acid amplification buffer and enzyme mixture in the reaction system.
  • a set of primers and probes needs to be developed corresponding to a set of nucleic acid amplification Buffer and enzyme mixtures cannot be mixed between reaction systems.
  • the RT-qPCR reaction system has poor applicability to various primer and probe reaction solutions.
  • the present disclosure provides a tube-type RT-qPCR fully premixed reaction reagent, which includes a Taq enzyme monoclonal antibody with double blocking capability.
  • amino acid sequence of the Taq enzyme monoclonal antibody with double blocking ability is shown in SEQ ID No. 1 and SEQ ID No. 2.
  • the content ratio (U/U) of the Taq enzyme monoclonal antibody with double blocking ability to Taq DNA polymerase is 1:3-9, preferably 1:5.
  • nucleic acid aptamers are also included.
  • the number of bases contained in the nucleic acid aptamer is 50 to 100 bp.
  • the concentration of the nucleic acid aptamer is 0.2-0.7ng/mL.
  • the concentration of the nucleic acid aptamer is 0.5ng/mL.
  • stabilizers and/or enhancers are also included.
  • the stabilizer includes 0.5-1 mg/mL BSA, 0.005%-0.008% (v/v) gelatin, 5%-10% (v/v) glycerol or 0.1%-0.2% (v/v) At least two of the non-ionic detergents.
  • the stabilizer includes 0.7 mg/mL BSA, 0.006% (v/v) gelatin and 7% (v/v) glycerol.
  • the enhancer includes 1% to 2% (v/v) formamide, 1% to 5% (v/v) DMSO, 0.1 to 0.3M betaine, 0.5 to 1.0g/L SSB single chain Binding protein, 0.3 ⁇ 0.8g/L pyrophosphatase, 0.1 ⁇ 0.3M trehalose and 0.05 ⁇ 0.1g/L sodium azide.
  • the enhancer includes 2% formamide, 2.5% DMSO, 0.2M betaine, 0.8g/L SSB single-chain binding protein, 0.2M trehalose and 0.08g/L sodium azide.
  • the present disclosure also provides the application of the one-tube RT-qPCR full premix reaction reagent described in any of the aforementioned items in preparing a one-tube RT-qPCR full premix reaction system.
  • the present disclosure also provides a tube-type RT-qPCR fully premixed reaction system, including a tube-type RT-qPCR fully premixed reaction reagent described in any of the preceding items;
  • DNA polymerase also includes DNA polymerase, reverse transcriptase, UNG enzyme, RNase inhibitor, buffer, stabilizer, enhancer, upstream and downstream primers and fluorescently labeled probe compositions.
  • the DNA polymerase is hot-start Taq DNA polymerase
  • the extension speed is 1.5 sec/kb
  • the optional addition amount is 3 to 6 U.
  • the reverse transcriptase includes M-MLV reverse transcriptase, with a temperature tolerance of 60° C. or above, and an added amount of 5 to 10 U.
  • the UNG enzyme is a thermosensitive UNG enzyme of 0.1 to 0.5 U.
  • the added amount of the RNase inhibitor is 2 to 3 U.
  • the buffer is a Tris-Hcl buffer solution of 100 to 500 mmol/L.
  • the buffer contains inorganic salts, and the inorganic salts are sulfate and/or chloride salts.
  • the inorganic salt is a chloride salt.
  • the upstream and downstream primers and fluorescently labeled probe compositions include a single detection composition, a dual detection composition, a triple detection composition or a quadruple detection composition. things.
  • the nucleic acid product is derived from a virus.
  • the virus is selected from at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • the single-plex detection composition is a norovirus primer-probe composition.
  • nucleotide sequence of the upstream primer in the norovirus primer-probe composition is as shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer in the norovirus primer-probe composition is as follows SEQ ID No. 4 is shown, and the nucleotide sequence of the probe in the norovirus primer-probe composition is shown in SEQ ID No. 5.
  • the dual detection composition is an influenza A and B virus primer-probe composition.
  • the nucleotide sequence of the influenza A virus upstream primer in the influenza A and B virus primer-probe composition is as shown in SEQ ID No. 6, and the nucleotide sequence of the influenza A virus downstream primer is as follows SEQ ID No.7 is shown.
  • the nucleotide sequence of the influenza A virus probe is shown in SEQ ID No.8.
  • the nucleotide sequence of the influenza B virus upstream primer is shown in SEQ ID No.9.
  • the nucleotide sequence of the influenza B virus probe is shown in SEQ ID No.9.
  • the nucleotide sequence of the influenza virus downstream primer is shown in SEQ ID No. 10
  • the nucleotide sequence of the influenza B virus probe is shown in SEQ ID No. 11.
  • the triple detection composition is a primer-probe composition for enterovirus type 71, enterovirus type 71, and coxsackie virus type 16.
  • the primer-probe composition for conventional enterovirus, enterovirus 71 and coxsackievirus 16 contains:
  • the universal upstream primer for enteric viruses the nucleotide sequence is shown in SEQ ID No. 12
  • the universal downstream primer for enteric viruses the nucleotide sequence is shown in SEQ ID No. 13
  • the universal probe for enteric viruses The nucleotide sequence is shown in SEQ ID No.14;
  • Enterovirus 71 upstream primer the nucleotide sequence is shown in SEQ ID No. 15, Enterovirus 71 downstream primer, the nucleotide sequence is shown in SEQ ID No. 16, Enterovirus 71 probe, The nucleotide sequence is shown in SEQ ID No.17;
  • Coxsackie virus type 16 upstream primer the nucleotide sequence is shown in SEQ ID No. 18, Coxsackie virus type 16 downstream primer, the nucleotide sequence is shown in SEQ ID No. 19, Coxsackie virus type 16 Probe, the nucleotide sequence is shown in SEQ ID No. 20.
  • the present disclosure also provides an RT-qPCR method.
  • the aforementioned one-tube RT-qPCR fully premixed reaction system is used to amplify the sample to be tested according to the following procedures: reverse transcription at 55-65°C for 1-5 minutes; pre-mixed reaction system at 94-98°C. Denaturation for 5 to 15 seconds; denaturation at 94 to 98°C for 1 to 5 seconds; annealing and extension at 55 to 65°C for 8 to 15 seconds; denaturation and annealing and extension cycles for 38 to 45 times.
  • the amplification program is: reverse transcription at 60°C for 2 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 1 s; annealing at 60°C for 10 s; extension at 60°C for 10 s; and 40 cycles of denaturation, annealing and extension.
  • the present disclosure also provides the use of the one-tube RT-qPCR full premix reaction reagent or the one-tube RT-qPCR full premix reaction system described in any one of the above for the diagnosis of viral infection.
  • the virus includes at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • the present disclosure also provides a method of diagnosing a disease associated with a viral infection in a subject, comprising:
  • the virus includes at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • the source of the sample is selected from feces, vomitus, nasal swabs, throat swabs, sputum, bronchoalveolar lavage fluid, cells, chicken embryo culture, herpes fluid, serum, cerebrospinal fluid or tissue culture .
  • Figure 1 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 1 of the present disclosure
  • Figure 2 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 2 of the present disclosure
  • Figure 3 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 2 of the present disclosure
  • Figure 4 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 2 of the present disclosure
  • Figure 5 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 3 of the present disclosure
  • Figure 6 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 4 of the present disclosure
  • Figure 7 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 5 of the present disclosure
  • Figure 8 shows the amplification results of the fully premixed RT-PCR reaction system and the control group in Example 6 of the present disclosure
  • Figure 9 is the long-term stability test result of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 1 month;
  • Figure 10 shows the long-term stability test results of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 3 months;
  • Figure 11 shows the long-term stability test results of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 6 months;
  • Figure 12 shows the long-term stability test results of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 9 months;
  • Figure 13 shows the long-term stability test results of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 12 months;
  • Figure 14 shows the long-term stability test results of the fully premixed RT-PCR reaction system of Example 6 of the present disclosure after being stored for 15 months.
  • the terms "subject,” “individual,” and “patient” are used interchangeably herein and refer to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, domestic animals, competitive animals, and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also included.
  • primer refers to an oligonucleotide that is placed under conditions that cause the synthesis of a primer extension product complementary to one nucleic acid strand (i.e., in the presence of a nucleotide and an inducing substance such as DNA polymerase) and at the appropriate temperature and pH) can serve as the starting point for synthesis.
  • the primer may be single-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of the inducing substance. The exact length of the primer will depend on numerous factors, including temperature, source of primer, and purpose of the method.
  • diagnosis refers to a medical determination of a person's mental and physical state. Specifically, the process of determining which disease or condition may explain a subject's symptoms and signs. For example, the presence of a disease related to a viral infection in a subject is determined by using a one-tube RT-qPCR complete master mix reaction reagent or a one-tube RT-qPCR complete master mix reaction system described herein.
  • One embodiment of the present disclosure provides a tube-type RT-qPCR fully premixed reaction reagent, which includes a Taq enzyme monoclonal antibody with double blocking ability.
  • the amino acid sequence of the Taq enzyme monoclonal antibody with double blocking ability is such as SEQ ID No. 1 (DIEMTQSSGAELARPGASVKMSCKASGTFRYSDYMHWVKQRPGQGLEWIGYRGPNDRYTKYNQKFKKATLTADKSSSTAYMQLSSLTSEDSAVYYCGRDDYYCSHDATWGQGTTLSRT) and SEQ ID No. 2
  • the content ratio (U/U) of the Taq enzyme monoclonal antibody with double blocking ability to Taq DNA polymerase is 1:3 ⁇ 9, for example, it can be 1:3.5 ⁇ 8.5, 1:4 ⁇ 8 or 1:5 ⁇ 7, such as 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9, or an interval value between any two endpoint values.
