CN111676326B - Human papilloma virus type 6 and/or 11 detection markers and detection reagents - Google Patents
Human papilloma virus type 6 and/or 11 detection markers and detection reagents Download PDFInfo
- Publication number
- CN111676326B CN111676326B CN202010645815.0A CN202010645815A CN111676326B CN 111676326 B CN111676326 B CN 111676326B CN 202010645815 A CN202010645815 A CN 202010645815A CN 111676326 B CN111676326 B CN 111676326B
- Authority
- CN
- China
- Prior art keywords
- seq
- probe
- detection
- primer
- papilloma virus
- 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.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 241000701828 Human papillomavirus type 11 Species 0.000 title claims abstract description 22
- 241001428582 Human papillomavirus type 6 Species 0.000 title claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 15
- 239000000523 sample Substances 0.000 claims abstract description 56
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 108020004414 DNA Proteins 0.000 claims description 16
- 239000012295 chemical reaction liquid Substances 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000011535 reaction buffer Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 6
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical group COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 108091005904 Hemoglobin subunit beta Proteins 0.000 claims description 2
- 102100021519 Hemoglobin subunit beta Human genes 0.000 claims description 2
- 241000701806 Human papillomavirus Species 0.000 abstract description 10
- 239000003550 marker Substances 0.000 abstract description 7
- 241000700605 Viruses Species 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 241000722343 Human papillomavirus types Species 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 206010059313 Anogenital warts Diseases 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000003908 quality control method Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 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 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 208000031504 Asymptomatic Infections Diseases 0.000 description 2
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 2
- 108700005307 Human papillomavirus HPV L1 Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 208000000260 Warts Diseases 0.000 description 2
- 238000001792 White test Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 210000005000 reproductive tract Anatomy 0.000 description 2
- 201000010153 skin papilloma Diseases 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010021079 Hypopnoea Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000009608 Papillomavirus Infections Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 208000019802 Sexually transmitted disease Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 238000002573 colposcopy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 208000001786 gonorrhea Diseases 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 208000003154 papilloma Diseases 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 208000012201 sexual and gender identity disease Diseases 0.000 description 1
- 208000015891 sexual disease Diseases 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000002477 vacuolizing effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- 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
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)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of detection reagents, in particular to a detection marker and a detection reagent for human papilloma virus type 6 and/or 11. The invention provides an application of BamHI (human papilloma virus) L1 region BamHI (human papilloma virus) sequence as a marker in preparing a detection reagent, and provides detection primers and probes for human papilloma virus types 6 and 11 and a kit. The primer probe has good sensitivity and specificity, does not interfere with each other when detecting two types of HPV, and can interfere with the results of avoiding other types of HPV viruses. And the reagent composition in the kit is reasonable, and the good stability of the reaction solution can be maintained.
Description
Technical Field
The invention relates to the technical field of detection reagents, in particular to a detection marker and a detection reagent for human papilloma virus type 6 and/or 11.
Background
Human papilloma virus (human papillomavirus, HPV) is a virus that has a high affinity for human skin and mucosal tissue cells, and can widely infect human skin, genital tract and respiratory epithelial tissue, and is closely related to various benign and malignant tumors in humans. Among the HPV types found, there are about 30 that can infect the genital tract. Low-risk HPV types (e.g., types 6, 11, 42, 43, 44) and high-risk HPV types (e.g., types 16, 18, 31, 33, 39, 45, 51, 52, 58, 59, 68) are classified according to their pathogenicity differences. Low-risk HPV mainly causes condyloma acuminatum (condylomata acuminate, CA), which is mainly manifested by skin lesions (e.g., warts at different sites), and can seriously cause precancerous lesions or tumors. The main infection group of the disease is young and middle-aged in the sexual activity period, and the incidence of condyloma acuminatum can be increased due to sexual disorder, more sexual partners, frequent smoking, long-term use of contraceptive or immunosuppressant, and the like. In addition, HPV infection has no strict age restriction, and the infection probability of age group population has no obvious difference. The most important transmission way of condyloma acuminatum is direct contact, the incubation period is 1-8 months, the average period is about 3 months, and the disease condition is not affected by seasons. About 90% of CA patients are infected with HPV types 6 and 11. Epidemiological data indicate that condyloma acuminatum has a minimum incidence of 9/10 ten thousand in spanish and 45/10 ten thousand in mexico. In the united states, the current figures indicate that approximately 1% of adults with frequent sexual activity have condyloma acuminatum, while at least 15% have subclinical infections. Condyloma acuminata is one of the main sexually transmitted diseases in China at present, is the second highest-incidence disease (second to gonorrhea), has 17.55/10 ten thousand people in 2000 nationwide, and is very easy to relapse. Has become one of the common venereal diseases which are harmful to human health.
