CN101017141A - Polymerase chain reaction (PCR) method for diagnosing human papillomavirus (HPV) and reagent kit thereof - Google Patents

Abstract

This invention relates to one polymer enzyme linkage reaction fluorescence test method to dialogue dangerous human body nipple shape virus and to isolate DNA sample HPV gene type to test one set of dangerous HPV infection from patient by the method, wherein, it belongs to life science and biological technique. This invention agent case comprises one fluorescence meter PCR technique as base of multi-layer polymer enzyme reaction composed of multiple HPV positive lead object, reaction lead object and fluorescence detector.

Description

The method and the kit of the fluorescent polyase chain reaction (PCR) that diagnosing human papillomavirus (HPV) infects

Technical field

The present invention relates to a kind of PCR (PCR) and fluorescence detection method of in clinical sample, diagnosing human papillomavirus (HPV) to infect and distinguish out one group of high-risk HPV, belong to life science and biological technical field.This method is particularly related to a kind ofly utilizes multiplex polymerase chain re-action (PCR) fluorescence detection method, and utilizes described method to detect the genotypic fluorescence detection reagent kit of multiple high-risk HPV simultaneously in single pipe PCR reaction tube.

Background technology

Studies show that human papillomavirus (HPV) has substantial connection with cervical carcinoma, is important carcinogen, also is simultaneously one of necessary condition that causes cervical carcinoma.The HPV (being referred to as high-risk HPV) of the certain type of the clear proof of molecular epidemiology is the main cause that causes cervical carcinoma.Cervical carcinoma has become No. second killer of women's cancer in the world.According to the World Health Organization (WHO) (WTO) report, the annual cervical carcinoma in the whole world causes 28.8 ten thousand women's death (http://www.who.int/vaccine_research/diseases/hpv/), increase morbidity example about 510,000 global every year newly, wherein 80% occur in (Africa 6.8 ten thousand people of developing country, Latin America 7.7 ten thousand people, Asia 24.5 ten thousand people).The incidence of disease of China's cervical carcinoma accounts for 1/4 (about 13.5 ten thousand) in the world, and annual new cases number surpasses 130,000, about 20,000 people so and dead.So prevention, diagnosis and treatment cervical carcinoma have crucial meaning to safeguarding WomanHealth.

Cervical carcinoma experience a series of precancerous lesion process, it is epithelium of cervix uteri atypical hyperplasia (claiming on the pathology that cervical intraepithelial neoplasia becomes (CIN)), be divided into three grades according to the order of severity: slight knurl becomes (CIN I) in the epithelium of cervix uteri, the interior moderate knurl of epithelium of cervix uteri becomes (CIN II) and epithelium of cervix uteri inner height knurl becomes (CIN III), and these precancerous lesions all might develop into invasive carcinoma of cervix.CIN is considered to mainly to be infected by HPV and causes, not all HPV the infected and CIN can make progress and be cancer, and the probability of progress is relevant with the cytopathy degree with the HPV type of infection, and the HPV infection of particular type can improve the generation probability of infection disease.Differentiated in more than the 90 kind of HPV genotype have kind more than 40 can feel genital atriium (de VilliersE-M.Taxonomic classification of papillomaviruses.Papillomavirus Rep 2001; 12:57-63).The HPV that can infect genital atriium at present is divided into two classes: with cervical carcinoma low relevant (low danger) type and height correlation (high-risk) type.Low risk HPV mainly finds in the wart of reproductive organs surface, the normal and wellability tumour association of high-risk-type.Disclose according to recent result of study, high-risk HPV has 13 to 19 types, wherein has only 11 types (HPV16,18,31,33,35,39,45,51,52,56 with 58 types) by the consistent high-risk-type that is defined as.Nubia

The result of study of delivering in 2003 Deng the people is thought 15 kinds of high-risk-types are arranged (what increase newly is 59,68,73,82 types) and three kinds of possible carcinogenic types (26,53 and 66 type) (Nubia

, et al, Epidemiologic Classification of Human PapillomavirusTypes Associated with Cervical Cancer, The new england journal of medicine 2003,5t8-527).Other are several also to be considered to high-risk-type and to be detected the HPV genotype that HPV67 (Martin Moberg is arranged, et al, Real-Time PCR-BasedSystem for Simultaneous Quantification of Human Papillomavirus Types Associated with High Riskof Cervical Cancer JOURNAL 0F CLINICAL MICROBIOLOGY, July 2003, p.3221-3228 Vol.41, No.7), HPV69 (Bosch FX, et al, Prevalence of Human papillomavirus in cervical cancer:a worldwideperspectlve.International Biological Study on Cervical Cancer (IBSCC) Study Group.J Natl CancerInst 1995,87:796-802), HPV70 (Eileen M.Burd, Human Papillomavirus and Cervical CancerCLINICAL MICROBIOLOGY REVIEWS, 2003,1-17.) etc.HPV-16 mainly finds in the cervical squamous cells cancer, and HPV-18 is mainly seen in adenocarcinoma of the uterine cervix.Research data shows, the HPVl6 type is just relevant with cervical carcinoma more than 50%, modal high-risk HPV 16, HPV18 only account for the 50-70% that can cause cervical carcinoma HPV, and 13 kinds of known type high-risk HPVs (comprise HPV16,18,31,33,35,39,45,51,52,56,58,59,68 etc.), HPV reaches concurrent infection separately can cause cervical carcinoma more than 98%.Based on above research, by the molecular biological characteristics of differentiating that HPV infects, invention complete detection fast and accurately comprises the method for high-risk HPV more than 13 kinds, and is significant for diagnosis and prevention cervical carcinoma.

Be diagnosing in early days of falling ill in cervical carcinoma, most widely used is traditional cervical cytology inspection technique-Pap smear (thePapanicolaou-stained (Pap) smear).This method is a cervical cytology inspection technique the earliest, invented by virologist GeorgePapanicolaou in 1949, method is: the outermost layer cell of collecting the cervix surface, spread upon on the microslide, after handling through dyeing, examine under a microscope and comprise the histopathology feature in being formed on of koilocyte, epithelial cell kernel Zhou Konghuan and make diagnosis.The advantage of Pap smear method is that method is easy, patient's no pain, and cost is lower, is widely used conventional cervical carcinoma screening method.Begin to be used for the cervical carcinoma examination from the forties in 20th century,, made the incidence of disease of cervical carcinoma and mortality ratio reduce by 1/2 even 2/3 more than by the examination of Pap smear.The shortcoming of this method is to draw materials, film-making, dye, read sheet etc. and be subjected to the influence of artificial subjective factor very big, and false negative rate is greater than 10%-20% even higher (20%-30%).Because the restriction of these shortcomings, the more reliable diagnostic method of using other is very necessary as vaginoscopy.

Traditional Pap smear legal person draws materials in order to reduce, film-making, dye, read sheet etc. and be the influence of factor, successively derive improved cytolgical examination method: computer is assisted cervical cytology detection system (X Song, 1998) be that a kind of that the American develops is called as " cranial nerve network simulation system ", be used to scan traditional Pap smear, be called for short CCT and check.But statistical research shows that the susceptibility of this system's primary dcreening operation is lower than experienced professional on the contrary, is used for breadboard quality control so food and drug administration (FDA) only ratifies it.The membrane liquid base thin-layer cell learn a skill (TCT) be present state-of-the-art cervical cytology detection technique, the existing how tame large hospital of China is introduced this system, method is that the doctor puts into the specimen bottle that cell-preservation liquid is housed with the cell that collects and sends to the laboratory, and the film-making process is controlled by computer program.Its advantage is to have removed impurity, forms a cell monolayer smear clearly, and pathologist can come into plain view.This method make cervical carcinoma especially the diagnosis of precancerous lesion significantly improve, higher because of expense, usually only in the people at highest risk, use.

The vaginoscopy method: gynecatoptron is a kind of endoscope, but the pathology of Direct observation uterine neck and following genital tract epithelium, and enlargement factor can reach 10-40 doubly, is one of important householder method of early diagnosis cervical carcinoma and precancerous lesion.Often advise carrying out vaginoscopy when clinical suspicious or cytolgical examination is unusual, gynecatoptron and cytology share and can reduce false-negative generation.Colposcopy is checked the recall rate of HPV infection up to 70%, although vaginoscopy in Europe as screening method by more use, but thinking that its specificity is lower, most of developed countries should not make screening method, then higher in developing country because of its device consumes and cost, colposcopic accuracy is influenced by himself and examiner's experience and technical merit usually, need the technical skill personnel of certain experience again, and can't be as the screening method widespread use.

The common drawback of above-mentioned several method is the operating personnel that need be skilled in technique, and can not differentiate the existence of HPV DNA (deoxyribonucleic acid) (DNA), more can not discern excessive risk and low-risk HPV and infect genotype.Can detect HPV and identify the genotypic various technology of HPV so make great efforts development all the time, so that Pap smear method and vaginoscopy are carried out well replenishing.

HPV has been confirmed to be the main virulence factor of cervical carcinoma, and HPV virus is difficult to especially in vitro culture, and not every the infected has detectable antibody response.Therefore, the DNA detection non-intervention type that is is measured HPV and is infected the detection method with maximum sensitivity that exists.Cytolgical examination of U.S. FDA approved and HPV detect the associating use as the cervical carcinoma screening method, show that detecting HPV gains public acceptance as the method for preventing cervical carcinoma.The detection method of HPV DNA mainly contains the DNA detection method of HPV based on the Protocols in Molecular Biology that uses nucleic acid probe.The DNA detection method of HPV mainly be divided three classes (Roger A.Hubbard, Human Papillomavirus Testing Methods ARCH PATHOL LAB MED2003127,940-945):

The first kind is direct probe combined techniques, Sourthern trace and some trace if any HPV type specific probe, methods such as (FISH) is filtered in situ hybridization, because its muting sensitivity, complex operation is time-consuming and need the HPV probe of large-scale purification, and these methods can not detect all carcinogenic HPV genotype, now seldom adopt, so do not promote widely.

Second class is a signal amplification method, as hybrid capture (Hybrid Capture kit, Digene Diagnostics, Silver Spring, MD, USA, http://www.digene.com) method (Digene company) and bDNA method (Bayer company).Hybrid capture (Hybrid Capture) method is the technology of the detection HPV DNA of U.S. Digene company, be that the present U.S. unique acquisition FDA approval can be in the detection technique of a kind of HPV of detection DNA of clinical use, HC2 can detect the various HPV from the high-risk-type to the low risk, what this kit was used is that a cover nucleic acid hybridization amplification of signal systematic cross is caught two generations test (HC2) HPV DNA detection reagent, can detect 13 kinds of high-risk HPV (HPV16 simultaneously, 18,31,33,35,39,45,51,52,56,58,59 and 68).In said method, utilize the liquid hybridization of Hybrid Capture and general primer PCR linear probe test afterwards to be considered to be directed to the only method of diagnostic purpose.Business-like Hybrid Capture kit need not pcr amplification can detect the HPV DNA in the clinical sample, and can distinguish out high-risk-type and low risk.But, use rna probe may influence the stability of kit, and can not get rid of the possibility of cross reaction.

The 3rd class methods are based on the target sequence fragment amplification technology (document 3-13) of PCR, use PCR Idiotype HPV target sequence fragment is increased, and differentiate the HPV type with the oligonucleotide probe of type specificity.

The advantage of HPV DNA detection method is: it is easy to take a sample, and detection and result report that artificial factor affecting is few, are adapted at carrying out among the cervical carcinoma people at highest risk large-scale primary dcreening operation.Such at present main detection method has:

Type specificity PCR (Type-specific PCR) method: this method is carried out pcr analysis (Walboomers according to the specific primer of region of variability design mode of E6, E7 gene, 1999), its sensitivity copies every reaction about 10-200 HPV greatly, and is different and variant slightly with the HPV type.But this method mainly is confined to research field at present, has limited high-throughout use because every increment originally all needs to make many parts of PCR simultaneously.

Degeneracy/universal primer PCR is by (Kleter etc. 1999 at the conservative L1 gene design primer of height, Gravitt PE, 2000), can in a tube reaction, detect a plurality of HPV types simultaneously, use the definite somatotype of type specificity probe hybridization subsequently, can distinguish about 40 kinds of types.Primer commonly used comprises degenerate primer MY09/11 (Bosch, 1995) and modified PGMY09/11 (Gravitt PE, 1998), SPF1/2 (Reesink-Peters N, 2001), universal primer GP5/6 (De Roda Husman AM, 1995) and GP5+/6+ (Hutchinson, 1994), the sensitivity of SPF1/2 reaches 0.5-10fg (10-200 copy), the PCR product also can further be analyzed by multiple means: for example sequential analysis, restricted length polymorphism analysis (RFLP, Wang TS, 1999), and use specific probe to carry out dot hybridization (LaconiS, 2001) etc.

It is the representative products of Roche company that the Amplicor microwell plate detects (Microtiter Well Plate), this product uses the fragment of 13 kinds of high-risk-type L1 of non-degenerate serial primer mixture specific amplification section length as 170bp, the respective capture molecule that product is fixed on the microwell plate is caught, and the colour developing by substrate and enzyme is subsequently detected product.This method has been used the TaqGold archaeal dna polymerase, has reduced non-specific amplification and the sensitivity of reaction is greatly improved.Since its at fragment shorter, this detection means is particularly effective for detect preserving the sample of being not good at, it is reported (Thomas Iftner, 2003) relatively its recall rate of PGMY09/11 (at the fragment of 450bp) to cervical smear can improve 13%.

HPV classification diagnosis chip is by development (the TS Hwang of company of company limited of biomedical laboratory (Biomedlab) of Korea S, 2003), can detect 22 kinds of HPV at present, method is, type specificity probe and contrast probe (Beta globulin) are fixed on the chip, primer comprises at the primer of Beta globulin and at the primer (doing further modification on the GP5/6 basis) in HPV L1 district, contain fluorescein-labeled nucleosides Cy3 or Cy5 in the PCR system, through the target sequence DNA of pcr amplification product, with chip hybridization, can obtain the result by laser scanner at last.For the sample of multiple infection, can detect a plurality of hybridization signals, although the sensitivity of this method and specificity are all very high, its practicality is still waiting further demonstration.

More than detecting several hybrid method methods of HPV gene utilizes GP5/6 or MY11/9 universal primer or its to improve primer, add the Idiotype probe sequence in polymorphic district, several shortcomings are all arranged, for example, carry out also needing to do the hypotype that further experiment could differentiate that HPV is different after the PCR reaction; And, as there being the mixed infection of different type HPV in the sample, different HPV types has different amplified reaction efficient, cause some type unbalanced amplification to be arranged with respect to other type, unbalanced amplification makes molecular balance trend towards height copy type in the sample, consumed the component of PCR reaction, made the few type of copy number be difficult for being detected.

(fluoresence PCR F-PCR), at first succeeds in developing (United States Patent (USP) 1994 U.S.Patent No.5538848 by U.S. PE company to TaqMan fluorescence probe round pcr; PNAS USA 88:7276-7280 (1991) such as Holland).The TaqMan probe is the two ends specific oligonucleotide strands of mark fluorescent group respectively, at 5 ' end mark fluorescent reporter group, at 3 ' end mark fluorescent quenching group.When PCR reaction beginning, primer combines with the target sequence specificity, and the Taq enzyme begins to synthesize to 3 ' end from 5 ' end along template, and this moment, probe was kept perfectly, and the fluorescence of fluorescence report group is not produced fluorescence signal by the cancellation of fluorescent quenching group; When the Taq enzyme is blended into when running into probe along the double-stranded DNA template, probe 5 ' end is cut off, the fluorescence report group that gets off that dissociates can send report fluorescence; Intensity of fluorescence strengthens with the PCR cycle index, and proportional with the quantity of PCR product.The fluoroscopic examination instrument sees through wavelength and the length variations that the PCR tube wall can directly detect fluorescence signal, so fluorescent PCR detects and can realize that stopped pipe monitors the variation of PCR process in real time, and the PCR product need not subsequent detection and processing.It has the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectral technique accurate quantification concurrently, operation fast, visual result, variation in the energy direct detection PCR process, its product detects not to be needed to do the PCR aftertreatment, need not to carry out gel electrophoresis, stopped pipe operation fully, effectively reduce the PCR product pollution, further reduced false positive rate.

The fluorescence probe of fluorescent PCR technology has now developed into polytype, as the TaqManMGB probe, and Beacon probe, Fret probe, Scorpion (scorpion) probe etc.The Taq-Man probe is divided into two kinds according to the difference of the fluorescent quenching group of its 3 ' end mark: common TaqMan probe and TaqManMGB probe.The TaqManMGB detecting probe method is the method for updating that produces after the TaqMan method, and the full name of MGB is Minor GrooveBinder, and it just was pushed out in calendar year 2001.Compare with the TaqMan probe, its principle be TaqManMGB probe one be probe 3 ' end mark self non-luminous cancellation fluorescence molecule MGB (Minor Groove Binder) modification group, to replace the TAMRA fluorescence labeling that routine can be luminous, can reduce the intensity of background signal greatly, the MGB group can improve about 10 ℃ with the Tm value of probe simultaneously, therefore in order to obtain same Tm value, the MGB probe can get shorter than general T aqMan probe design, both reduced synthetic cost, increase the hybrid stability of probe greatly, also made the success ratio of probe design greatly improve.This method has following advantage: 1, design easily; 2, probe is short; 3, the Tm value difference that improves between pairing and non-matching template is different; 4, high specific and high precision; 5, Za Jiao good stability; 6, low background has been improved signal to noise ratio (S/N ratio); 7, favorable reproducibility as a result.

Taq-Man fluorescence probe round pcr has been used to detect the HPV type, (Journal of Clinicai Microbiology 35 (4): 886-891 such as Swan, (1997)) primer of usefulness type specificity and multiplex PCR amplification HPV16,18, the L1 district dna segment of 31,33,35 5 type HPV, with the reaction signal of terminal point behind the certain PCR circular response of type specific fluorescence probe in detecting, but this method is not in real time-PCR (real-time).(Journal of Clinical Microbiology 37 (3): 490-496 such as Josefsson, (1999)) reported the primer PCR amplification HPV16 that uses type specificity, 18,31, the E1 district dna segment of 33,3 five type HPV detects this five kinds of HPV hypotypes in real time with different fluorescently-labeled type specific probes, different with the method for Swan etc., this method has been used Real-time PCR principle.(Molecular Diagnosis 6 (1): 39-47 (2001)) reported with the primer of type specificity and the conserved region of multiplex PCR amplification HPV DNAE6/E7 bonding pad, detected HPV in real time with different fluorescently-labeled type specific probes for Tucker etc.(Journal of Clinical Microbiology, 39 (9): 3204-3212 (Vol.39, No.2001)) report divides three pipes to detect HPV6,11,16,18,31,33,39,45,51,560 kinds of types with the Scorpions fluorescence probe to HART etc.

The world and the European patent of HPV genetic test have:

1.Fluorescent multiplex hpv pcr assays using multiple fluorophores CA2457888, EP 1421200 (2001), WO03019143 (2003), US2005175987 (2005) (real-time PCR), adopt three site methods to detect E6/E7 gene and the L1 of HPV, adopt four kinds of taqman probe in detecting HPV6,11,16,18, seven kinds of HPV types such as 31,33,45 grades.

2.Virus detection method, primers therefor and screening kit.CA 2450164, the every type of WO 02103050 (2002) (real-time PCR) adopts a kind of Scorpion probe in detecting HPV6,11,16,18 4 kinds of HPV types.

Above-mentioned international monopoly and fluorescent PCR technology, but these methods detect less HPV type only simultaneously, but also the real-time fluorescence PCR detection method of TaqManMGB probe is not applied to the report and the patented claim of the detection of HPV DNA.

Based on the above, although the detection of HPV DNA has developed many kinds of technology, develop a kind of simple, accurately, good reproducibility be used to detect that multiple type HPV infects and and the method for distinguishing out high-risk HPV be very important.

Summary of the invention

Known have 20 types approximately with the high-risk HPV cervical carcinoma height correlation, the clinical diagnosis needs are a kind of simply, accurate, the method for good reproducibility, can detect a series of multiple HPV simultaneously, detect particularly that HPV infects and and distinguish out the method for high-risk HPV.Fluorescent real time PCR (Fluorescent real-time PCR) technology is in conjunction with advantages such as the specificity of the sensitivity of PCR, DNA hybridization and spectral technique accurate quantifications, have simple to operate, result and judge characteristics such as directly perceived and pollution-free, in clinical diagnosis, be widely used in multiple viral pathogen DNA detection.

This method is particularly related to a kind of multi-PCR detection method that utilizes, and utilizes described method to detect the multiple common genotypic fluorescence detection reagent kit of high-risk HPV fast, abreast simultaneously in single pipe PCR reaction tube.Fundamental purpose of the present invention provides the genotyping kit that a kind of HPV of diagnosis infects, and diagnoses HPV to infect easily, measures the genotype of high-risk HPV exactly.

General PCR only adopts a pair of primer, produces a nucleic acid fragment by pcr amplification.It is the multiple nucleic acid TRAP that the present invention then adopts multiplex PCR method (multiplexPCR), in same PCR reaction system, add primer more than two pairs, each carries out the PCR reaction to primer at special separately target sequence, its reaction principle, reaction reagent is identical with general PCR with operating process.Adopt the reason of multiplex PCR that multiple advantage is arranged: multiplex PCR has characteristics such as high efficiency systematicness economical and convenient, can in same reaction tube, detect multiple high-risk HPV type simultaneously, can save time greatly, save reagent, the reduction of expenditure spending, provide more diagnostic messages more accurately for clinical, more press close to the clinical examination demand.Emphasize oligonucleotide sequence primer and probe and corresponding HPV type specific bond among the present invention at each HPV type, key is to contain one group of probe, a group-specific forward primer and a reverse primer in the reaction system, at 18 kinds of high-risk HPV types, can combine respectively with corresponding high-risk HPV type target sequence specificity.HPV genotype detection kit by this law preparation can detect the HPV infection easy, accurately, identifies that the HPV that infects belongs to high-risk genotype.

Method step of the present invention comprises: (1) PCR in the presence of polymerase and one group of Auele Specific Primer various at HPV carries out the amplified target sequence, produce the target sequence amplified production, at the primer sequence of each HPV type to including: one at first with the forward primer of first binding site combination of target sequence upstream of corresponding HPV type, reverse primer with second the binding site combination in downstream of corresponding HPV type target sequence, forward primer and reverse primer can be the sequences that the sequence table of this instructions is listed.(2) TaqMan fluorescence labeling probes at corresponding HPV type target sequence sequence between forward primer and reverse primer; The TaqMan probe is divided into two kinds according to the difference of the fluorescent quenching group of its 3 ' end mark: common TaqMan probe and TaqManMGB probe.Method of the present invention can adopt common TaqMan probe and TaqManMGB probe.The quenching group of TaqManMGB probe adopts non-fluorescent quenching group (Non-Fluorescent Quencher), and itself does not produce fluorescence, can reduce the intensity of background signal greatly.Also be connected with MGB (Minor Groove Binder) modification group on the probe simultaneously, the Tm value of probe can be improved about 10 ℃.Therefore in order to obtain same Tm value, the MGB probe can get shorter than general T aqMan probe design, has both reduced synthetic cost, also makes the success ratio of probe design greatly improve.Probe can be the sequence that the sequence table of this instructions is listed.(3) when PCR reaction beginning, dna fragmentation specificity at the pcr amplification in the primer tasteless nucleotide sequence of each type HPV and the clinical sample combines hybridization, primer combines with the target sequence specificity, the Taq enzyme begins to synthesize to 3 ' end from 5 ' end along template, the probe that not combine with target sequence this moment is kept perfectly, the fluorescence of fluorescence report group is not produced fluorescence signal by the cancellation of fluorescent quenching group; When Taq enzymic synthesis double-stranded DNA moves when running into probe along dna profiling, probe 5 ' end to be sheared removed, the fluorescence report group that gets off that dissociates sends report fluorescence; Intensity of fluorescence strengthens with the PCR cycle index, and is ratio with the quantity of PCR product and increases.Fluorescence probe can be one or more in double-tagging probe (double-labeled probe), molecular beacon (molecular beacon), the NE BY ENERGY TRANSFER fluorescence probe (Fluorescent resonance energy transfer), and the fluorescence that is used for label probe can be one or two or more kinds of FAM, TET, JOE, ViC, HEX, ROX TAMPA, CY3, CY3.5, CY5, CY5.5, OregonGreenTM, CALRedTM, Red 640, Texas Red.3 ' end of probe can be to have DNA ditch compound (MGB, Minor Groove Bingding) mark.Adopt one or more different fluorescence that one or more different probes are carried out mark.(4) the fluoroscopic examination instrument sees through the PCR tube wall and directly detects next or a plurality of different fluorescence signals at specific wavelength, and the quantity that fluorescence intensity changes the target sequence amplified production of the corresponding HPV type of representative increases, and corresponding HPV type is positive in sample.

The invention is characterized in that adopting single stage method or two-step approach is template with HPV DNA, viral specific fragment in the amplification sample carries out the quantitative or qualitative fluoroscopic examination of HPV simultaneously under the effect of archaeal dna polymerase.The sample of described extraction is the sample that uterine cervix obtains.This patent is a kind of PCR method of multi-primers, by more than 2 pairs to from 18 pairs of multi-primerses, at 18 kinds of HPV genotype, each product detects more than 2 kinds DNA (comprising HPV16,18,31,33,35,39,45,51,52,56,58,59,66,67,68,69,73,82 types) to 18 kinds of HPV types simultaneously by fluorescence signal in a sample hose.

Preparation process of the present invention is described in detail in following step.The present invention has done to further specify in following examples, but scope of the present invention is not subjected to the restriction of embodiment.Although particularly the kit for preparing among the embodiment has 18 pairs of primers and degeneracy probe and combination thereof; but can not be interpreted as that the present invention just is confined to the type and the quantity of probe, multiplex polymerase chain re-action (PCR) fluorescence PCR detecting method of every use HPV DNA derivatized nucleotide sequence and the similar approach and the kit of detection kit and the described DNA fluorescence PCR detection reagent kit of any use all belong to protection domain of the present invention.

Description of drawings

The initial copy number that the fluorescent PCR amplification curve of Fig. 1 .HPV16 type recombinant plasmid standard items gradient dilution, curve are from left to right corresponding is respectively (1 * 10 ⁶, 1 * 10 ⁵, 1 * 10 ⁴, 1 * 10 ³, 1 * 10 ², 1 * 10 ¹)

The typical curve of Fig. 2 .HPV16 type recombinant plasmid standard items gradient dilution, R ²=0.994, illustrate that linear relationship is very good; Slope=-3.35, pcr amplification efficient are 98.8% (ideally slope=-3.32, PCR efficient is 100%)

Fig. 3. the fluorescent PCR detection curve synoptic diagram of cervical cell sample HPV

Embodiment

Embodiment 1: primer and probe design

Studies show that between the various genomic L1 region sequence of HPV, existing certain homology is a HPV conservative property sequence, having certain polymorphism again is type specificity, can the design mode Auele Specific Primer according to the type specificity sequence.Primer and probe design principle that present embodiment detects 18 kinds of high-risk HPV types are: select adjacent polymorphic sequence design specificity forward primer and reverse primer in the genomic L1 of HPV district (the target sequence position is about L1 district bp800-bp1200), and probe sequence, carry out multiplex PCR at a reaction tube, use 18 kinds of high-risk HPVs of general TAQMAN-MGB probe in detecting simultaneously at 18 high-risk HPVs.The template denaturation temperature is at 92-97 ℃; The primer annealing temperature is at 50-65 ℃; Synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down, and the temperature of extending synthetic complementary dna chain is at 56-65 ℃; The period of carrying out amplified reaction is 30-45.Table 1 has been listed the position and the maximum length of target sequence amplified production.Forward primer can be the primer and the combination thereof of the corresponding 18 kinds of HPV types of SEQ ID NO.1-SEQ ID NO.90 in the sequence table, reverse primer can be the primer and the combination thereof of the corresponding 18 kinds of HPV types of sequence table SEQ ID NO.91-SEQ ID NO.180, and probe can be the probe sequence and the combination thereof of the corresponding 18 kinds of HPV types of SEQ ID NO.181-SEQ ID NO.270 in the sequence table.Above-mentioned primer and probe also can be that suitable sequence between this patent claim 1 described forward primer and the reverse primer is as the sequence of primer and probe, the sequence that is not limited only to list in the sequence table.

The gene pool numbering and the position of table 1.HPV target sequence amplified production institute foundation

The HPV type	The gene pool numbering	The target sequence position	Maximum amplified production length
				HPV16	NC_001526	6538-6745	120-208
HPV18	NC_001357	6514-6724	120-211
				HPV31	NC_001527	6456-6663	120-208
HPV33	NC_001528	6495-6699	120-205
				HPV35	M74117	6478-6685	120-208
HPV39	NC_001535	6541-6751	120-211
				HPV45	NC_001590	6518-6728	120-211
HPV51	NC_001533	6422-6629	120-208
				HPV52	NC_001592	6559-6763	120-205
HPV56	NC_001594	6495-6699	120-205
				HPB58	NC_001443	6544-6748	120-205
HPB59	NC_001635	6507-6717	120-211
				HPV66	U31794	6545-6749	120-205
HPV67	D21208	6520-6724	120-205
				HPV68	M73258	2518-2728	120-211
HPV69	NC_002171	6445-6655	120-211
				HPV73	NC_006165	6389-6602	120-214
HPV82	NC_002172	6472-6682	120-211

Embodiment 2: specimen preparation

For the HPV that detects human woman cervix uteri assay specimen infects, from described sample, extract DNA, and purifying.For whether specimen DNA amount is enough, described purify DNA carries out pcr amplification by detecting the distinctive gene of people's gene group, selects the DNA sample that those show the distinctive gene DNA amplification of people's gene group, is used for the analysis of next step HPV DNA.The distinctive gene of people's gene group can select pearl beta-globin gene and HMBS gene primer to carry out pcr amplification.

The preparation of positive control plasmid and negative control plasmid

The HPV sequence that obtains from the listed source of table 1, the recombinant plasmid dna that contains HPV L1 district target sequence that makes up in this laboratory uses Promega as the positive control standard items ^PGEM-T easy vector system connects kit, purified PCR product is connected on the pGEM-Teasy carrier, transform then and enter in the escherichia coli jm109 competent cell, make up 18 kinds of HPV types (HPV16,18,31,33,35,39,45,51,52,56,58,59,66,67,68,69,73,82) recombinant plasmid dna as the positive reference substance standard items, and make up 5 kinds of HPV types (HPV6,11,42,43,44) recombinant plasmid dna as the negative control standard items.The HPV recombinant plasmid that makes up is identified through order-checking.In addition, from the DNA of following clone separation and purification as positive control: SiHa cell line (HPV 16) and Hila cell line.The above-mentioned sample DNA of selecting uses one group of primer sets in the sequence table to carry out pcr amplification.

Embodiment 3 optimizes multiplex PCR fluoroscopic examination reaction

Primer and concentration and probe concentration be optimized make and in a pipe PCR reaction, can detect 18 kinds of high-risk HPVs simultaneously, use ABI PRISM ^18 kinds of high-risk-types ( HPV type 16,18,31,33,35,39,45,51,52,56,58,59,66,67,68,69,73,82) the positive control plasmid assessment Ct value and the Rn value of 7000 pairs 10-1000000 copy, optimize primer and concentration and probe concentration with the variation of primer and probe, with 5 kinds of low risks ( HPV type 6,11,42,43,44) negative control plasmid detection specificity, 18 kinds of high-risk-type positive control plasmids that are 10-10000000 copy can be detected, and the negative control plasmid can not be detected.PCR is reflected at 40 microlitres and sends out should mix in the liquid and carry out, and reaction mixture includes the TAQ polymerase of 1-2 unit, the primer of 20-200nM at each high-risk HPV, and the concentration of probe is 0.02-0.6pM.

Embodiment 4 uses this kit to detect HPV infection in the clinical sample

Embodiment 4-1 uses this kit to detect positive control plasmid and property control plasmid

Use this kit to detect and comprise 18 kinds of high-risk-types ( HPV type 16,18,31,33,35,39,45,51,52,56,58,59,66,67,68,69,73,82) according to positive control plasmid and 5 kinds of low risks ( HPV type 6,11,42,43,44) negative control plasmid, the result meets expection fully.The HPV16 standard items had good detection dynamics range and linear relationship (10-1000000 copy) (Fig. 1-2).

Embodiment 4-2 uses this kit to detect HPV infection in the clinical sample

For detecting accuracy and the validity that kit diagnosis HPV of the present invention infects, 102 routine clinical samples are carried out pcr amplification and fluoroscopic examination, to detect the HPV infection and to determine that high-risk type HPV infects.102 examples prepare the DNA lamina membranacea at the sample that uterine cervix obtains by method described in the embodiment 2, and then the DNA cloning of optimizing with the present invention described in the embodiment 3 and analytical approach detect HPV and infect (the part testing result is seen Fig. 3), comparison with DIGENE HC2 method, and carry out dna sequence analysis and identify the HPV type, with the validity (the results are shown in Table 2) of identifying detection method clinical diagnosis of the present invention.

Detection/identified gene type result that table 2. adopts the method for determined dna sequence as a result of kit of the present invention and contrast clinical diagnosis and DIGENE HC2 method to obtain compares.

Sample number into spectrum	Digene HC2 result	Fluorescent PCR testing result of the present invention	The HPV type
				1026	-	+	HPV45
1038	-	+	HPV31
				1049	-	+	HPV58
11	+	+	HPV58
				18	+	+	HPV59
19	-	+	HPV16
				32	-	+	HPV68
43	+	+	HPV68
				23521	+	+	HPV18
1026	+	+	HPV45
				1038	-	+	HPV31
1149	-	+	HPV58
				1137	+	+	HPV18
7272	+	+	HPV18
				7295	+	+	HPV68
7326	+	+	HPV16
				7297	+	+	HPV16
7425	+	+	HPV16
				7416	+	+	HPV16
7417	+	+	HPV16
				7328	+	+	HPV18
7354	+	+	HPV18

7277	+	+	HPV18
				7289	+	+	HPV18
7404	+	+	HPV31
				7308	+	+	HPV33
7431	+	+	HPV39
				7444	+	+	HPV45
7392	+	+	HPV45
				7437	+	+	HPV45
7443	+	+	HPV52
				7403	+	+	HPV56
7326	+	+	HPV16
				7297	+	+	HPV16
7425	+	+	HPV16
				7416	+	+	HPV16
7417	+	+	HPV16
				7364	+	+	HPV16
7390	+	+	HPV16
				7272	+	+	HPV18
7328	+	+	HPV18
				7354	+	+	HPV18
7277	+	+	HPV18
				7289	+	+	HPV18
7404	+	+	HPV31
				7308	+	+	HPV33
7431	+	+	HPV39
				7444	+	+	HPV45
7392	+	+	HPV45
				7437	+	+	HPV45
7434	+	-	HPV45
				7295	+	+	HPV68
7314	+	+	HPV68
				7318	+	+	HPV52
7333	+	+	HPV58
				7336	+	+	HPV51
7343	+	+	HPV68
				7278	+	+	HPV68
7298	+	+	HPV59
				7368	+	+	HPV45
7408	+	+	HPV18
				7410	+	+	HPV31
7415	+	+	HPV58
				7441	+	+	HPV68
7445	+	+	HPV45
				7449	+	+	HPV52

7366	+	+	HPV58
				7372	+	+	HPV52
7385	+	+	HPV58
				7393	+	+	HPV18，42
7397	+	+	HPV51
				7402	+	+	HPV18
7405	+	+	HPV45
				7426	+	+	HPV58
7363	+	+	HPV30，16
				1029	-	+	HPV66
1035	-	+	HPV66
				23087	-	+	HPV53
1029	-	+	HPV66
				1035	-	+	HPV66
7352	-	+	HPV66
				7283	-	+	HPV66
7317	-	+	HPV66
				7414	-	+	HPV66
7442	-	+	HPV66
				7401	-	+	HPV66
7352	-	+	HPV66
				7283	-	+	HPV66
7317	-	+	HPV66
				7414	-	+	HPV66
7435	-	+	HPV67
				7442	-	+	HPV66
7401	-	+	HPV66
				2010	-	+	HPV67
80054	-	+	HPV66
				47192	-	+	HPV16
98600	-	+	-
				81196	-	+	-
75962	-	+	-
				77770	+	+	HPV45
90611	+	-	HPV45
				90746	+	-	HPV45

In the table 2, compare with DigeneHC2 reagent, the present invention can detect 13 kinds of high-risk HPV types high-risk HPV in addition of DigeneHC2 report.To the sequencing result of 102 routine samples, detect the result relatively with this kit, accuracy rate of the present invention is 95.1%.Fluorescent polyase chain reaction (PCR) kit that diagnosing human papillomavirus of the present invention (HPV) infects, testing process simple, fast can detect the several genes type easily, has more advantages with other correlation technique than existing type specificity PCR.Can improve more extensive case research.FDA (Food andDrug Administration) confirms: be used for the recent utilization rate rising of Hybrid Capture kit that quick diagnosis HPV infects.It is reported: infect for detecting and distinguish high or low risk HPV.Its accuracy rate is 98%, and analytical approach of the present invention is compared the advantage of competitive efficient and extra identified gene type with Hybrid Capture kit.The above demonstration: use HPV fluorescence detection reagent kit of the present invention to be better than the method for tradition diagnosis HPV in many aspects in diagnosis HPV infection method.

Below clear interpretation explanation: a kind of PCR (PCR) fluorescence detection method that is used to diagnose high-risk-type human papillomavirus (HPV) infection of the present invention, and utilize kit that this method makes to detect in patient's body HPV genotype in the isolated DNA sample, infect to diagnose one group of high-risk HPV.Kit of the present invention be a kind of can be easy, accurately detect the instrument that HPV infects and identify the HPV infection type, for the early diagnosis of cervix cancer, prevention and treatment have effect very much.

Sequence table detects primer and the sequence table of TAQMAN-MGB probe oligonucleotides of high-risk HPV

Sequence numbering	The sequence title	Oligonucleotide sequence (5 '-3 ')
			SEQ ID NO.1	HPV16 forward primer 1	CCTCTGATGCCCAAATATTCAATAAACCT
SEQ ID NO.2	HPV16 forward primer 2	CCTCTGATGCCCAAATATTCAATAAACC
			SEQ ID NO.3	HPV16 forward primer 3	CCTCTGATGCCCAAATATTCAATAAAC
SEQ ID NO.4	HPV16 forward primer 4	CCTCTGATGCCCAAATATTCAATAAA
			SEQ ID NO.5	HPV16 forward primer 5	CCTCTGATGCCCAAATATTCAATAA
SEQ ID NO.6	HPV18 forward primer 1	CCTCTGACTCCCAGTTGTTTAATAAACCA
			SEQ ID NO.7	HPV18 forward primer 2	CCTCTGACTCCCAGTTGTTTAATAAACC
SEQ ID NO.8	HPV18 forward primer 3	CCTCTGACTCCCAGTTGTTTAATAAAC
			SEQ ID NO.9	HPV18 forward primer 4	CCTCTGACTCCCAGTTGTTTAATAAA
SEQ ID NO.10	HPV18 forward primer 5	CCTCTGACTCCCAGTTGTTTAATAA
			SEQ ID NO.11	HPV31 forward primer 1	CTTCAGATGCACAAATTTTTAATAAACCA
SEQ ID NO.12	HPV31 forward primer 2	CTTCAGATGCACAAATTTTTAATAAACC
			SEQ ID NO.13	HPV31 forward primer 3	CTTCAGATGCACAAATTTTTAATAAAC
SEQ ID NO.14	HPV31 forward primer 4	CTTCAGATGCACAAATTTTTAATAAA
			SEQ ID NO.15	HPV31 forward primer 5	CTTCAGATGCACAAATTTTTAATAA
SEQ ID NO.16	HPV33 forward primer 1	CTTCCGAATCTCAGTTATTTAATAAGCCA
			SEQ ID NO.17	HPV33 forward primer 2	CTTCCGAATCTCAGTTATTTAATAAGCC
SEQ ID NO.18	HPV33 forward primer 3	CTTCCGAATCTCAGTTATTTAATAAGC
			SEQ ID NO.19	HPV33 forward primer 4	CTTCCGAATCTCAGTTATTTAATAAG
SEQ ID NO.20	HPV33 forward primer 5	CTTCCGAATCTCAGTTATTTAATAA
			SEQ ID NO.21	HPV35 forward primer 1	CCTCCGATGCACAAATATTTAATAAACCT
SEQ ID NO.22	HPV35 forward primer 2	CCTCCGATGCACAAATATTTAATAAACC
			SEQ ID NO.23	HPV35 forward primer 3	CCTCCGATGCACAAATATTTAATAAAC
SEQ ID NO.24	HPV35 forward primer 4	CCTCCGATGCACAAATATTTAATAAA
			SEQ ID NO.25	HPV35 forward primer 5	CCTCCGATGCACAAATATTTAATAA
SEQ ID NO.26	HPV39 forward primer 1	CCTCTGATTCCCAGTTATTTAATAAGCCT
			SEQ ID NO.27	HPV39 forward primer 2	CCTCTGATTCCCAGTTATTTAATAAGCC
SEQ ID NO.28	HPV39 forward primer 3	CCTCTGATTCCCAGTTATTTAATAAGC
			SEQ ID NO.29	HPV39 forward primer 4	CCTCTGATTCCCAGTTATTTAATAAG
SEQ ID NO.30	HPV39 forward primer 5	CCTCTGATTCCCAGTTATTTAATAA
			SEQ ID NO.31	HPV45 forward primer 1	CTTCTGATTCTCAATTATTTAATAAGCCA
SEQ ID NO.32	HPV45 forward primer 2	CTTCTGATTCTCAATTATTTAATAAGCC
			SEQ ID NO.33	HPV45 forward primer 3	CTTCTGATTCTCAATTATTTAATAAGC
SEQ ID NO.34	HPV45 forward primer 4	CTTCTGATTCTCAATTATTTAATAAG
			SEQ ID NO.35	HPV45 forward primer 5	CTTCTGATTCTCAATTATTTAATAA
SEQ ID NO.36	HPV51 forward primer 1	CATCTGATTCTCAAATTTTTAATAAGCCT
			SEQ ID NO.37	HPV51 forward primer 2	CATCTGATTCTCAAATTTTTAATAAGCC
SEQ ID NO.38	HPV51 forward primer 3	CATCTGATTCTCAAATTTTTAATAAGC
			SEQ ID NO.39	HPV51 forward primer 4	CATCTGATTCTCAAATTTTTAATAAG
SEQ ID NO.40	HPV51 forward primer 5	CATCTGATTCTCAAATTTTTAATAA
			SEQ ID NO.41	HPV52 forward primer 1	CCTCAGAATCCCAATTATTTAATAAACCG

SEQ ID NO.42	HPV52 forward primer 2	CCTCAGAATCCCAATTATTTAATAAACC
			SEQ ID NO.43	HPV52 forward primer 3	CCTCAGAATCCCAATTATTTAATAAAC
SEQ ID NO.44	HPV52 forward primer 4	CCTCAGAATCCCAATTATTTAATAAA
			SEQ ID NO.45	HPV52 forward primer 5	CCTCAGAATCCCAATTATTTAATAA
SEQ ID NO.46	HPV56 forward primer 1	CGTCTGAGGCACAGTTATTTAATAAACCT
			SEQ ID NO.47	HPV56 forward primer 2	CGTCTGAGGCACAGTTATTTAATAAACC
SEQ ID NO.48	HPV56 forward primer 3	CGTCTGAGGCACAGTTATTTAATAAAC
			SEQ ID NO.49	HPV56 forward primer 4	CGTCTGAGGCACAGTTATTTAATAAA
SEQ ID NO.50	HPV56 forward primer 5	CGTCTGAGGCACAGTTATTTAATAA
			SEQ ID NO.51	HPB58 forward primer 1	CCTCAGAATCACAATTATTTAATAAGCCT
SEQ ID NO.52	HPB58 forward primer 2	CCTCAGAATCACAATTATTTAATAAGCC
			SEQ ID NO.53	HPB58 forward primer 3	CCTCAGAATCACAATTATTTAATAAGC
SEQ ID NO.54	HPB58 forward primer 4	CCTCAGAATCACAATTATTTAATAAG
			SEQ ID NO.55	HPB58 forward primer 5	CCTCAGAATCACAATTATTTAATAA
SEQ ID NO.56	HPB59 forward primer 1	CTTCTGATTCACAATTATTTAATAAACCA
			SEQ ID NO.57	HPB59 forward primer 2	CTTCTGATTCACAATTATTTAATAAACC
SEQ ID NO.58	HPB59 forward primer 3	CTTCTGATTCACAATTATTTAATAAAC
			SEQ ID NO.59	HPB59 forward primer 4	CTTCTGATTCACAATTATTTAATAAA
SEQ ID NO.60	HPB59 forward primer 5	CTTCTGATTCACAATTATTTAATAA
			SEQ ID NO.61	HPV66 forward primer 1	CCTCTGAGGCCCAATTATTTAATAAACCT
SEQ ID NO.62	HPV66 forward primer 2	CCTCTGAGGCCCAATTATTTAATAAACC
			SEQ ID NO.63	HPV66 forward primer 3	CCTCTGAGGCCCAATTATTTAATAAAC
SEQ ID NO.64	HPV66 forward primer 4	CCTCTGAGGCCCAATTATTTAATAAA
			SEQ ID NO.65	HPV66 forward primer 5	CCTCTGAGGCCCAATTATTTAATAA
SEQ ID NO.66	HPV67 forward primer 1	CCTCAGAATCTCAATTATTTAATAAACCA
			SEQ ID NO.67	HPV67 forward primer 2	CCTCAGAATCTCAATTATTTAATAAACC
SEQ ID NO.68	HPV67 forward primer 3	CCTCAGAATCTCAATTATTTAATAAAC
			SEQ ID NO.69	HPV67 forward primer 4	CCTCAGAATCTCAATTATTTAATAAA
SEQ ID NO.70	HPV67 forward primer 5	CCTCAGAATCTCAATTATTTAATAA
			SEQ ID NO.71	HPV68 forward primer 1	CCTCAGACTCCCAGTTATTTAACAAGCCC
SEQ ID NO.72	HPV68 forward primer 2	CCTCAGACTCCCAGTTATTTAACAAGCC
			SEQ ID NO.73	HPV68 forward primer 3	CCTCAGACTCCCAGTTATTTAACAAGC
SEQ ID NO.74	HPV68 forward primer 4	CCTCAGACTCCCAGTTATTTAACAAG
			SEQ ID NO.75	HPV68 forward primer 5	CCTCAGACTCCCAGTTATTTAACAA
SEQ ID NO.76	HPV69 forward primer 1	CCTCTGATGCTCAATTGTTTAATAAACCT
			SEQ ID NO.77	HPV69 forward primer 2	CCTCTGATGCTCAATTGTTTAATAAACC
SEQ ID NO.78	HPV69 forward primer 3	CCTCTGATGCTCAATTGTTTAATAAAC
			SEQ ID NO.79	HPV69 forward primer 4	CCTCTGATGCTCAATTGTTTAATAAA
SEQ ID NO.80	HPV69 forward primer 5	CCTCTGATGCTCAATTGTTTAATAA
			SEQ ID NO.81	HPV73 forward primer 1	CTTCAGATGCACAGTTGTTTAATAAACCT
SEQ ID NO.82	HPV73 forward primer 2	CTTCAGATGCACAGTTGTTTAATAAACC
			SEQ ID NO.83	HPV73 forward primer 3	CTTCAGATGCACAGTTGTTTAATAAAC
SEQ ID NO.84	HPV73 forward primer 4	CTTCAGATGCACAGTTGTTTAATAAA
			SEQ ID NO.85	HPV73 forward primer 5	CTTCAGATGCACAGTTGTTTAATAA

SEQ ID NO.86	HPV82 forward primer 1	CCTCTGATTCTCAGATTTTTAATAAGCCT
			SEQ ID NO.87	HPV82 forward primer 2	CCTCTGATTCTCAGATTTTTAATAAGCC
SEQ ID NO.88	HPV82 forward primer 3	CCTCTGATTCTCAGATTTTTAATAAGC
			SEQ ID NO.89	HPV82 forward primer 4	CCTCTGATTCTCAGATTTTTAATAAG
SEQ ID NO.90	HPV82 forward primer 5	CCTCTGATTCTCAGATTTTTAATAA
			SEQ ID NO.91	HPV16 reverse primer 1	TATTCCTCCCCATGTCGTAGGTACTCC
SEQ ID NO.92	HPV16 reverse primer 2	TATTCCTCCCCATGTCGTAGGTACTC
			SEQ ID NO.93	HPV16 reverse primer 3	TATTCCTCCCCATGTCGTAGGTACT
SEQ ID NO.94	HPV16 reverse primer 4	TATTCCTCCCCATGTCGTAGGTAC
			SEQ ID NO.95	HPV16 reverse primer 5	TATTCCTCCCCATGTCGTAGGTA
SEQ ID NO.96	HPV18 reverse primer 1	TATTCCTCAACATGTCTGCTATACTGC
			SEQ ID NO.97	HPV18 reverse primer 2	TATTCCTCAACATGTCTGCTATACTG
SEQ ID NO.98	HPV18 reverse primer 3	TATTCCTCAACATGTCTGCTATACT
			SEQ ID NO.99	HPV18 reverse primer 4	TATTCCTCAACATGTCTGCTATAC
SEQ ID NO.100	HPV18 reverse primer 5	TATTCCTCAACATGTCTGCTATAC
			SEQ ID NO.101	HPV31 reverse primer 1	AATTCCTCACCATGTCTTAAATACTCT
SEQ ID NO.102	HPV31 reverse primer 2	AATTCCTCACCATGTCTTAAATACTC
			SEQ ID NO.103	HPV31 reverse primer 3	AATTCCTCACCATGTCTTAAATACT
SEQ ID NO.104	HPV31 reverse primer 4	AATTCCTCACCATGTCTTAAATAC
			SEQ ID NO.105	HPV31 reverse primer 5	AATTCCTCACCATGTCTTAAATA
SEQ ID NO.106	HPV33 reverse primer 1	TATTCTTCAACATGTCTTATATATTCT
			SEQ ID NO.107	HPV33 reverse primer 2	TATTCTTCAACATGTCTTATATATTC
SEQ ID NO.108	HPV33 reverse primer 3	TATTCTTCAACATGTCTTATATATT
			SEQ ID NO.109	HPV33 reverse primer 4	TATTCTTCAACATGTCTTATATAT
SEQ ID NO.110	HPV33 reverse primer 5	TATTCTTCAACATGTCTTATATA
			SEQ ID NO.111	HPV35 reverse primer 1	TATTCTTCACCATGCCTTAAATATTCC
SEQ ID NO.112	HPV35 reverse primer 2	TATTCTTCACCATGCCTTAAATATTC
			SEQ ID NO.113	HPV35 reverse primer 3	TATTCTTCACCATGCCTTAAATATT
SEQ ID NO.114	HPV35 reverse primer 4	TATTCTTCACCATGCCTTAAATAT
			SEQ ID NO.115	HPV35 reverse primer 5	TATTCTTCACCATGCCTTAAATA
SEQ ID NO.116	HPV39 reverse primer 1	TACTCCTCCACGTGCCTGGTATATTCC
			SEQ ID NO.117	HPV39 reverse primer 2	TACTCCTCCACGTGCCTGGTATATTC
SEQ ID NO.118	HPV39 reverse primer 3	TACTCCTCCACGTGCCTGGTATATT
			SEQ ID NO.119	HPV39 reverse primer 4	TACTCCTCCACGTGCCTGGTATAT
SEQ ID NO.120	HPV39 reverse primer 5	TACTCCTCCACGTGCCTGGTATA
			SEQ ID NO.121	HPV45 reverse primer 1	TATTCCTCCACATGTCTACTATACTGC
SEQ ID NO.122	HPV45 reverse primer 2	TATTCCTCCACATGTCTACTATACTG
			SEQ ID NO.123	HPV45 reverse primer 3	TATTCCTCCACATGTCTACTATACT
SEQ ID NO.124	HPV45 reverse primer 4	TATTCCTCCACATGTCTACTATAC
			SEQ ID NO.125	HPV45 reverse primer 5	TATTCCTCCACATGTCTACTATA
SEQ ID NO.126	HPV51 reverse primer 1	TACTCTTCCCCATGCCTAATATATTGC
			SEQ ID NO.127	HPV51 reverse primer 2	TACTCTTCCCCATGCCTAATATATTG
SEQ ID NO.128	HPVS1 reverse primer 3	TACTCTTCCCCATGCCTAATATATT
			SEQ ID NO.129	HPV51 reverse primer 4	TACTCTTCCCCATGCCTAATATAT

SEQ ID NO.130	HPV51 reverse primer 5	TACTCTTCCCCATGCCTAATATA
			SEQ ID NO.131	HPV52 reverse primer 1	AATTCCTCGCCATGACGAAGGTATTCC
SEQ ID NO.132	HPV52 reverse primer 2	AATTCCTCGCCATGACGAAGGTATTC
			SEQ ID NO.133	HPV52 reverse primer 3	AATTCCTCGCCATGACGAAGGTATT
SEQ ID NO.134	HPV52 reverse primer 4	AATTCCTCGCCATGACGAAGGTAT
			SEQ ID NO.135	HPV52 reverse primer 5	AATTCCTCGCCATGACGAAGGTA
SEQ ID NO.136	HPV56 reverse primer 1	TATTCCTCCACATGTCTAAGGTACTGA
			SEQ ID NO.137	HPV56 reverse primer 2	TATTCCTCCACATGTCTAAGGTACTG
SEQ ID NO.138	HPV56 reverse primer 3	TATTCCTCCACATGTCTAAGGTACT
			SEQ ID NO.139	HPV56 reverse primer 4	TATTCCTCCACATGTCTAAGGTAC
SEQ ID NO.140	HPV56 reverse primer 5	TATTCCTCCACATGTCTAAGGTA
			SEQ ID NO.141	HPB58 reverse primer 1	TATTCTTCAACATGACGTACATATTCC
SEQ ID NO.142	HPB58 reverse primer 2	TATTCTTCAACATGACGTACATATTC
			SEQ ID NO.143	HPB58 reverse primer 3	TATTCTTCAACATGACGTACATATT
SEQ ID NO.144	HPB58 reverse primer 4	TATTCTTCAACATGACGTACATAT
			SEQ ID NO.145	HPB58 reverse primer 5	TATTCTTCAACATGACGTACATA
SEQ ID NO.146	HPB59 reverse primer 1	AATTCCTCCACATGTCTGGCATATTCT
			SEQ ID NO.147	HPB59 reverse primer 2	AATTCCTCCACATGTCTGGCATATTC
SEQ ID NO.148	HPB59 reverse primer 3	AATTCCTCCACATGTCTGGCATATT
			SEQ ID NO.149	HPB59 reverse primer 4	AATTCCTCCACATGTCTGGCATAT
SEQ ID NO.150	HPB59 reverse primer 5	AATTCCTCCACATGTCTGGCATA
			SEQ ID NO.151	HPV66 reverse primer 1	TATTCCTCCACATGGCGAAGGTATTGA
SEQ ID NO.152	HPV66 reverse primer 2	TATTCCTCCACATGGCGAAGGTATTG
			SEQ ID NO.153	HPV66 reverse primer 3	TATTCCTCCACATGGCGAAGGTATT
SEQ ID NO.154	HPV66 reverse primer 4	TATTCCTCCACATGGCGAAGGTAT
			SEQ ID NO.155	HPV66 reverse primer 5	TATTCCTCCACATGGCGAAGGTA
SEQ ID NO.156	HPV67 reverse primer 1	TATTCTTCCACATGTCTAAGGTATTCC
			SEQ ID NO.157	HPV67 reverse primer 2	TATTCTTCCACATGTCTAAGGTATTC
SEQ ID NO.158	HPV67 reverse primer 3	TATTCTTCCACATGTCTAAGGTATT
			SEQ ID NO.159	HPV67 reverse primer 4	TATTCTTCCACATGTCTAAGGTAT
SEQ ID NO.160	HPV67 reverse primer 5	TATTCTTCCACATGTCTAAGGTA
			SEQ ID NO.161	HPV68 reverse primer 1	TATTCCTCAACATGCCTAATATATTCC
SEQ ID NO.162	HPV68 reverse primer 2	TATTCCTCAACATGCCTAATATATTC
			SEQ ID NO.163	HPV68 reverse primer 3	TATTCCTCAACATGCCTAATATATT
SEQ ID NO.164	HPV68 reverse primer 4	TATTCCTCAACATGCCTAATATAT
			SEQ ID NO.165	HPV68 reverse primer 5	TATTCCTCAACATGCCTAATATA
SEQ ID NO.166	HPV69 reverse primer 1	TATTCCTCACCATGCCTTATAAACTGC
			SEQ ID NO.167	HPV69 reverse primer 2	TATTCCTCACCATGCCTTATAAACTG
SEQ ID NO.168	HPV69 reverse primer 3	TATTCCTCACCATGCCTTATAAACT
			SEQ ID NO.169	HPV69 reverse primer 4	TATTCCTCACCATGCCTTATAAAC
SEQ ID NO.170	HPV69 reverse primer 5	TATTCCTCACCATGCCTTATAAA
			SEQ ID NO.171	HPV73 reverse primer 1	AACTCTTCTGCATGTCTTAAATATTCC
SEQ ID NO.172	HPV73 reverse primer 2	AACTCTTCTGCATGTCTTAAATATTC
			SEQ ID NO.173	HPV73 reverse primer 3	AACTCTTCTGCATGTCTTAAATATT

SEQ ID NO.174	HPV73 reverse primer 4	AACTCTTCTGCATGTCTTAAATAT
			SEQ ID NO.175	HPV73 reverse primer 5	AACTCTTCTGCATGTCTTAAATA
SEQ ID NO.176	HPV82 reverse primer 1	TATTCTTCCCCATGCCTAATGTACTGC
			SEQ ID NO.177	HPV82 reverse primer 2	TATTCTTCCCCATGCCTAATGTACTG
SEQ ID NO.178	HPV82 reverse primer 3	TATTCTTCCCCATGCCTAATGTACT
			SEQ ID NO.179	HPV82 reverse primer 4	TATTCTTCCCCATGCCTAATGTAC
SEQ ID NO.180	HPV82 reverse primer 5	TATTCTTCCCCATGCCTAATGTA
			SEQ ID NO.181	HPV16 probe 1	TTTGTTACTGTTGTTGATACTACACGC
SEQ ID NO.182	HPV16 probe 2	TTTGTTACTGTTGTTGATACTACACG
			SEQ ID NO.183	HPV16 probe 3	TTTGTTACTGTTGTTGATACTACAC
SEQ ID NO.184	HPV16 probe 4	TTTGTTACTGTTGTTGATACTACA
			SEQ ID NO.185	HPV16 probe 5	TTTGTTACTGTTGTTGATACTAC
SEQ ID NO.186	HPV18 probe 1	TTTGTTACTGTGGTAGATACCACTCCC
			SEQ ID NO.187	HPV18 probe 2	TTTGTTACTGTGGTAGATACCACTCC
SEQ ID NO.188	HPV18 probe 3	TTTGTTACTGTGGTAGATACCACTC
			SEQ ID NO.189	HPV18 probe 4	TTTGTTACTGTGGTAGATACCACT
SEQ ID NO.190	HPV18 probe 5	TTTGTTACTGTGGTAGATACCAC
			SEQ ID NO.191	HPV31 probe 1	TTTGTTACTGTGGTAGATACCACACGT
SEQ ID NO.192	HPV31 probe 2	TTTGTTACTGTGGTAGATACCACACG
			SEQ ID NO.193	HPV31 probe 3	TTTGTTACTGTGGTAGATACCACAC
SEQ ID NO.194	HPV31 probe 4	TTTGTTACTGTGGTAGATACCACA
			SEQ ID NO.195	HPV31 probe 5	TTTGTTACTGTGGTAGATACCAC
SEQ ID NO.196	HPV33 probe 1	TTTGTTACTGTGGTAGATACCACTCGC
			SEQ ID NO.197	HPV33 probe 2	TTTGTTACTGTGGTAGATACCACTCG
SEQ ID NO.198	HPV33 probe 3	TTTGTTACTGTGGTAGATACCAACTC
			SEQ ID NO.199	HPV33 probe 4	TTTGTTACTGTGGTAGATACCACT
SEQ ID NO.200	HPV33 probe 5	TTTGTTACTGTGGTAGATACCAC
			SEQ ID NO.201	HPV35 probe 1	TTTGTTACTGTAGTTGATACAACCCGT
SEQ ID NO.202	HPV35 probe 2	TTTGTTACTGTAGTTGATACAACCCG
			SEQ ID NO.203	HPV35 probe 3	TTTGTTACTGTAGTTGATACAACCC
SEQ ID NO.204	HPV35 probe 4	TTTGTTACTGTAGTTGATACAACC
			SEQ ID NO.205	HPV35 probe 5	TTTGTTACTGTAGTTGATACAAC
SEQ ID NO.206	HPV39 probe 1	TTTCTTACTGTTGTGGACACTACCCGT
			SEQ ID NO.207	HPV39 probe 2	TTTCTTACTGTTGTGGACACTACCCG
SEQ ID NO.208	HPV39 probe 3	TTTCTTACTGTTGTGGACACTACCC
			SEQ ID NO.209	HPV39 probe 4	TTTCTTACTGTTGTGGACACTACC
SEQ ID NO.210	HPV39 probe 5	TTTCTTACTGTTGTGGACACTAC
			SEQ ID NO.211	HPV45 probe 1	TTTGTTACTGTAGTGGACACTACCCGC
SEQ ID NO.212	HPV45 probe 2	TTTGTTACTGTAGTGGACACTACCCG
			SEQ ID NO.213	HPV45 probe 3	TTTGTTACTGTAGTGGACACTACCC
SEQ ID NO.214	HPV45 probe 4	TTTGTTACTGTAGTGGACACTACC
			SEQ ID NO.215	HPV45 probe 5	TTTGTTACTGTAGTGGACACTAC
SEQ ID NO.216	HPV51 probe 1	TTTATTACCTGTGTTGATACTACCAGA
			SEQ ID NO.217	HPV51 probe 2	TTTATTACCTGTGTTGATACTACCAG

SEQ ID NO.218	HPV51 probe 3	TTTATTACCTGTGTTGATACTACCA
			SEQ ID NO.219	HPV51 probe 4	TTTATTACCTGTGTTGATACTACC
SEQ ID NO.220	HPV51 probe 5	TTTATTACCTGTGTTGATACTAC
			SEQ ID NO.221	HPV52 probe 1	TTTGTCACAGTTGTGGATACCACTCGT
SEQ ID NO.222	HPV52 probe 2	TTTGTCACAGTTGTGGATACCACTCG
			SEQID NO.223	HPV52 probe 3	TTTGTCACAGTTGTGGATACCACTC
SEQ ID NO.224	HPV52 probe 4	TTTGTCACAGTTGTGGATACCACT
			SEQ ID NO.225	HPV52 probe 5	TTTGTCACAGTTGTGGATACCAC
SEQ ID NO.226	HPV56 probe 1	TTTGTTACTGTAGTAGATACTACTAGA
			SEQ ID NO.227	HPV56 probe 2	TTTGTTACTGTAGTAGATACTACTAG
SEQ ID NO.228	HPV56 probe 3	TTTGTTACTGTAGTAGATACTACTA
			SEQ ID NO.229	HPV56 probe 4	TTTGTTACTGTAGTAGATACTACT
SEQ ID NO.230	HPV56 probe 5	TTTGTTACTGTAGTAGATACTAC
			SEQ ID NO.231	HPB58 probe 1	TTTGTTACCGTGGTTGATACCACTGCT
SEQ ID NO.232	HPB58 probe 2	TTTGTTACCGTGGTTGATACCACTGC
			SEQ ID NO.233	HPB58 probe 3	TTTGTTACCGTGGTTGATACCACTG
SEQ ID NO.234	HPB58 probe 4	TTTGTTACCGTGGTTGATACCACT
			SEQ ID NO.235	HPB58 probe 5	TTTGTTACCGTGGTTGATACCAC
SEQ ID NO.236	HPB59 probe 1	TTTTTAACAGTTGTAGATACTACTCGC
			SEQ ID NO.237	HPB59 probe 2	TTTTTAACAGTTGTAGATACTACTCG
SEQ ID NO.238	HPB59 probe 3	TTTTTAACAGTTGTAGATACTACTC
			SEQ ID NO.239	HPB59 probe 4	TTTTTAACAGTTGTAGATACTACT
SEQ ID NO.240	HPB59 probe 5	TTTTTAACAGTTGTAGATACTAC
			SEQ ID NO.241	HPV66 probe 1	TTTGTTACTGTTGTGGATACTACCAGA
SEQ ID NO.242	HPV66 probe 2	TTTGTTACTGTTGTGGATACTACCAG
			SEQ ID NO.243	HPV66 probe 3	TTTGTTACTGTTGTGGATACTACCA
SEQ ID NO.244	HPV66 probe 4	TTTGTTACTGTTGTGGATACTACC
			SEQ ID NO.245	HPV66 probe 5	TTTGTTACTGTTGTGGATACTAC
SEQ ID NO.246	HPV67 probe 1	TTTGTTACTGTTGTAGACACTACACGT
			SEQ ID NO.247	HPV67 probe 2	TTTGTTACTGTTGTAGACACTACACG
SEQ ID NO.248	HPV67 probe 3	TTTGTTACTGTTGTAGACACTACAC
			SEQ ID NO.249	HPV67 probe 4	TTTGTTACTGTTGTAGACACTACA
SBQ ID NO.250	HPV67 probe 5	TTTGTTACTGTTGTAGACACTAC
			SEQ ID NO.251	HPV68 probe 1	TTTCTTACTGTTGTGGATACCACTCGC
SEQ ID NO.252	HPV68 probe 2	TTTCTTACTGTTGTGGATACCACTCG
			SEQ ID NO.253	HPV68 probe 3	TTTCTTACTGTTGTGGATACCACTC
SEQ ID NO.254	HPV68 probe 4	TTTCTTACTGTTGTGGATACCACT
			SEQ ID NO.255	HPV68 probe 5	TTTCTTACTGTTGTGGATACCAC
SEQ ID NO.256	HPV69 probe 1	TTTGTTACTTGTGTAGATACTACCCGC
			SEQ ID NO.257	HPV69 probe 2	TTTGTTACTTGTGTAGATACTACCCG
SEQ ID NO.258	HPV69 probe 3	TTTGTTACTTGTGTAGATACTACCC
			SEQ ID NO.259	HPV69 probe 4	TTTGTTACTTGTGTAGATACTACC
SEQ ID NO.260	HPV69 probe 5	TTTGTTACTTGTGTAGATACTAC
			SEQ ID NO.261	HPV73 probe 1	TTTTTAACTGTTGTAGATACTACTAGA

SEQ ID NO.262	HPV73 probe 2	TTTTTAACTGTTGTAGATACTACTAG
			SEQ ID NO.263	HPV73 probe 3	TTTTTAACTGTTGTAGATACTACTA
SEQ ID NO.264	HPV73 probe 4	TTTTTAACTGTTGTAGATACTACT
			SEQ ID NO.265	HPV73 probe 5	TTTTTAACTGTTGTAGATACTAC
SEQ ID NO.266	HPV82 probe 1	TTTATTACTTGTGTTGACACTACTAAA
			SEQ ID NO.267	HPV82 probe 2	TTTATTACTTGTGTTGACACTACTAA
SEQ ID NO.268	HPV82 probe 3	TTTATTACTTGTGTTGACACTACTA
			SEQ ID NO.269	HPV82 probe 4	TTTATTACTTGTGTTGACACTACT
SEQ ID NO.270	HPV82 probe 5	TTTATTACTTGTGTTGACACTAC

Claims (5)

1. the method for the fluorescent polyase chain reaction (PCR) that infects of a diagnosing human papillomavirus (HPV) may further comprise the steps:

(1) oligonucleotide sequence with HPV DNA complementation is used for the primer that the pcr amplification clinical sample obtains DNA; Comprise one group of amplified production that has target sequence (target gene segment) at the primer sequence and the polymerase of each type of HPV, here, the primer sequence of each type comprises: (a) forward primer of energy and a kind of HPV type gene pairs first site hybridization of target sequence upstream of answering, (b) reverse primer of second the site hybridization in the target sequence downstream that energy is corresponding with being positioned at a kind of HPV type.Forward primer and reverse primer can be the sequences that the sequence table of this instructions is listed.

(2) be used to detect one group of oligonucleotide sequence probe of fluorescence labeling of HPV, this probe can and in target sequence the HPV sequence hybridization between the hybridization of above-mentioned first and second sites.Probe can be the sequence that the sequence table of this instructions is listed.

(3) sample of obtaining is carried out pcr amplification, it is characterized in that with HPV DNA, the one or more HPV DNA target sequence fragments of amplification in the reaction system as template.

(4) adopt one or more different fluorescence that one or more different probes are carried out mark,, determine whether infect HPV in the sample by detecting the variation of one or more different fluorescence signals in the PCR reaction system, and the content of HPV.

2. fluoroscopic examination according to claim 1, it is characterized in that the described fluorescence probe that is used for compound detection can be double-tagging probe (double-labeled probe), molecular beacon (molecular beacon), in the NE BY ENERGY TRANSFER fluorescence probe (Fluorescent resonance energytransfer) one or more, the fluorescence that is used for label probe can be FAM, TET, JOE, ViC, HEX, ROXTAMPA, CY3, CY3.5, CY5, CY5.5, OregonGreenTM, CALRedTM, Red 640, among the Texas Red one or two or more kinds.3 ' end of probe can be to have DNA ditch compound (MGB, Minor Groove Bingding) mark.

3. detect according to claim 1,2 described HPV FLuorescents, it is characterized in that, adopting single stage method or two-step approach is template with HPV DNA, and viral specific fragment in the amplification sample carries out the quantitative or qualitative fluoroscopic examination of HPV simultaneously under the effect of archaeal dna polymerase.The sample of described extraction is the sample that uterine cervix obtains.

4. according to claim 3, this patent is a kind of polymerase chain reaction method of multi-primers, carries out following reaction at the template DNA chain simultaneously by primer more than 2 pairs:

(1) template sex change is characterized in that, the template denaturation temperature is at 92-97 ℃;

(2) primer annealing is characterized in that, the primer annealing temperature is at 50-65 ℃;

(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down, it is characterized in that the temperature of extending synthetic complementary dna chain is at 56-65 ℃;

(4) amplified reaction is carried out in (1) one (3) circulation set by step, it is characterized in that, the period that amplified reaction is carried out in circulation is 30-45.

5. polymerase chain reaction method according to claim 1, it is characterized in that the reaction in primer to for 2-18 to multi-primers, at 18 kinds of HPV genotype, and the application in HPV genotype detection kit, it is characterized in that each product detects more than 2 kinds DNA (comprising HPV16,18,31,33,35,39,45,51,52,56,58,59,66,67,68,69,73,82 types) to 18 kinds of HPV types simultaneously by fluorescence signal in a sample hose.

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