  • the content ratio (U/U) of the Taq enzyme monoclonal antibody with double blocking ability to Taq DNA polymerase is 1:5.
  • nucleic acid aptamers are also included.
  • the nucleic acid aptamer contains a number of bases of 50 to 100 bp, such as 55 to 90 bp, 65 to 80 bp or 75 to 80 bp, such as 50 bp, 60 bp, 70 bp, 80 bp, 90 bp or 100 bp, or any The interval value between two endpoint values.
  • the concentration of the nucleic acid aptamer is 0.2-0.7ng/mL, such as 0.2ng/mL, 0.3ng/mL, 0.4ng/mL, 0.5ng/mL, 0.6ng/mL or 0.7ng/mL, or The interval value between any two endpoint values.
  • the concentration of nucleic acid aptamer is 0.5ng/mL.
  • stabilizers and/or enhancers are also included.
  • stabilizers include 0.5-1 mg/mL BSA, 0.005%-0.008% (v/v) gelatin, 5%-10% (v/v) glycerol or 0.1%-0.2% (v/v) nonionic At least two types of detergents.
  • the BSA content is 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, or 1 mg/mL, or an interval value between any two endpoint values.
  • the gelatin content (v/v) is 0.005%, 0.006%, 0.007% or 0.008%, or an interval value between any two endpoint values.
  • the glycerol content (v/v) is 5%, 6%, 7%, 8%, 9% or 10%, or an interval value between any two endpoint values.
  • the non-ionic detergent content (v/v) is 0.1%, 0.14%, 0.18% or 0.2%, or an interval value between any two endpoint values.
  • stabilizers include 0.7 mg/mL BSA, 0.006% (v/v) gelatin, and 7% (v/v) glycerol.
  • the enhancer includes 1% ⁇ 2% (v/v) formamide, 1% ⁇ 5% (v/v) DMSO, 0.1 ⁇ 0.3M betaine, 0.5 ⁇ 1.0g/L SSB single chain binding protein , 0.3 ⁇ 0.8g/L pyrophosphatase, 0.1 ⁇ 0.3M trehalose and 0.05 ⁇ 0.1g/L sodium azide.
  • the formamide content (v/v) is 1%, 1.5% or 2%, or an interval between any two endpoint values.
  • the DMSO content (v/v) is 1%, 2%, 3%, 4% or 5%, or an interval value between any two endpoint values.
  • the content of betaine is 0.1M, 0.2M or 0.3M, or an interval value between any two endpoint values.
  • the content of SSB single-chain binding protein is 0.5g/L, 0.7g/L, 0.9g/L or 1.0g/L, or an interval value between any two endpoint values.
  • the content of pyrophosphatase is 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L or 0.8g/L, or an interval value between any two endpoint values.
  • the content of trehalose can be 0.1M, 0.2M or 0.3M, or an interval value between any two endpoint values.
  • the content of sodium azide can be 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, or 1.0g/L, or an interval between any two endpoint values value.
  • enhancers include 2% formamide, 2.5% DMSO, 0.2M betaine, 0.8g/L SSB single-chain binding protein, 0.2M trehalose, and 0.08g/L sodium azide.
  • One embodiment of the present disclosure also provides the application of the one-tube RT-qPCR fully premixed reaction reagent described in any one of the preceding embodiments in preparing a one-tube RT-qPCR fully premixed reaction system.
  • One embodiment of the present disclosure also provides a tube-type RT-qPCR fully premixed reaction system, including a tube-type RT-qPCR fully premixed reaction reagent according to any one of the preceding embodiments;
  • DNA polymerase also includes DNA polymerase, reverse transcriptase, UNG enzyme, RNase inhibitor, buffer, stabilizer, enhancer, upstream and downstream primers and fluorescently labeled probe compositions.
  • the DNA polymerase is hot-start Taq DNA polymerase, and the extension speed is 1.5 sec/kb.
  • the amount of DNA polymerase added is 3 to 6 U, such as 3 U, 4 U, 5 U or 6 U, or any two. The interval value between endpoint values.
  • the reverse transcriptase includes M-MLV reverse transcriptase, with a temperature tolerance of 60°C or above, and an added amount of 5 to 10 U.
  • the UNG enzyme is a thermosensitive UNG enzyme of 0.1 to 0.5 U (such as 0.1 U, 0.2 U, 0.3 U, 0.4 U or 0.5 U, or an interval value between any two endpoint values).
  • the amount of RNase inhibitor added is 2 to 3 U.
  • the buffer is a Tris-Hcl buffer solution of 100 to 500 mmol/L.
  • the buffer contains inorganic salts, which are sulfate and/or chloride salts, optionally chloride salts.
  • the upstream and downstream primers and fluorescently labeled probe compositions include a single detection composition, a double detection composition, a triple detection composition or a quadruple detection composition. Test composition.
  • the nucleic acid product is derived from a virus.
  • the virus is selected from at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • the single-plex detection composition is a norovirus primer-probe composition.
  • nucleotide sequence of the upstream primer in the norovirus primer-probe composition is as shown in SEQ ID No. 3 (CTGGTGGATTGGAAATGTA), and the nucleotide sequence of the downstream primer in the norovirus primer-probe composition is as shown in SEQ ID No. 4 is shown (AATTCGGGCAGAAGATTG), and the nucleotide sequence of the probe in the norovirus primer-probe composition is shown in SEQ ID No. 5 (CTGTCCACAATCCGAGGTCAT);
  • the dual detection composition is an influenza A and B virus primer and probe composition
  • the nucleotide sequence of the influenza A virus upstream primer in the influenza A and B virus primer-probe composition is shown in SEQ ID No. 6 (AGTCTTCTAACCGAGGTC), and the nucleotide sequence of the influenza A virus downstream primer is As shown in SEQ ID No.7 (TCGAGATCTGCGTTCTTC), the nucleotide sequence of the influenza A virus probe is shown in SEQ ID No.8 (CATCCTCAAGTCTCTGTGCGA), and the nucleotide sequence of the influenza B virus upstream primer is as SEQ ID No.
  • GGAGTCTTATCCCAATTTG the nucleotide sequence of the influenza B virus downstream primer is shown in SEQ ID No.10 (CTTCTGTGACCAGTCTAA), and the nucleotide sequence of the influenza B virus probe is shown in SEQ ID No.11 (CAAGAGCACCGATTATCACCAGAA);
  • the triple detection composition is a primer-probe composition for enterovirus type 71, enterovirus type 71, and coxsackievirus type 16.
  • enteroviruses include poliovirus, coxsackievirus and orphan viruses that are cytopathic in the human intestine (ECHO virus).
  • ECHO virus cytopathic in the human intestine
  • the International Committee on Viral Nomenclature classified these viruses into the genus Enterovirus of the family Picornaviridae.
  • the primer-probe composition for enterovirus, enterovirus 71 and coxsackievirus 16 contains:
  • the universal upstream primer for enteric conventional viruses the nucleotide sequence is shown in SEQ ID No. 12 (GCAAGTAGTCACAGATTAG), the universal downstream primer for enteric conventional viruses, the nucleotide sequence is shown in SEQ ID No. 13 (GATTCTTGTCACTAGCATT), the intestinal tract Conventional virus universal probe, the nucleotide sequence is shown in SEQ ID No. 14 (TACCAGCACTACAAGCCGC);
  • Enterovirus 71 upstream primer the nucleotide sequence is shown in SEQ ID No. 15 (CACAGGTGAGCAGTCATC), enterovirus 71 downstream primer, the nucleotide sequence is shown in SEQ ID No. 16 (CGCCCTACTGAAGAAACT), intestinal Virus type 71 probe, the nucleotide sequence is shown in SEQ ID No. 17 (TACAGGCAAGGTTCCAGCAC);
  • Coxsackie virus type 16 upstream primer the nucleotide sequence is shown in SEQ ID No. 18 (CTTCCAGAGATTCGTTTG), Coxsackie virus type 16 downstream primer, the nucleotide sequence is shown in SEQ ID No. 19 (CATATTATTCGGGGCATTGA), Coxsackie virus type 16 probe, the nucleotide sequence is shown in SEQ ID No. 20 (CAGACAGCCACCAACCCAT).
  • One embodiment of the present disclosure also provides an RT-qPCR method, using the one-tube RT-qPCR fully premixed reaction system described in the previous embodiment to amplify the sample to be tested according to the following procedure: reverse transcription at 55-65°C for 1-5 minutes; 94 Pre-denaturation at ⁇ 98°C for 5-15 seconds; denaturation at 94-98°C for 1-5 seconds; annealing and extension at 55-65°C for 8-15 seconds; denaturation and annealing extension cycles for 38-45 times.
  • the amplification program is: reverse transcription at 60°C for 2 min; pre-denaturation at 95°C for 10 s; denaturation at 95°C for 1 s; annealing at 60°C for 10 s; extension at 60°C for 10 s; and 40 cycles of denaturation, annealing and extension.
