CN116855642A - Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs - Google Patents
Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs Download PDFInfo
- Publication number
- CN116855642A CN116855642A CN202311031974.1A CN202311031974A CN116855642A CN 116855642 A CN116855642 A CN 116855642A CN 202311031974 A CN202311031974 A CN 202311031974A CN 116855642 A CN116855642 A CN 116855642A
- Authority
- CN
- China
- Prior art keywords
- primer
- hpv
- target
- detection
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 190
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 78
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 76
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 76
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 28
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 title abstract description 158
- 230000008696 hypoxemic pulmonary vasoconstriction Effects 0.000 title abstract 5
- 239000000523 sample Substances 0.000 claims abstract description 174
- 230000003321 amplification Effects 0.000 claims abstract description 81
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 81
- 238000006243 chemical reaction Methods 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 64
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 58
- 241000701806 Human papillomavirus Species 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 21
- 239000011324 bead Substances 0.000 claims description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 13
- 238000003556 assay Methods 0.000 claims description 13
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 12
- 239000007995 HEPES buffer Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 12
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 11
- 238000001917 fluorescence detection Methods 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 9
- 239000013641 positive control Substances 0.000 claims description 9
- 125000006853 reporter group Chemical group 0.000 claims description 8
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims description 7
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims description 7
- 102100034343 Integrase Human genes 0.000 claims description 7
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 7
- 239000013642 negative control Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- DNXKVOCRSQBZHO-UHFFFAOYSA-M lithium;dodecane-1-sulfonate Chemical compound [Li+].CCCCCCCCCCCCS([O-])(=O)=O DNXKVOCRSQBZHO-UHFFFAOYSA-M 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000002480 mineral oil Substances 0.000 claims description 5
- 235000010446 mineral oil Nutrition 0.000 claims description 5
- 238000013518 transcription Methods 0.000 claims description 5
- 230000035897 transcription Effects 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 3
- 108091034117 Oligonucleotide Proteins 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- 239000010836 blood and blood product Substances 0.000 claims description 3
- 229940125691 blood product Drugs 0.000 claims description 3
- 235000013365 dairy product Nutrition 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000004246 zinc acetate Substances 0.000 claims description 3
- 238000002405 diagnostic procedure Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims 1
- 230000000171 quenching effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 18
- 241000341655 Human papillomavirus type 16 Species 0.000 description 13
- 238000012545 processing Methods 0.000 description 8
- 239000012085 test solution Substances 0.000 description 8
- 238000013461 design Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 206010008342 Cervix carcinoma Diseases 0.000 description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 3
- 201000010881 cervical cancer Diseases 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000003761 preservation solution Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 238000002573 colposcopy Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 101150071673 E6 gene Proteins 0.000 description 1
- 101150013359 E7 gene Proteins 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012026 site acceptance test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for synchronously detecting 14 high-risk HPVs, and a special primer and a special probe thereof, belonging to the technical field of biomedical detection. The kit provided by the invention comprises nucleic acid extracting solutions, detection solutions and SAT enzyme solutions of two systems for 14 high-risk HPVs, primers and probes which are more suitable for detecting the high-risk HPVs are optimally designed, the detection systems are reasonably distributed, and each component is added step by step in the detection process to carry out step reaction, so that the real-time and rapid detection of the 14 high-risk HPVs can be synchronously realized, the sensitivity is high, the specificity is good, the RNA of an amplification product is easy to degrade, the environmental pollution is not caused, and the cross pollution of a sample is not caused in the detection process.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a real-time fluorescent nucleic acid isothermal amplification detection kit capable of detecting 14 high-risk HPVs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), a special primer and a probe for detecting the 14 high-risk HPVs, and particularly relates to a primer, a probe and a related kit used in the real-time fluorescent nucleic acid isothermal amplification detection of the 14 high-risk HPVs by combining a specific target capturing technology and a real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Test, SAT).
Background
Persistent infection with HPV is a major cause of cervical cancer in women, especially 14 of over 200 HPV subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) are considered to be high-risk HPV subtypes responsible for cervical cancer and precancerous lesions. Early diagnosis and early treatment are main means for preventing and treating cervical cancer.
The diagnosis of HPV at present mainly comprises colposcopy examination, cervical biopsy and PCR amplification detection. However, none of the colposology, colposcopy and cervical biopsy, among others, determine whether the infected HPV is a high risk HPV, which may be confusing for clinical administration and may create unnecessary mental stress on the patient. The PCR amplification detection method is a method commonly used for detecting high risk type HPV at present, for example, patent document CN107988432A (hereinafter referred to as document 1) discloses a kit for simultaneously detecting a plurality of high risk type human papillomaviruses in a single tube reaction, which at least comprises specific probes SEQ ID NOs 1-14 corresponding to 14 genotypes of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, respectively, and 6 forward primers and 6 reverse primers corresponding to the 14 genotypes are synthesized, and the minimum detection limit of the kit for 14 HPV genotypes can reach 100 copies/reaction (wherein the minimum detection limit for HPV16 and 18 can reach 10 copies/reaction). However, the method disclosed in the document 1 is based on the basic principle of PCR, which requires temperature rise and fall and circulation during the PCR reaction, and thus requires a long detection time (generally, 2 to 3 hours), and is inefficient; in addition, the reaction product of PCR is DNA, which is not easy to degrade and can easily cause sample cross contamination and experimental environment pollution. In order to overcome the defect of the PCR method in detecting HPV, patent document CN106834547A (hereinafter referred to as document 2) discloses a kit for detecting the E6/E7 gene of human papilloma virus based on real-time isothermal amplification, which comprises primers and probes designed for 14 high-risk HPVs, and can specifically detect and identify the mRNA of HPV E6/E7. However, the lowest limit of detection of RNA templates against 14 high-risk HPV viruses in this document 2 is only 100 copies/. Mu.l (i.e., 500 copies/reaction), and the detection sensitivity is low.