The current detection method of human papilloma viruses 6 and 11 comprises the following steps: pathological examination, i.e. visual observation of histopathological changes, the epidermis shows papilloma-like hyperplasia and the acantha layer is hypertrophic. There is mild hyperkeratosis and hypopnea on the surface. The vacuolated cells are visible in the acantha cells and the granular layers, the cell bodies are larger, a round deeply-dyed nucleus is formed, perinuclear vacuolation and light dyeing are realized, and filaments are connected between the nuclear membrane and the serosa, so that the cells are in a cat eye shape. The acetic acid white test, soaking gauze with 3% -5% acetic acid solution to wrap or apply on suspicious skin or mucosa surface, tearing off after 3-5 minutes, typical condyloma acuminatum damage will appear as white pimple or wart, while subclinical infection appears as white patch or spot; colposcopy is mainly used for observing mucous membrane of cervical and vaginal part, and performing acetic acid white test.
The PCR reaction is utilized to detect the human papilloma virus, so that the detection efficiency can be improved, and the detection flow can be simplified. However, PCR detection has high requirements for specificity of detection markers and detection reagents.
Disclosure of Invention
In view of the above, the present invention aims to provide a detection marker and a detection reagent for human papilloma virus type 6 and/or type 11, so that the detection of human papilloma virus type 6 and/or type 11 has higher sensitivity and accuracy.
The invention provides an application of BamHI W sequence of human papillomavirus L1 region as a marker in preparing detection reagent.
Experiments show that the BamHI W sequence is used as a detection marker, so that various types of human papillomaviruses can be distinguished specifically.
In the present invention, the human papillomavirus is human papillomavirus type 6 and/or human papillomavirus type 11.
The invention also provides a primer and probe combination for detecting human papillomavirus type 11, which comprises the following steps: two primers of the nucleotide sequence shown in SEQ ID NO. 1-2, and a probe of the nucleotide sequence shown in SEQ ID NO. 7.
The invention also provides a primer and probe combination for detecting human papillomavirus type 6, which comprises the following steps: two primers of the nucleotide sequence shown in SEQ ID NO. 3-4, and a probe of the nucleotide sequence shown in SEQ ID NO. 8.
In the invention, different fluorescent labels are respectively used for the probe with the nucleotide sequence shown in SEQ ID NO.7 and the probe with the nucleotide sequence shown in SEQ ID NO.8, so that HPV6/11 type can be typed while being detected;
the invention relates to a primer and probe combination for detecting human papilloma virus type 6, and application of the primer and probe combination for detecting human papilloma virus type 11 in preparing a human papilloma virus type 6 and human papilloma virus type 11 co-detection kit.
The invention also provides a kit for co-detecting human papillomavirus type 6 and human papillomavirus type 11, which comprises the primer and probe combination for detecting human papillomavirus type 6 and the primer and probe combination for detecting human papillomavirus type 11.
The kit also comprises a detection primer and a probe of an internal reference, wherein the internal reference is beta-globin; the nucleotide sequence of the primer is shown in SEQ ID NO. 5-6; the nucleotide sequence of the probe is shown as SEQ ID NO. 9.