  • One embodiment of the present disclosure also provides a tube-type RT-qPCR fully premixed reaction reagent according to any of the preceding embodiments, a tube-type RT-qPCR fully premixed reaction system according to any one of the preceding embodiments, or, Application of the RT-qPCR method described in the aforementioned embodiments in the diagnosis of viral infection.
  • One embodiment of the present disclosure also provides a tube-type RT-qPCR fully premixed reaction reagent according to any one of the preceding embodiments or a one-tube RT-qPCR fully premixed reaction system according to any one of the preceding embodiments. Use in the diagnosis of viral infections.
  • the virus includes at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • One embodiment of the present disclosure also provides a method for diagnosing diseases related to viral infection in a subject, including:
  • One embodiment of the present disclosure also provides a diagnostic method for viral infection.
  • the diagnostic method includes using the one-tube RT-qPCR fully premixed reaction reagent configuration according to any one of the aforementioned embodiments to obtain a tube according to any one of the aforementioned embodiments.
  • the RT-qPCR fully premixed reaction system is used to amplify the sample to be tested according to the RT-qPCR method described in the previous embodiment, and determine the source of the virus of the amplified product.
  • the virus includes at least one of norovirus, influenza A virus, influenza B virus, enterovirus, enterovirus 71 or coxsackievirus 16.
  • the source of the sample to be tested is selected from feces, vomitus, nasal swabs, throat swabs, sputum, bronchoalveolar lavage fluid, cells, chicken embryo culture, herpes fluid, serum, cerebrospinal fluid or tissue culture.
  • the source of the sample to be tested is selected from feces, vomitus, nasal swabs, throat swabs, sputum, bronchoalveolar lavage fluid, cells, chicken embryo culture, herpes fluid, serum, cerebrospinal fluid or At least one of tissue culture.
  • the throat swab includes a human throat swab or an avian throat swab.
  • samples to be tested for norovirus can be samples such as patient feces or vomitus, samples to be tested for influenza A virus or influenza B virus.
  • the samples to be tested for conventional enterovirus, enterovirus 71 or coxsackievirus 16 can be selected from the patient's herpes fluid, throat secretions, stool, serum, cerebrospinal fluid or isolated tissue culture.
  • a specific amplification method is as follows:
  • step S300 place the PCR reaction tube obtained in step S200 in a fluorescence quantitative PCR instrument for amplification and fluorescence signal detection;
  • the amplification and fluorescence signal detection cycle parameters of the fluorescence quantitative PCR instrument in step S300 are as follows in Table 1:
  • the present disclosure discloses a one-tube rapid RT-qPCR fully premixed reaction system.
  • This disclosure mainly addresses three problems existing in the existing RT-qPCR reaction system: (1) The total reaction system is divided into multiple tubes for storage. Each component needs to be prepared for each use, and the primers and probes must be stored separately or combined with the amplification tubes. The amplification reaction solution is stored together, and premixing each component in advance will lead to stability and specificity problems of the reaction system; (2) The RT-qPCR reaction system cannot adapt to the rapid amplification procedure, and the reaction time is too long, making it unsuitable for rapid detection. ; (3) The compatibility between the RT-qPCR reaction system and primer probes is insufficient, and the formula of the reaction system needs to be adjusted according to different primer and probe combinations.
  • the one-tube RT-qPCR fully premixed reaction reagent provided by this disclosure can be used to prepare a one-tube RT-qPCR fully premixed reaction system and then used for RT-qPCR reactions to solve various problems in the existing RT-qPCR reaction systems. They are stored separately and need to be prepared and used immediately. After full premixing, the sensitivity decreases, non-specificity increases, storage stability decreases, and the applicability of primers and probes is low.
  • This disclosure provides a fully premixed RT-qPCR reaction system and its application.
  • the hot-start Taq DNA polymerase that supports ultra-fast extension is selected, which greatly reduces the running time of the reaction system, and the entire reaction time is controlled within 30 minutes;
  • a high-temperature resistant reverse transcriptase was selected to improve the adaptability of the RT-qPCR reaction system to primers and probes, allowing different combinations of primers and probes to be plug-and-play;
  • the RT-qPCR reaction system was added with double blocking capabilities Taq enzyme monoclonal antibodies (Double-Block anti-Taq DNA Polymerase Antibody) and nucleic acid aptamers ensure that they can effectively inhibit the enzyme activity of Taq DNA polymerase and reverse transcriptase at low temperatures and reduce primers during high temperature pressurization and long-term storage.
  • the generation of dimers and the shearing effect of Taq DNA polymerase on the probe significantly improve the specificity of the reaction system and avoid false positives caused by the reaction system; the RT-qPCR reaction system adds stabilizers and enhancers. It protects the activities of Taq DNA polymerase, reverse transcriptase and other enzymes in the reaction system to the greatest extent, improves the stability of high-temperature pressurization and long-term storage, and has better detection capabilities.
  • the fully premixed RT-qPCR reaction system can be pressurized at 37°C for 7 days without affecting the detection ability, and can be stored stably for 12 to 15 months at conditions below -20°C. At the same time, the reaction system can be directly packaged and used after adding nucleic acid, which greatly reduces the workload of inspection personnel and is especially suitable for automated platforms.
  • the one-tube rapid RT-qPCR fully premixed reaction system of this example includes: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2 , 0.25mmol/LdN (U )TPs, 4U Taq DNA polymerase, 6U M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, 0.5uM norovirus upstream primer and downstream primer, 0.55uM probe, primer
  • the probe sequence is: the upstream primer sequence is: CTGGTGGATTGGAAATGTA (SEQ ID No. 3), the downstream primer sequence is: AATTCGGGCAGAAGATTG (SEQ ID No. 4), and the probe sequence is: CTGTCCACAATCCGAGGTCAT (SEQ ID No. 5).
  • 4U Taq DNA polymerase is a Taq DNA polymerase with an extension speed of 1.5sec/kb.
  • Example 1 lies in the one-tube fast RT-qPCR fully premixed reaction system, including: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2. 0.25mmol/LdN(U)TPs, 4U Taq DNA polymerase, 6U high temperature resistant M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, different combinations of primer probes.
  • Tris-Hcl buffer solution pH8.4
  • 50mmol/LKCl 1.5mmol/L MgCl 2.
  • 0.25mmol/LdN(U)TPs 4U Taq DNA polymerase
  • 6U high temperature resistant M-MLV reverse transcriptase 0.25U thermosensitive UNG enzyme
  • 2.5U nuclease inhibitor different combinations of primer probes.
  • 6U M-MLV reverse transcriptase is M-MLV reverse transcriptase that can withstand high temperatures of 60 degrees.
  • Example 2 lies in the one-tube rapid RT-qPCR fully premixed reaction system, which includes: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2 , 0.25mmol/LdN(U)TPs, 4U fast Taq DNA polymerase, 6U M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, 5ug/mL Double-Block anti- Taq DNA Polymerase Antibody, 0.5uM norovirus upstream primer and downstream primer, 0.55uM probe, the primer probe sequence is: the upstream primer sequence is: CTGGTGGATTGGAAATGTA (SEQ ID No. 3), the downstream primer sequence is: AATTCGGGCAGAAGATTG (SEQ ID No. 4), the probe sequence is: CTGTCCACAATCCGAGGTCAT (SEQ ID No. 5).
  • the Double-Block anti-Taq DNA Polymerase Antibody is a Taq enzyme monoclonal antibody with double blocking ability, and the usage ratio of Taq DNA polymerase is 1:5 (content ratio: U/U).
  • Example 3 lies in the one-tube rapid RT-qPCR fully premixed reaction system, which includes: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2 , 0.25mmol/LdN(U)TPs, 4U fast Taq DNA polymerase, 6U M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, Double-Block anti-Taq DNA Polymerase Antibody, 0.5ng/mL nucleic acid aptamer, 0.5uM norovirus upstream primer and downstream primer, 0.55uM probe, the primer probe sequence is: upstream primer sequence: CTGGTGGATTGGAAATGTA (SEQ ID No. 3), downstream primer The sequence is: AATTCGGGCAGAAGATTG (SEQ ID No. 4), and the probe sequence is: CTGTCCACAATCCGAGGTCAT (SEQ ID No. 5).
  • the nucleic acid aptamer is a single stranded oligonucleotide of 50-100 bp.
  • Example 4 lies in the one-tube rapid RT-qPCR fully premixed reaction system, which includes: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2 , 0.25mmol/LdN(U)TPs, 4U fast Taq DNA polymerase, 6U M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, Double-Block anti-Taq DNA Polymerase Antibody, 0.5ng/mL nucleic acid aptamer, PCR system stabilizer, 0.5uM norovirus upstream primer and downstream primer, 0.55uM probe, the primer probe sequence is: The upstream primer sequence is: CTGGTGGATTGGAAATGTA (SEQ ID No. 3), the downstream primer sequence is: AATTCGGGCAGAAGATTG (SEQ ID No. 4), and the probe sequence is: CTGTCCACAATCCGAGGTCAT (SEQ ID No. 3
  • the stabilizers are 0.7 mg/mLBSA, 0.006% gelatin, and 7.0% glycerin, where the percentages are volume percentages.
  • Example 5 lies in the one-tube rapid RT-qPCR fully premixed reaction system, which includes: 200mmol/L Tris-Hcl buffer solution (pH8.4), 50mmol/LKCl, 1.5mmol/L MgCl 2 , 0.25mmol/LdN(U)TPs, 4U fast Taq DNA polymerase, 6U M-MLV reverse transcriptase, 0.25U thermosensitive UNG enzyme, 2.5U nuclease inhibitor, Double-Block anti-Taq DNA Polymerase Antibody, 0.5ng/mL nucleic acid aptamer, PCR system stabilizer, PCR system enhancer, 0.5uM norovirus upstream primer and downstream primer, 0.55uM probe, the primer probe sequence is: The upstream primer sequence is: CTGGTGGATTGGAAATGTA (SEQ ID No. 3), the downstream primer sequence is: AATTCGGGCAGAAGATTG (SEQ ID No. 4), and the probe sequence is: CTGTCCACAATCCGA
  • the enhancers are 2% formamide, 2.5% DMSO, 0.2M betaine, 0.8g/LSSB single-chain binding protein, 0.2M trehalose, and 0.08g/L sodium azide.
  • Applicability evaluation Take 15 ⁇ L of the premixed reaction solution in Example 2 and aliquot it, and then add 5 ⁇ L of extracted RNA (1000 copies/mL) (standard corresponding to the pathogen target) as a template, where the single-plex primer probe
  • the needle is a Noro primer probe, consistent with Example 1, and the dual primer probe combination is an influenza A and B virus primer probe (A flow upstream primer sequence: AGTCTTCTAACCGAGGTC (SEQ ID No.
  • a flow downstream primer sequence TCGAGATCTGCGTTCTTC (SEQ ID No.7), stream A probe sequence: CATCCTCAAGTCTCTGTGCGA (SEQ ID No.8), stream B upstream primer sequence: GGAGTCTTATCCCAATTTG (SEQ ID No.9), stream B downstream primer sequence: CTTCTGTGACCAGTCTAA (SEQ ID No.
  • the triple primer probe combination is enterovirus universal enterovirus 71 coxsackievirus type 16 primer probe (intestinal universal upstream Primer sequence: GCAAGTAGTCACAGATTAG (SEQ ID No.12), intestinal universal downstream primer sequence: GATTCTTGTCACTAGCATT (SEQ ID No.13), intestinal universal probe sequence: TACCAGCACTACAAGCCGC (SEQ ID No.14), enterovirus 71 upstream Primer sequence: CACAGGTGAGCAGTCATC (SEQ ID No.15), Enterovirus 71 downstream primer sequence: CGCCCTACTGAAGAAACT (SEQ ID No.16), Enterovirus 71 probe sequence: TACAGGCAAGGTTCCAGCAC (SEQ ID No.17), Xhosa Strange virus type 16 upstream primer sequence: CTTCCAGAGATTCGTTTG (SEQ ID No.
  • Coxsackie virus type 16 downstream primer sequence CATATTATTCGGGCATTGA (SEQ ID No. 19)
  • Coxsackie virus type 16 probe sequence CAGACAGCCACCAACCCAT (SEQ ID No. 20)
  • Amplification and detection were performed according to the RT-qPCR procedure in Example 1. The detection results are shown in Figures 2 to 4.
  • Sensitivity evaluation Dispense 15 ⁇ L of the premixed reaction solution in Example 6, add 5 ⁇ L of extracted RNA (400 copies/mL) as a template, and amplify and detect according to RT-qPCR.
  • the fully premixed RT-PCR reaction system without PCR enhancer was used as the control group.
  • the fluorescence quantitative PCR detection results are shown in Figure 8.
  • the fully premixed components of the qPCR reaction system can be used directly by end customers without preparation, and the fully premixed system can be stored stably for a long time, which solves the problems of stability and specificity of the long-term storage of the fully premixed system; in When using fast DNA polymerase, the entire reaction time is shortened to less than 30 minutes, which greatly reduces the experimental time and increases the detection throughput in the same time; when using high-temperature resistant reverse transcriptase, the reaction system and primers The adaptability of the probe has been greatly improved. Primer probes designed by different people can be used directly without the need to design primer probes again.
  • the disclosure provides one-tube RT-qPCR fully premixed reaction reagents, applications and RT-qPCR methods.
  • the present disclosure uses this reagent to prepare a tube-type RT-qPCR fully premixed reaction reagent or its system, and then uses it for RT-qPCR reaction, which solves the problem that each component of the existing RT-qPCR reaction system is stored separately and needs to be prepared and used immediately.
  • the problems of decreased sensitivity, increased non-specificity, decreased storage stability, and low applicability of primers and probes have significantly reduced the running time of the reaction system and improved the suitability of the RT-qPCR reaction system for primers and probes. .
  • the disclosed fully premixed RT-qPCR reaction system can be pressurized at high temperature of 37°C for 7 days without affecting the detection ability, and can be stably stored under conditions below -20°C for 12 to 15 months.
  • the reaction system can be directly packaged and used after adding nucleic acid, which greatly reduces the workload of inspection personnel. It is especially suitable for automated platforms and therefore has excellent practical performance.

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Abstract

涉及反转录荧光PCR技术领域的一管式 RT-qPCR 全预混反应试剂、应用及RT-qPCR 方法和一管式快速RT-qPCR 全预混反应体系。其针对现有RT-qPCR反应体系存在的三个问题:(1)总反应体系分成多管保存，每次使用时各组分需要现配现用，引物探针必需单独保存或与扩增反应液一起保存，将各组分提前预混后会导致反应体系稳定性和特异性问题:(2)RT-qPCR 反应体系无法适应快速扩增程序,反应时间过长，不适用于快速检测;(3)RT-qPCR反应体系与引物探针的适配性不足，需要根据不同的引物探针组合调整反应体系的配方。

Description

一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法
相关申请的交叉引用
本公开要求于2022年08月30日提交中国专利局的申请号为CN202211045852.3、名称为“一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及反转录荧光PCR技术领域，尤其是涉及一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法。
背景技术
聚合酶链式反应(polymerase chain reation PCR)是利用单链募核苷酸引物对特异DNA片段进行体外快速扩增的一种方法，该反应是一个指数式反应，可在短时间内使极微量的目的DNA片段特异地扩增上百万倍。
RT-qPCR则是在PCR的基础上加入了反转录酶，以RNA为原始模板进行扩增得到DNA的方法。RT-qPCR根据反应体系中加入的荧光基团所积累的荧光信号强度变化，对PCR反应中的每一个循环扩增产物进行实时监控。
目前常用的RT-qPCR反应体系由核酸扩增缓冲液、酶混合液、引物探针反应液等组成且需要单独保存，在使用时需要将各组分室温溶解充分混匀后待用，使用时将各组分按照固定比例加入混匀后再分装到pcr管中，整个流程需要进行计算和仔细操作，会经常出现配错的情况，而且反应体系需要现配现用，如提前将核酸扩增缓冲液、酶混合液、引物探针反应液预混，冷冻或冷藏保存后会出现稳定性问题，具体表现为灵敏度下降和阴性样本中出现非特异性扩增。
RT-qPCR反应体系中引物探针在快速程序下的扩增效率与反应体系中的核酸扩增缓冲液和酶混合液有直接关系，一般情况下一组引物探针需要对应开发一组核酸扩增缓冲液和酶混合液，反应体系之间不能混用，RT-qPCR反应体系对各种引物探针反应液的适用性较差。
发明内容
本公开提供一管式RT-qPCR全预混反应试剂，所述试剂包括带有双封闭能力Taq酶单克隆抗体。
可选地，所述带有双封闭能力Taq酶单克隆抗体的氨基酸序列如SEQ ID No.1和SEQ ID No.2所示。
可选地，所述带有双封闭能力Taq酶单克隆抗体与Taq DNA聚合酶的含量比(U/U)为1:3～9，优选为1:5。
可选地，还包括核酸适配体。
可选地，所述核酸适配体含有的碱基数量为50～100bp。
可选地，所述核酸适配体的浓度为0.2～0.7ng/mL。可选地，所述核酸适配体的浓度为0.5ng/mL。
可选地，还包括稳定剂和/或增强剂。
可选地，所述稳定剂包括0.5～1mg/mL BSA、0.005％～0.008％(v/v)明胶、5％～10％(v/v)甘油或0.1％～0.2％(v/v)非离子型去垢剂中至少两种。
可选地，所述稳定剂包括0.7mg/mL BSA、0.006％(v/v)明胶和7％(v/v)甘油。
可选地，所述增强剂包括1％～2％(v/v)甲酰胺、1％～5％(v/v)DMSO、0.1～0.3M甜菜碱、0.5～1.0g/L SSB单链结合蛋白、0.3～0.8g/L焦磷酸酶、0.1～0.3M海藻糖和0.05～0.1g/L叠氮钠。
可选地，所述增强剂包括2％甲酰胺、2.5％DMSO、0.2M甜菜碱、0.8g/L SSB单链结合蛋白、0.2M海藻糖和0.08g/L叠氮钠。
本公开还提供前述任一项所述的一管式RT-qPCR全预混反应试剂在制备一管式RT-qPCR全预混反应体系中的应用。
本公开还提供一管式RT-qPCR全预混反应体系，包括前述任一项所述的一管式RT-qPCR全预混反应试剂；
还包括DNA聚合酶、逆转录酶、UNG酶、RNA酶抑制剂、缓冲液、稳定剂、增强剂、上下游引物和荧光标记的探针组合物。
可选地，所述DNA聚合酶为热启动Taq DNA聚合酶，延伸速度为1.5sec/kb，可选地添加量为3～6U。
可选地，所述逆转录酶包括M-MLV逆转录酶，耐受温度为60℃以上，添加量为5～10U。
可选地，所述UNG酶为0.1～0.5U的热敏UNG酶。
可选地，所述RNA酶抑制剂添加量为2～3U。
可选地，所述缓冲液为100～500mmol/L的Tris-Hcl缓冲溶液。
可选地，所述缓冲液含有无机盐，所述无机盐为硫酸盐和/或氯化盐。可选地，所述无机盐为氯化盐。
可选地，按照单次扩增核酸产物种类数量的区别，所述上下游引物和荧光标记的探针组合物包括单重检测组合物、双重检测组合物、三重检测组合物或四重检测组合物。
可选地，所述核酸产物来源于病毒。
可选地，所述病毒选自诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
可选地，所述单重检测组合物为诺如病毒引物探针组合物。
可选地，所述诺如病毒引物探针组合物中上游引物的核苷酸序列如SEQ ID No.3所示，所述诺如病毒引物探针组合物中下游引物的核苷酸序列如SEQ ID No.4所示，所述诺如病毒引物探针组合物中探针的核苷酸序列如SEQ ID No.5所示。
可选地，所述双重检测组合物为甲型乙型流感病毒引物探针组合物。
可选地，所述甲型乙型流感病毒引物探针组合物中甲型流感病毒上游引物的核苷酸序列如SEQ ID No.6所示，甲型流感病毒下游引物的核苷酸序列如SEQ ID No.7所示，甲型流感病毒探针的核苷酸序列如SEQ ID No.8所示，乙型流感病毒上游引物的核苷酸序列如SEQ ID No.9所示，乙型流感病毒下游引物的核苷酸序列如SEQ ID No.10所示，乙型流感病毒探针的核苷酸序列如SEQ ID No.11所示。
可选地，所述三重检测组合物为肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物。
可选地，所述肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物中含有：
肠道常规病毒通用上游引物，核苷酸序列如SEQ ID No.12所示，肠道常规病毒通用下游引物，核苷酸序列如SEQ ID No.13所示，肠道常规病毒通用探针，核苷酸序列如SEQ ID No.14所示；
肠道病毒71型上游引物，核苷酸序列如SEQ ID No.15所示，肠道病毒71型下游引物，核苷酸序列如SEQ ID No.16所示，肠道病毒71型探针，核苷酸序列如SEQ ID No.17所示；
柯萨奇病毒16型上游引物，核苷酸序列如SEQ ID No.18所示，柯萨奇病毒16型下游引物，核苷酸序列如SEQ ID No.19所示，柯萨奇病毒16型探针，核苷酸序列如SEQ ID No.20所示。
本公开还提供RT-qPCR方法，使用前述所述一管式RT-qPCR全预混反应体系对待测样本按照如下程序扩增：55～65℃下逆转录1～5min；94～98℃下预变性5～15s；94～98℃下变性1～5s；55～65℃下退火延伸8～15s；变性、退火延伸循环38～45次。
可选地，扩增程序为：60℃下逆转录2min；95℃下预变性10s；95℃下变性1s；60℃下退火10s；60℃下延伸10s；变性、退火和延伸循环40次。
本公开还提供上文任一项所述的一管式RT-qPCR全预混反应试剂或者所述一管式RT-qPCR全预混反应体系用于病毒感染诊断中的用途。
可选地，所述病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
本公开还提供一种诊断受试者中与病毒感染相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使上文任一项所述的一管式RT-qPCR全预混反应试剂或者所述一管式RT-qPCR全预混反应体系对来自所述受试者的样品经由RT-qPCR方法进行扩增；以及
B)确定扩增产物的病毒来源。
可选地，所述病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
可选地，所述样品的来源选自粪便、呕吐物、鼻拭子、咽拭子、痰液、支气管肺泡灌洗液、细胞、鸡胚培养物、疱疹液、血清、脑脊液或组织培养物。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开实施例1全预混RT-PCR反应体系和对照组的扩增结果；
图2为本公开实施例2全预混RT-PCR反应体系和对照组的扩增结果；
图3为本公开实施例2全预混RT-PCR反应体系和对照组的扩增结果；
图4为本公开实施例2全预混RT-PCR反应体系和对照组的扩增结果；
图5为本公开实施例3全预混RT-PCR反应体系和对照组的扩增结果；
图6为本公开实施例4全预混RT-PCR反应体系和对照组的扩增结果；
图7为本公开实施例5全预混RT-PCR反应体系和对照组的扩增结果；
图8为本公开实施例6全预混RT-PCR反应体系和对照组的扩增结果；
图9为本公开实施例6全预混RT-PCR反应体系保存1个月后的长期稳定性检测结果；
图10为本公开实施例6全预混RT-PCR反应体系保存3个月后的长期稳定性检测结果；
图11为本公开实施例6全预混RT-PCR反应体系保存6个月后的长期稳定性检测结果；
图12为本公开实施例6全预混RT-PCR反应体系保存9个月后的长期稳定性检测结果；
图13为本公开实施例6全预混RT-PCR反应体系保存12个月后的长期稳定性检测结果；
图14为本公开实施例6全预混RT-PCR反应体系保存15个月后的长期稳定性检测结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例中的附图，对本公开实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本公开实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本公开的实施例的详细描述并非旨在限制要求保护的本公开的范围，而是仅仅表示本公开的选定实施例。基于本公开中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
术语定义
如本文所使用，术语“受试者”、“个体”和“患者”在本文可以互换使用，是指脊椎动物，优选地是哺乳动物，最优选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
如本文所用，术语“引物”指一种寡核苷酸，所述寡核苷酸在置于引起与一条核酸链互补的引物延伸产物合成的条件下(即存在核苷酸和诱导物质(如DNA聚合酶)并在适宜的温度和pH)时能够充 当合成的起始点。引物可以是单链的并且必须足够长到在诱导物质存在下引发所需延伸产物的合成。引物的确切长度将取决于众多因素，包括温度、引物来源和方法的用途。
如本文所用，术语“诊断”或“医学诊断”是指从医学角度对人们的精神和体质状态作出的判断。具体来说是一种确定那种疾病或病症可以解释受试者的症状和体征的过程。例如，通过使用本文所述的一管式RT-qPCR全预混反应试剂或一管式RT-qPCR全预混反应体系来确定受试者中与病毒感染相关的疾病的存在。
本公开一实施方式提供一管式RT-qPCR全预混反应试剂，试剂包括带有双封闭能力Taq酶单克隆抗体。
在可选的实施方式中，带有双封闭能力Taq酶单克隆抗体的氨基酸序列如SEQ ID No.1(DIEMTQSSGAELARPGASVKMSCKASGTFRYSDYMHWVKQRPGQGLEWIGYRGPNDRYTKYNQKFKKATLTADKSSSTAYMQLSSLTSEDSAVYYCGRDDYYCSHDATWGQGTTLSRT)和SEQ ID No.2
(VQLVESGGMSASPGEKVTMTCAASSVYSSMHWYQQKSGTSPKRWIYDYQKLASGVPARFSGSGSGTSYSLTISSMEAETPETYYCEDWNPSDTFFGSGTTISRDN)所示。
在可选的实施方式中，带有双封闭能力Taq酶单克隆抗体与Taq DNA聚合酶的含量比(U/U)为1:3～9，例如可以为1:3.5～8.5、1:4～8或1:5～7，诸如1:3、1:4、1:5、1:6、1:7、1:8或1:9，或者任意两个端点值之间的区间值。可选地，带有双封闭能力Taq酶单克隆抗体与Taq DNA聚合酶的含量比(U/U)为1:5。
在可选的实施方式中，还包括核酸适配体。
在可选的实施方式中，核酸适配体含有的碱基数量为50～100bp，诸如55～90bp、65～80bp或75～80bp，诸如50bp、60bp、70bp、80bp、90bp或100bp，或者任意两个端点值之间的区间值。
可选地，核酸适配体的浓度为0.2～0.7ng/mL，诸如0.2ng/mL、0.3ng/mL、0.4ng/mL、0.5ng/mL、0.6ng/mL或0.7ng/mL，或者任意两个端点值之间的区间值。可选地，核酸适配体的浓度为0.5ng/mL。
在可选的实施方式中，还包括稳定剂和/或增强剂。
可选地，稳定剂包括0.5～1mg/mL BSA、0.005％～0.008％(v/v)明胶、5％～10％(v/v)甘油或0.1％～0.2％(v/v)非离子型去垢剂中至少两种。例如，BSA含量为0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1mg/mL，或者任意两个端点值之间的区间值。例如，明胶的含量(v/v)为0.005％、0.006％、0.007％或0.008％，或者任意两个端点值之间的区间值。例如，甘油的含量(v/v)为5％、6％、7％、8％、9％或10％，或者任意两个端点值之间的区间值。例如，非离子型去垢剂的含量(v/v)为0.1％、0.14％、0.18％或0.2％，或者任意两个端点值之间的区间值。
可选地，稳定剂包括0.7mg/mL BSA、0.006％(v/v)明胶和7％(v/v)甘油。
可选地，增强剂包括1％～2％(v/v)甲酰胺、1％～5％(v/v)DMSO、0.1～0.3M甜菜碱、0.5～1.0g/L SSB单链结合蛋白、0.3～0.8g/L焦磷酸酶、0.1～0.3M海藻糖和0.05～0.1g/L叠氮钠。例如，甲酰 胺的含量(v/v)为1％、1.5％或2％，或者任意两个端点值之间的区间值。例如，DMSO的含量(v/v)为1％、2％、3％、4％或5％，或者任意两个端点值之间的区间值。例如，甜菜碱的含量为0.1M、0.2M或0.3M，或者任意两个端点值之间的区间值。例如，SSB单链结合蛋白的含量为0.5g/L、0.7g/L、0.9g/L或1.0g/L，或者任意两个端点值之间的区间值。例如，焦磷酸酶的含量为0.3g/L、0.4g/L、0.5g/L、0.6g/L、0.7g/L或0.8g/L，或者任意两个端点值之间的区间值。例如，海藻糖的含量可以为0.1M、0.2M或0.3M，或者任意两个端点值之间的区间值。例如，叠氮钠的含量可以为0.05g/L、0.06g/L、0.07g/L、0.08g/L、0.09g/L、或1.0g/L，或者任意两个端点值之间的区间值。
可选地，增强剂包括2％甲酰胺、2.5％DMSO、0.2M甜菜碱、0.8g/L SSB单链结合蛋白、0.2M海藻糖和0.08g/L叠氮钠。
本公开一实施方式还提供前述实施方式任一项所述的一管式RT-qPCR全预混反应试剂在制备一管式RT-qPCR全预混反应体系中的应用。
本公开一实施方式还提供一管式RT-qPCR全预混反应体系，包括前述实施方式任一项所述的一管式RT-qPCR全预混反应试剂；
还包括DNA聚合酶、逆转录酶、UNG酶、RNA酶抑制剂、缓冲液、稳定剂、增强剂、上下游引物和荧光标记的探针组合物。
可选地，DNA聚合酶为热启动Taq DNA聚合酶，延伸速度为1.5sec/kb，可选地，DNA聚合酶的添加量为3～6U，诸如3U、4U、5U或6U，或者任意两个端点值之间的区间值。
可选地，逆转录酶包括M-MLV逆转录酶，耐受温度为60℃以上，添加量为5～10U。
可选地，UNG酶为0.1～0.5U(诸如0.1U、0.2U、0.3U、0.4U或0.5U，或者任意两个端点值之间的区间值)的热敏UNG酶。
可选地，RNA酶抑制剂添加量为2～3U。
可选地，缓冲液为100～500mmol/L的Tris-Hcl缓冲溶液。
可选地，缓冲液含有无机盐，无机盐为硫酸盐和/或氯化盐，可选地为氯化盐。
在可选的实施方式中，按照单次扩增核酸产物种类数量的区别，上下游引物和荧光标记的探针组合物包括单重检测组合物、双重检测组合物、三重检测组合物或四重检测组合物。
可选地，核酸产物来源于病毒。
可选地，病毒选自诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
可选地，单重检测组合物为诺如病毒引物探针组合物。
可选地，诺如病毒引物探针组合物中上游引物的核苷酸序列如SEQ ID No.3所示(CTGGTGGATTGGAAATGTA)，诺如病毒引物探针组合物中下游引物的核苷酸序列如SEQ ID No.4所示(AATTCGGGCAGAAGATTG)，诺如病毒引物探针组合物中探针的核苷酸序列如SEQ ID No.5所示(CTGTCCACAATCCGAGGTCAT)；
可选地，双重检测组合物为甲型乙型流感病毒引物探针组合物；
可选地，甲型乙型流感病毒引物探针组合物中甲型流感病毒上游引物的核苷酸序列如SEQ ID No.6所示(AGTCTTCTAACCGAGGTC)，甲型流感病毒下游引物的核苷酸序列如SEQ ID No.7所示(TCGAGATCTGCGTTCTTC)，甲型流感病毒探针的核苷酸序列如SEQ ID No.8所示(CATCCTCAAGTCTCTGTGCGA)，乙型流感病毒上游引物的核苷酸序列如SEQ ID No.9所示(GGAGTCTTATCCCAATTTG)，乙型流感病毒下游引物的核苷酸序列如SEQ ID No.10所示(CTTCTGTGACCAGTCTAA)，乙型流感病毒探针的核苷酸序列如SEQ ID No.11所示(CAAGAGCACCGATTATCACCAGAA)；
可选地，三重检测组合物为肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物。
肠道常规病毒是指包括脊髓灰质炎病毒、柯萨奇病毒和在人类肠道致细胞病变的孤儿病毒(ECHO病毒)。1970年国际病毒命名委员会将这些病毒归属于微小核糖核酸病毒科的肠道病毒属。
可选地，肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物中含有：
肠道常规病毒通用上游引物，核苷酸序列如SEQ ID No.12所示(GCAAGTAGTCACAGATTAG)，肠道常规病毒通用下游引物，核苷酸序列如SEQ ID No.13所示(GATTCTTGTCACTAGCATT)，肠道常规病毒通用探针，核苷酸序列如SEQ ID No.14所示(TACCAGCACTACAAGCCGC)；
肠道病毒71型上游引物，核苷酸序列如SEQ ID No.15所示(CACAGGTGAGCAGTCATC)，肠道病毒71型下游引物，核苷酸序列如SEQ ID No.16所示(CGCCCTACTGAAGAAACT)，肠道病毒71型探针，核苷酸序列如SEQ ID No.17所示(TACAGGCAAGGTTCCAGCAC)；
柯萨奇病毒16型上游引物，核苷酸序列如SEQ ID No.18所示(CTTCCAGAGATTCGTTTG)，柯萨奇病毒16型下游引物，核苷酸序列如SEQ ID No.19所示(CATATTATTCGGGCATTGA)，柯萨奇病毒16型探针，核苷酸序列如SEQ ID No.20所示(CAGACAGCCACCAACCCAT)。
本公开一实施方式还提供RT-qPCR方法，使用前述实施方式所述一管式RT-qPCR全预混反应体系对待测样本按照如下程序扩增：55～65℃下逆转录1～5min；94～98℃下预变性5～15s；94～98℃下变性1～5s；55～65℃下退火延伸8～15s；变性、退火延伸循环38～45次。
可选地，扩增程序为：60℃下逆转录2min；95℃下预变性10s；95℃下变性1s；60℃下退火10s；60℃下延伸10s；变性、退火和延伸循环40次。
本公开一实施方式还提供了前述实施方式任一项所述一管式RT-qPCR全预混反应试剂，前述实施方式任一项所述一管式RT-qPCR全预混反应体系，或者，前述实施方式所述RT-qPCR方法在病毒感染诊断中的应用。
本公开一实施方式还提供了前述实施方式任一项所述的一管式RT-qPCR全预混反应试剂或者前述实施方式任一项所述一管式RT-qPCR全预混反应体系用于病毒感染诊断中的用途。
可选地，病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
本公开一实施方式还提供了一种诊断受试者中与病毒感染相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使前述实施方式任一项所述的一管式RT-qPCR全预混反应试剂或者前述实施方式任一项所述一管式RT-qPCR全预混反应体系对来自所述受试者的样品经由RT-qPCR方法进行扩增；以及
B)确定扩增产物的病毒来源。
本公开一实施方式还提供了病毒感染的诊断方法，诊断方法包括，使用前述实施方式任一项所述一管式RT-qPCR全预混反应试剂配置得到前述实施方式任一项所述一管式RT-qPCR全预混反应体系对待测样本按照前述实施方式所述RT-qPCR方法进行扩增，确定扩增产物的病毒来源。
可选地，病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
可选地，待测样本来源选自粪便、呕吐物、鼻拭子、咽拭子、痰液、支气管肺泡灌洗液、细胞、鸡胚培养物、疱疹液、血清、脑脊液或组织培养物。可选地，可选地，待测样本来源选自粪便、呕吐物、鼻拭子、咽拭子、痰液、支气管肺泡灌洗液、细胞、鸡胚培养物、疱疹液、血清、脑脊液或组织培养物中的至少一种。
可选地，咽拭子包括人类咽拭子或禽类咽拭子。
对于上述待测样本来源，本领域技术人员能够根据实际需求进行选择，例如，诺如病毒的待测样本可以选择患者粪便或呕吐物等样品，甲型流感病毒或乙型流感病毒的待测样本可以选择患者的鼻拭子、咽拭子、痰液、支气管肺泡灌洗液；或者，环境采样得到的离体细胞、鸡胚培养物，以及禽类咽喉拭子等样品。而肠道常规病毒、肠道病毒71型或柯萨奇病毒16型的待测样本可以选择患者的疱疹液、咽喉分泌物、粪便、血清、脑脊液或离体组织培养物。
例如，某次具体扩增方法如下：
S100、预处理，取出RT-qPCR全预混反应体系，并在室温下融化，充分振荡混匀后瞬间离心；
S200、加样：取出装有RT-qPCR全预混的预分装八连管，加入待测样本、阳性对照和阴性对照核酸各10μL，盖紧管盖，振荡均匀后瞬时离心；
S300、扩增和荧光信号检测，将步骤S200得到的PCR反应管放置在荧光定量PCR仪中进行扩增和荧光信号检测；
可选的，根据上述RT-qPCR方法，步骤S300中的荧光定量PCR仪的扩增和荧光信号检测循环参数如下表1：
表1
Figure PCTCN2022144290-appb-000001
Figure PCTCN2022144290-appb-000002
本公开的一种一管式快速RT-qPCR全预混反应体系。本公开主要针对现有RT-qPCR反应体系存在的三个问题：(1)总反应体系分成多管保存，每次使用时各组分需要现配现用，引物探针必需单独保存或与扩增反应液一起保存，将各组分提前预混后会导致反应体系稳定性和特异性问题；(2)RT-qPCR反应体系无法适应快速扩增程序，反应时间过长，不适用于快速检测；(3)RT-qPCR反应体系与引物探针的适配性不足，需要根据不同的引物探针组合调整反应体系的配方。
本公开提供的一管式RT-qPCR全预混反应试剂，使用该试剂制备一管式RT-qPCR全预混反应体系后，用于RT-qPCR反应，解决现有RT-qPCR反应体系各组分单独保存、需要现配现用，全预混后灵敏度下降、非特异性增加、保存稳定性下降，引物探针适用性低的问题。
本公开提供了一种全预混RT-qPCR反应体系及其应用，选择了支持超快速延伸的热启动Taq DNA聚合酶，大幅度减少了反应体系的运行时间，整个反应时间控制在30min以内；同时选择了耐高温的逆转录酶，提高了RT-qPCR反应体系对引物探针的适配性，做到不同组合引物探针即插即用；RT-qPCR反应体系添加了带有双封闭能力Taq酶单克隆抗体(Double-Block anti-Taq DNA Polymerase Antibody)、核酸适配体保证在低温情况下有效抑制Taq DNA聚合酶和逆转录酶的酶活，减少在高温加压和长期保存时引物二聚体的产生和Taq DNA聚合酶对探针的剪切作用，从而显著提高反应体系的特异性，避免了因反应体系导致的假阳性；RT-qPCR反应体系添加了稳定剂、增强剂，最大程度保护反应体系中Taq DNA聚合酶、逆转录酶及其它酶的活性，提高了高温加压和长效保存的稳定性，检测能力更好。全预混RT-qPCR反应体系可在37℃高温加压7天对检测能力无影响，-20℃以下条件稳定保存12～15个月。同时反应体系可直接分装后加核酸后使用，大幅度减少了检验人员的工作量，特别适用于自动化平台。
实施例
下面结合附图，对本公开的一些实施方式作详细说明。在不冲突的情况下，下述的实施例及实施例中的特征可以相互组合。
实施例1
本实施例的一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U Taq DNA聚合酶、6U M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂，0.5uM的诺如病毒上游引物和下游引物、0.55uM探针，引物探针序列为：上游引物序列为：CTGGTGGATTGGAAATGTA(SEQ ID No.3)，下游引物序列为：AATTCGGGCAGAAGATTG(SEQ ID No.4)，探针序列为：CTGTCCACAATCCGAGGTCAT(SEQ ID No.5)。
其中4U Taq DNA聚合酶为延伸速度1.5sec/kb的Taq DNA聚合酶。
实施例2
本实施例与实施例1的不同之处在于一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U Taq DNA聚合酶、6U耐高温M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂，不同组合引物探针。
其中6U M-MLV逆转录酶为耐60度高温M-MLV逆转录酶。
实施例3
本实施例与实施例2的不同之处在于的一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U快速Taq DNA聚合酶、6U M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂、5ug/mL Double-Block anti-Taq DNA Polymerase Antibody、0.5uM的诺如病毒上游引物和下游引物、0.55uM探针，引物探针序列为：上游引物序列为：CTGGTGGATTGGAAATGTA(SEQ ID No.3)，下游引物序列为：AATTCGGGCAGAAGATTG(SEQ ID No.4)，探针序列为：CTGTCCACAATCCGAGGTCAT(SEQ ID No.5)。
其中所述Double-Block anti-Taq DNA Polymerase Antibody为带有双封闭能力Taq酶单克隆抗体，与Taq DNA聚合酶的使用比例为1:5(含量比：U/U)。
实施例4
本实施例与实施例3的不同之处在于的一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U快速Taq DNA聚合酶、6U M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂、Double-Block anti-Taq DNA Polymerase Antibody、0.5ng/mL核酸适配体、0.5uM的诺如病毒上游引物和下游引物、0.55uM探针，引物探针序列为：上游引物序列为：CTGGTGGATTGGAAATGTA(SEQ ID No.3)，下游引物序列为：AATTCGGGCAGAAGATTG(SEQ ID No.4)，探针序列为：CTGTCCACAATCCGAGGTCAT(SEQ ID No.5)。
其中所述核酸适配体为50-100bp的寡核苷酸单链。
实施例5
本实施例与实施例4的不同之处在于的一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U快速Taq DNA聚合酶、6U M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂、Double-Block anti-Taq  DNA Polymerase Antibody、0.5ng/mL核酸适配体、PCR体系稳定剂、0.5uM的诺如病毒上游引物和下游引物、0.55uM探针，引物探针序列为：上游引物序列为：CTGGTGGATTGGAAATGTA(SEQ ID No.3)，下游引物序列为：AATTCGGGCAGAAGATTG(SEQ ID No.4)，探针序列为：CTGTCCACAATCCGAGGTCAT(SEQ ID No.5)。
其中稳定剂为0.7mg/mLBSA、0.006％明胶、7.0％甘油、其中所述百分比为体积百分比。
实施例6
本实施例与实施例5的不同之处在于的一管式快速RT-qPCR全预混反应体系，包括：200mmol/L Tris-Hcl缓冲溶液(pH8.4)、50mmol/LKCl、1.5mmol/L MgCl 2、0.25mmol/LdN(U)TPs、4U快速Taq DNA聚合酶、6U M-MLV逆转录酶、0.25U热敏UNG酶、2.5U的核酸酶抑制剂、Double-Block anti-Taq DNA Polymerase Antibody、0.5ng/mL核酸适配体、PCR体系稳定剂、PCR体系增强剂、0.5uM的诺如病毒上游引物和下游引物、0.55uM探针，引物探针序列为：上游引物序列为：CTGGTGGATTGGAAATGTA(SEQ ID No.3)，下游引物序列为：AATTCGGGCAGAAGATTG(SEQ ID No.4)，探针序列为：CTGTCCACAATCCGAGGTCAT(SEQ ID No.5)。
其中增强剂为2％甲酰胺、2.5％DMSO、0.2M甜菜碱、0.8g/LSSB单链结合蛋白、0.2M海藻糖、0.08g/L叠氮钠。
实验例
反应速度评价：将实施例1中预混好的反应液，取15μL进行分装，再加入5μL提取的RNA/DNA(5000拷贝/mL)作为模板，按照下述RT-qPCR进行扩增并检测。以使用常规Taq DNA聚合酶的全预混RT-qPCR反应体系作为对照组。检测结果如图1。
其中扩增程序如下表2：
表2
Figure PCTCN2022144290-appb-000003
适用性评价：将实施例2中预混好的反应液，取15μL进行分装，再加入5μL提取的RNA(1000 拷贝/mL)(病原体靶标对应标准品)作为模板，其中，单重引物探针为诺如引物探针，与实施例1一致，双重引物探针组合为甲型乙型流感病毒引物探针(甲流上游引物序列：AGTCTTCTAACCGAGGTC(SEQ ID No.6)、甲流下游引物序列：TCGAGATCTGCGTTCTTC(SEQ ID No.7)、甲流探针序列：CATCCTCAAGTCTCTGTGCGA(SEQ ID No.8)，乙流上游引物序列：GGAGTCTTATCCCAATTTG(SEQ ID No.9)、乙流下游引物序列：CTTCTGTGACCAGTCTAA(SEQ ID No.10)、乙流探针序列：CAAGAGCACCGATTATCACCAGAA)(SEQ ID No.11)，三重引物探针组合为肠道病毒通用肠道病毒71型柯萨奇病毒16型引物探针(肠道通用上游引物序列：GCAAGTAGTCACAGATTAG(SEQ ID No.12)、肠道通用下游引物序列：GATTCTTGTCACTAGCATT(SEQ ID No.13)、肠道通用探针序列：TACCAGCACTACAAGCCGC(SEQ ID No.14)，肠道病毒71型上游引物序列：CACAGGTGAGCAGTCATC(SEQ ID No.15)、肠道病毒71型下游引物序列：CGCCCTACTGAAGAAACT(SEQ ID No.16)、肠道病毒71型探针序列：TACAGGCAAGGTTCCAGCAC(SEQ ID No.17)，柯萨奇病毒16型上游引物序列：CTTCCAGAGATTCGTTTG(SEQ ID No.18)、柯萨奇病毒16型下游引物序列：CATATTATTCGGGCATTGA(SEQ ID No.19)、柯萨奇病毒16型探针序列：CAGACAGCCACCAACCCAT(SEQ ID No.20)，以使用常规M-MLV逆转录酶的全预混RT-qPCR反应体系作为对照组。按照实施例1中RT-qPCR程序进行扩增并检测。检测结果如图2～4。
特异性评价：将实施例3～4中预混好的反应液，取15μL进行分装，再加入5μL提取的人基因组核酸(阴性样本)作为模板，加样后2～8℃保存12小时后按照RT-qPCR进行扩增并检测。以不含Double-Block anti-Taq DNA Polymerase Antibody、核酸适配体作为对照，检测结果如图5～6。
稳定性评价：将实施例5预混好的反应液，取15μL进行分装，再加入5μL提取的RNA(5000拷贝/mL)作为模板，加样后37℃保存7天后，按照RT-qPCR进行扩增并检测。以不含PCR稳定剂的全预混RT-PCR反应体系作为对照组。其荧光定量PCR检测结果如图7所示。
灵敏度评价：将实施例6预混好的反应液，取15μL进行分装，再加入5μL提取的RNA(400拷贝/mL)作为模板，按照RT-qPCR进行扩增并检测。以不含PCR增强剂的全预混RT-PCR反应体系作为对照组。其荧光定量PCR检测结果如图8所示。
长期稳定性评价：将实施例6预混好的反应液，-20℃保存1个月、3个月、6个月、9个月、12个月、15个月时进行检测，具体的实验方法为：取15μL进行分装，再加入5μL提取的RNA(5000拷贝/mL)作为模板，按照RT-qPCR进行扩增并检测。以不含Double-Block anti-Taq DNA Polymerase Antibody、核酸适配体、PCR体系稳定剂、PCR体系增强剂作为对照，检测结果分别如图9～14，分别对应保存-20℃保存1个月、3个月、6个月、9个月、12个月、15个月。
由上述结果可以看出，本公开通过组分的筛选、Double-Block anti-Taq DNA Polymerase Antibody、核酸适配体的制备及使用、PCR体系稳定剂、PCR体系增强剂的使用，实现了RT-qPCR反应体系各组分的全预混，终端客户可在不配制的情况下直接使用，且全预混体系可长期稳定保存，解决了全 预混体系长期保存的稳定性和特异性问题；在使用快速DNA聚合酶的情况下，整个反应时间缩短到30分钟以内，大幅度减少了实验时间，增加了等同时间内的检测通量；在使用耐高温逆转录酶的情况下，反应体系与引物探针的适配性有巨大提升，不同人设计的引物探针可直接使用，无需二次设计引物探针。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的一管式RT-qPCR全预混反应试剂、应用及RT-qPCR方法。本公开使用该试剂制备一管式RT-qPCR全预混反应试剂或其体系后，用于RT-qPCR反应，解决了现有RT-qPCR反应体系各组分单独保存、需要现配现用，全预混后灵敏度下降、非特异性增加、保存稳定性下降，引物探针适用性低的问题，大幅度减少了反应体系的运行时间，提高了RT-qPCR反应体系对引物探针的适配性。本公开的全预混RT-qPCR反应体系可在37℃高温加压7天对检测能力无影响，-20℃以下条件稳定保存12～15个月。同时反应体系可直接分装后加核酸后使用，大幅度减少了检验人员的工作量，特别适用于自动化平台，因此具有优异的实用性能。

Claims (15)

  1. 一管式RT-qPCR全预混反应试剂，其特征在于，所述试剂包括带有双封闭能力Taq酶单克隆抗体。
  2. 根据权利要求1所述的一管式RT-qPCR全预混反应试剂，其特征在于，所述带有双封闭能力Taq酶单克隆抗体的氨基酸序列如SEQ ID No.1和SEQ ID No.2所示。
  3. 根据权利要求2所述的一管式RT-qPCR全预混反应试剂，其特征在于，所述带有双封闭能力Taq酶单克隆抗体与Taq DNA聚合酶的含量比(U/U)为1:3～9，优选为1:5。
  4. 根据权利要求1所述的一管式RT-qPCR全预混反应试剂，其特征在于，还包括核酸适配体。
  5. 根据权利要求4所述的一管式RT-qPCR全预混反应试剂，其特征在于，所述核酸适配体含有的碱基数量为50～100bp；
    优选地，所述核酸适配体的浓度为0.2～0.7ng/mL，优选为0.5ng/mL。
  6. 根据权利要求1～5任一项所述的一管式RT-qPCR全预混反应试剂，其特征在于，还包括稳定剂和/或增强剂；
    优选地，所述稳定剂包括0.5～1mg/mL BSA、0.005％～0.008％(v/v)明胶、5％～10％(v/v)甘油或0.1％～0.2％(v/v)非离子型去垢剂中至少两种；
    优选地，所述稳定剂包括0.7mg/mL BSA、0.006％(v/v)明胶和7％(v/v)甘油；
    优选地，所述增强剂包括1％～2％(v/v)甲酰胺、1％～5％(v/v)DMSO、0.1～0.3M甜菜碱、0.5～1.0g/L SSB单链结合蛋白、0.3～0.8g/L焦磷酸酶、0.1～0.3M海藻糖和0.05～0.1g/L叠氮钠；
    优选地，所述增强剂包括2％甲酰胺、2.5％DMSO、0.2M甜菜碱、0.8g/L SSB单链结合蛋白、0.2M海藻糖和0.08g/L叠氮钠。
  7. 权利要求1～6任一项所述的一管式RT-qPCR全预混反应试剂在制备一管式RT-qPCR全预混反应体系中的应用。
  8. 一管式RT-qPCR全预混反应体系，其特征在于，包括权利要求1～6任一项所述的一管式RT-qPCR全预混反应试剂；
    还包括DNA聚合酶、逆转录酶、UNG酶、RNA酶抑制剂、缓冲液、稳定剂、增强剂、上下游引物和荧光标记的探针组合物；
    优选地，所述DNA聚合酶为热启动Taq DNA聚合酶，延伸速度为1.5sec/kb，优选添加量为3～6U；
    优选地，所述逆转录酶包括M-MLV逆转录酶，耐受温度为60℃以上，添加量为5～10U；
    优选地，所述UNG酶为0.1～0.5U的热敏UNG酶；
    优选地，所述RNA酶抑制剂添加量为2～3U；
    优选地，所述缓冲液为100～500mmol/L的Tris-Hcl缓冲溶液；
    优选地，所述缓冲液含有无机盐，所述无机盐为硫酸盐和/或氯化盐，更优选为氯化盐。
  9. 根据权利要求8所述的一管式RT-qPCR全预混反应体系，其特征在于，按照单次扩增核酸产物种类数量的区别，所述上下游引物和荧光标记的探针组合物包括单重检测组合物、双重检测组合物、三重检测组合物或四重检测组合物；
    优选地，所述核酸产物来源于病毒；
    优选地，所述病毒选自诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种；
    优选地，所述单重检测组合物为诺如病毒引物探针组合物；
    优选地，所述诺如病毒引物探针组合物中上游引物的核苷酸序列如SEQ ID No.3所示，所述诺如病毒引物探针组合物中下游引物的核苷酸序列如SEQ ID No.4所示，所述诺如病毒引物探针组合物中探针的核苷酸序列如SEQ ID No.5所示；
    优选地，所述双重检测组合物为甲型乙型流感病毒引物探针组合物；
    优选地，所述甲型乙型流感病毒引物探针组合物中甲型流感病毒上游引物的核苷酸序列如SEQ ID No.6所示，甲型流感病毒下游引物的核苷酸序列如SEQ ID No.7所示，甲型流感病毒探针的核苷酸序列如SEQ ID No.8所示，乙型流感病毒上游引物的核苷酸序列如SEQ ID No.9所示，乙型流感病毒下游引物的核苷酸序列如SEQ ID No.10所示，乙型流感病毒探针的核苷酸序列如SEQ ID No.11所示；
    优选地，所述三重检测组合物为肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物；
    优选地，所述肠道常规病毒、肠道病毒71型和柯萨奇病毒16型引物探针组合物中含有：
    肠道常规病毒通用上游引物，核苷酸序列如SEQ ID No.12所示，肠道常规病毒通用下游引物，核苷酸序列如SEQ ID No.13所示，肠道常规病毒通用探针，核苷酸序列如SEQ ID No.14所示；
    肠道病毒71型上游引物，核苷酸序列如SEQ ID No.15所示，肠道病毒71型下游引物，核苷酸序列如SEQ ID No.16所示，肠道病毒71型探针，核苷酸序列如SEQ ID No.17所示；
    柯萨奇病毒16型上游引物，核苷酸序列如SEQ ID No.18所示，柯萨奇病毒16型下游引物，核苷酸序列如SEQ ID No.19所示，柯萨奇病毒16型探针，核苷酸序列如SEQ ID No.20所示。
  10. RT-qPCR方法，其特征在于，使用权利要求8或9所述一管式RT-qPCR全预混反应体系对待测样本按照如下程序扩增：55～65℃下逆转录1～5min；94～98℃下预变性5～15s；94～98℃下变性1～5s；55～65℃下退火延伸8～15s；变性、退火延伸循环38～45次；
    优选地，扩增程序为：60℃下逆转录2min；95℃下预变性10s；95℃下变性1s；60℃下退火延伸10s；变性、退火和延伸循环40次。
  11. 权利要求1～6任一项所述的一管式RT-qPCR全预混反应试剂或者权利要求8或9所述一管式RT-qPCR全预混反应体系用于病毒感染诊断中的用途。
  12. 根据权利要求11所述的用途，其特征在于，所述病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
  13. 一种诊断受试者中与病毒感染相关的疾病的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1～6任一项所述的一管式RT-qPCR全预混反应试剂或者权利要求8或9所述一管式RT-qPCR全预混反应体系对来自所述受试者的样品经由RT-qPCR方法进行扩增；以及
    B)确定扩增产物的病毒来源。
  14. 根据权利要求13所述的方法，其特征在于，所述病毒包括诺如病毒、甲型流感病毒、乙型流感病毒、肠道常规病毒、肠道病毒71型或柯萨奇病毒16型中至少一种。
  15. 根据权利要求13或14所述的方法，其特征在于，所述样品的来源选自粪便、呕吐物、鼻拭子、咽拭子、痰液、支气管肺泡灌洗液、细胞、鸡胚培养物、疱疹液、血清、脑脊液或组织培养物。
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