Disclosure of Invention
In view of one or more problems in the prior art, one aspect of the present invention provides a real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV types, comprising:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe and a second specific capture probe for capturing detection sequences of 9 high-risk HPV types, and 9 primers for specifically binding to target sequences of 9 high-risk HPV types, respectively, wherein the 9 high-risk HPV types comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer for specifically binding to target sequences of HPV 16, a sixth primer for specifically binding to target sequences of HPV 31, a seventh primer for specifically binding to target sequences of HPV 33, an eighth primer for specifically binding to target sequences of HPV 35, a ninth primer for specifically binding to target sequences of HPV 51, an eleventh primer for specifically binding to target sequences of HPV 52, an eleventh primer for specifically binding to target sequences of HPV 58, a twelfth primer for specifically binding to sequences of HPV 56 and HPV 66, and a universal primer;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe and a fourth specific capture probe for capturing detection sequences of 5 high-risk HPV types, and 4 primers for specifically binding to target sequences of 5 high-risk HPV types, respectively, wherein the 5 high-risk HPV types comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer for specifically binding to target sequences of HPV 18, an eighteenth primer for specifically binding to target sequences of HPV 45, a nineteenth primer for specifically binding to target sequences of HPV 59, a twentieth primer for specifically binding to target sequences of HPV 39 and HPV 68, and one of the universal primers;
(T3) amplification assay 1.1: it comprises a first primer, a second primer, a third primer and a fourth primer, wherein the first primer is matched with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is matched with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is matched with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is matched with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35 and 51 respectively;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 51, a second target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 16, 31, 35, a third target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 33, 52, 58, and a fourth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 56, 66;
(T5) amplification assay solution 2.1: comprising a thirteenth primer, a fourteenth primer, a fifteenth primer and a sixteenth primer, wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and a universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and a universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and a universal primer for amplifying a target sequence of HPV 39 and HPV 68;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 18, a sixth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 45, a seventh target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 59, and an eighth target detection probe that specifically binds to amplified product RNA copies of target sequences of HPV 39 and HPV 68;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
wherein:
the first specific capture probe to the fourth specific capture probe respectively comprise nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
the first primer to the twentieth primer respectively comprise nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 20;
the universal primer comprises a nucleotide sequence shown as SEQ ID NO. 21; and the sequences of the 5' end of the fifth primer to the twelfth primer and the seventeenth primer to the twentieth primer are the sequences of the universal primers;
The first target detection probe to the eighth target detection probe respectively comprise nucleotide sequences shown as SEQ ID NO. 22-SEQ ID NO. 29.
In some embodiments, the first to eighth target detection probes each carry a fluorescent reporter group and a quencher group at both ends of the nucleotide sequence, and the fluorescent reporter groups carried by the eighth target detection probes are the same or different.
In some embodiments, the kit further comprises:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: a system for E6/E7 in vitro transcription of RNA comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68; and/or
(M4) negative control: a system that does not contain human papillomavirus target nucleic acid.
In some embodiments, the components of the nucleic acid extraction solution 1 include: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μm of the first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the fifth to twelfth primers, 25-150pmol/mL of the universal primers.
In some embodiments, the components of the nucleic acid extraction solution 2 include: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μm of the third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the seventeenth to twentieth primers, 25-150pmol/mL of the universal primers.
In some embodiments, the components of the amplification test solution 1.1 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the first primer to the fourth primer.
In some embodiments, the components of the amplification test solution 1.2 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the first target detection probe to the fourth target detection probe.
In some embodiments, the components of the amplification detection solution 2.1 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the thirteenth to sixteenth primers.
In some embodiments, the components of the amplification detection solution 2.2 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the fifth target detection probe to the eighth target detection probe.
In some embodiments, the components of the SAT enzyme solution include: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
In another aspect, the invention provides an oligonucleotide combination for detecting 14 high risk HPV types and suitable for use in a real-time fluorescent nucleic acid isothermal amplification detection system, comprising:
(i) A specific capture probe combination comprising: first to fourth specific capture probes respectively comprising the nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
(ii) A primer combination comprising: first to twentieth primers comprising the nucleotide sequences shown as SEQ ID NO. 1 to SEQ ID NO. 20, respectively;
(iii) A universal primer comprising the nucleotide sequence set forth in SEQ ID NO. 21;
(iv) A target detection probe combination comprising: first to eighth target detection probes respectively comprising nucleotide sequences as shown in SEQ ID NO. 22-SEQ ID NO. 29.
In some embodiments, the first to eighth target detection probes each carry a fluorescent reporter group and a quencher group at both ends of the nucleotide sequence, and the fluorescent reporter groups carried by the eighth target detection probes are the same or different.
In yet another aspect, the present invention provides a non-disease diagnostic method for detecting 14 high-risk HPV types, the 14 high-risk HPV types including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, the method comprising the steps of:
1) Sub-packaging a sample to be tested into a tube 1 and a tube 2, adding a nucleic acid extracting solution 1 into the tube 1 for nucleic acid extraction, and adding a nucleic acid extracting solution 2 into the tube 2 for nucleic acid extraction to obtain analysis detection samples 1 and 2 respectively;
2) Adding an amplification detection liquid 1.1 into the analysis detection sample 1 to perform a first-step reaction to obtain a first-step reaction liquid 1, and adding an amplification detection liquid 2.1 into the analysis detection sample 2 to perform a first-step reaction to obtain a first-step reaction liquid 2;
3) Adding SAT enzyme solution into the first-step reaction solution 1 to perform a second-step reaction to obtain a second-step reaction solution 1, and adding SAT enzyme solution into the first-step reaction solution 2 to perform a second-step reaction to obtain a second-step reaction solution 2;
4) Adding an amplification detection liquid 1.2 into the second-step reaction liquid 1 for a third-step reaction to obtain an amplification system 1, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 1, and adding an amplification detection liquid 2.2 into the second-step reaction liquid 2 for a third-step reaction to obtain an amplification system 2, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 2;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if the channel corresponding to the amplification system 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected of the tube 1 contains HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested of the tube 1 does not contain HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids;
if an S-shaped amplification curve appears in a channel corresponding to the amplification system 2 and dt is less than or equal to 35, the sample to be detected of the tube 2 contains HPV18, 35, 45, 59 and 68 genotype nucleic acids; if the "S" type amplification curve does not appear, or the "S" type amplification curve appears, but dt >35, then HPV18, 35, 45, 59, 68 genotype nucleic acids are not contained in the sample to be tested of tube 2.
In some embodiments, the conditions of the first step reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15 minutes.
In some embodiments, the SAT enzyme solution in step 3) is preheated prior to use at a temperature of 41-43 ℃.
In some embodiments, the conditions of the second reaction step in step 3) are 41 ℃ to 43 ℃ for 30 to 50 minutes.
In some embodiments, the sample to be tested comprises medical samples and non-medical samples of sources including food, blood products, dairy products.
The primers and probes for detecting the real-time fluorescent nucleic acid isothermal amplification of 14 high-risk HPVs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) based on the technical scheme are obtained based on the optimized design of the 14 high-risk HPV E6/E7 RNA, a section of universal sequence is inserted into a downstream primer, and the universal primer (the sequence of which is the universal sequence) is added into the subsequent step-by-step reaction for detection, so that the inhibition effect in the reaction process can be greatly reduced, and the detection sensitivity is improved. The real-time fluorescent nucleic acid isothermal amplification detection kit provided based on the primer and the probe comprises a nucleic acid extracting solution 1, a nucleic acid extracting solution 2, an amplification detection solution 1.1, an amplification detection solution 1.2, an amplification detection solution 2.1, an amplification detection solution 2.2, a SAT enzyme solution and the like. The nucleic acid extract 1 may contain specific capture probes for capturing the detection sequences of HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the nucleic acid extract 2 may contain specific capture probes for capturing the detection sequences of HPV18, 45, 39, 59, 68, the amplification detection solution 1.1 contains primers for specifically binding to the target sequences of HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the amplification detection solution 1.2 contains target detection probes for respectively detecting HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the amplification detection solution 2.1 contains primers for specifically binding to the target sequences of HPV18, 45, 39, 59, 68, the amplification detection solution 2.2 contains target detection probes for respectively detecting HPV18, 45, 39, 59, 68, and the SAT enzyme solution contains RNA polymerase, reverse transcriptase, and the like necessary for the reaction. Based on SAT isothermal amplification detection technology, through stepwise reaction of the reaction liquid in the use process, mutual interference between different primers and probes can be further avoided, the system reaction is simpler, and high-sensitivity detection of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 can be realized by using only two reaction systems. The invention can be used for detecting non-medical samples, such as food, blood products, dairy products and the like, besides HPV medical samples, and is suitable for being widely popularized and used.
Compared with the existing detection method, the method has the following advantages:
(1) The primer and the probe provided by the invention are obtained by optimizing design according to HPV E6/E7 RNA, a section of general sequence (namely the sequence of the general primer) is inserted into the downstream primer, 14 high-risk HPVs are divided into two tubes for detection, and higher sensitivity detection can be realized by adding different components step by step in the detection process. The results of the examples show that the detection limit of the kit and the method provided by the invention on 14 high-risk HPVs can reach 10 copies/reaction, which is obviously lower than the detection limit (100 copies/reaction) of the method disclosed in the above document 1 and is obviously lower than the detection limit (100 copies/μl, namely 500 copies/reaction) of the method disclosed in the above document 2.
(2) The invention adopts the real-time fluorescent nucleic acid isothermal amplification detection technology to detect 14 high-risk HPV RNAs, and the whole process has no temperature rise and fall and circulation, so compared with the above-mentioned document 1, the invention can avoid the operations of heating, centrifugation, in vitro reverse transcription and the like of extracting RNA in the traditional PCR, has simpler experimental steps and reaction systems, is easy to realize automation, reduces the error caused by personnel operation, reduces the risk of infection of operators, greatly shortens the required time, can improve the detection efficiency, and reduces the design and production cost of the PCR instrument used.
(3) The amplified product of the invention is RNA which is easy to degrade in nature, and compared with the PCR amplified DNA in the document 1, the amplified product has the advantages of easy control of pollution, small cross influence and no environmental pollution.
Drawings
FIG. 1 is a graph showing the detection of HPV16 and HPV18 genotype nucleic acids, respectively, using the 3-lot kit of example 3.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings.
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: ALaboratory Manual) Sambrook, j., russell, david w., molecular Cloning: ALaboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The various biomaterials described in the examples were obtained by merely providing a means of experimental acquisition for the purposes of specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
All primers, specific capture probes, target detection probes and in vitro transcribed RNA products mentioned in the present invention are synthesized using the prior art.
Example 1: special primer and probe design for detecting 14 high-risk HPVs (human papilloma Virus) by real-time fluorescent nucleic acid isothermal amplification
The inventor starts from 14 high-risk type HPV E6/E7 RNA nucleic acid sequences (wherein HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) disclosed in Genbank databases, and designs primers and probe sequences which can simultaneously perform real-time fluorescent nucleic acid isothermal amplification detection on 9 high-risk type HPVs (HPV 16, 31, 33, 35, 51, 52, 56, 58, 66) in the same detection system (referred to herein as "system 1") according to primer and probe design principles, and can simultaneously perform amplification on the high-risk type HPVs in the same detection system (HPV 18, 39, 45, 59, 68) in the same detection system (referred to herein as "system 1"), and insert primers and probe sequences which can simultaneously perform high-sensitivity fluorescent amplification on the high-risk type HPVs in the same detection system (HPV 18, 39, 45, 59, 68) in the same system) in the same detection system (referred to herein as "system 2"), in the same high-risk type HPV 14 ") according to primer and probe design principles.
This example designed multiple sets of downstream primers for each of the 14 high-risk HPV types, two of which are shown in tables 1 and 2 below (the two sets of downstream primers respectively cooperate with the corresponding upstream primers to have high detection sensitivity for the corresponding HPV genotypes when each HPV genotype is detected individually, each up to 1 copy/reaction), and sequence information of specific capture probes, upstream primers, universal primers and target detection probes designed for the 14 high-risk HPV types.
Table 1: primer and probe sequence information in System 1
Table 2: primer and probe sequence information in System 2
In this example, based on the primer and probe sequence information listed in tables 1 and 2 above, various combinations of primers and probes were performed, 4 combinations (group 1 to group 4) were listed for system 1 and system 2, respectively, in table 3 below, and the 14 high-risk HPV positive controls (for in vitro transcription of RNA, described in detail below) and negative controls (without the 14 HPV high-risk target nucleic acid sequences, such as deionized water or sample preservation solution (commercially available)) were used in both systems to perform real-time fluorescent nucleic acid isothermal amplification detection (specific detection methods can be seen in example 4 below) to screen out primer and probe combinations that can be used to detect 14 high-risk HPV types with good sensitivity and specificity, respectively.
Table 3: primer and probe combination information in system 1 and system 2 for detecting 14 high-risk HPVs
The positive control in this example was prepared by the following steps:
(1) E6/E7 RNA fragments of HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 (corresponding to KP677556.1, KC662604.1, KC662597.1, KC662602.1, GQ465863.1, KT943747.1, KC662608.1, KF436866.1, KY077836.1, KX645763.1, JX896422.1, KU195240.1, JN661482.1, KU195243.1, respectively) were synthesized by chemical synthesis and constructed on a conventional plasmid vector containing a T7 promoter sequence;
(2) RNA fragments were transcribed using a commercial T7 promoter external transcription kit (sigma), and after purification, RNA copy numbers were calculated by UV and in vitro transcribed RNA was used as positive controls, respectively.
The positive control samples (in vitro transcribed RNA gradient concentrations were 10 each) containing each HPV RNA (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) were each subjected to the respective gradient concentration using the primers and probes of each set (set 1 to set 4) shown in Table 3 above 5 cobies/reaction, 10 4 cobies/reaction, 10 3 cobies/reaction, 10 2 cobies/reaction, 10 1 cobies/reaction, 10 0 cobies/reactions; each system contains RNA templates of HPV types) and negative controls. The results are shown in tables 4-7 below, wherein "+" indicates detected and "-" indicates undetected.
Table 4: detection results of primers and probes shown in group 1 respectively aiming at HPV nucleic acids with different concentrations and different types
Table 5: detection results of primers and probes shown in group 2 respectively aiming at HPV nucleic acids with different concentrations and different types
Table 6: detection results of primers and probes shown in group 3 respectively aiming at HPV nucleic acids with different concentrations and different types
/>
Table 7: the primer and the probe shown in the group 4 are respectively aimed at detection results of HPV nucleic acids with different concentrations and different types
As can be seen from the sensitivity and specificity results of each set of primer probes shown in tables 4-7 above for the detection of 14 high-risk HPV nucleic acids, respectively, different combinations of primers (downstream primers) may have an effect on the sensitivity and specificity results of the detection. Specifically, for 9 HPV detection in system 1, the use of the downstream primers of group 2 and group 3 will detect the target (HPV 39, HPV 68) in system 2, and the use of the downstream primer of group 4 will result in a minimum detection limit of HPV16, 31, 35, 52, 33, 58 of not reaching 10 copies/reaction. For 5 HPV detection in system 2, the use of the downstream primer of group 2 resulted in the lowest detection limit of HPV 45, 39, 68 not reaching 10 copies/reaction, the use of the downstream primer of group 3 detected the target in system 1 (HPV 51), and the use of the downstream primer of group 4 detected the target in system 1 (HPV 16, 31, 35). It can be seen that, of the four sets of primers and probes above, only the specific combination (set 1) has higher sensitivity and specificity when HPV is detected for system 1 and system 2, while the primers and/or probes in other combinations may interfere with each other to affect the sensitivity and specificity of detection. Therefore, the invention determines the primer and probe combination of the group 1 to be the optimal combination for detecting 14 high-risk HPVs and suitable for a real-time fluorescent nucleic acid isothermal amplification detection method.
In the embodiment, the primer probe combinations of the group 1 (the primer probe combinations of the system 1 and the system 2) are used for respectively detecting 5 low-risk type HPV nucleic acids (HPV 6, 11, 42, 43 and 44), and the results are negative, so that the primer probe combinations of the group 1 provided by the invention have better specificity when aiming at the low-risk type HPV detection, and can not obtain non-specific detection results.
In this example, the primer probe combination of group 1 was also used to perform multiplex real-time fluorescent nucleic acid isothermal amplification detection on mixed forms of HPV RNA templates, wherein HPV RNA templates for system 1 were mixed with equal concentrations of HPV16, 31, 33, 35, 51, 52, 56, 58, 66 in vitro transcribed RNA, the final RNA concentration was 10 copies/reaction, and HPV RNA templates for system 2 were mixed with equal concentrations of HPV18, 39, 45, 59, 68 in vitro transcribed RNA, the final RNA concentration was 10 copies/reaction. The results are shown in Table 8 below, where "+" indicates detected and "-" indicates undetected. As can be seen from the results shown in Table 8, even when the primer probe of the group 1 provided by the invention performs multiplex detection on HPV RNA templates in a mixed form, target RNA with a final RNA concentration of 10 copies/reaction can be detected, and the detection specificity is still good.
Table 8: detection results of primers and probes shown in group 1 against HPV RNA templates in mixed form
Example 2: real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV RNAs
The real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk type HPV RNAs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) provided in this embodiment is a kit based on the RNA nucleic acid isothermal synchronous amplification detection principle, and specifically may include the following components:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe (SEQ ID NO: 42) and a second specific capture probe (SEQ ID NO: 43) for capturing 9 high-risk HPVs, and 9 primers for specifically binding to target sequences of 9 high-risk HPVs, respectively, wherein the 9 high-risk HPVs comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer (SEQ ID NO: 5) for specifically binding to target sequences of HPV 16, a sixth primer (SEQ ID NO: 6) for specifically binding to target sequences of HPV 31, a seventh primer (SEQ ID NO: 7) for specifically binding to target sequences of HPV 33, an eighth primer (SEQ ID NO: 8) for specifically binding to target sequences of HPV 51, a ninth primer (SEQ ID NO: 9) for specifically binding to target sequences of HPV 51, a tenth primer (SEQ ID NO: 10) for specifically binding to sequences of HPV 52, an eleventh primer (SEQ ID NO: 12) for specifically binding to target sequences of HPV 31, an eleventh primer (SEQ ID NO: 8) for binding to target sequences of HPV 35; specifically, the nucleic acid extraction solution 1 may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 mu m of first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL fifth to twelfth primers, 25-150pmol/mL universal primers;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe (SEQ ID NO: 44) and a fourth specific capture probe (SEQ ID NO: 45) for capturing 5 high-risk HPVs, and 4 primers for specifically binding to target sequences of 5 high-risk HPVs, respectively, wherein the 5 high-risk HPVs comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer (SEQ ID NO: 17) for specifically binding to target sequences of HPV 18, an eighteenth primer (SEQ ID NO: 18) for specifically binding to target sequences of HPV 45, a nineteenth primer (SEQ ID NO: 19) for specifically binding to target sequences of HPV 59, a twentieth primer (SEQ ID NO: 20) for specifically binding to target sequences of HPV 39 and HPV 68, respectively, and a universal primer (SEQ ID NO: 21); specifically, the nucleic acid extraction solution 2 may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 mu m of third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL seventeenth to twentieth primers, 25-150pmol/mL universal primers;
(T3) amplification assay 1.1: comprising a first primer (SEQ ID NO: 1), a second primer (SEQ ID NO: 2), a third primer (SEQ ID NO: 3) and a fourth primer (SEQ ID NO: 4), wherein the first primer is complexed with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is complexed with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is complexed with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is complexed with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35, 51; specifically, the amplification detection solution 1.1 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of first primer to fourth primer;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe (SEQ ID NO: 22), a second target detection probe (SEQ ID NO: 23), and a third target detection probe (SEQ ID NO: 24)And a fourth target detection probe (SEQ ID NO: 25), wherein the first target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 51, the second target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 16, 31, 35, the third target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 33, 52, 58, and the fourth target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 56, 66; specifically, the amplification detection solution 1.2 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL universal primer, 143-857pmol/mL primary target detection probes to fourth target detection probes;
(T5) amplification assay solution 2.1: comprising a thirteenth primer (SEQ ID NO: 13), a fourteenth primer (SEQ ID NO: 14), a fifteenth primer (SEQ ID NO: 15) and a sixteenth primer (SEQ ID NO: 16), wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and the universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and the universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and the universal primer for amplifying a target sequence of HPV 39 and HPV 68; specifically, the amplification detection solution 2.1 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL thirteenth to sixteenth primers;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe (SEQ ID NO: 26), a sixth target detection probe (SEQ ID NO: 27), a seventh target detection probe (SEQ ID NO: 28), and an eighth target detection probe (SEQ ID NO: 29), wherein the fifth target detection probe specifically binds to an amplified product RNA copy of a target sequence of HPV 18, the sixth target detection probe specifically binds to an amplified product RNA copy of a target sequence of HPV 45, and the seventh target detection probe specifically binds to a target sequence of HPV 59The eighth target detection probe specifically binds to amplified product RNA copies of the target sequences of HPV 39 and HPV 68; specifically, the amplification detection solution 2.2 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL universal primer, 143-857pmol/mL fifth target detection probes to eighth target detection probes;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase; specifically, the SAT enzyme solution may comprise: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
For ease of detection, this example provides a kit further comprising the following components:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: E6/E7 in vitro RNA transcription system containing HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 (prepared in example 1); and/or
(M4) negative control: systems that do not contain human papillomavirus target nucleic acids, such as deionized water or sample preservation solutions (which contain high concentrations of detergent and physiological saline).
Example 3: stability of real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV RNAs
To verify the stability of the kit of the present invention (provided in example 2), 3 sets of different batches of kits were prepared using 3 sets of different batches of raw material reagents in this example, and the resulting kits were used to simultaneously detect HPV positive controls at two concentrations (see example 4 below for specific detection methods). The results obtained showed that the detection results of the 3 batches of kits were very similar, as shown in Table 9 and FIG. 1 (HPV 16, 18 are examples).
Table 9: detection of two-concentration HPV positive control results by three different batches of kit
Example 4: method for detecting 14 high-risk HPV genotypes by real-time fluorescent nucleic acid isothermal amplification
The method of this embodiment is based on the principle of isothermal amplification detection of RNA, and comprises the steps of synchronously detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 by two tubes, and detecting whether the sample contains the 14 high-risk HPV nucleic acids by using the kit provided in the above embodiment 2:
4.1, sample preparation
Taking 2mL of human cervical exfoliated cell sample stored in Hao Luo Jie company ThinPrep PreservCyt Solution, and uniformly mixing with 2mL of sample storage solution (Shanghai Reed degree biotechnology Co., ltd.) in a ratio of 1:1, and taking the mixture as a sample to be tested; 400. Mu.L of sample preservation solution was placed in a sample processing tube as a negative control.
4.2 nucleic acid extraction
(1) Respectively taking 400 mu L of a sample to be tested into a sample processing tube 1 and a sample processing tube 2, adding 100 mu L-800 mu L of a nucleic acid extracting solution 1 into the sample processing tube 1, adding 100 mu L-800 mu L of a nucleic acid extracting solution 2 into the sample processing tube 2, uniformly mixing, preserving heat at 60 ℃ for 10 minutes, and standing at room temperature for 10 minutes;
(2) And placing each sample treatment tube on a magnetic bead separation device, and standing for 3-5 minutes. After the magnetic beads are adsorbed on the tube wall, the sample treatment tube is kept on the magnetic bead separation device, waste liquid is sucked and discarded, and the magnetic beads are reserved. Adding 1mL of washing liquid, shaking uniformly, standing for 3-5 minutes, adding 800 mu L of washing liquid and 200 mu L of mineral oil, shaking uniformly, standing for 3-5 minutes, sucking waste liquid, and retaining magnetic beads;
(3) The sample processing tube is removed from the magnetic bead separation device and the tube is filled with a magnetic bead-nucleic acid complex for use (the beads should be clearly visible in this step).
4.3 SAT amplification assay
(1) Adding 40 mu L of amplification detection solution 1.1 into the sample treatment tube 1 (the magnetic bead-nucleic acid compound in the tube) treated in the step 4.2, adding 40 mu L of amplification detection solution 2.1 into the sample treatment tube 2 (the magnetic bead-nucleic acid compound in the tube), and oscillating to re-suspend the magnetic beads;
(2) Taking 40 mu L of detection liquid 1 (amplification detection liquid 1.1 in a sample treatment tube 1) and detection liquid 2 (amplification detection liquid 2.1 in a sample treatment tube 2) which are uniformly mixed by vibration, respectively adding 50 mu L of mineral oil into a micro-reaction tube, preheating the micro-reaction tube for 5-10min under the reaction condition of 42 ℃, adding 25 mu L of SAT enzyme liquid preheated to 42 ℃, and continuously preserving at the constant temperature of 42 ℃ for 5-10min;
(3) To a micro-reaction tube containing the amplification test solution 1.1 (test solution 1), 35. Mu.L of the amplification test solution 1.2 preheated to 42℃was added, to a micro-reaction tube containing the amplification test solution 2.1 (test solution 2), 35. Mu.L of the amplification test solution 2.2 preheated to 42℃was added, the micro-reaction tube was rapidly transferred to a constant temperature fluorescence detection apparatus, reacted at 42℃for 40 minutes, and the collection of a fluorescence signal every minute was set. Negative controls were processed and tested synchronously as above.
4.4, result determination
And automatically reading the dt value by software according to a curve obtained by the SAT amplification result, and judging the result.
The result judgment criteria were:
if the corresponding channel in the sample processing tube 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains HPV16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acid (any one or more); if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be detected does not contain HPV16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acid;
if the corresponding channel in the sample processing tube 2 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains HPV18, 35, 45, 59 and 68 genotype nucleic acid (any one or more); if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested does not contain HPV18, 35, 45, 59 and 68 genotype nucleic acids.
By using the method, 500 clinical samples are taken as detection objects, and the kit provided by the invention is compared with an HPV kit of Hao Luo Jie company. Similar to the kit of the invention, the Haoro kit also utilizes TMA isothermal detection technology to detect the 14 high-risk HPV genotypes. The detection of these samples was first completed by the state intellectual property test according to the protocol of the Hao Luo Jie panther kit. The remaining samples were tested by the kit of the invention. The comparison result shows that the positive consistency rate of the detection results of the two kits is 94.4%, the negative consistency rate is 96.5%, and the total consistency rate is 96.0% (table 10), so that the kit provided by the invention has higher accuracy in detecting clinical samples, and is basically equivalent to the detection results of the conventional HPV kit of Hao Luo Jie company.
Table 10: the kit and the Hao Luo Jie HPV kit of the invention take clinical samples as the objects to compare and detect results
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any equivalent substitutions, modifications and the like made within the spirit and principles of the present invention should fall within the present disclosure.
Claims (10)
1. A real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV types, comprising:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe and a second specific capture probe for capturing detection sequences of 9 high-risk HPV types, and 9 primers for specifically binding to target sequences of 9 high-risk HPV types, respectively, wherein the 9 high-risk HPV types comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer for specifically binding to target sequences of HPV 16, a sixth primer for specifically binding to target sequences of HPV 31, a seventh primer for specifically binding to target sequences of HPV 33, an eighth primer for specifically binding to target sequences of HPV 35, a ninth primer for specifically binding to target sequences of HPV 51, an eleventh primer for specifically binding to target sequences of HPV 52, an eleventh primer for specifically binding to target sequences of HPV 58, a twelfth primer for specifically binding to sequences of HPV 56 and HPV 66, and a universal primer;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe and a fourth specific capture probe for capturing detection sequences of 5 high-risk HPV types, and 4 primers for specifically binding to target sequences of 5 high-risk HPV types, respectively, wherein the 5 high-risk HPV types comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer for specifically binding to target sequences of HPV 18, an eighteenth primer for specifically binding to target sequences of HPV 45, a nineteenth primer for specifically binding to target sequences of HPV 59, a twentieth primer for specifically binding to target sequences of HPV 39 and HPV 68, and one of the universal primers;
(T3) amplification assay 1.1: it comprises a first primer, a second primer, a third primer and a fourth primer, wherein the first primer is matched with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is matched with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is matched with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is matched with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35 and 51 respectively;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 51, a second target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 16, 31, 35, a third target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 33, 52, 58, and a fourth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 56, 66;
(T5) amplification assay solution 2.1: comprising a thirteenth primer, a fourteenth primer, a fifteenth primer and a sixteenth primer, wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and a universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and a universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and a universal primer for amplifying a target sequence of HPV 39 and HPV 68;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 18, a sixth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 45, a seventh target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 59, and an eighth target detection probe that specifically binds to amplified product RNA copies of target sequences of HPV 39 and HPV 68;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
wherein:
the first specific capture probe to the fourth specific capture probe respectively comprise nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
the first primer to the twentieth primer respectively comprise nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 20;
the universal primer comprises a nucleotide sequence shown as SEQ ID NO. 21; and the sequences of the 5' end of the fifth primer to the twelfth primer and the seventeenth primer to the twentieth primer are the sequences of the universal primers;
The first target detection probe to the eighth target detection probe respectively comprise nucleotide sequences shown as SEQ ID NO. 22-SEQ ID NO. 29.
2. The kit for isothermal amplification detection of real-time fluorescent nucleic acids according to claim 1, wherein fluorescent reporter groups and quenching groups are respectively carried at both ends of the nucleotide sequences of the first target detection probe to the eighth target detection probe, and the fluorescent reporter groups carried by the eighth target detection probe are the same or different.
3. The real-time fluorescent nucleic acid isothermal amplification detection kit according to claim 1 or 2, further comprising:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: a system for E6/E7 in vitro transcription of RNA comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68; and/or
(M4) negative control: a system that does not contain human papillomavirus target nucleic acid.
4. The kit for real-time fluorescent nucleic acid isothermal amplification detection according to any one of claims 1-3,
the nucleic acid extraction solution 1 comprises the following components: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μΜ of the first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the fifth to twelfth primers, 25-150pmol/mL of the universal primers;
The nucleic acid extraction solution 2 comprises the following components: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μΜ of the third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the seventeenth to twentieth primers, 25-150pmol/mL of the universal primers;
the components of the amplification detection liquid 1.1 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the first primer to the fourth primer;
the components of the amplification detection liquid 1.2 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the first target detection probe to fourth target detection probe;
the components of the amplification detection liquid 2.1 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the thirteenth primer to sixteenth primer;
the components of the amplification detection liquid 2.2 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the fifth target detection probe through eighth target detection probe;
The SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
5. An oligonucleotide combination for detecting 14 high-risk HPV types and suitable for use in a real-time fluorescent nucleic acid isothermal amplification detection system, comprising:
(i) A specific capture probe combination comprising: first to fourth specific capture probes respectively comprising the nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
(ii) A primer combination comprising: first to twentieth primers comprising the nucleotide sequences shown as SEQ ID NO. 1 to SEQ ID NO. 20, respectively;
(iii) A universal primer comprising the nucleotide sequence set forth in SEQ ID NO. 21;
(iv) A target detection probe combination comprising: first to eighth target detection probes respectively comprising nucleotide sequences as shown in SEQ ID NO. 22-SEQ ID NO. 29.
6. The oligonucleotide combination according to claim 5, wherein a fluorescent reporter group and a quencher group are carried at both ends of the nucleotide sequence of the first target detection probe to the eighth target detection probe, respectively, and the fluorescent reporter groups carried by the eighth target detection probe are the same or different.
7. A non-disease diagnostic method for detecting 14 high-risk HPV types, the 14 high-risk HPV types comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, the method comprising the steps of:
1) Sub-packaging a sample to be tested into a tube 1 and a tube 2, adding a nucleic acid extracting solution 1 into the tube 1 for nucleic acid extraction, and adding a nucleic acid extracting solution 2 into the tube 2 for nucleic acid extraction to obtain analysis detection samples 1 and 2 respectively;
2) Adding an amplification detection liquid 1.1 into the analysis detection sample 1 to perform a first-step reaction to obtain a first-step reaction liquid 1, and adding an amplification detection liquid 2.1 into the analysis detection sample 2 to perform a first-step reaction to obtain a first-step reaction liquid 2;
3) Adding SAT enzyme solution into the first-step reaction solution 1 to perform a second-step reaction to obtain a second-step reaction solution 1, and adding SAT enzyme solution into the first-step reaction solution 2 to perform a second-step reaction to obtain a second-step reaction solution 2;
4) Adding an amplification detection liquid 1.2 into the second-step reaction liquid 1 for a third-step reaction to obtain an amplification system 1, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 1, and adding an amplification detection liquid 2.2 into the second-step reaction liquid 2 for a third-step reaction to obtain an amplification system 2, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 2;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if the channel corresponding to the amplification system 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected of the tube 1 contains HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested of the tube 1 does not contain HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids;
if an S-shaped amplification curve appears in a channel corresponding to the amplification system 2 and dt is less than or equal to 35, the sample to be detected of the tube 2 contains HPV18, 35, 45, 59 and 68 genotype nucleic acids; if the "S" type amplification curve does not appear, or the "S" type amplification curve appears, but dt >35, then HPV18, 35, 45, 59, 68 genotype nucleic acids are not contained in the sample to be tested of tube 2.
8. The method according to claim 7, wherein the conditions of the first reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15min.
9. The method according to claim 7 or 8, wherein the SAT enzyme solution in step 3) is preheated before use at a temperature of 41-43 ℃; and/or
The condition of the second step reaction in the step 3) is 41-43 ℃ for 30-50min.
10. The method according to any of claims 7-9, wherein the sample to be tested comprises medical samples and non-medical samples of sources including food, blood products, dairy products.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311031974.1A CN116855642A (en) | 2023-08-16 | 2023-08-16 | Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311031974.1A CN116855642A (en) | 2023-08-16 | 2023-08-16 | Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116855642A true CN116855642A (en) | 2023-10-10 |
Family
ID=88227136
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311031974.1A Pending CN116855642A (en) | 2023-08-16 | 2023-08-16 | Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116855642A (en) |
-
2023
- 2023-08-16 CN CN202311031974.1A patent/CN116855642A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108060269B (en) | DPO primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus and application thereof | |
CN111560482A (en) | Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit | |
CN109487008B (en) | Multiplex PCR detection kit for respiratory pathogens, application and use method thereof | |
CN110273027B (en) | Nucleic acid typing detection kit and detection method for norovirus GII, GII and GIV | |
CN108220480B (en) | RPA fluorescent quantitative primer pair, probe and kit for specific detection of HPV18 | |
CN110885906A (en) | Compositions and methods for detecting human papillomavirus nucleic acids | |
CN111926114A (en) | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application | |
CN108070636A (en) | A kind of processing method and kit of fluorescent PCR amplified sample | |
WO2013189306A1 (en) | Methods and compositions for assessing copy number of target polynecleotides | |
CN110607398B (en) | RT-LAMP kit for fluorescent visual rapid detection of porcine epidemic diarrhea virus | |
CA2501030C (en) | Method and kit for quantitative and qualitative determination of human papillomavirus | |
JPWO2020149301A1 (en) | Preparation method of liquid biopsy using PVA sponge | |
WO2006132601A1 (en) | Diagnostic primers and method for detecting avian influenza virus subtype h5 and h5n1 | |
KR101912488B1 (en) | Molecular detection assay | |
CN113502341B (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for treponema pallidum 16s RNA and special primer and probe thereof | |
CN106929601B (en) | High-sensitivity human papilloma virus 6,11 type nucleic acid detection kit | |
Wu et al. | Detection of five types of HPV genotypes causing Anogenital warts (Condyloma Acuminatum) using PCR-Tm analysis technology | |
CN116855642A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs | |
CN109355362B (en) | High-sensitivity SNPs detection system and application | |
CN111378724A (en) | RNA amplification detection method and detection kit | |
CN111206117A (en) | Kit for detecting human immunodeficiency virus | |
CN106676163B (en) | Primer and method for detecting pichia pastoris cell DNA | |
JP6318239B2 (en) | Method for extracting fungal nucleic acid | |
CN116144843A (en) | Real-time fluorescent isothermal amplification detection kit for HPV16 and/or HPV18 genotypes and special primer and probe thereof | |
RU2795016C1 (en) | OLIGONUCLEOTIDES FOR DETECTION OF MUTATION S:delVYY143-145 OF SARS-CoV-2 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |