WO2016029867A1 - 一种高危型人乳头瘤病毒检测及分型方法 - Google Patents

一种高危型人乳头瘤病毒检测及分型方法 Download PDF

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WO2016029867A1
WO2016029867A1 PCT/CN2015/088325 CN2015088325W WO2016029867A1 WO 2016029867 A1 WO2016029867 A1 WO 2016029867A1 CN 2015088325 W CN2015088325 W CN 2015088325W WO 2016029867 A1 WO2016029867 A1 WO 2016029867A1
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sequence
rna
hpv
dna
dna sequence
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华绍炳
胡杰锋
竺旭峰
寿莹佳
李德强
魏栓林
韩斌
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杭州德同生物技术有限公司
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Priority to EP15836767.2A priority Critical patent/EP3187596B1/en
Priority to US15/506,988 priority patent/US10465254B2/en
Publication of WO2016029867A1 publication Critical patent/WO2016029867A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of biomedicine, and more particularly, to a HPV DNA typing detection technique and application based on nucleic acid hybridization chemiluminescence immunoassay.
  • HPV Human papillomavirus
  • ORFs open reading frames
  • HPV is classified into different types according to the homology of genomic nucleic acid sequences of less than 90%. In the same type, the difference of viral genomic nucleic acid sequences is regarded as different subtypes in 2-10%, while in the same subtype, viruses A genomic nucleic acid sequence difference of less than 2% is referred to as a variant of the subtype. So far, researchers have found nearly 100 HPV types.
  • the skin type includes HPV 1, 2, 3, 4, 7, 10, 12, 15 and the like associated with common warts, flat warts, warts, etc., and also includes HPV 5, 8, 14, 17 related to verrucous epidermal dysplasia. 20, 36, 38, etc.; mucosal type includes HPV6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54 etc. related to infections such as genitals, anus, oropharynx, esophageal mucosa, etc. HPV16, 18, 30, 31, 33, 35, 39, etc. related to cervical cancer, rectal cancer, oral cancer, tonsillar cancer, etc.
  • HPV can be divided into “high-risk” and "low-risk” HPV.
  • Low-risk HPV such as HPV6, 11, 42, 53 and other infections related to genitals, anus, oropharynx, esophageal mucosa, etc.
  • high-risk HPV is the main cause of cervical cancer.
  • HPV infection is the main cause of cervical cancer.
  • the persistent infection of HPV has been considered as the necessary and most important cause of cervical cancer.
  • Numerous studies have shown that most of the cervical intraepithelial neoplasia (CIN) and almost all cervical cancer lesions have high-risk HPV infection (Remmink AJ et al. 1995, The presence of persistent high-risk HPV genotypes in dysplastic Cervical lesions are associated with progressive disease: natural history up to 36months. Int J Cancer, 61(3): 306-11).
  • cervical cancer The onset of the disease generally needs to undergo continuous infection from HPV to varying degrees of CIN (CIN1-CIN3), and finally develop into cervical cancer (Bosch FX et al. 2002, The causal relation between human papillomavirus and cervical cancer. J Clin Pathol, 55 (4) :244-65). This process is generally lengthy and takes an average of 10-15 years, which provides sufficient time for early detection and blockade of cervical cancer and its precancerous lesions.
  • HPV 16 and 18 infections are of great significance in the prevention of cervical cancer.
  • HPV cannot be cultured in vitro.
  • high-risk HPV testing has been used to assess the risk of cervical cancer, in order to be diagnosed early and treated in time.
  • cervical diseases including cervical cancer
  • HPV antigen After HPV infects the human epidermis, it synthesizes a capsid protein in the cell to become an HPV antigen component. Immunoenzyme staining detects HPV antigenic protein L1 in infected tissue cells for HPV infection. However, because the HPV antigen immunohistochemistry method can only confirm the capsid protein of the nucleus, and this capsid protein only appears in a stage of the HPV life cycle (produced in the late viral particles), the degree of lesions is different from the antigen expression. At the same time, this method requires a large amount of virus particles to produce a positive reaction, so the detection rate is low (Dillner J. 1999, The serological response to papillomaviruses. Semin Cancer Biol, 9: 423-30).
  • Detection of HPV antibodies After HPV is infected, the body induces anti-HPV antibodies, so anti-HPV antibodies in serum can be detected. Detection antibodies are mainly detected against HPV16E6 and E7 antibodies, because their detection sensitivity is not high, and antibodies appear in the late stages of the disease, it is not suitable for early diagnosis of the disease. Moreover, after human infection with HPV, the antibodies produced by the body can exist for a long time, and the serological detection of HPV antibodies cannot determine whether it is a recent infection or a previous infection. At the same time, due to the large number of HPV types and inconsistent immune responses, HPV antibody detection is rarely used (Frazer IH. 2010, Measuring serum antibody to human papillomavirus following infection or vaccination. Gynecol Oncol, 118 (1 Suppl): S8-11).
  • the newly developed detection methods mainly use molecular biological methods to detect HPV, including signal amplification methods and template-based amplification methods. Based on the template amplification method, PCR is used to combine different detection technologies, including fluorescent PCR, PCR combined with membrane hybridization, PCR combined with reverse dot blot hybridization, or PCR combined with quantum dot technology (Anco Molijn et al. 2005, Molecular diagnosis of human papillomavirus (HPV) infections. J Clin Virol, 32 Suppl 1: S43-51).
  • the PCR-based template-based amplification method mainly has the following problems: (1) PCR technology is performed by amplifying a target DNA fragment, which requires a high laboratory environment and a relatively strict experimental operation level; Generally speaking, The virus detected in the PCR laboratory of the hospital is not only one kind of HPV, so in the process of amplifying the target DNA, a large number of different kinds of viral DNA amplified fragments are prone to occur, and these fragments may be leaked due to laboratory environment and operational errors.
  • a patient When a patient has multiple subtypes of infection, it may only amplify a subtype with a higher viral load, and a subtype with a smaller number of viruses will result in a false negative result due to relatively less amplification; that is, if the patient is infected If the number of high-risk subtype viruses is lower than that of low-risk subtypes, the result will be a negative result, which will lead to misdiagnosis of patients; (4)
  • the sensitivity and specificity of PCR detection methods are mainly affected by sample transport and storage conditions, the same HPV sub- The variation of the DNA sequence in the type, the loss of HPV DNA during the extraction process, the design of the primer pair, the size of the PCR product, and the PCR program design.
  • the object of the present invention is to provide a high-risk human papillomavirus detection and typing method.
  • kits for high-risk HPV virus detection comprising: for HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56 , 58, 59, 66 and 68 virus-specific RNA probes.
  • the RNA probe specific for HPV16 is an RNA sequence comprising or encoding the DNA sequence of positions 83-1558, 3193-4628, 5559-7154 in the sequence of GenBank Accession No. K02718 Complementary RNA sequence;
  • RNA probe specific for HPV18 is an RNA sequence of a DNA sequence including positions 105-1593, 2376-3867, 5430-7136 in the sequence of GenBank Accession No. X05015 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV31 is an RNA sequence comprising a DNA sequence of positions 108-1463, 3564-4982, 5558-7072 in the sequence of GenBank Accession No. HQ537666 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV33 is an RNA sequence comprising a DNA sequence of positions 109-1654, 3789-5087, 5594-7093 in the sequence of GenBank Accession No. M12732 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV35 is an RNA sequence comprising a DNA sequence of positions 110-1476, 3452-4897, 5601-7109 in the sequence of GenBank Accession No. X74477 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV39 is an RNA sequence comprising a DNA sequence of positions 107-1608, 2654-4187, 5643-7160 in the sequence of GenBank Accession No. M62849 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV45 is an RNA sequence comprising a DNA sequence of positions 102-1543, 3421-4872, 5530-7149 in the sequence of GenBank Accession No. X74479 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV51 is an RNA sequence comprising a DNA sequence of positions 68-1476, 2875-4452, 5794-7431 in the sequence of GenBank Accession No. M62877 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV52 is an RNA sequence of a DNA sequence including positions 89-1565, 2459-4098, 5565-7154 in the sequence of GenBank Accession No. X74481 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV56 is an RNA sequence of a DNA sequence including positions 102-1567, 2432-3896, 5492-7096 in the sequence of GenBank Accession No. X74483 or an RNA sequence complementary to the DNA sequence;
  • the HPV58-specific RNA probe is an RNA sequence comprising a DNA sequence of positions 110-1542, 2763-4389, 5565-7139 in the sequence of GenBank Accession No. D90400 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV59 is an RNA sequence comprising a DNA sequence of positions 55-1398, 3567-4896, 5606-7132 in the sequence of GenBank Accession No. X77858 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV66 is an RNA sequence of a DNA sequence including positions 67-1534, 2679-4231, 5647-7158 in the sequence of GenBank Accession No. U31794 or an RNA sequence complementary to the DNA sequence;
  • RNA probe specific for HPV68 is an RNA sequence comprising the DNA sequence of positions 150-1678, 2986-4476, 5508-7025 in the sequence of GenBank Accession No. FR751039 or an RNA sequence complementary to the DNA sequence.
  • each of the probes may be present separately or may be mixed as needed for detection.
  • the kit includes:
  • Probe reagent A which is an RNA probe containing HPV16, type 18 specificity
  • Probe reagent B which is an RNA probe specific for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68;
  • a capture antibody which is a specific antibody against a DNA-RNA hybrid, which is contained in a solution or immobilized on a solid support;
  • An antibody is detected which is a specific antibody against an anti-DNA-RNA hybrid linked to a detectable signal.
  • the solid phase carrier includes, but is not limited to, a multiwell plate, a microsphere, a test paper, and a slide.
  • the probe is with or without a label (e.g., a fluorescent label).
  • a label e.g., a fluorescent label
  • the kit further comprises one or more reagents selected from the group consisting of:
  • An agent that identifies a detectable signal An agent that identifies a detectable signal
  • the detectable signal is selected from, but not limited to, alkaline phosphatase, horseradish peroxidase, glucose oxidase, ⁇ -D-galactosidase, urease, catalase or Glucose amylase; the reagent for identifying a detectable signal is the alkaline phosphatase, horseradish peroxidase, glucose oxidase, ⁇ -D-galactosidase, urease, catalase or glucose starch The substrate for the enzyme.
  • the high risk HPV control comprises:
  • High-risk HPV low-value control A a solution containing 1pg/ml HPV16DNA
  • high-risk HPV low-value control B a solution containing 1pg/ml HPV58DNA
  • high-risk HPV high-value control A a solution of 5 pg/ml HPV16 DNA
  • a high-risk HPV high-value control B a solution containing 5 pg/ml HPV58 DNA
  • the low-risk HPV control is a solution containing 5 pg/ml HPV6 DNA.
  • kits for the detection of high-risk HPV viruses HPV 16, 18, 31, 33, 35, 39, 45, 51, 52 , 56, 58, 59, 66, 68 viruses.
  • the use is non-disease diagnostic use.
  • kits for the classification of high-risk HPV viruses dividing high-risk HPV viruses into HPV 16, 18-type groups, or HPV 31, 33, 35, 39, Groups 45, 51, 52, 56, 58, 59, 66, 68.
  • the use is non-disease diagnostic use.
  • a high-risk HPV virus detection or typing method comprising: detecting a sample to be tested by using the kit, thereby identifying whether a high risk is present in the sample to be tested Type HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 viruses; or
  • the kit of claim 3 is used to detect a sample to be tested, thereby classifying high-risk HPV viruses into HPV 16, 18-type groups, or HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 , 66, 68 type group.
  • the detecting or typing method comprises:
  • the hybridization solution A and the hybridization solution B are applied to the solid phase carrier A and the solid phase carrier B coated with the capture antibody, Forming a capture compound and a DNA-RNA hybrid binary complex on the solid phase carriers A and B;
  • the detectable signals on the solid phase carriers A and B are both "positive", it indicates that the sample to be tested contains HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 One or several, also containing one or two of HPV types 16, 18;
  • the method is a non-disease diagnostic method, or the test object (sample to be tested) for which the method is directed is isolated, including samples from public or laboratory samples.
  • FIG. 1 Schematic diagram of the detection principle of the method of the present invention.
  • HPV human papillomavirus
  • sample to be tested or “sample of nucleic acid (DNA) to be tested” refers to a nucleic acid sample to be detected containing one nucleic acid or a plurality of nucleic acids, and it is necessary to know whether or not a target nucleic acid is present therein.
  • target nucleic acid refers to a nucleic acid of interest, for example, which is a marker or disease associated.
  • DNA-RNA hybrid refers to a nucleic acid comprising two strands, one of which is a DNA strand and the other of which is an RNA strand, the nucleotide sequence of the DNA strand and the RNA strand It is basically complementary. “Substantially complementary” means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner to form a double chain.
  • At least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • probe refers to a single-stranded nucleic acid having a known nucleotide sequence (preferably RNA in the present invention) having a nucleotide sequence structure substantially complementary to a target nucleic acid, which can be associated with a "target”
  • the nucleic acid forms a double strand.
  • the "probe” can carry a label or not.
  • the label can be attached to the 5' or 3' end of the probe.
  • capture antibody refers to an antibody that can be coated on a solid support, specifically recognizing and binding the DNA-RNA hybrid, which does not bind to a single-stranded nucleic acid (including DNA or RNA). Wait).
  • the "capture antibody” achieves binding to a double-stranded hybrid by recognizing the double-stranded structure of the double-stranded hybrid, and is not base sequence specific. Coating the antibody on a solid support is a technique well known to those skilled in the art.
  • the term “capture antibody” can be used interchangeably with “coated antibody.”
  • detection antibody refers to an antibody that specifically recognizes and binds to the DNA-RNA hybrid, which does not bind to a single-stranded nucleic acid (including DNA or RNA, etc.).
  • the “detection antibody” achieves binding to a double-stranded hybrid by recognizing the double-stranded structure of the double-stranded hybrid, and is not base sequence-specific.
  • the “detection antibody” carries a detectable signal for reporting the capture of a double-stranded hybrid.
  • HPV viruses have a large number of subtypes and variant strains, up to hundreds, some of which are high-risk types, and some subtypes or variants are low-risk. Therefore, identifying whether the HPV virus is present in the sample to be tested and whether the HPV virus is of high risk type is critical for clinical testing, prognosis, and knowing the drug regimen.
  • identifying whether the HPV virus is present in the sample to be tested and whether the HPV virus is of high risk type is critical for clinical testing, prognosis, and knowing the drug regimen.
  • the high number of HPV virus subtypes and variants and the homology of the genome sequences between different plant types is also very high, how to accurately and conveniently (preferably in real time) the high-risk HPV has been There is a high degree of false positive and false negative in the clinical methods currently used in the field.
  • the present inventors prepared a detection kit capable of specifically detecting 14 high-risk HPV viruses, and the kit includes an RNA probe capable of specifically recognizing 14 high-risk HPV viruses.
  • the probes were obtained after extensive screening by the present inventors, were of suitable length, were easy to synthesize, had good specificity, and did not cross-react with any HPV strains other than 14 high-risk HPV types.
  • the probes are mixed, and HPV16 and 18 type specific RNA probes are mixed to form probe reagent A; HPV 31, 33, 35, 39, 45, 51, The 52, 56, 58, 59, 66, 68 type specific RNA probes are mixed to form probe reagent B.
  • HPV 16, 18-type groups or HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 type groups.
  • HPV 16 and 18 viruses that cause 70% of cervical cancer in women and other HPV viruses can be achieved, which is very useful for clinical applications.
  • the current kit is divided into 2 groups. : Type 2 (HPV 16, 18) and Type 12 (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68); or can also be divided into 3 groups: Type 1 (HPV16), Type 1 (HPV18) and Type 12 (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and the like. Or other combinations are also possible.
  • the grouping of Type 2 (HPV 16, 18) and Type 12 HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) is most preferred.
  • the probe reagent includes a certain concentration of a salt ion component, such as trisodium citrate, sodium dihydrogen phosphate, etc., in addition to a specific RNA probe, to ensure specific RNA detection.
  • a salt ion component such as trisodium citrate, sodium dihydrogen phosphate, etc.
  • the needle and single-stranded DNA hybridize according to the base pairing pairing at a concentration containing a certain salt ion.
  • the HPV RNA probe in the probe reagent may also be a carrier-carrying label, such as biotin, digoxin, tacrolimus; the corresponding capture microplate is coated with avidin. , digoxin antibody, tacrolimus binding protein; or avidin-labeled enzyme, digoxin antibody-labeled enzyme, tacrolimus-binding protein-labeled enzyme in the corresponding detection reagent.
  • a carrier-carrying label such as biotin, digoxin, tacrolimus
  • the corresponding capture microplate is coated with avidin. , digoxin antibody, tacrolimus binding protein; or avidin-labeled enzyme, digoxin antibody-labeled enzyme, tacrolimus-binding protein-labeled enzyme in the corresponding detection reagent.
  • the kit further includes a capture antibody and a detection antibody.
  • the capture antibody can be prepared using the same antibody or a different antibody as the detection antibody, that is, the detection antibody can be the same or different from the capture antibody without carrying a detectable signal.
  • the detection antibody can be the same or different from the capture antibody without carrying a detectable signal.
  • antibodies produced by anti-DNA-RNA hybrids have no specific sequence or antigenic determinant, and specifically recognize the double helix structure unique to double-stranded hybrids.
  • Anti-DNA-RNA antibodies (whether monoclonal or polyclonal) bind to any DNA-RNA double-stranded hybrid.
  • a method of preparing an antibody against the double-stranded hybrid using a specific double-stranded hybrid is a technique known in the art, for example, according to the method of Kitagawa & Stollar (Kitagawa Y, Stollar BD, Mol Immunol 1982, 19: 413-420).
  • Kitagawa & Stollar Kitagawa Y, Stollar BD, Mol Immunol 1982, 19: 413-420.
  • a polyclonal antibody or a monoclonal antibody can be prepared according to the method of Fliss et al. (Fliss I, Laurent M, Emond E, et al., Appl Environ Microbiol, 1993, 59(8): 2698-2705).
  • the solution for detecting the antibody further contains a certain concentration of NaCl, MgCl2 and Tween-20 to ensure that the detection antibody binds to the DNA-RNA hybrid in a solution containing a certain salt ion to make the non-specificity of the detection antibody. Adsorption is minimized.
  • the solid phase carrier to be employed in the present invention is not particularly limited as long as it can bind (couple, connect) to the coated antibody.
  • the solid support is selected from the group consisting of a microtiter plate (such as a 96-well plate), a slide, a test strip, or a microsphere. Techniques for coating antibodies onto a solid support are also well known to those skilled in the art.
  • the detectable signal is a reporter molecule that is ligated or coupled to a detection antibody for reporting the binding of the detection antibody.
  • the detectable signal is selected from the group consisting of: alkaline phosphatase (AP), horseradish peroxidase (HRP), glucose oxidase, ⁇ -D-galactosidase, urease, catalase or Glucose amylase.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • glucose oxidase ⁇ -D-galactosidase
  • urease catalase
  • catalase catalase
  • Glucose amylase Glucose amylase
  • the substrate is, for example, o-phenylenediamine (OPD) for horseradish peroxidase, tetramethylbenzidine (TMB), ABTS; p-nitrophenyl phosphate for alkaline phosphatase ( P-nitrophenyl phosphate, p-NPP), CDP-Star; and so on.
  • OPD o-phenylenediamine
  • TMB tetramethylbenzidine
  • ABTS p-nitrophenyl phosphate for alkaline phosphatase
  • P-nitrophenyl phosphate p-NPP
  • CDP-Star CDP-Star
  • the kit may further comprise a negative control (NC), a low-risk and high-risk HPV control (including a high-risk HPV low-value control A (HLC-A).
  • a negative control NC
  • a low-risk and high-risk HPV control including a high-risk HPV low-value control A (HLC-A).
  • High-risk HPV low-value control product B HLC-B
  • HHC-A high-risk HPV high-value control product A
  • HHC-B high-risk HPV high-value control product B
  • LC low-risk HPV Quality Control
  • the kit may also contain reagents required for nucleic acid melting, washing, color development, and the like.
  • a denaturing reagent such as an alkali treatment reagent
  • an indicator dye for color development treatment.
  • the kit further includes a sample preserving solution, wherein the main component of the sample preserving solution is a TE buffer containing a certain concentration of salt, and the nucleic acid is stably stored in the TE buffer.
  • a sample preserving solution wherein the main component of the sample preserving solution is a TE buffer containing a certain concentration of salt, and the nucleic acid is stably stored in the TE buffer.
  • the washing liquid is a buffer having a certain pH value and has an effect of reducing non-specific adsorption.
  • the detection kit of the invention has high specificity, high sensitivity, low cost, no need for DNA amplification, no special test conditions, simple and easy operation, and minimizes experimental contamination, and can realize 2 of 14 high-risk HPVs.
  • High-risk HPV (HPV16, 18) and 12 high-risk HPV (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) were type tested.
  • kits may also be included in the kit.
  • the method of the invention is based on the principle of an ELISA reaction.
  • the object captured by the antibody is specific, that is, a DNA-RNA hybrid formed by complementation of DNA and RNA, rather than a conventional protein.
  • the specific principle (Fig. 1) is: the HPV virus double-stranded DNA in the sample is decomposed into a single strand, the single-stranded DNA is combined with a specific RNA probe into a DNA-RNA hybrid, and the DNA-RNA hybrid is coated with a capture antibody.
  • the capture antibody on the solid phase carrier (such as a microplate) binds to the detection antibody coupled to the detectable signal, detects the detectable signal, qualitatively detects the HPV viral nucleic acid in the sample, and achieves typing.
  • the hybridized hybridized solution is transferred to a solid phase carrier and subjected to an oscillating reaction, since the hybridized solution contains a salt in a certain concentration of the sample preservation solution and a rapid oscillating reaction, thereby promoting coating in the microcapsule Specific high affinity monoclonal antibodies against DNA-RNA hybrids on the well plates capture as much as possible of more than 90% of the paired DNA-RNA hybrids in the hybridization solution.
  • the method of the present invention provides a technique for typing two high-risk HPV (HPV16, 18) causing 70% of cervical cancer, overcoming the high-risk HPV type in the existing detection technology. Defects of insufficient type. Other combinations are also possible depending on the requirements of the test. Of course, the grouping of Type 2 (HPV 16, 18) and Type 12 (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) is most preferred, and the inventors have found that This combination has an excellent detection effect and basically has no possibility of cross-reacting with other viruses.
  • the method of the invention does not need to adopt the PCR technology with high requirements on the laboratory environment, and overcomes the shortcomings of the PCR technology that the experimental pollution is difficult to control, the false negative is high, the competition inhibition exists, and the sample storage and the DNA extraction process are affected.
  • the preparation method is as follows:
  • HPV16 No. 83-1558, 3193-4628, 5559-7154 in the sequence of GenBank accession number K02718;
  • HPV18 GenBank accession number X05015 in the sequence of 105-1593, 2376-3867, 5430-7136;
  • HPV31 No. 108-1463, 3564-4982, 5558-7072 in the sequence of GenBank accession number HQ537666;
  • HPV33 GenBank accession number M12732 in the sequence of 109-1654, 3789-5087, 5594-7093;
  • HPV35 GenBank accession number X74477 in the sequence of 110-1476, 3452-4897, 5601-7109;
  • HPV39 No. 107-1608, 2654-4187, 5643-7160 in the sequence of GenBank accession number M62849;
  • HPV45 No. 102-1543, 3421-4872, 5530-7149 in the sequence of GenBank accession number X74479;
  • HPV51 No. 68-1476, 2875-4452, 5794-7431 in the sequence of GenBank accession number M62877;
  • HPV52 GenBank accession number X74481 in the sequence of 89-1565, 2459-4098, 5565-7154;
  • HPV56 No. 102-1567, 2432-3896, 5492-7096 in the sequence of GenBank accession number X74483;
  • HPV58 GenBank accession number D90400 in the sequence of 110-1542, 2763-4389, 5565-7139;
  • HPV59 No. 55-1398, 3567-4896, 5606-7132 in the sequence of GenBank accession number X77858;
  • HPV66 No. 67-1534, 2679-4231, 5647-7158 in the sequence of GenBank accession number U31794;
  • HPV68 No. 150-1678, 2986-4476, 5508-7025 in the sequence of GenBank accession number FR751039;
  • GCTAGC NheI site sequence
  • GCGGCCGC NotI site sequence
  • T7 is attached upstream of the above DNA sequence.
  • template DNA template DNA
  • T7 RNA polymerase T7 RNA polymerase
  • Reaction conditions for RNA preparation using T7 RNA polymerase 10 ⁇ l 5 ⁇ transcription buffer, 10 ⁇ l 10 mM 4 NTP (ATP, CTP, GTP, UTP) mixture, 1 ⁇ g template DNA, 50 U RNase inhibitor, 1.5 ⁇ l T7 RNA polymerase (20 U/ Ll), add DEPC-treated water to a total volume of 50 ⁇ l. After the above mixture was incubated at 37 ° C for 100 minutes, the reaction was terminated by adding 2 ⁇ l of 0.5 M EDTA.
  • RNA probe reagent A was a solution containing HPV16 and 18 RNA probes; in the solution, the HPV16 RNA probe concentration was 0.5 ⁇ g/ml, and the HPV18 RNA probe concentration was 0.5 ⁇ g/ml. Also included in the solution are: 0.125 M trisodium citrate, 0.125 M sodium dihydrogen phosphate.
  • probe reagent B which is an RNA probe containing HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
  • concentration of each RNA probe in the solution was 0.5 ⁇ g/ml.
  • 0.125 M trisodium citrate, 0.125 M sodium dihydrogen phosphate are also included in the solution.
  • the obtained recombinant plasmid DNA can be used as a quality control product.
  • the concentration of the HPV6 DNA plasmid is 5 pg/ml; in the high-risk HPV high-value control product, the concentration of the HPV16 DNA plasmid is 5 pg/ml, HPV58 DNA
  • the concentration of the plasmid was 5 pg/ml; in the high-risk HPV low-value control, the concentration of the HPV16 DNA plasmid was 1 pg/ml, and the concentration of the HPV58 DNA plasmid was 1 pg/ml, which were independently placed in each control tube.
  • the control solution also included: 10 mM Tris, 10 mM EDTA, pH 7.2 ⁇ 0.2.
  • the probe reagent A and the probe reagent B prepared in Example 1 were placed in a kit to obtain a detection kit 1.
  • the probe reagent A, the probe reagent B and the control prepared in Example 1 were placed in separate containers and placed in a kit. And also loaded in the kit:
  • Coated antibody ATCC #HB-8730, dissolved in carbonate buffer (pH 9.6), can be used to coat microplates.
  • Detection antibody a solution (antibody concentration: 100 ng/ml) of a highly specific antibody (ATCC #HB8730) containing an anti-DNA-RNA hybrid conjugated with alkaline phosphatase.
  • the solution also contained: 0.6 M NaCl, 0.1 mM MgCl 2 , 0.25% Tween-20.
  • a test kit 2 was obtained.
  • each reagent is separately installed in a separate container:
  • Denaturation reagent a solution containing 1.75 M sodium hydroxide.
  • Capture microplate a chemiluminescent plate coated with a specific antibody against DNA-RNA hybrids, coated with antibody (ATCC#HB-8730, dissolved in carbonate buffer (pH 9.6)) The 96-well plate was coated at 0.1 ⁇ g/ml and 100 ⁇ l/well.
  • Detection reagent a solution (antibody concentration: 100 ng/ml) of a highly specific antibody (ATCC #HB8730) containing an anti-DNA-RNA hybrid conjugated with alkaline phosphatase.
  • the solution also contained: 0.6 M NaCl, 0.1 mM MgCl 2 , 0.25% Tween-20.
  • Substrate reagent an alkaline phosphatase chemiluminescent substrate, which is a 0.4 mM CDP-Star solution.
  • Negative control sample preservation solution (10 mM Tris, 10 mM EDTA, pH 7.2 ⁇ 0.2).
  • High-risk HPV low-value control product A (HLC-A): a solution containing 1 pg/ml HPV16 DNA.
  • High-risk HPV low-value control product B (HLC-B): a solution containing 1 pg/ml HPV58 DNA.
  • High-risk HPV high-value control product A (HHC-A): a solution containing 5 pg/ml HPV16 DNA.
  • High-risk HPV high-value control product B (HHC-B): a solution containing 5 pg/ml HPV58 DNA.
  • Low-risk HPV control a solution containing 5 pg/ml HPV6 DNA.
  • HPV DNA typing detection procedure is as follows:
  • Denaturation 0.5 ml of denaturing reagent was added to the test tube (1 ml) storage tube and each control (1 ml) tube, and incubated in a 65 ° C water bath for 45 minutes to degrade the RNA in the sample and decompose the double-stranded DNA into Single-stranded DNA.
  • Hybridization Prepare two 96-well microplates, one labeled "hybridization microplate A” and the other labeled "hybridization microplate B”. Add hybridization microplate A to probe reagent A (25 ⁇ l/well). ), the probe reagent B (25 ⁇ l/well) was added to the hybrid microplate B, and the denatured control substance and the sample to be tested were taken out from the water bath, and returned to room temperature, respectively, 75 ⁇ l was added to the hybrid microplate A and hybridized.
  • the hybrid microplate was sealed with a sealing membrane, and the hybrid microplate was placed on a microplate heating shaker, shaken at 1100 rpm for 3 minutes, the shaking was stopped, and the mixture was incubated at 65 ° C for 60 minutes.
  • the hybrid microplate A and the hybrid microplate B were taken out from the microplate heating shaker and lowered to room temperature.
  • Capture Prepare two capture microplates, mark one of the two capture microplates as “capture microplate A” and the other as “capture microplate B", which will hybridize the liquid in the microplate ( About 100 ⁇ l/well) completely transferred to the capture microplate pair
  • the wells were sealed; the capture microplate was sealed with a sealing film, placed on a microplate heating shaker that had been cooled to room temperature, and shaken at 1100 rpm for 60 minutes at room temperature.
  • the captured microplate was taken out, the liquid in the capture microplate (about 100 ⁇ l/well) was removed; the detection reagent (75 ⁇ l/well) was added to the capture microplate, and incubated for 45-60 minutes at room temperature.
  • Chemiluminescence Remove the detection reagent (about 75 ⁇ l/well) from the capture microplate, wash the capture microplate with 1 ⁇ wash solution, add substrate reagent (75 ⁇ l/well) to the capture microplate, and avoid the light at room temperature. Incubate for 15-30 minutes and read on a chemiluminescent immunoassay analyzer.
  • the determination of the validity of the test is used to judge whether the reagent and the operation are effective, whether the reference value can be accurately given, and the validity of the test must be determined for each test.
  • HPV16, 18 Two types were used to detect two types (HPV16, 18) and 12 (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) high-risk individuals.
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 high-risk individuals.
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 high-risk individuals.
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 high-risk individuals.
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 high-risk individuals.
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 high-risk individuals.
  • the results of capture microplate A and capture microplate B are both “positive”, indicating that one or both of the high-risk human papilloma viruses (HPV16, 18) contained in the sample also contained One or more of 12 high-risk human papillomaviruses (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68).
  • the detection result of the microplate A is “positive”, and the detection result of the microplate B is “negative”, indicating that the sample contains one of two (HPV16, 18) high-risk human papillomaviruses or Two.
  • the detection result of the microplate A was “negative”, and the detection result of the microplate B was “positive”, indicating that the sample contained 12 kinds (HPV31, 33, 35, 39, 45, 51, 52, 56, 58).
  • Types 59, 66, 68 One or more of the high-risk human papillomaviruses.
  • the minimum detection limit of HPV16 and 18 DNA was performed, and the ratio S/N of the measured value of the HPV DNA concentration to the zero concentration was determined.
  • the specific method is: using the kit Probe reagent A in different concentrations (0.5pg/ml, 1pg/ml, 2pg/ml, 5pg/ml, 10pg/ml, 50pg/ml, 100pg/ml; dissolved in sample preservation solution (10mM Tris, 10mM EDTA) The HPV16, 18 DNA with a pH of 7.2 ⁇ 0.2) was detected, and the steps of denaturation, hybridization, capture and detection during the detection were the same as those in Example 3.
  • HPV16DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.08 4.48 9.08 22.11 44.75 289.00 580.26
  • HPV18DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.08 4.47 9.67 24.04 47.06 300.44 588.96
  • the minimum detection limit of HPV16 and 18 DNA is 0.5 pg/ml, and it can be seen that the kit of the present invention can detect virus in a very low concentration of the sample, and the sensitivity is ideal.
  • the lowest detection limit of HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 DNA was performed using the probe reagent B in the kit 3 of Example 2, and different HPV DNA concentration measurements were made.
  • the ratio of the value to the zero concentration measurement value S/N is determined by using the probe reagent B in the kit for different concentrations (0.5pg/ml, 1pg/ml, 2pg/ml, 5pg/ml, 10pg/ml).
  • sample preservation solution (10mM Tris, 10mM EDTA, pH 7.2 ⁇ 0.2) 66, 68 DNA was detected, and the steps of denaturation, hybridization, capture and detection during the detection were the same as those in Example 3.
  • HPV31DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.19 4.19 8.76 21.26 42.48 203.39 406.51
  • HPV33DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.18 4.20 8.22 20.68 42.17 203.39 393.10
  • HPV35DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.31 4.45 8.81 20.95 42.87 212.32 402.34
  • HPV39DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.18 4.24 8.46 20.78 42.52 207.76 408.62
  • HPV45DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.03 3.99 7.88 19.31 37.21 190.88 360.75
  • HPV51DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.07 4.03 7.78 19.58 37.90 190.88 373.96
  • HPV52DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.03 3.66 7.80 19.03 37.88 195.97 368.83
  • HPV56DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.03 3.87 7.91 18.88 37.62 198.44 371.01
  • HPV58DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.12 4.20 8.44 21.62 42.38 213.81 415.44
  • HPV59DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.13 4.26 8.62 23.07 41.84 218.42 412.26
  • HPV66DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.15 4.19 8.54 23.39 41.32 227.49 400.69
  • HPV68DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml S/N 2.11 4.20 8.57 23.01 40.61 228.68 399.92
  • HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 DNA is the most The low detection limit was 0.5 pg/ml, and it was found that the kit of the present invention can detect viruses in samples at extremely low concentrations, and the sensitivity is ideal.
  • Type HPV HPV6, 11, 16, 26, 61, 67, 69, 71, 73, 81 was subjected to cross-reactivity studies. The steps of denaturation, hybridization, capture and detection during the detection were the same as in Example 3.
  • the samples to be cross-reactive were dissolved in the sample preservation solution (10 mM Tris, 10 mM EDTA, pH 7.2 ⁇ 0.2) at 25 pg/ml, and the measurement per well was calculated based on the detection efficiency of the kit.
  • the ratio of the value/reference value (S/Cutoff), the ratio ⁇ 1, is a cross-reaction, the ratio is ⁇ 1, and there is no cross-reaction.
  • test data is shown in Table 16 and Table 17.
  • Sample collection for testing Samples of cervical exfoliated cells from clinical patients were collected from the Obstetrics and Gynecology Hospital of Zhejiang University School of Medicine. Samples of cervical exfoliated cells are collected by a professional gynaecologist in a conventional manner and placed in a sample storage tube containing 1 ml of sample preservation solution, numbered 1-8.
  • the reagent used was tested against the cervical exfoliated cell sample in the same manner as in the kit 3 of Example 2.
  • the test procedure and the result of the sample were determined in the same manner as in Example 3.
  • the specific test results are shown in Table 18.
  • type 2 represents: the virus is at least one of HPV16 or HPV18;
  • the virus is at least one of HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
  • the results of the kit of the present invention are completely consistent with the results of the final diagnosis of the hospital, indicating that the specificity is good and the accuracy is good. Moreover, the kit of the present invention is also capable of achieving two high-risk HPVs (HPV16, 18) and 12 high-risk HPVs among 14 high-risk HPVs (HPV31, 33, 35, 39, 45, 51, 52, 56). , 58, 59, 66, 68) for typing detection.
  • the inventors performed "1" as in the present example for 1404 cases of cervical exfoliated cell samples collected from clinical patients from the Obstetrics and Gynecology Hospital of Zhejiang University School of Medicine, Qilu Hospital of Shandong University, and the First affiliated Hospital of Wenzhou Medical College. The same method of detection. The statistical analysis of the test result data was carried out. The results are shown in Table 19.
  • the Kappa coefficient is 0.9662, and the 95% CI is 95.14% to 98.09%, and the consistency is good.
  • the positive consensus percentage was 98.68%, the negative consensus percentage was 98.33%, and the total agreement rate was 98.58%.
  • the comprehensive analysis showed that the test product had a high coincidence rate with the test results confirmed by the hospital.

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Abstract

本发明提供了一种高危型人乳头瘤病毒检测及分型方法。本发明揭示了对于14种HPV高危型病毒具有特异性检测功能的探针，并基于核酸杂交化学发光免疫技术，开发了可实现对高危型人乳头瘤病毒（HPV）DNA进行检测和分型的技术。

Description

一种高危型人乳头瘤病毒检测及分型方法 技术领域
本发明涉及生物医学领域，更具体地，本发明涉及一种基于核酸杂交化学发光免疫技术的HPV DNA分型检测技术及应用。
背景技术
人乳头瘤病毒(Human papillomaviruses，简称HPV)是一种属于乳多空病毒科的乳头瘤空泡病毒A属的二十面体DNA病毒。HPV的基因组是包含约8000个碱基对的双链环状DNA，全部的开放阅读框(ORFs)均有一条DNA链编码。HPV根据基因组核酸序列的同源性小于90％被分为不同的型别，在同一型内病毒基因组核酸序列差异在2-10％被视为不同的亚型，而在同一亚型内，病毒基因组核酸序列差异小于2％，则称为该亚型的变异株。到目前为止，研究人员发现有近百种HPV型别。不同的型别引起不同的临床表现，根据侵犯的组织部位不同可分为：皮肤型和黏膜型。皮肤型包括与寻常疣、扁平疣、跖疣等相关的HPV1、2、3、4、7、10、12、15等，也包括与疣状表皮发育不良有关的HPV5、8、14、17、20、36、38等；黏膜型包括与生殖器、肛门、口咽部、食道黏膜等感染相关的HPV6、11、13、32、34、40、42、43、44、53、54等，也包括与子宫颈癌、直肠癌、口腔癌、扁桃体癌等相关的HPV16、18、30、31、33、35、39等(Jenson AB et al.1984，Human papillomavirus.In:Belshe RB，editor.Textbook of Human Virology.Littleton，MA:PSG-Wright；p951-68)。根据HPV对人肿瘤的发生危害性，HPV可分为“高危型”和“低危型”HPV。低危型HPV，比如HPV6，11，42，53等与生殖器、肛门、口咽部、食道黏膜等感染相关，高危型HPV是引起宫颈癌等的主要原因。
德国著名医学科学家Harald zur Hausen早在上世纪70年代研究发现高危型HPV感染是宫颈癌的成因(zur Hausen H.1977，Human papillomaviruses and their possible role in squamous cell carcinomas.Curr.Top.Microbiol.Immunol.，78:1-30)，他通过研究证实了两者间的直接关联性，并因此荣获2008年度诺贝尔生理学和医学奖。据中国医学科学院肿瘤医院肿瘤研究所我国著名肿瘤流行病专家乔友林教授及其科研团队对中国的肿瘤流行病调查研究发现，中国宫颈疾病(≥CIN2)与HPV感染的相关性达96-100％(Jing Li et al.2013，Epidemiological Features of Human Papillomavirus(HPV)Infection among Women Living in Mainland China.Asian Pac J Cancer Prev，14(7):4015-4023)。自1995年国际癌症协会(IARC)专题讨论会认为HPV感染是宫颈癌的主要病因，HPV的持续感染已被认为是宫颈癌发病的必要因素和最主要病因。大量研究表明，多数宫颈上皮内瘤变(cervical intraepithelial neoplasia，CIN)和几乎所有的宫颈癌病变中都存在高危型HPV感染(Remmink AJ et al.1995，The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease:natural history up to 36months.Int J Cancer，61(3):306-11)。值得注意的是，宫颈癌的 发病一般需要经历从HPV持续感染到不同程度的CIN(CIN1-CIN3)，最后发展成为宫颈癌(Bosch FX et al.2002，The causal relation between human papillomavirus and cervical cancer.J Clin Pathol，55(4):244-65)。这个过程一般比较漫长，平均需要10-15年时间，这为宫颈癌及其癌前病变的早期发现和阻断提供了足够的时间。
目前，世界卫生组织(WHO)和国际癌症研究机构(IARC)将14种HPV亚型(HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68)确定为高危型HPV型别，并且大量研究发现，70％宫颈癌是由HPV16，18引起的(Khan MJ et al.2005，The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus(HPV)type 16or 18and the possible utility of type-specific HPV testing in clinical practice.J Natl Cancer Inst，97(14):1072-9)。因而，检测高危型HPV16和18感染对预防宫颈癌具有十分重要的意义。
迄今为止，HPV不能在体外进行培养，随着高危型HPV与子宫颈癌的病因学关系广泛受到重视，高危型HPV检测已经用于评估发生子宫颈癌的风险，为了能早期诊断并及时处理，起到预防宫颈疾病(包括子宫颈癌)的发生，有必要进行HPV检测的研究。
HPV抗原的检测：HPV感染人体表皮后，在细胞内增殖合成衣壳蛋白而成为HPV抗原成分。免疫酶染色可检测感染组织细胞内的HPV抗原蛋白L1，以了解有无HPV感染。但由于HPV抗原免疫组化方法只能确认细胞核的衣壳蛋白，而此衣壳蛋白仅出现在HPV生活周期中的一个阶段(在后期病毒颗粒中产生)，病变程度不同抗原表达量也不同，同时，这种方法需要大量的病毒颗粒才出现阳性反应，故此法检出率较低(Dillner J.1999，The serological response to papillomaviruses.Semin Cancer Biol，9:423-30)。
HPV抗体的检测：HPV感染人体后，机体诱导产生抗HPV抗体，故可检测血清中抗HPV抗体。检测抗体主要检测针对HPV16E6和E7抗体，由于其检测灵敏度不高，而且抗体出现在疾病的晚期，不适合对疾病进行早期诊断。而且人感染HPV后，机体产生的抗体可长期存在，血清学方法检测HPV抗体不能确定是近期感染还是既往感染。同时由于HPV型别很多，且免疫反应不一致，所以HPV抗体检测很少使用(Frazer IH.2010，Measuring serum antibody to human papillomavirus following infection or vaccination.Gynecol Oncol，118(1Suppl):S8-11)。
传统的形态学方法和免疫学方法不能充分证明是否存在HPV感染，且临床灵敏度和特异度均不够理想，会引起较高的假阳性率和假阴性率。新发展的检测方法主要用分子生物学方法对HPV进行检测，包括基于信号扩增方法和基于模板扩增方法。基于模板扩增方法以PCR为基础结合不同的检测技术，主要包括荧光PCR方法，PCR结合膜杂交法，PCR结合反向点杂交法、或PCR结合量子点技术等(Anco Molijn et al.2005，Molecular diagnosis of human papillomavirus(HPV)infections.J Clin Virol，32Suppl 1:S43-51)。
以PCR为基础的基于模板扩增的方法主要存在以下几点问题：(1)PCR技术是通过扩增目标DNA片段来进行检测的，需要较高的实验室环境和较严格的实验操作水平；一般来说， 医院的PCR实验室中进行检测的病毒不止HPV一种，故在扩增目标DNA过程中，容易出现大量不同种类的病毒DNA扩增片段，这些片段会因实验室环境的纰漏和操作上的误差而相互反应，引起实验室环境污染的同时，出现假阳性或假阴性结果，导致患者的误诊或漏诊；(2)基于PCR技术的很多产品一般仅检测HPV基因组的L1区，研究表明，这样检测会漏诊5-10％的宫颈癌，从而出现假阴性结果(Walboomers et al.1999，Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol，189:12-19)；(3)PCR反应存在竞争性抑制。当患者存在多种亚型感染时，可能会仅扩增病毒量较高的亚型，病毒数较少的亚型会因扩增相对少而导致假阴性结果；也就是说，如果患者感染的高危亚型病毒数量不及低危亚型，那么出来的结果便会是阴性结果，从而导致患者误诊；(4)PCR技术的检测方法的灵敏度和特异性主要受样本运输及保存条件，同一HPV亚型中DNA序列的变异，HPV DNA在提取过程中的损失，引物对设计，PCR产物大小以及PCR程序设计等因素的影响。
综上，本领域还需要进一步优化HPV的检测方法，开发出适用于临床的便捷、准确的产品。
发明内容
本发明的目的在于提供一种高危型人乳头瘤病毒检测及分型方法。
在本发明的第一方面，提供一种用于高危型HPV病毒检测的试剂盒，所述的试剂盒中包括：针对HPV16、18、31、33、35、39、45、51、52、56、58、59、66和68型病毒特异性的RNA探针。
在一个优选例中，所述的针对HPV16特异性的RNA探针是包括GenBank登录号K02718的序列中第83-1558，3193-4628，5559-7154位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV18特异性的RNA探针是包括GenBank登录号X05015的序列中第105-1593，2376-3867，5430-7136位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV31特异性的RNA探针是包括GenBank登录号HQ537666的序列中第108-1463，3564-4982，5558-7072位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV33特异性的RNA探针是包括GenBank登录号M12732的序列中第109-1654，3789-5087，5594-7093位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV35特异性的RNA探针是包括GenBank登录号X74477的序列中第110-1476，3452-4897，5601-7109位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV39特异性的RNA探针是包括GenBank登录号M62849的序列中第107-1608，2654-4187，5643-7160位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV45特异性的RNA探针是包括GenBank登录号X74479的序列中第102-1543，3421-4872，5530-7149位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV51特异性的RNA探针是包括GenBank登录号M62877的序列中第68-1476，2875-4452，5894-7431位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV52特异性的RNA探针是包括GenBank登录号X74481的序列中第89-1565，2459-4098，5565-7154位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV56特异性的RNA探针是包括GenBank登录号X74483的序列中第102-1567，2432-3896，5492-7096位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的HPV58特异性的RNA探针是包括GenBank登录号D90400的序列中第110-1542，2763-4389，5565-7139位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV59特异性的RNA探针是包括GenBank登录号X77858的序列中第55-1398，3567-4896，5606-7132位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV66特异性的RNA探针是包括GenBank登录号U31794的序列中第67-1534，2679-4231，5647-7158位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
所述的针对HPV68特异性的RNA探针是包括GenBank登录号FR751039的序列中第150-1678，2986-4476，5508-7025位的DNA序列的RNA序列或与该DNA序列互补的RNA序列。
在另一优选例中，所述的各探针可以分离地存在，或可以按照检测需要进行混合。
在另一优选例中，所述的试剂盒中包括：
探针试剂A，其是含HPV16、18型特异性的RNA探针；
探针试剂B，其是含HPV31、33、35、39、45、51、52、56、58、59、66、68型特异性的RNA探针；
捕获抗体，其是抗DNA-RNA杂合体的特异性抗体，其包含于溶液中或被固定于固相载体上；
检测抗体，其是连接有可检测信号的抗DNA-RNA杂合体的特异性抗体。
在另一优选例中，所述的固相载体包括但不限于：多孔板、微球、试纸、玻片。
在另一优选例中，所述的探针带有或不带有标记物(如荧光标记)。
在另一优选例中，所述的试剂盒中还包括选自下组的一种或多种试剂：
高危型HPV质控品；
低危型HPV质控品；
洗涤液；
变性液；
鉴定可检测信号的试剂；和/或
微孔板；
较佳地，所述的可检测信号选自(但不限于)：碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶；所述的鉴定可检测信号的试剂是所述碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶的底物。
在另一优选例中，所述的高危型HPV质控品包括：
高危型HPV低值质控品A：为含1pg/ml HPV16DNA的溶液；高危型HPV低值质控品B：为含1pg/ml HPV58DNA的溶液；高危型HPV高值质控品A：为含5pg/ml HPV16DNA的溶液；和高危型HPV高值质控品B：为含5pg/ml HPV58DNA的溶液；或
所述的低危型HPV质控品为含5pg/ml HPV6DNA的溶液。
在本发明的另一方面，提供所述的试剂盒的用途，用于高危型HPV病毒检测，所述的高危型HPV病毒是HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68型病毒。在一个优选例中，所述的用途是非疾病诊断性的用途。
在本发明的另一方面，提供所述的试剂盒的用途，用于高危型HPV病毒的分型，将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。在一个优选例中，所述的用途是非疾病诊断性的用途。
在本发明的另一方面，提供一种高危型HPV病毒检测或分型方法，所述方法包括：利用所述的试剂盒来检测待测样品，从而鉴定出所述待测样品中是否存在高危型HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68型病毒；或
利用权利要求3所述的试剂盒来检测待测样品，从而将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。
在一个优选例中，所述的检测或分型方法包括：
(1)将待测DNA样本(较佳地，还包含阳性质控品和阴性质控品)解链为单链(较佳地，通过变性解链)后，分别与探针试剂A和探针试剂B混合，获得杂交液A和杂交液B；
(2)将杂交液A和杂交液B分别加样到包被有捕获抗体的固相载体A和固相载体B上， 在固相载体A和B上形成捕获抗体与DNA-RNA杂合体二元复合物；
(2)将检测抗体加样于固相载体，在固相载体A和B上形成捕获抗体、DNA-RNA杂合体和检测抗体三元复合物；
(3)鉴定固相载体A和B上的可检测信号，确定待测样品中是否存在高危型HPV病毒；将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。
在另一优选例中，如下分型：
若固相载体A和B上的可检测信号均为“阳性”，则表明待测样本中含有HPV31、33、35、39、45、51、52、56、58、59、66、68型的一种或几种，也含有HPV16、18型的一种或两种；
若仅固相载体A上的可检测信号为“阳性”，则表明待测样本中含有HPV16、18型的一种或两种；和
若仅固相载体B上的可检测信号为“阳性”，则表明待测样本中含有HPV31、33、35、39、45、51、52、56、58、59、66、68型的一种或几种。
在另一优选例中，所述的方法是非疾病诊断性的方法，或所述的方法针对的检测对象(待测样品)是分离的，包括来自公共场合的样品或实验室样品。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、本发明的方法的检测原理示意图。
具体实施方式
本发明人经过深入的研究和广泛的试验，揭示了对于14种HPV高危型病毒具有特异性检测功能的探针，并基于核酸杂交化学发光免疫技术，开发了可实现对高危型人乳头瘤病毒(HPV)DNA进行检测和分型的技术。
术语
如本文所用，“待测样本”或“待测核酸(DNA)样本”是指待检测的核酸样本，其中含有一种核酸或多种核酸，需要了解其中是否存在目标核酸。
如本文所用，“目标核酸”是指感兴趣的核酸，例如其是一种标志物或是疾病相关的。
如本文所用，“DNA-RNA杂合体”是指一种核酸，其含有两条链，其中一条链是DNA链，另一条链是RNA链，所述的DNA链和RNA链的核苷酸序列是基本上互补的。“基本上互补”是指核苷酸的序列是足够互补的，可以以一种可预见的方式发生相互作用，形成双 链。通常，两条“基本上互补”的核苷酸序列互相之间至少有70％的核苷酸是互补的；优选的，至少有80％的核苷酸是互补的；更优选的，至少有90％的核苷酸是互补的；进一步优选的，至少有95％的核苷酸是互补的；如98％、99％或100％。
如本文所用，“探针”是指一种具有已知核苷酸序列的单链核酸(本发明中优选RNA)，其具有与目标核酸基本上互补的核苷酸序列结构，可以与“目标核酸”形成双链。所述的“探针”可以携带标记物或不携带标记物。例如，标记物可以连接在探针的5’末端或3’末端。
如本文所用，“捕获抗体”是指可被包被在固相载体上的，特异性地识别和结合所述的DNA-RNA杂合体的抗体，其不结合单链的核酸(包括DNA或RNA等)。所述的“捕获抗体”通过识别双链杂交体的双螺旋结构而实现与双链杂交体的结合，而并非是碱基序列特异性的。将抗体包被在固相载体上是本领域技术人员熟知的技术。术语“捕获抗体”可以与“包被抗体”互换使用。
如本文所用，“检测抗体”是指特异性地识别和结合所述的DNA-RNA杂合体的抗体，其不结合单链的核酸(包括DNA或RNA等)。所述的“检测抗体”通过识别双链杂交体的双螺旋结构而实现与双链杂交体的结合，而并非是碱基序列特异性的。所述的“检测抗体”携带有可检测信号，用于报告双链杂交体的捕获情况。
试剂盒
众所周知，HPV病毒具有非常多的亚型和变异株，多达上百种，其中有的亚型或变异株是高危型，有的亚型或变异株是低危型的。因此，鉴定待测样本中是否存在HPV病毒以及HPV病毒是否是高危型的，对于临床检测、预后、知道用药方案是非常关键的。但是，鉴于HPV病毒的亚型和变异株非常多，且不同株型之间基因组序列的同源性也非常高，如何准确地、便捷地(最好实时地)判断出高危型的HPV一直是本领域高度关注的课题，目前临床所用方法实际上存在较高比例的假阳性和假阴性现象。
本发明人经过长期的研究后，制备了可特异性检测出14种高危型HPV病毒的检测试剂盒，所述试剂盒中，包含可特异性识别14种高危型HPV病毒的RNA探针。所述的探针是在经过本发明人广泛筛选后获得的，长度合适且易于人工合成，特异性良好，不会与14种高危型HPV以外的其它任何HPV株型发生交叉反应。
作为本发明的优选方式，所述试剂盒中，所述的探针被混合，HPV16、18型特异性的RNA探针混合形成探针试剂A；HPV31、33、35、39、45、51、52、56、58、59、66、68型特异性的RNA探针混合形成探针试剂B。从而，可实现将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。如此设置，可实现对引起女性70％宫颈癌的HPV16、18型病毒与其它HPV病毒的准确区分，这对于临床应用是非常有用的。
作为本发明的其它实施方式，可采取其它高危型HPV分型检测形式，根据探针试剂中 特异性HPV RNA探针组份调整，可以对14种高危型HPV按14种型别进行分型检测，也可以对14种高危型HPV按组进行分型检测，比如目前的试剂盒分2组：2型(HPV16、18)和12型(HPV31、33、35、39、45、51、52、56、58、59、66、68)；或也可分3组：1型(HPV16)、1型(HPV18)和12型(HPV31、33、35、39、45、51、52、56、58、59、66、68)等。或也可进行其它的组合。当然，2型(HPV16、18)和12型(HPV31、33、35、39、45、51、52、56、58、59、66、68)的分组方式是最为优选的。
作为本发明的优选方式，所述的探针试剂中除了含有特异性RNA探针之外还包括一定浓度的盐离子组份，如柠檬酸三钠，磷酸二氢钠等，保证特异性RNA探针和单链DNA在含有一定盐离子浓度下根据碱基互补配对进行杂交。
作为可选择的方式，探针试剂中的HPV RNA探针也可以是携带标记物的，如生物素，地高辛，他克莫司；相应的捕获微孔板上包被的为亲和素，地高辛抗体，他克莫司结合蛋白；或者相应检测试剂中为亲和素标记的酶，地高辛抗体标记的酶，他克莫司结合蛋白标记的酶。
作为本发明的优选方式，所述的试剂盒中，还包括捕获抗体和检测抗体。
所述的捕获抗体与所述的检测抗体可以应用相同的抗体或不同的抗体来制备，即检测抗体在不携带可检测信号的情况下可以是与捕获抗体相同的或不同的。以DNA-RNA杂合体作为抗原与以蛋白质作为抗原不同，抗DNA-RNA杂合体所产生的抗体无特定的序列或抗原决定簇的要求，其特异性识别双链杂交体所特有的双螺旋结构。抗DNA-RNA抗体(无论是单克隆或多克隆抗体)能与任何DNA-RNA双链杂交体相结合。利用特定的双链杂交体来制备抗所述双链杂交体的抗体的方法是本领域已知的技术，例如可以按照Kitagawa&Stollar的方法(Kitagawa Y,Stollar BD,Mol Immunol 1982，19：413-420)来制备多克隆抗体；或可以按照Fliss等的方法(Fliss I,Laurent M,Emond E,et al.,Appl Environ Microbiol,1993,59(8):2698-2705)来制备单克隆抗体。
作为本发明的优选方式，检测抗体的溶液中还含有一定浓度的NaCl、MgCl2和Tween-20，保证检测抗体在含有一定盐离子的溶液中和DNA-RNA杂合体结合时使检测抗体的非特异性吸附降到最低。
本发明对所采用的固相载体没有特别的限制，只要其能够与包被抗体相结合(偶联、连接)即可。例如，所述的固相载体选自：微量滴定板(如96孔板)、载玻片、试纸或微球。将抗体包被到固相载体上的技术也是本领域技术人员所熟知的。
所述的可检测信号是连接或偶联于检测抗体上的、用于报告检测抗体的结合情况的报告分子。优选的，所述的可检测信号选自：碱性磷酸酶(AP)，辣根过氧化物酶(HRP)，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶。这些可检测信号具有特定的底物，在与底物接触后可以发生显色反应或其它可被检测到或可见的反应，从而报告检测抗体的结合情况。所述的底物比如：用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS；用于碱性磷酸酯酶的对硝基苯磷酸酯(p-nitrophenyl phosphate，p-NPP)、 CDP-Star；等等。
作为本发明的优选方式，所述试剂盒中，还可包含阴性质控品(NC)、低危和高危型HPV质控品(如包括高危型HPV低值质控品A(HLC-A)、高危型HPV低值质控品B(HLC-B)、高危型HPV高值质控品A(HHC-A)、高危型HPV高值质控品B(HHC-B)、低危型HPV质控品(LC))。
为了便于操作，所述的试剂盒中还可含有进行核酸解链处理、洗涤、显色等操作所需要的试剂。用于解链处理的如变性试剂(如碱处理试剂)，用于显色处理的如指示剂染料。
作为本发明的优选方式，所述试剂盒中，还包括样本保存液，所述的样本保存液主要成份为含有一定浓度盐的TE缓冲液，使核酸稳定地保存在TE缓冲液中。
作为本发明的优选方式，洗涤液为一定pH值的缓冲液，具有减少非特异性吸附的作用。
本发明的检测试剂盒特异性高、灵敏度高，成本低廉，无需DNA扩增，无需特殊试验条件，操作简单易培训，使实验污染的降到最低，可实现对14种高危型HPV中的2种高危型HPV(HPV16、18)和12种高危型HPV(HPV31、33、35、39、45、51、52、56、58、59、66、68)进行分型检测。
此外，所述的试剂盒中还可包括使用说明书等。
检测方法
本发明的方法基于ELISA反应原理。但是，本发明人设计的方法中，抗体捕获的对象是特殊的，即由DNA与RNA互补形成的DNA-RNA杂合体，而非常规的蛋白质。具体原理(图1)是：样本中的HPV病毒双链DNA变性分解成单链，单链DNA与特异性RNA探针结合为DNA-RNA杂合体，DNA-RNA杂合体与包被有捕获抗体的固相载体(如微孔板)上的捕获抗体结合，再与偶联有可检测信号的检测抗体结合，通过检测可检测信号，定性检测样本中的HPV病毒核酸，以及实现分型。
作为本发明的优选方式，杂交后的杂交液转移至固相载体上并且进行振荡反应，由于杂交液中含有一定浓度的样本保存液中带来的盐以及快速振荡反应，促使包被在捕获微孔板上的抗DNA-RNA杂合体的特异性高亲合力单克隆抗体尽量对杂交液中的90％以上配对的DNA-RNA杂合体进行捕获。
作为本发明的优选方式，本发明的方法提供了对引起70％宫颈癌的两种高危型HPV(HPV16，18)进行分型的技术，克服了现有检测技术中对高危型HPV型别分型不足的缺陷。根据检测所需，也可进行其它的组合。当然，2型(HPV16、18)和12型(HPV31、33、35、39、45、51、52、56、58、59、66、68)的分组方式是最为优选的，本发明人发现，这种组合的检测效果非常理想，基本上没有与其它病毒发生交叉反应的可能性。
本发明的方法无需采用对实验室环境要求高的PCR技术，克服了PCR技术中实验污染不易控制、假阴性高、存在的竞争抑制以及受样本保存及DNA提取过程等影响的缺点。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、探针试剂和质控品的制备
1.探针试剂A和探针试剂B的制备
制备方法如下：
(1)首先合成下列DNA序列片段(SEQ ID NO:1)：
Figure PCTCN2015088325-appb-000001
(2)将上述合成DNA序列克隆到pUC19载体的KpnⅠ和XbaⅠ位点中，从而得到经修饰的pUC19载体。
(3)分别合成HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68亚型的基因组DNA的片段，各DNA的序列片段分别是：
HPV16：GenBank登录号K02718的序列中第83-1558，3193-4628，5559-7154位；
HPV18：GenBank登录号X05015的序列中第105-1593，2376-3867，5430-7136位；
HPV31：GenBank登录号HQ537666的序列中第108-1463，3564-4982，5558-7072位；
HPV33：GenBank登录号M12732的序列中第109-1654，3789-5087，5594-7093位；
HPV35：GenBank登录号X74477的序列中第110-1476，3452-4897，5601-7109位；
HPV39：GenBank登录号M62849的序列中第107-1608，2654-4187，5643-7160位；
HPV45：GenBank登录号X74479的序列中第102-1543，3421-4872，5530-7149位；
HPV51：GenBank登录号M62877的序列中第68-1476，2875-4452，5894-7431位；
HPV52：GenBank登录号X74481的序列中第89-1565，2459-4098，5565-7154位；
HPV56：GenBank登录号X74483的序列中第102-1567，2432-3896，5492-7096位；
HPV58：GenBank登录号D90400的序列中第110-1542，2763-4389，5565-7139位；
HPV59：GenBank登录号X77858的序列中第55-1398，3567-4896，5606-7132位；
HPV66：GenBank登录号U31794的序列中第67-1534，2679-4231，5647-7158位；
HPV68：GenBank登录号FR751039的序列中第150-1678，2986-4476，5508-7025位；
并且，在上述序列的5’端加上NheⅠ位点序列(GCTAGC)，3’端加上NotⅠ位点序列(GCGGCCGC)。
(4)合成后的上述DNA序列分别克隆到所述经修饰的pUC19载体的NheⅠ/NotⅠ位点中。
(5)上述DNA序列的上游连接有T7。分别以构建后的载体作为模板(模板DNA)，经NotⅠ酶切后，应用T7RNA聚合酶制备HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68的RNA探针。应用T7RNA聚合酶制备RNA的反应条件：10μl 5×转录缓冲液，10μl 10mM 4NTP(ATP，CTP，GTP，UTP)混合液，1μg模板DNA，50U RNA酶抑制剂，1.5μl T7RNA聚合酶(20U/μl)，加DEPC-处理水至总体积50μl。上述混合液在37℃保温100分钟后，加入2μl 0.5M EDTA终止反应。
(6)将HPV16、HPV18RNA探针混合，获得探针试剂A，即为含有HPV16、18RNA探针的溶液；溶液中，HPV16RNA探针浓度0.5μg/ml，HPV18RNA探针浓度0.5μg/ml。溶液中还包括：0.125M柠檬酸三钠、0.125M磷酸二氢钠。
(7)将HPV16、HPV18RNA探针以外的其它探针混合，获得探针试剂B，即为含有HPV31、33、35、39、45、51、52、56、58、59、66、68RNA探针的溶液。溶液中，各RNA探针的浓度为0.5μg/ml。溶液中还包括：0.125M柠檬酸三钠、0.125M磷酸二氢钠。
2.质控品的制备
制备含有HPV6DNA、HPV16DNA、HPV58DNA的质控品，方法如下：
(1)用化学合成法合成HPV6(GenBank登录号FR751337的序列中第1-8031位)、HPV16(GenBank登录号K02718的序列中第1-7904位)、HPV58序列(GenBank登录号D90400的序列中第1-7824位)；在两端分别加上KpnⅠ和XbaⅠ位点。
(2)分别将上述序列克隆到pUC18载体KpnⅠ和XbaⅠ位点中，构建HPV质粒；
(3)HPV质粒转化到大肠杆菌DH5α中；
(4)在含有氨苄抗性的固体琼脂糖培养平板上挑选单克隆菌株；
(5)单克隆菌株在LB培养基中扩大培养；
(6)提取质粒DNA，并进行鉴定、定量。获得的重组质粒DNA即可作为质控品，低危型HPV质控品中，HPV6DNA质粒的浓度为5pg/ml；高危型HPV高值质控品中，HPV16DNA质粒的浓度为5pg/ml，HPV58DNA质粒的浓度为5pg/ml；高危型HPV低值质控品中，HPV16DNA质粒的浓度为1pg/ml，HPV58DNA质粒的浓度为1pg/ml，均为独立放置于每个质控品管中。质控品稀释溶液中还包括：10mM Tris，10mM EDTA，pH为7.2±0.2。
实施例2、试剂盒的组装
试剂盒1：
将实施例1制备的探针试剂A、探针试剂B，装于试剂盒中，获得检测试剂盒1。
试剂盒2：
将实施例1制备的探针试剂A、探针试剂B和质控品分别置于不同的容器中，装于试剂盒中。并且在试剂盒中还装入：
包被抗体：ATCC#HB-8730，溶于碳酸盐缓冲液(pH9.6)中，可用于包被微孔板。
检测抗体：为含偶联有碱性磷酸酶的抗DNA-RNA杂合体的高特异性抗体(ATCC#HB8730)的溶液(抗体浓度：100ng/ml)。溶液中还含有：0.6M NaCl、0.1mM MgCl2、0.25％Tween-20。
获得检测试剂盒2。
试剂盒3：
在试剂盒1的基础上，再包含如下试剂(各试剂分别装于独立的容器中)：
1、变性试剂(变性液)：为含1.75M氢氧化钠的溶液。
2、捕获微孔板：为包被有抗DNA-RNA杂合体的特异性抗体的化学发光板，将包被抗体(ATCC#HB-8730，溶于碳酸盐缓冲液(pH9.6)中)以0.1μg/ml、100μl/孔对96孔板进行包被。
3、检测试剂：为含偶联有碱性磷酸酶的抗DNA-RNA杂合体的高特异性抗体(ATCC#HB8730)的溶液(抗体浓度：100ng/ml)。溶液中还含有：0.6M NaCl、0.1mM MgCl2、0.25％Tween-20。
4、底物试剂：为碱性磷酸酶化学发光底物，为0.4mM CDP-Star溶液。
5、10×浓缩洗涤液：为10×TBS缓冲液。
6、阴性质控品(NC)：样本保存液(10mM Tris，10mM EDTA，pH为7.2±0.2)。
7、高危型HPV低值质控品A(HLC-A)：为含1pg/ml HPV16DNA的溶液。
8、高危型HPV低值质控品B(HLC-B)：为含1pg/ml HPV58DNA的溶液。
9、高危型HPV高值质控品A(HHC-A)：为含5pg/ml HPV16DNA的溶液。
10、高危型HPV高值质控品B(HHC-B)：为含5pg/ml HPV58DNA的溶液。
11、低危型HPV质控品(LC)：为含5pg/ml HPV6DNA的溶液。
实施例3、HPV DNA分型检测
利用实施例2中试剂盒3，对HPV DNA分型检测步骤如下：
变性：在待测样本(1ml)保存管和各质控品(1ml)管中加入0.5ml的变性试剂，在65℃水浴中孵育45分钟，使样本中的RNA降解，双链DNA变性分解成单链DNA。
杂交：准备二块96-孔微孔板，一块标记为“杂交微孔板A”、另一块标记为“杂交微孔板B”，杂交微孔板A中加入探针试剂A(25μl/孔)，杂交微孔板B中加入探针试剂B(25μl/孔)，从水浴中取出变性后的质控品和待测样本，恢复至室温，分别取75μl加至杂交微孔板A和杂交微孔板B中，用封板膜封住杂交微孔板，将杂交微孔板放在微孔板加热振荡器上，1100rpm振荡3分钟，停止振荡，在65℃条件下孵育60分钟，从微孔板加热振荡器上取出杂交微孔板A和杂交微孔板B，降至室温。
捕获：准备二块捕获微孔板，将二块捕获微孔板中一块标记为“捕获微孔板A”、另一块标记为“捕获微孔板B”，将杂交微孔板中的液体(约100μl/孔)完全转移至捕获微孔板对 应的孔中；用封板膜封住捕获微孔板，放至已降至室温的微孔板加热振荡器上，在室温条件下1100rpm振荡60分钟。
检测：取出完成捕获的捕获微孔板，将捕获微孔板中的液体(约100μl/孔)移除；捕获微孔板中加入检测试剂(75μl/孔)，室温孵育45-60分钟。
化学发光：将捕获微孔板中的检测试剂(约75μl/孔)移除，用1×洗涤液清洗捕获微孔板；捕获微孔板中加入底物试剂(75μl/孔)，室温避光孵育15-30分钟，在化学发光免疫分析仪上读数。
参考值：
检测有效性的判定是用来判断试剂以及操作是否有效，能否精确的给定检测参考值，每次检测都必须进行检测有效性的判定。
高危型HPV低值质控品均值/阴性质控品均值≥2.0时，此次检测有效，高危型HPV低值质控品均值即为此次检测的参考值。所有的检测都以样本测量值/参考值(S/Cutoff)的比值表示，比值≥1.0，结果为“阳性”；比值＜1.0，结果为“阴性”。
检验结果如下判断：
1.应用两组微孔板分别检测2种(HPV16、18型)和12种(HPV31、33、35、39、45、51、52、56、58、59、66、68型)高危型人乳头状瘤病毒(HPV)核酸。
2.捕获微孔板A和捕获微孔板B检测结果均为“阳性”，说明样本中既含有2种(HPV16、18型)高危型人乳头状瘤病毒中的一种或两种也含有12种(HPV31、33、35、39、45、51、52、56、58、59、66、68型)高危型人乳头状瘤病毒中的一种或几种。
3.捕获微孔板A检测结果为“阳性”，捕获微孔板B检测结果为“阴性”，说明样本中含有2种(HPV16、18型)高危型人乳头状瘤病毒中的一种或两种。
4.捕获微孔板A检测结果为“阴性”，捕获微孔板B检测结果为“阳性”，说明样本中含有12种(HPV31、33、35、39、45、51、52、56、58、59、66、68型)高危型人乳头状瘤病毒中的一种或几种。
实施例4、最低检测限以及交叉反应性研究
1、最低检测限研究
利用实施例2中试剂盒3中的探针试剂A对HPV16、18DNA进行最低检测限研究，做不同HPV DNA浓度测量值与零浓度测量值的比值S/N，具体方法为：利用本试剂盒中的探针试剂A对不同浓度(0.5pg/ml，1pg/ml，2pg/ml，5pg/ml，10pg/ml，50pg/ml，100pg/ml；溶于样本保存液(10mM Tris，10mM EDTA，pH为7.2±0.2)中)的HPV16、18DNA进行检测，检测过程中变性、杂交、捕获、检测步骤同实施例3。
结果如表2和表3。
表2
HPV16DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.08 4.48 9.08 22.11 44.75 289.00 580.26
表3
HPV18DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.08 4.47 9.67 24.04 47.06 300.44 588.96
由表2和表3可见，HPV16、18DNA的最低检测限均为0.5pg/ml，可见本发明的试剂盒可以检测到极低浓度的样品中的病毒，敏感性很理想。
利用实施例2中试剂盒3中的探针试剂B对HPV31、33、35、39、45、51、52、56、58、59、66、68DNA进行最低检测限研究，做不同HPV DNA浓度测量值与零浓度测量值的比值S/N，具体方法为：利用本试剂盒中的探针试剂B对不同浓度(0.5pg/ml，1pg/ml，2pg/ml，5pg/ml，10pg/ml，50pg/ml，100pg/ml；溶于样本保存液(10mM Tris，10mM EDTA，pH为7.2±0.2)中)的HPV31、33、35、39、45、51、52、56、58、59、66、68DNA进行检测，检测过程中变性、杂交、捕获、检测步骤同实施例3。
结果如表4-15。
表4
HPV31DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.19 4.19 8.76 21.26 42.48 203.39 406.51
表5
HPV33DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.18 4.20 8.22 20.68 42.17 203.39 393.10
表6
HPV35DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.31 4.45 8.81 20.95 42.87 212.32 402.34
表7
HPV39DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.18 4.24 8.46 20.78 42.52 207.76 408.62
表8
HPV45DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.03 3.99 7.88 19.31 37.21 190.88 360.75
表9
HPV51DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.07 4.03 7.78 19.58 37.90 190.88 373.96
表10
HPV52DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.03 3.66 7.80 19.03 37.88 195.97 368.83
表11
HPV56DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.03 3.87 7.91 18.88 37.62 198.44 371.01
表12
HPV58DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.12 4.20 8.44 21.62 42.38 213.81 415.44
表13
HPV59DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.13 4.26 8.62 23.07 41.84 218.42 412.26
表14
HPV66DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.15 4.19 8.54 23.39 41.32 227.49 400.69
表15
HPV68DNA 0.5pg/ml 1pg/ml 2pg/ml 5pg/ml 10pg/ml 50pg/ml 100pg/ml
S/N 2.11 4.20 8.57 23.01 40.61 228.68 399.92
由表4-15可见，HPV31、33、35、39、45、51、52、56、58、59、66、68DNA进行最 低检测限均为0.5pg/ml，可见本发明的试剂盒可以检测到极低浓度的样品中的病毒，敏感性很理想。
2、交叉反应性研究
对实施例2中试剂盒3进行交叉反应研究，探针试剂A对CT(Chlamydia trachomatis，衣原体-沙眼衣原体)、NG(Neisseria gonorrhoeae，细菌-奈瑟淋病双球菌)、UU(Ureaplasmaurealyticum，支原体-解脲支原体)、CMV(Human cytomegalovirus，巨细胞病毒)、HSV(Human Simplex Virus，人单纯疱疹病毒)以及2种HPV(HPV16、18)以外的其他型别HPV(HPV6、11、26、31、33、35、39、51、52、56、58、59、61、66、67、68、69、71、73、81)进行交叉反应研究，在检测有效的基础上，计算每孔测量值/参考值的比值；探针试剂B对CT、NG、UU、CMV、HSV以及12种HPV(HPV31、33、35、39、45、51、52、56、58、59、66、68)以外的其他型别HPV(HPV6、11、16、26、61、67、69、71、73、81)进行交叉反应研究。检测过程中变性、杂交、捕获、检测步骤同实施例3。
以上方法中，各种待进行交叉反应研究样本均以25pg/ml溶于样本保存液(10mM Tris，10mM EDTA，pH为7.2±0.2)中，在试剂盒检测有效性基础上，计算每孔测量值/参考值(S/Cutoff)的比值，比值≥1，为具有交叉反应，比值＜1，为无交叉反应。
检测数据如表16、表17。
表16
Figure PCTCN2015088325-appb-000002
Figure PCTCN2015088325-appb-000003
表17
Figure PCTCN2015088325-appb-000004
由上结果分析：三批试剂盒的交叉反应研究结果分析，捕获微孔板A和捕获微孔板B每孔测量值与参考值(S/Cutoff)的比值均＜1.0，检测结果均为阴性，说明本试剂盒的探针A对CT、NG、UU、CMV、HSV以及2种HPV(HPV16、18)以外的其他型别HPV(HPV6、11、26、31、33、35、39、51、52、56、58、59、61、66、67、68、69、71、73、81)无交叉反应，本试剂盒的探针B对CT、NG、UU、CMV、HSV以及12种HPV(HPV31、33、35、39、45、51、52、56、58、59、66、68)以外的其他型别HPV(HPV6、11、16、26、61、67、69、71、73、81)无交叉反应。
实施例5、临床子宫颈脱落细胞样本的检测
1、8个临床样本的检测
待测样本收集：从浙江大学医学院附属妇产科医院收集临床患者的子宫颈脱落细胞样本。由专业妇科医生以常规方法采集子宫颈脱落细胞样本后，将其放入含有1ml样本保存液的样本保存管中，编号为1-8。
所用试剂同实施例2中试剂盒3，对子宫颈脱落细胞样本进行检测。样本的检测步骤及结果判定同实施例3。具体检测结果如表18。
表18
Figure PCTCN2015088325-appb-000005
其中2型代表：病毒为HPV16或HPV18型中至少一种；
其中12型代表：病毒为HPV31、33、35、39、45、51、52、56、58、59、66、68型中的至少一种。
由上述表18结果可见，本发明的试剂盒的结果与该医院最终确诊的检测结果完全一致，说明其特异性良好，准确性好。并且，本发明的试剂盒还能够实现对14种高危型HPV中的2种高危型HPV(HPV16、18)和12种高危型HPV(HPV31、33、35、39、45、51、52、56、58、59、66、68)进行分型检测。
2、更多临床样本的检测数据统计
针对1404例从浙江大学医学院附属妇产科医院、山东大学齐鲁医院、温州医学院附属第一医院收集临床患者的子宫颈脱落细胞样本，本发明人进行了如本实施例中“1”的方法相同的检测。并对检测结果数据进行了统计分析。结果如表19。
表19
Figure PCTCN2015088325-appb-000006
由表19可见，与医院最终确诊的检测结果相比，Kappa系数0.9662，95％CI为95.14％～98.09％，一致性好。阳性一致百分比为98.68％，阴性一致百分比为98.33％，总一致率为98.58％。综合分析显示本试验产品与医院确诊的检测结果符合率高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于高危型HPV病毒检测的试剂盒，其特征在于，所述的试剂盒中包括：针对HPV16、18、31、33、35、39、45、51、52、56、58、59、66和68型病毒特异性的RNA探针。
  2. 如权利要求1所述的试剂盒，其特征在于，所述的针对HPV16特异性的RNA探针是包括GenBank登录号K02718的序列中第83-1558，3193-4628，5559-7154位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV18特异性的RNA探针是包括GenBank登录号X05015的序列中第105-1593，2376-3867，5430-7136位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV31特异性的RNA探针是包括GenBank登录号HQ537666的序列中第108-1463，3564-4982，5558-7072位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV33特异性的RNA探针是包括GenBank登录号M12732的序列中第109-1654，3789-5087，5594-7093位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV35特异性的RNA探针是包括GenBank登录号X74477的序列中第110-1476，3452-4897，5601-7109位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV39特异性的RNA探针是包括GenBank登录号M62849的序列中第107-1608，2654-4187，5643-7160位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV45特异性的RNA探针是包括GenBank登录号X74479的序列中第102-1543，3421-4872，5530-7149位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV51特异性的RNA探针是包括GenBank登录号M62877的序列中第68-1476，2875-4452，5894-7431位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV52特异性的RNA探针是包括GenBank登录号X74481的序列中第89-1565，2459-4098，5565-7154位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV56特异性的RNA探针是包括GenBank登录号X74483的序列中第 102-1567，2432-3896，5492-7096位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的HPV58特异性的RNA探针是包括GenBank登录号D90400的序列中第110-1542，2763-4389，5565-7139位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV59特异性的RNA探针是包括GenBank登录号X77858的序列中第55-1398，3567-4896，5606-7132位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV66特异性的RNA探针是包括GenBank登录号U31794的序列中第67-1534，2679-4231，5647-7158位的DNA序列的RNA序列或与该DNA序列互补的RNA序列；
    所述的针对HPV68特异性的RNA探针是包括GenBank登录号FR751039的序列中第150-1678，2986-4476，5508-7025位的DNA序列的RNA序列或与该DNA序列互补的RNA序列。
  3. 如权利要求1所述的试剂盒，其特征在于，所述的试剂盒中包括：
    探针试剂A，其是含HPV16、18型特异性的RNA探针；
    探针试剂B，其是含HPV31、33、35、39、45、51、52、56、58、59、66、68型特异性的RNA探针；
    捕获抗体，其是抗DNA-RNA杂合体的特异性抗体，其包含于溶液中或被固定于固相载体上；
    检测抗体，其是连接有可检测信号的抗DNA-RNA杂合体的特异性抗体。
  4. 如权利要求1所述的试剂盒，其特征在于，所述的试剂盒中还包括选自下组的一种或多种试剂：
    高危型HPV质控品；
    低危型HPV质控品；
    洗涤液；
    变性液；
    鉴定可检测信号的试剂；和/或
    微孔板；
    较佳地，所述的可检测信号选自：碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀粉酶；所述的鉴定可检测信号的试剂是所述碱性磷酸酶，辣根过氧化物酶，葡萄糖氧化酶，β–D-半乳糖苷酶，脲酶，过氧化氢酶或葡萄糖淀 粉酶的底物。
  5. 如权利要求4所述的试剂盒，其特征在于，所述的高危型HPV质控品包括：
    高危型HPV低值质控品A：为含1pg/ml HPV16DNA的溶液；高危型HPV低值质控品B：为含1pg/ml HPV58DNA的溶液；高危型HPV高值质控品A：为含5pg/ml HPV16DNA的溶液；和高危型HPV高值质控品B：为含5pg/ml HPV58DNA的溶液；或
    所述的低危型HPV质控品为含5pg/ml HPV6DNA的溶液。
  6. 权利要求1-5任一所述的试剂盒的用途，用于高危型HPV病毒检测，所述的高危型HPV病毒是HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68型病毒。
  7. 权利要求3所述的试剂盒的用途，用于高危型HPV病毒的分型，将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。
  8. 一种高危型HPV病毒检测或分型方法，其特征在于，所述方法包括：利用权利要求1-6任一所述的试剂盒来检测待测样品，从而鉴定出所述待测样品中是否存在高危型HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68型病毒；或
    利用权利要求3所述的试剂盒来检测待测样品，从而将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。
  9. 如权利要求8所述的方法，其特征在于，所述的检测或分型方法包括：
    (1)将待测DNA样本解链为单链后，分别与探针试剂A和探针试剂B混合，获得杂交液A和杂交液B；
    (2)将杂交液A和杂交液B分别加样到包被有捕获抗体的固相载体A和固相载体B上，在固相载体A和B上形成捕获抗体与DNA-RNA杂合体二元复合物；
    (2)将检测抗体加样于固相载体，在固相载体A和B上形成捕获抗体、DNA-RNA杂合体和检测抗体三元复合物；
    (3)鉴定固相载体A和B上的可检测信号，确定待测样品中是否存在高危型HPV病毒；将高危型HPV病毒分为HPV16、18型群组，或HPV31、33、35、39、45、51、52、56、58、59、66、68型群组。
  10. 如权利要求8或9所述的方法，其特征在于，如下分型：
    若固相载体A和B上的可检测信号均为“阳性”，则表明待测样本中含有HPV31、33、 35、39、45、51、52、56、58、59、66、68型的一种或几种，也含有HPV16、18型的一种或两种；
    若仅固相载体A上的可检测信号为“阳性”，则表明待测样本中含有HPV16、18型的一种或两种；和
    若仅固相载体B上的可检测信号为“阳性”，则表明待测样本中含有HPV31、33、35、39、45、51、52、56、58、59、66、68型的一种或几种。
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