Specifically, the kit comprises a PCR reaction liquid 1 and a PCR reaction liquid 2;
the PCR reaction liquid 1 comprises: 0.1M Tris-HCl, 10 XPCR reaction buffer, 1 pmol/. Mu. L, taq polymerase 5-10U/. Mu. L, UNG enzyme 0.1-0.5U/. Mu.L of 3 probes of the nucleotide sequence shown in SEQ ID NO. 7-9 and dNTP20mM each;
the PCR reaction liquid 2 comprises water and: PCR reaction buffer solution and 6 primers of nucleotide sequences shown in SEQ ID No. 1-6 are 4 pmol/. Mu.L respectively, 20-30 mM MgCl 2 And 0.09wt% sodium azide.
In some embodiments of the present invention, in some embodiments,
the PCR reaction liquid 1 comprises: 0.1M Tris-HCl, 10 XPCR reaction buffer, 1 pmol/. Mu. L, taq polymerase 10U/. Mu. L, UNG enzyme 0.1U/. Mu.L each of 3 probes of the nucleotide sequences shown in SEQ ID NOS.7-9 and dNTP20mM;
the PCR reaction liquid 2 comprises water and: PCR reaction buffer and 6 primers of the nucleotide sequences shown in SEQ ID No.1 to 6 each 4 pmol/. Mu.L, 25mM MgCl 2 And 0.09wt% sodium azide.
In the kit provided by the invention, the reaction liquid 1 and the reaction liquid 2 have stable properties, can be stored for 12 months at the temperature of 2-8 ℃, and avoid the severe condition that other kits are required to be stored for 12 months at the temperature of-20 ℃. Experiments show that the kit provided by the invention can still realize a good detection effect after accelerated investigation for 28 days at 37 ℃.
The 5 '-end of the probe of the nucleic acid sequence shown in SEQ ID NO.7 is modified with a FAM group, and the 3' -end is modified with a BHQ1 group;
a ROX group is modified at the 5 'end of the probe of the nucleic acid sequence shown in SEQ ID NO.8, and a BHQ2 group is modified at the 3' end;
the 5 '-end of the probe of the nucleic acid sequence shown in SEQ ID NO.9 is modified with a CY5 group, and the 3' -end is modified with a BHQ3 group;
the invention also provides a method for detecting human papilloma virus type 6 and human papilloma virus type 11 for non-diagnostic purposes, which uses the kit of the invention to amplify DNA of a sample to be detected.
The amplified system comprises: 120 mu l of PCR reaction liquid, 210 mu l of PCR reaction liquid and 20-50 mu l of DNA of a sample to be detected.
The amplification procedure includes: 50℃2min at 94℃2min, (94℃5s at 55℃30 s). Times.40 cycles.
The amplification result judgment includes: the negative quality control product should have CY5 with Ct value less than or equal to 40 and FAM with No Ct; the Ct value of the positive quality control product is between 20 and 30.
The invention provides an application of BamHI (human papilloma virus) L1 region BamHI (human papilloma virus) sequence as a marker in preparing a detection reagent, and provides detection primers and probes for human papilloma virus types 6 and 11 and a kit. The primer probe has good sensitivity and specificity, does not interfere with each other when detecting two types of HPV, and can interfere with the results of avoiding other types of HPV viruses. And the reagent composition in the kit is reasonable, and the good stability of the reaction solution can be maintained.
Drawings
FIG. 1 shows HPV type 11 detection limit assay;
FIG. 2 shows HPV type 6 detection limit assay.
Detailed Description
The present invention provides human papillomavirus type 6 and/or 11 detection reagents, and one skilled in the art can, given the benefit of this disclosure, suitably modify the process parameters to achieve. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
Preparation of a polymerase chain reaction system: 20ul of reaction liquid 1, 10ul of reaction liquid 2, 600ul of negative property control and 600ul of positive property control.
(1) Reaction liquid 1: the base solution was pH8.00.1M Tris-HCl,20ul, containing 1 pmol/. Mu.l probe 1, 1 pmol/. Mu.l probe 2, 1 pmol/. Mu.l probe 3, 10UTaq enzyme, 0.1 UUUNG and 10 XPCR reaction buffer, the three probes being:
probe 1: FAM-AAGGTTGACCCCTGCCTA-BHQ1 (SEQ ID NO. 7);
probe 2: ROX-ATGCCCATACCAAACGT-BHQ2 (SEQ ID NO. 8);
probe 3: CY5-CTTGAGGTTGTCCAGGTGAGCCAG-BHQ3 (SEQ ID NO. 9).
(2) Reaction liquid 2: the base liquid is H 2 O,10ul, containing 25mM MgCl 2 0.09% sodium azide, 4 pmol/. Mu.l primer 1, 4 pmol/. Mu.l primer 2, 4 pmol/. Mu.l primer 3, 4 pmol/. Mu.l primer 4, 4 pmol/. Mu.l primer 5, 4 pmol/. Mu.l primer 6, six primers:
primer 1:5'-CACAGCGTTTAGTATGGGCG-3' (SEQ ID No. 1);
primer 2:5'-AGCAATGGATGCCCACTAACA-3' (SEQ ID No. 2);
primer 3:5'-TTGCATTGCCTGACTCGTCT-3' (SEQ ID No. 3);
primer 4:5'-ACACCCACACCTAATGGCTG-3' (SEQ ID No. 4);
primer 5:5'-GAAGGCTCATGGCAAGAAAG-3' (SEQ ID No. 5);
primer 6:5'-CTCACTCAGTGTGGCAAAGG-3' (SEQ ID No. 6).
(3) Negative quality control: the base fluid is TE,600ul, containing human beta globin gene plasmid;
(4) Controlling the nature of yang: the base solution is TE,600ul, contains HPV6 type DNA fragment plasmid with tracing source of 5E+7Copies/ml, HPV11 type DNA fragment plasmid with tracing source of 1.75E+7Copies/ml, human beta globin gene plasmid with tracing source of 8.0E+05Copies/ml;
the human papillomavirus type 6 and type 11 were qualitatively detected using the above reaction system as follows. The sample to be tested in this embodiment is a swab sample containing human papilloma virus type 6 or type 11, but may be any other sample type that may contain human papilloma virus type 6 or type 11.
1. Template DNA extraction:
template DNA was extracted using a commercial extraction kit for use in a subsequent PCR reaction.
1) Adding 100 mu L of magnetic bead suspension into the pretreated sample to be detected and the positive quality control product and the negative quality control product
Magnetic bead suspension: the base liquid is H 2 O, comprising ultrapure magnetic silica nano magnetic beads with the diameter of 0.5-1 nm;
2) Adding 1.2mL of lysate to a sample to be detected, and performing room temperature pyrolysis for 3min;
lysate: the base liquid is H 2 O, comprising 3-5M guanidine isothiocyanate and 0.5% Tween-20
3) Using a magnetic plate to magnetically treat a sample to be detected for 30s, and removing supernatant;
4) Adding 2ml of washing liquid A into the centrifuge tube, and blowing and uniformly mixing;
washing liquid A: the base liquid is H 2 O, comprising 1-3M sodium perchlorate, 0.1-0.5M potassium acetate and 25% ethanol
5) Using a magnetic plate to magnetically treat a sample to be detected for 30s, and removing supernatant;
6) Adding 2ml of washing liquid B into the centrifuge tube, and blowing and uniformly mixing;
washing liquid B: the base liquid is H 2 O, which contains 70-85% ethanol
7) Using a magnetic plate to magnetically treat a sample to be detected for 30s, and removing supernatant;
8) Adding 0.1ml of eluent into the centrifuge tube, blowing and uniformly mixing;
eluent: pH8.0 TE
9) Dissociation at 80 ℃ is carried out, and then supernatant fluid is taken out by magnetism for standby.
2. Polymerase chain reaction: the reaction system was prepared in a 100. Mu.l PCR tube: 120. Mu.l of the PCR reaction solution, 210. Mu.l of the PCR reaction solution and 20 to 50. Mu.l of the template DNA were reacted under the conditions of 50℃2min,94℃2min, (94℃5s,55℃30 s). Times.40 cycles of amplification.
3. And (3) judging results: the negative quality control product should have CY5 with Ct value less than or equal to 40 and FAM with No Ct; the Ct value of the positive quality control should be between 20 and 30. Under the above conditions, the results of the sample to be measured are explained as follows:
TABLE 1
The 20 lowest limit samples were tested as described above, the resulting amplification curves are shown in FIGS. 1 and 2, and the data are shown in Table 2.
TABLE 2
| 11 lowest detection limit sample number | FAMCt | 6 lowest detection limit sample number | ROXCt |
| 1 | 34.24 | 1 | 34.9 |
| 2 | 34.76 | 2 | 34.23 |
| 3 | 34.8 | 3 | 34.44 |
| 4 | 33.92 | 4 | 34.48 |
| 5 | 34.53 | 5 | 34.21 |
| 6 | 32.52 | 6 | 34.09 |
| 7 | 34.16 | 7 | 34.42 |
| 8 | 34.3 | 8 | 34.7 |
| 9 | 34.44 | 9 | 33.95 |
| 10 | 34.37 | 10 | 34.56 |
| 11 | 34.28 | 11 | 33.16 |
| 12 | 34.53 | 12 | 34.33 |
| 13 | 34.57 | 13 | 34.39 |
| 14 | 34.35 | 14 | 35.44 |
| 15 | 34.7 | 15 | 34.88 |
| 16 | 34.17 | 16 | 34.45 |
| 17 | 33.94 | 17 | 34.79 |
| 18 | 34.07 | 18 | 35.27 |
| 19 | 33.87 | 19 | 34.23 |
| 20 | 34.12 | 20 | 33.54 |
Conclusion: the kit detects 20 cases of positive clinical samples of 200copies/mL, and the detection limit of the kit can reach 200copies/mL.
Specific detection
Cross verification shows that the kit provided by the invention can not detect HPV6 type and HPV11 type in the 'human papillomavirus L1 genotyping reference product'.
TABLE 3 Table 3
Repeatability detection
Verifying total inaccuracy, setting 2 concentration median and low value samples of HPV6 type and HPV11 type respectively, making 2 times daily 2 compound holes each time continuously for 20 days to obtain 40 data, calculating respective total CV, and requiring CV% to be less than or equal to 5%
TABLE 4 Table 4
The mean calculation and analysis results are shown in Table 5:
TABLE 5
| Mean value of | SD | CV | |
| 6 low value | 31.27 | 1.42 | 0.045 |
| Median value of 6 | 24.94 | 0.42 | 0.017 |
| 11 low value | 28.99 | 0.41 | 0.014 |
| Median value of 11 | 26.12 | 0.46 | 0.018 |
Acceleration stability
The kit is placed at 37 ℃ to accelerate the placement stability, accelerate for 28 days, check 10 samples of HPV6 type and HPV11 type weak positive precision, 1 sample of detection limit, 5 samples of positive samples, 10 negative samples and stable check data. The results are shown in Table 6
TABLE 6
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Zhengzhou Anji bioengineering Co., ltd
<120> human papillomavirus type 6 and/or 11 detection markers and detection reagents
<130> MP2012976
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
agcaatggat gcccactaac a 21
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
aaggttgacc cctgccta 18
<210> 8
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgcccatac caaacgt 17
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cttgaggttg tccaggtgag ccag 24
Claims (5)
1. A kit for co-detecting human papilloma virus type 6 and human papilloma virus type 11, characterized in that,
comprises a primer and probe combination for detecting human papillomavirus type 6, and a primer and probe combination for detecting human papillomavirus type 11;
a primer and probe combination for detecting human papillomavirus type 11, comprising: two primers of the nucleotide sequence shown in SEQ ID NO. 1-2, and a probe of the nucleotide sequence shown in SEQ ID NO. 7;
a primer and probe combination for detecting human papillomavirus type 6, comprising: two primers of the nucleotide sequence shown in SEQ ID NO. 3-4, and a probe of the nucleotide sequence shown in SEQ ID NO. 8.
2. The kit of claim 1, further comprising a detection primer and probe for an internal reference, the internal reference being β -globin; the nucleotide sequence of the primer is shown in SEQ ID NO. 5-6; the nucleotide sequence of the probe is shown as SEQ ID NO. 9.
3. The kit according to claim 1 or 2, comprising a PCR reaction solution 1 and a PCR reaction solution 2;
the PCR reaction liquid 1 comprises: 0.1MTris-HCl, 10 XPCR reaction buffer, 1 pmol/. Mu. L, taq polymerase 5-10U/. Mu. L, UNG enzyme 0.1-0.5U/. Mu.L of 3 probes of the nucleotide sequence shown in SEQ ID NO. 7-9 and dNTP20mM each;
the PCR reaction liquid 2 comprises water and: PCR reaction buffer solution and 6 primers of nucleotide sequences shown in SEQ ID No. 1-6, 4 pmol/. Mu.L, 20-30 mM MgCl respectively 2 And 0.09wt% sodium azide.
4. The kit according to claim 3, wherein,
the 5 '-end of the probe of the nucleic acid sequence shown in SEQ ID NO.7 is modified with a FAM group, and the 3' -end is modified with a BHQ1 group;
a ROX group is modified at the 5 'end of the probe of the nucleic acid sequence shown in SEQ ID NO.8, and a BHQ2 group is modified at the 3' end;
and the 5 '-end of the probe of the nucleic acid sequence shown in SEQ ID NO.9 is modified with a CY5 group, and the 3' -end of the probe is modified with a BHQ3 group.
5. A method for detecting human papillomavirus type 6 and human papillomavirus type 11 for non-diagnostic purposes, characterized in that the DNA of a sample to be detected is amplified with a kit according to any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010645815.0A CN111676326B (en) | 2020-07-07 | 2020-07-07 | Human papilloma virus type 6 and/or 11 detection markers and detection reagents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010645815.0A CN111676326B (en) | 2020-07-07 | 2020-07-07 | Human papilloma virus type 6 and/or 11 detection markers and detection reagents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111676326A CN111676326A (en) | 2020-09-18 |
| CN111676326B true CN111676326B (en) | 2023-05-23 |
Family
ID=72438114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010645815.0A Active CN111676326B (en) | 2020-07-07 | 2020-07-07 | Human papilloma virus type 6 and/or 11 detection markers and detection reagents |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111676326B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584229B (en) * | 2021-08-10 | 2023-08-22 | 中国疾病预防控制中心病毒病预防控制所 | Primer probe combination, kit and application for isothermal nucleic acid amplification detection of human papilloma virus type 6 and/or type 11 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5447839A (en) * | 1988-09-09 | 1995-09-05 | Hoffmann-La Roche Inc. | Detection of human papillomavirus by the polymerase chain reaction |
| WO2008074182A1 (en) * | 2006-12-18 | 2008-06-26 | Shanghai Tellgen Life Science Co., Ltd. | Method for the detection and typing of human papillomavirus, and the reagent kit thereof |
| CN101676408A (en) * | 2008-09-19 | 2010-03-24 | 扬子江药业集团北京海燕药业有限公司 | 6,11 type human papillomavirus fluorescence quantitative PCR detection method and kit thereof |
| CN105506173A (en) * | 2014-09-25 | 2016-04-20 | 苏州新波生物技术有限公司 | Nucleic acid detection kit for human papilloma virus, use method and application thereof |
| CN106929601A (en) * | 2015-12-30 | 2017-07-07 | 上海星耀医学科技发展有限公司 | A kind of type kit for detecting nucleic acid of highly sensitive HPV 6,11 |
| CN109234455A (en) * | 2018-10-10 | 2019-01-18 | 上海裕隆神光医学检验实验室有限公司 | The DNA typing detection kit of human papilloma virus |
-
2020
- 2020-07-07 CN CN202010645815.0A patent/CN111676326B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5447839A (en) * | 1988-09-09 | 1995-09-05 | Hoffmann-La Roche Inc. | Detection of human papillomavirus by the polymerase chain reaction |
| WO2008074182A1 (en) * | 2006-12-18 | 2008-06-26 | Shanghai Tellgen Life Science Co., Ltd. | Method for the detection and typing of human papillomavirus, and the reagent kit thereof |
| CN101676408A (en) * | 2008-09-19 | 2010-03-24 | 扬子江药业集团北京海燕药业有限公司 | 6,11 type human papillomavirus fluorescence quantitative PCR detection method and kit thereof |
| CN105506173A (en) * | 2014-09-25 | 2016-04-20 | 苏州新波生物技术有限公司 | Nucleic acid detection kit for human papilloma virus, use method and application thereof |
| CN106929601A (en) * | 2015-12-30 | 2017-07-07 | 上海星耀医学科技发展有限公司 | A kind of type kit for detecting nucleic acid of highly sensitive HPV 6,11 |
| CN109234455A (en) * | 2018-10-10 | 2019-01-18 | 上海裕隆神光医学检验实验室有限公司 | The DNA typing detection kit of human papilloma virus |
Non-Patent Citations (2)
| Title |
|---|
| 庄敏等.尖锐湿疣组织中HPV型别检测及HPV11型L1基因的克隆与测序.《中国皮肤性病学杂志》.2005,第19卷(第9期),第519-521页. * |
| 欧琴等.尖锐湿疣组织HPV6b 和11 型L1基因多态性及蛋白特性分析.《温州医学院学报》.2008,第38卷(第5期),第405-408页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111676326A (en) | 2020-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zambrano et al. | Detection of human polyomaviruses and papillomaviruses in prostatic tissue reveals the prostate as a habitat for multiple viral infections | |
| WO2022179494A1 (en) | Salmonella typhi detection kit, preparation method therefor and application thereof | |
| CN105755169B (en) | Detection and typing kit for high-risk human papilloma virus and application thereof | |
| CN112538550B (en) | RT-RPA and CRISPR/Cas-based DHAV-1 and DHAV-3 detection system and application | |
| CN101781686A (en) | Fluorescent PCR (Polymerase Chain Reaction) kit for quantitively detecting HPV16/18 type infection | |
| CN111676326B (en) | Human papilloma virus type 6 and/or 11 detection markers and detection reagents | |
| Syrjänen et al. | Colposcopy, punch biopsy, in situ DNA hybridization, and the polymerase chain reaction in searching for genital human papillomavirus (HPV) infections in women with normal PAP smears | |
| CN114214415A (en) | Primer probe combination for methylation detection of cervical cancer related genes and application thereof | |
| CN112391495A (en) | High-risk human papilloma virus typing detection method and kit | |
| CN108179226B (en) | Nucleic acid composition for detecting human papilloma virus, application thereof and kit | |
| CN114457174A (en) | Multiplex fluorescence quantitative probe method PCR kit for detecting urinary tract pathogen infection | |
| CN101812538B (en) | Enterovirus 71-detecting fluorescent quantitative RT-PCR kit | |
| CN111471800B (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| CN116411141A (en) | RPA-CRISPR/Cas12 a-based visualized detection kit for porcine group A rotavirus and application | |
| CN115747379A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for herpes simplex virus types 1 and 2 and special primer and probe thereof | |
| CN112195276B (en) | Kit and method for simultaneously detecting herpes simplex virus, kaposi sarcoma-associated herpes virus, JC virus and EB virus | |
| CN110387436A (en) | Human papilloma virus primer and molecular beacon combination, kit and detection method based on constant-temperature amplification | |
| TWI816964B (en) | Compositions and methods for detecting human papillomavirus | |
| CN107828920B (en) | Kit for realizing typing of multiple high-risk human papilloma viruses | |
| CN112795677A (en) | Kit for identifying skin leishmania species | |
| RU2532344C2 (en) | Diagnostic technique for hyperproliferative conditions and method for assessing risk of cervical cancer accompanying cervical intraepithelial neoplasia and papilloma virus carrier state by determinig mrna of functional human gene complex | |
| CN112126713A (en) | Coronavirus and influenza virus combined detection product and application thereof | |
| CN113444821B (en) | Kit and method for synchronously detecting various genital tract pathogens | |
| CN112280902B (en) | Method for detecting molluscum virus by TaqMan probe fluorescent quantitative PCR and application | |
| KR102584987B1 (en) | A RPA kit for detecting coronavirus and use thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |