CN105603121A - Method, oligonucleotide and kit for detecting high-risk HPV (human papilloma viruses) - Google Patents

Method, oligonucleotide and kit for detecting high-risk HPV (human papilloma viruses) Download PDF

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CN105603121A
CN105603121A CN201610027771.9A CN201610027771A CN105603121A CN 105603121 A CN105603121 A CN 105603121A CN 201610027771 A CN201610027771 A CN 201610027771A CN 105603121 A CN105603121 A CN 105603121A
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probe
primer
detection
base sequence
respectively seqidno
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CN105603121B (en
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刘学锋
张太松
吕娜
朱丽敏
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Acon Biotech Hangzhou Co Ltd
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Abstract

The invention provides a method, oligonucleotide and kit for detecting common high-risk HPV (human papilloma viruses). A fluorescence PCR (polymerase chain reaction) technology is adopted, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73 and 82 which possibly exist in a sample are subjected to initial detection and type identification. By the method, oligonucleotide and kit for detecting the common high-risk HPV, 18 high-risk types can be detected simultaneously, and the HPV 16 and 18 can be subjected to genetic typing simultaneously. On the basis of 18 types of high-risk HPV and affinity of other types on a phylogenetic tree, a high-risk HPV primer and a probe are designed, a background fluorescence value is reduced remarkably while detection accuracy, sensitivity and specificity are guaranteed, detection flux is increased remarkably, and detection cost is greatly reduced.

Description

For detection of method, oligonucleotides and the kit of high-risk HPV
Technical field
The invention belongs to bioscience and biological technical field, particularly a kind of detect 18 kinds common high-riskMethod, oligonucleotides and the kit of type HPV, adopt Fluorescence PCR assay, to existing in clinical sampleHPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and 82 carry out Preliminary detection and type qualification.
Background technology
HPV (humanpapillomavirus, HPV) is that a class can infect people's epidermis and mucous membraneThe small DNA virus of scaly epithelium, can cause the disease such as condyloma acuminatum and cervical carcinoma. The mankind are the unique of HPVHost. HPV genome is double-stranded cyclic DNA, is about 7900 pairs of bases (bp), containing 8 ORFs (openReadingframes, ORFs), formed by 3 gene regions, comprise early stage district (E district), late region (LDistrict) and noncoding region or upstream regulatory region. E district can be divided into E6, E7, E1, E2, E3, E4 in orderWith E5 totally 7 genes, wherein E6 and E7 are the Analyses of major carcinogens in mainstream genes of HPV, with the cell transformation merit of HPVCan be relevant with carcinogenicity. L district mainly contains L1 and two genes of L2.
Scientists has been found 120 kinds of above HPV hypotypes at present. Different HPV Subtypes cause differenceWhether pathology, can cause cancer according to it, and HPV hypotype can be divided into: high-risk HPV, and with cervical carcinoma and palaceThe generation of neck intraepithelial neoplasia (cin) (CINI/II/III) is relevant, common are HPV16,18,31,33,35,45, the hypotypes such as 51,52,56,58,29,68; Low risk HPV, general and condyloma acuminatum or low squamousUpper intracutaneous pathology is relevant, seldom causes infiltrating carcinoma, common are as HPV6,11,42,43, the hypotypes such as 44.About the division of high-risk HPV and low risk HPV, many mechanisms have all provided reference proposition, foundation in the worldThe achievement in research of WHO international cancer research institution (IARC) and other international organizations, by HPV16,18,31,33,35,39,45,51,52,56,58,59 and 68 totally 13 kinds of hypotypes classify high-risk HPV as, and willTotally 5 kinds of hypotypes of HPV26,53,66,73 and 82 classifies medium risk HPV as. In the time that reality detects, usually willMedium risk HPV hypotype is also attributed to high-risk HPV.
80% women can infect HPV virus in life, and virus meeting nature in 6～8 months is clear conventionallyRemove, only 25% can there is precancerous lesion. High-risk HPV repeatedly or persistent infection be regarded as nearly all palaceThe necessary condition of neck carcinogenesis. Existing result of study demonstration, cervical lesions degree is heavier, high-risk HPV senseThe rate of dying is higher; In CINI, CINII and CINIII patient, high-risk HPV infection rate is respectively 30%,55% and 65%, and in more than 99.8% cervical cancer patient body, can detect that high-risk HPV infects, HPVCan there is hardly cervical carcinoma in negative patient. In these high-risk HPVs, the most commonly HPV16, secondlyFor HPV18, the infection rate of these two kinds of high-risk HPV hypotypes is the highest, can reach 70%; If add HPV45 andHPV31, the infection rate of these 4 kinds of high-risk HPVs can reach 80% left and right. One, China is sent out across 7 cervical carcinomas19 hospitals of sick rate different regions, include altogether the relevant high-risk HPV of cervix cancer in China of 1244 cases inSubtype distribution (taking hospital as basis) multicenter study confirmation, cervical carcinoma and the uterine neck of Chinese zones of different are highDegree pathology is all that to infect HPV16/18 be main, and about 85% cervical carcinoma confirmed cases belong to this two kinds of hypotypes,And the infection rate of HPV16/18 and carcinogenic rate are all apparently higher than other high-risk HPVs (ChenWetal.Humanpapillomavirustype-distributionincervicalcancerinChina:theimportanceofHPV16and18.CancerCausesandControl,2009,20:1705-1713)。
In addition, there are some researches show the women of HPV16/18 of infection, no matter be recent or at a specified future date, made progress and beThe risk of height cervical lesions is all far away higher than other high-risk HPV. Based on this, U.S.'s colposcopy in 2013Looking into will be to cytology ASC-US (by uterine neck liquid base thin layer with the special proposition of cervix disease meeting of science (ASCCP)On the cytology that cytolgical examination is found without the change of the ASC of its meaning) negative, HPV is positiveThe more than 30 years old women of property carries out the detection of HPV16/18 Genotyping. Pathogenic due to different HPV hypotypesAnd the difference of prognosis, HPV16/18 being carried out to somatotype, will contribute to better High risk group to be carried out to riskMulti-zone supervision, finds to suffer from the normal result of cytology the people at highest risk of CIN and the ASC-US positive in timeIn result, need the crowd who more closely follows up a case by regular visits to.
Cervical carcinoma early symptom is also not obvious, has long, reversible precancerous lesion phase in evolution.Develop into cervical carcinoma from general precancerous lesions of uterine cervix and approximately needed for 10 years. If at precancerous stageCarry out medical intervention, cervical carcinoma cure rate can reach 98%. Therefore, high-risk HPV being carried out to examination is still at presentThe key of prevention cervical carcinoma.
Common high-risk HPV detection method has fluorescent PCR method, hybrid capture and PCR-reversal point at presentHybrid method etc. The shortcoming of hybrid capture is without interior mark, detection time long (generally needing 5-6 hour), spiritSensitivity is low by (3.5 × 105Copies/ml), complicated operation, detects genotype on the low side, and false positive rate is higher.Although PCR-reverse hybridization can detect many HPV hypotypes, its shortcoming is that detection time is long (extremelyNeed less 4 hours), testing cost is high, and detection sensitivity is low by (104Copies/ml), complicated operation, noCan meet the needs of clinical high flux examination.
Fluorescent PCR method has higher sensitivity and specificity, and can carry out real-time online inspection to amplified productionSurvey, can reduce detection time significantly, also avoid the generation of residual contamination simultaneously. Common fluorescent PCRMethod has SYBRGreenI dye method, two Probe Hybridization methods and Taqman sonde method etc. Wherein SYBRGreenI is owing to being non-saturable dye, and specificity, not as two Probe Hybridization methods and Taqman sonde method, must be passed throughObserve solubility curve and judge its specificity; And two probe method hybrid method cost is comparatively expensive.
On domestic market, existing many producers develop the product that detects high-risk HPV based on fluorescent PCR methodProduct, and be to detect 13,14 or 15 kind of high-risk HPV mostly, only have the minority producer to utilize fluorescence. simultaneouslyPCR method develop detect simultaneously 18 kinds of common high-risk HPVs (HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and 82) product, but, at theseIn product, have plenty of for every kind of high-risk HPV and design a pair of type specificity primer and a type specificity spyPin, this can sharply increase testing cost, and so the existence of multiprobe also can significantly increase background fluorescence signal simultaneously;What have still carries out multitube detection, and the consumption that this can significantly increase sample and detect reagent, increases testing cost,And can not carry out somatotype to HPV16 and HPV18; What is more, designs for different high-risk HPVsPrimer and/or probe are confined on same gene region, and base sequence difference is too little, can cause intercrossingAmplified reaction, produces false positive, can not distinguish exactly its concrete type.
Summary of the invention
For the deficiencies in the prior art part, the invention provides a kind of (especially normal for detection of high-risk HPV18 kinds of high-risk HPVs seeing, HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and 82) method, oligonucleotides and kit, described method, few coreThe primer that thuja acid and kit are related and probe are the chadograms by building high-risk HPV, based on these heightThe affinity of danger type HPV on chadogram, allows some high-risk HPVs share upstream or downstream primer and probe,Ensureing detection accuracy, sensitivity and specific while, significantly increase and detect flux, and greatly reduceTesting cost.
If the base sequence of the primer in the present invention and/or probe contains letter " Y ", " S ", " R " or " M ",Show that this primer and/or probe are degenerate primer or degeneracy probe, wherein Y represents C or T, and S represents GOr C, R represents A or G, M represents A or C.
The invention provides the oligonucleotides that detects high-risk HPV for fluorescent PCR, described oligonucleotides comprises:(1) for detection of primer and the probe of HPV16, its base sequence is respectively SEQIDNO:1～3; (2)For detection of primer and the probe of HPV18, its base sequence is respectively SEQIDNO:4～6.
Further, described oligonucleotides also comprises at least one in (3) and (4): (3) for detection ofThe primer of HPV31 and probe, its base sequence is respectively SEQIDNO:7,9 and 14; (4) for detection ofThe primer of HPV45 and probe, its base sequence is respectively SEQIDNO:15,16 and 19.
Further, described oligonucleotides also comprises at least one in (5)～(16): (5) are for inspectionPrimer and the probe of surveying HPV35, its base sequence is respectively SEQIDNO:8,9 and 14; (6) for inspectionPrimer and the probe of surveying HPV33, its base sequence is respectively SEQIDNO:10,13 and 14; (7) forThe primer and the probe that detect HPV52, its base sequence is respectively SEQIDNO:11,13 and 14; (8) useIn the primer and the probe that detect HPV58, its base sequence is respectively SEQIDNO:12～14; (9) forThe primer and the probe that detect HPV59, its base sequence is respectively SEQIDNO:17～19; (10) for inspectionPrimer and the probe of surveying HPV39 and HPV68, its base sequence is respectively SEQIDNO:20～22; (11)For detection of primer and the probe of HPV53, its base sequence is respectively SEQIDNO:23,24 and 28; (12)For detection of primer and the probe of HPV56, its base sequence is respectively SEQIDNO:25,26 and 28; (13)For detection of primer and the probe of HPV66, its base sequence is respectively SEQIDNO:25,27 and 28; (14)For detection of primer and the probe of HPV26, its base sequence is respectively SEQIDNO:29,31 and 32; (15)For detection of primer and the probe of HPV51 and HPV82, its base sequence is respectively SEQIDNO:30～32;(16) for detection of primer and the probe of HPV73, its base sequence is respectively SEQIDNO:33～35.
Further, described oligonucleotides also comprises: (17) are for detection of interior target primer and probe, its alkaliBasic sequence is respectively SEQIDNO:36～38.
The present invention also provides a kind of method of utilizing fluorescent PCR to detect 18 kinds of high-risk HPVs, described method bagDraw together: (1) extracts the DNA in sample; (2) utilize fluorescent PCR in single tube, the DNA in (1) to be carried outAmplification; (3) determine whether to exist at least one high-risk HPV in described 18 kinds of high-risk HPVs; (4)If described at least one high-risk HPV be only HPV16, HPV18 in described 18 kinds of high-risk HPVs orHPV16 and HPV18 exist simultaneously, detect and finish; If described high-risk HPV comprises except HPV16 and HPV18Outside at least one high-risk HPV, alternatively, utilize described except HPV16 and HPV18 at leastA kind of specific primer of high-risk HPV and probe carry out fluorescent PCR amplification again, determine its concrete type, itsAmplification in middle step (2) is carried out under one group of primer and probe existence, described one group of primer and probeBase sequence be SEQIDNO:1～38.
The present invention also provides a kind of kit that utilizes fluorescent PCR to detect high-risk HPV, and described kit comprisesSample nucleic acid extracting reagent and Fluorescence PCR liquid, described Fluorescence PCR liquid comprises oligonucleotides, described inOligonucleotides comprises: (1), for detection of primer and the probe of HPV16, its base sequence is respectively SEQIDNO:1～3; (2) for detection of primer and the probe of HPV18, its base sequence be respectively SEQIDNO:4～6。
Further, described oligonucleotides also comprises at least one in (3) and (4): (3) for detection ofThe primer of HPV31 and probe, its base sequence is respectively SEQIDNO:7,9 and 14; (4) for detection ofThe primer of HPV45 and probe, its base sequence is respectively SEQIDNO:15,16 and 19.
Further, described oligonucleotides also comprises at least one in (5)～(16): (5) are for inspectionPrimer and the probe of surveying HPV35, its base sequence is respectively SEQIDNO:8,9 and 14; (6) for inspectionPrimer and the probe of surveying HPV33, its base sequence is respectively SEQIDNO:10,13 and 14; (7) forThe primer and the probe that detect HPV52, its base sequence is respectively SEQIDNO:11,13 and 14; (8) useIn the primer and the probe that detect HPV58, its base sequence is respectively SEQIDNO:12～14; (9) forThe primer and the probe that detect HPV59, its base sequence is respectively SEQIDNO:17～19; (10) for inspectionPrimer and the probe of surveying HPV39 and HPV68, its base sequence is respectively SEQIDNO:20～22; (11)For detection of primer and the probe of HPV53, its base sequence is respectively SEQIDNO:23,24 and 28; (12)For detection of primer and the probe of HPV56, its base sequence is respectively SEQIDNO:25,26 and 28; (13)For detection of primer and the probe of HPV66, its base sequence is respectively SEQIDNO:25,27 and 28; (14)For detection of primer and the probe of HPV26, its base sequence is respectively SEQIDNO:29,31 and 32; (15)For detection of primer and the probe of HPV51 and HPV82, its base sequence is respectively SEQIDNO:30～32;(16) for detection of primer and the probe of HPV73, its base sequence is respectively SEQIDNO:33～35.
Further, described oligonucleotides also comprises: (17) are for detection of interior target primer and probe, its alkaliBasic sequence is respectively SEQIDNO:36～38.
Further, described Fluorescence PCR liquid also comprises TaqDNA polymerase and anti-TaqDNA polymerase.
Further, described kit also contains positive control solution and negative controls.
Beneficial effect: the affinity on chadogram based on 18 kinds of high-risk HPVs and other types, the present inventionDesign high-risk HPV primer and probe, allow some high-risk HPV hypotypes share upstream/downstream primer and probe,Thereby significantly reduce the usage quantity of primer and probe, ensureing detection accuracy, sensitivity and specific sameTime, significantly reduce background fluorescence value, and significantly increase and detect flux and greatly reduce testing cost; In addition,Primer and the probe of the present invention's design are aimed at the higher region of homology of selecting multiple genes, instead ofBe confined in individual gene region, this helps avoid cross reaction and further carries out type qualification.
Brief description of the drawings
Fig. 1 is 18 kinds of common high-risk HPV evolution tree graphs.
Fig. 2 is that clinical uterine neck cotton swab sample VIC passage (detect in sample and whether have HPV16) detects knotFruit figure.
Fig. 3 is clinical uterine neck cotton swab sample TexasRed passage (detects in sample and whether have HPV18)Testing result figure.
Fig. 4 is whether clinical uterine neck cotton swab sample F AM passage (detects in sample and exist except HPV16 and HPV18Outside other 16 kinds of high-risk HPVs at least one) testing result figure.
Fig. 5 is clinical uterine neck cotton swab sample Cy5 passage (mark β-globin in detecting) testing result figure.
Fig. 6 is the positive reference material VIC of HPV16 passage testing result figure.
Fig. 7 is the positive reference material TexasRed of HPV18 passage testing result figure.
Fig. 8 is HPV26,31,33,35,45,56,58,59,66,73,82 positive reference material FAMPassage testing result figure.
Fig. 9 is mark Cy5 passage testing result figure in HPV National reference.
Figure 10 is the positive reference material sensitivity of 13 kinds of high-risk HPVs testing result figure.
Figure 11 is the comparing result figure of background fluorescence signal.
Detailed description of the invention
Detect 18 kinds of common high-risk HPVs (HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and 82) in, due to the infection rate of HPV16/18 and carcinogenicWhether rate, all apparently higher than other high-risk HPV hypotypes, is therefore necessary to exist in detection sample at least oneWhen planting the existence of high-risk HPV hypotype, also tackle HPV16 and HPV18 simultaneously and carry out somatotype.
In invention, issuable non-specific amplification while amplification in order to reduce as much as possible fluorescent PCR,In Fluorescence PCR liquid, add anti-TaqDNA polymerase antibody, what this antibody can be in conjunction with TaqDNA polymeraseAvtive spot, thus TaqDNA polymerase can not be played a role, only in the sex change stage, anti-TaqDNAAt high temperature inactivation and not can be incorporated on the avtive spot of TaqDNA polymerase of polymerase antibody, thus makeTaqDNA polymerase can be brought into play catalytic action, the synthetic DNA chain making new advances.
Primer of the present invention and probe design and Genotyping thinking thereof are as follows: first according to these 18 kinds of high-risk-typesThe DNA sequence dna of HPV hypotype, draws their chadogram (as shown in Figure 1), according to them on chadogramAffiliation, allow some high-risk HPV hypotypes share upstream/downstream primer and probe, thus significantly simultaneouslyThe quantity that reduces primer and probe, not only significantly reduces testing cost, also significantly reduces background fluorescence signal simultaneously,Improve detection accuracy.
Poor in view of the L1 DNA homolog of HPV, be usually used in somatotype, the present invention successfully selects some high-riskThe genome area that type HPV homology is higher designs some shared primer and probes as target, whereinHPV31,33,35,52 and 58 has higher homology region on E1 gene; HPV45 and 59On raq gene, there is higher homology region; HPV39 and 68 has higher on raq geneHomology region; HPV26,51 and 82 has higher homology region on raq gene; HPV53,56 and 66 have higher homology region on E7 gene. Based on this, in the time of design primer and probe,The present invention allows HPV31 and HPV35 share downstream primer (HPV31/35R), HPV33, HPV52 and HPV58Share downstream primer (HPV33/52/58R), these 5 kinds of high-risk HPVs share a probe (Probe3) simultaneously;HPV45 and HPV59 share a probe (Probe4); HPV39 and HPV68 share upstream primer(HPV39/68F), downstream primer (HPV39/68R) and a probe (Probe5); HPV56 and HPV66Share upstream primer (HPV56/66F), these two kinds of high-risk HPV hypotypes also share a probe with HPV53(Probe6); HPV51 and HPV82 share upstream primer (HPV51/82F), these two kinds of high-risk HPV AsiasType also shares downstream primer (HPV26/51/82R) and a probe (Probe7) with HPV26, and this just significantlyReduce the quantity of primer and probe, simultaneously in view of other the 16 kinds of high-risk HPVs except HPV16 and HPV18The infection proportion of hypotype is corresponding lower, allows probe Probe3, Probe4, Probe5, Probe6, Probe7Share identical fluorescence report group with Probe8, these all help and reduce reagent development cost, are convenient to productExploitation.
The more important thing is, the present invention is in the time detecting cervical carcinoma excessive risk clinical sample, and each single tube detects just can be trueWhether fixed every duplicate samples contains HPV16 and HPV18, also can judge whether to exist other 16 kinds of high-risk-types simultaneouslyAt least one in HPV hypotype, if there is at least one in other 16 kinds of high-risk HPV hypotypes, also canSelect Auele Specific Primer and the probe of every kind of hypotype in other 16 kinds of high-risk HPV hypotypes again to carry out fluorescencePCR detects, and further determines its type. Infection rate based on HPV16 and HPV18 is 70%, utilizes thisThe bright primer providing and probe, single tube detect once just can get rid of in most of sample, do not infect other 16Plant high-risk HPV hypotype, detect and needn't carry out follow-up type again, this has not only saved testing cost, withTime also increased the detection flux of batch. Moreover the present invention is some products on market not,For the same gene of HPV (being mainly L1 gene) upper design primer and probe, but 4 genes (L1,E1, E2 and E7) upper design primer and probe, so just can avoid as far as possible designing not on same geneDuring with high-risk HPV primer and probe because the base sequence otherness of some type specificity primers and probe is too smallAnd cause cross reaction to produce, thereby can not distinguish exactly high-risk HPV (especially HPV16 and HPV18)Type. Finally, the present invention also introduces interior mark and detects, and this can increase detection accuracy and reliability.
The present invention uses 9 TaqMan probes, and 5 ' end of every probe is fluorescence report group, and 3 ' end is for sending outGo out fluorescence or do not send the quenching group of fluorescence, its middle probe Probe1 and Probe2 use different fluorescence reportsAccuse group; Probe3, Probe4, Probe5, Probe6, Probe7 and Probe8 use identical fluorescenceReporter group; The fluorescence report group that Probe1, Probe2 and Probe9 use is different, and this threeThe fluorescence report group also using with Probe3, Probe4, Probe5, Probe6, Probe7 and Probe8Not identical yet. The fluorescence report group of these 9 probes can be selected from FAM, TET, VIC, JOE, HEX, Cy3,Cy3.5, Cy5, Cy5.5, TAMRA, ROX, TexasRed, LCRED640 and LCRED705 etc.; QuenchThe group that goes out can be selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and NFQ etc., quenching groupSelection principle be that the fluorescent absorption spectrum of quenching group and the emission spectrum of fluorescence report group exist overlapping.
For convenience of explanation, the primer providing taking table 1 and probe describe as example. Like this at single reaction pipeIn, clinical sample is carried out to fluorescent PCR amplification, point four looks detect HPV16 (VIC passage), HPV18 (TexasRed passage), other the 16 kinds of high-risk HPVs (FAM passage) except HPV16 and HPV18, interior mark β-globin (Cy5 passage), not only can detect in sample, whether there is at least one high-risk HPV hypotype,And can also carry out somatotype to HPV16 and HPV18.
Table 1. primer and probe
Following examples further illustrate the present invention. These embodiment are not for limiting the scope of the invention, butA further understanding of the present invention is provided.
Embodiment 1: sample DNA extracts
For convenience of explanation, the present embodiment contains with uterine neck cotton swab sample and sample nucleic acid extracting reagent usedHaving lysate, isopropyl alcohol and 1 × TE buffer solution is that example describes:
(1) take out uterine neck cotton swab sample, after concussion mixes, draw 1mL sample in 1.5mL centrifuge tube,The centrifugal 1min of 12000rpm, careful suction abandoned supernatant, retains precipitation;
(2) add 500 μ l lysates toward the precipitation in (1), concussion mixes, 100 DEG C of water-baths or dryBathe 10min, wherein lysate for containing 0.2MNaCl, 10mMNaOH, 0.1%SDS, 0.5%NP-40 and1 × TE buffer solution of 0.5%Tween-20;
(3) add 500 μ l isopropyl alcohols, concussion mixes, and room temperature is placed 2min, the centrifugal 5min of 12000rpm,Abandon supernatant, room temperature is placed 2min;
(4) add 50 μ l1 × TE buffer solutions, fully dissolve, room temperature leaves standstill 5min, and 12000rpm is centrifugal1min, supernatant is DNA solution, for subsequent use.
Embodiment 2: Fluorescence PCR step
(1) preparation high-risk type HPV fluorescence PCR reactant liquor: 4 μ l10 × PCRBuffer (100mMTris-HCl,500mMKCl)；0.08μldATP(0.4mM)、0.08μldTTP(0.4mM)、0.08μldCTP(0.4mM)、0.08μldGTP(0.4mM)；0.8μlBSA(10mg/mL)；6.4μlMgCl2(25mM)；0.05μlHPV16F(100μM)、0.08μlHPV16F(100μM)、0.08μlProbe1(100μM)；0.05μlHPV18F(100μM)、0.08μlHPV18F(100μM)、0.08μlProbe2(100μM)；0.08μlHPV31F(100μM)、0.08μlHPV35F(100μM)、0.08μlHPV31/35R(100μM)、0.08μlHPV33F(100μM)、0.08μlHPV52F(100μM)、0.08μlHPV58F(100μM)、0.08μlHPV33/52/58R(100μM)、0.06μlProbe3(100μM)；0.08μlHPV45F(100μM)、0.06μlHPV45R(100μM)、0.08μlHPV59F(100μM)、0.06μlHPV59R(100μM)、0.06μlProbe4(100μM)；0.08μlHPV39/68F(100μM)、0.08μlHPV39/68R(100μM)、0.06μlProbe5(100μM)；0.08μlHPV53F(100μM)、0.08μlHPV53R(100μM)、0.08μlHPV56/66F(100μM)、0.08μlHPV56R(100μM)、0.08μlHPV66R(100μM)、0.06μlProbe6(100μM)；0.08μlHPV26F(100μM)、0.06μlHPV51/82F(100μM)、0.08μlHPV26/51/82R(100μM)、0.06μlProbe7(100μM)；0.08μlHPV73F(100μM)、0.08μlHPV73R(100μM)、0.05μlProbe8(100μM)；0.04μlIC-F(100μM)、0.04 μ lIC-R (100 μ M), 0.03 μ lProbe9 (100 μ M); 0.4 μ lTaqDNA polymerase (is purchasedBuy from Promega), the anti-TaqDNA polymerase of 0.067 μ l antibody; The remaining ddH that adds2O makes its volumeBe 36 μ l, wherein said TaqDNA polymerase and anti-TaqDNA polymerase antibody preferably add before useIn high-risk type HPV fluorescence PCR reactant liquor;
(2) [pipe positive control solution+1, n=clinical sample number+1 pipe is negative right to calculate the sample number n part that need prepareAccording to liquid], the high-risk type HPV fluorescence PCR reactant liquor of preparation in 36 μ l (1) is joined to different reaction tubesIn, then in different reaction tubes, add DNA solution, 4 μ l the moon of in 4 μ l embodiment 1, extracting respectivelyProperty contrast solution and 4 μ l positive control solution, wherein positive control solution is contain high-risk HPV nucleic acid moltenLiquid, negative controls is the solution that does not contain high-risk HPV nucleic acid.
(3) each reaction tube is put on fluorescent PCR instrument in certain sequence, is carried out pcr amplification by following program,Amplification program is as shown in table 2:
Table 2 fluorescent PCR amplification program
(4) after step (3) finishes, according to FAM passage, VIC passage, TexasRed passage, Cy5Whether the Ct value of passage detects high-risk HPV and exists, and its testing result judges as shown in table 3:
The judgement of table 3 testing result
Embodiment 3: clinical sample detects
Extract the DNA in 140 routine HPV uterine neck cotton swab samples by the method in embodiment 1, then according to realityThe method of executing in example 2 is carried out fluorescent PCR amplification to the DNA in this 140 routine HPV Cervical scrapes sample respectively.Meanwhile, for the ease of relatively, utilize RocheCombas48014 kind high-risk HPV (be HPV16,18,31,33,35,39,45,51,52,56,58,59,68 and 66) detection kit to this 140DNA in example HPV Cervical scrapes sample detects, and testing result is as shown in table 4.
Table 4 testing result
Result result in table 4 shows, in this 140 routine clinical samples, the present invention is to 134 routine clinical samplesConsistent with Roche kit of the testing result of product, but also have 6 routine clinical samples, the present invention can detectGo out the existence of high-risk HPV, Roche kit can not detect the existence of high-risk HPV. Pass throughThe confirmation of Sanger PCR sequencing PCR, this 6 routine sample contains respectively HPV53, HPV82, HPV73, HPV53, HPV26And HPV26. This has further verified detection accuracy of the present invention. In addition, the present invention is at single reaction tubeIn may exist in clinical sample 18 kinds of high-risk HPVs are carried out to the detection of four-way fluorescent PCR, Qi Zhongtu2 is VIC passage (detect in clinical sample and whether have HPV16) testing result; Fig. 3 is TexasRedPassage (detect in clinical sample and whether have HPV18) testing result; Fig. 4 is that FAM passage (detects clinicalIn sample, whether there is at least one in other the 16 kinds of high-risk HPVs except HPV16 and HPV18)Testing result; Fig. 5 is Cy5 passage (mark β-globin in detecting) testing result. Preferably, for thatIn fluorescent PCR amplification for the first time, determine at least one high-risk-type containing except HPV16 and HPV18 a bitThe clinical sample of HPV, the present invention is recycling other 16 kinds of high-risk-types except HPV16 and HPV18 alsoThe Auele Specific Primer of HPV hypotype and probe carry out fluorescent PCR amplification again, thereby determine its concrete type.
In addition, with respect to 14 kinds of high-risk HPV detection kit of Roche, the present invention can detect moreHigh-risk HPV type, the more important thing is, for the high-risk HPV type of many increases, the present invention has more thanBe simply for the each type in them designs specific primer and probe, and consider they and otherThe affiliation of type, allows HPV53 and HPV56 and HPV66 share same probe, allows HPV82 and HPV51Share upstream primer, allow HPV82, HPV51 and HPV26 share downstream primer and a probe, also allow simultaneouslyDetect HPV53,73,82,26 and HPV31,33,35,39,45,51,52,56,58,59,68Share same sense channel with the probe using at 66 o'clock, this can reduce primer, probe and detection effectivelyThe quantity of passage, thus testing cost and background fluorescence signal effectively reduced.
Embodiment 4: specific detection result
HPV National reference arranges as follows:
5 kinds of negative reference material N1-N5: be followed successively by chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), gonorrhoeaNeisser's coccus (NG), herpes simplex virus type II (HSV-2), the negative uterine neck cotton swab of HPV nucleic acid sample;
20 kinds of positive reference materials: be followed successively by HPV6,11,61,67,69,71,81,16,18,26,31,33,35,45,56,58,59,66,73 and 82 positive reference materials.
According to the method in embodiment 2, each HPV National reference is carried out to fluorescent PCR amplification to each HPVNational reference carries out fluorescent PCR detection, and result is as follows:
Negative reference material N1-N5 and HPV6,11,61,67,69,71,5 kinds of positive reference materials such as 81FAM, VIC and TexasRed passage testing result are all negative; The VIC passage inspection of the positive reference material of HPV16Survey result is positive, as shown in Figure 6; The TexasRed passage testing result of the positive reference material of HPV18 is sunProperty, as shown in Figure 7; HPV26,31,33,35,45,56,58,59,66,73,82 positive referencesProduct FAM passage testing result is positive, as shown in Figure 8; Interior mark Cy5 passage testing result is positive, as figureShown in 9.
Embodiment 5: sensitivity testing result
Respectively to the HPV16 in embodiment 4,18,26,31,33,35,45,56,58,59,66,It is 1 × 10 that 73 and 82 positive reference materials are carried out gradient dilution to concentration3Copies/ml, in embodiment 2Method each positive reference material is carried out to fluorescent PCR amplification, test result is all positive, i.e. inspection of the present inventionSurveying sensitivity is 1 × 103Copies/ml. Result as shown in figure 10.
Embodiment 6: background fluorescence signal detecting result
Respectively to the HPV16 in embodiment 4,18,26,31,33,35,45,56,58,59,66,73According to the method in the embodiment of the present invention 2, each positive reference material is carried out to fluorescent PCR expansion with 82 positive reference materialsIncrease; In order to contrast, 18 kinds of common high-risk HPVs that can detect for the present invention, for each simultaneouslyThe high-risk HPV a pair of specific primer of design and a specific probe, also right respectively under the same conditionsSame positive reference material is carried out fluorescent PCR amplification, detects as example taking FAM passage, carries out background fluorescence signalContrast, comparing result is as shown in figure 11. As shown in Figure 11, the present invention is because the number of probes using significantly subtractsFew, background fluorescence signal significantly reduces, and is convenient to result interpretation.

Claims (10)

1. the oligonucleotides that detects high-risk HPV for fluorescent PCR, is characterized in that, described oligonucleotides comprises:(1) for detection of primer and the probe of HPV16, its base sequence is respectively SEQIDNO:1～3; (2)For detection of primer and the probe of HPV18, its base sequence is respectively SEQIDNO:4～6.
2. the oligonucleotides as shown in claim 1, is characterized in that, described oligonucleotides also comprises (3) and (4)In at least one: (3), for detection of primer and the probe of HPV31, its base sequence is respectively SEQIDNO:7,9 and 14; (4) for detection of primer and the probe of HPV45, its base sequence is respectively SEQIDNO:15,16 and 19.
3. the oligonucleotides as shown in claim 1, is characterized in that, described oligonucleotides also comprises (5)～(16)In at least one: (5), for detection of primer and the probe of HPV35, its base sequence is respectively SEQIDNO:8,9 and 14; (6) for detection of primer and the probe of HPV33, its base sequence is respectively SEQIDNO:10,13 and 14; (7) for detection of primer and the probe of HPV52, its base sequence is respectively SEQIDNO:11,13 and 14; (8) for detection of primer and the probe of HPV58, its base sequence is respectively SEQIDNO:12～14; (9) for detection of primer and the probe of HPV59, its base sequence be respectively SEQIDNO:17～19; (10) for detection of primer and the probe of HPV39 and HPV68, its base sequence is respectively SEQIDNO:20～22; (11) for detection of primer and the probe of HPV53, its base sequence be respectively SEQIDNO:23,24 and 28; (12) for detection of primer and the probe of HPV56, its base sequence be respectively SEQIDNO:25,26 and 28; (13) for detection of primer and the probe of HPV66, its base sequence be respectively SEQIDNO:25,27 and 28; (14) for detection of primer and the probe of HPV26, its base sequence be respectively SEQIDNO:29,31 and 32; (15) for detection of primer and the probe of HPV51 and HPV82, its base sequence is respectively SEQIDNO:30～32; (16) for detection of primer and the probe of HPV73, its base sequence is respectively SEQIDNO:33～35。
4. the oligonucleotides as shown in one of claim 1-3, is characterized in that, described oligonucleotides also comprises: (17)For detection of interior target primer and probe, its base sequence is respectively SEQIDNO:36～38.
5. utilize fluorescent PCR to detect a method for 18 kinds of high-risk HPVs, described method comprises: (1) extracts sampleDNA in product; (2) under one group of primer and probe existence, in single tube, carry out glimmering to the DNA in (1)Light pcr amplification; (3) determine whether to exist at least one high-risk HPV in described 18 kinds of high-risk HPVs,If described at least one high-risk HPV comprises HPV16 and/or HPV18, simultaneously can also be to HPV16 and/orHPV18 carries out somatotype; (4) if described at least one high-risk HPV comprise except HPV16 and HPV18One in other 16 kinds of high-risk HPVs, alternatively, utilizes in described other 16 kinds of high-risk HPVs everyA kind of specific primer of high-risk HPV and probe carry out fluorescent PCR amplification again, further determine its type,It is characterized in that: the base sequence of described one group of primer and probe is SEQIDNO:1～38.
6. utilize fluorescent PCR to detect a kit for high-risk HPV, described kit comprises Fluorescence PCRLiquid, described Fluorescence PCR liquid comprises oligonucleotides, it is characterized in that, described oligonucleotides comprises: (1)For detection of primer and the probe of HPV16, its base sequence is respectively SEQIDNO:1～3; (2) forThe primer and the probe that detect HPV18, its base sequence is respectively SEQIDNO:4～6.
7. the kit as shown in claim 6, is characterized in that, described oligonucleotides also comprises (3) and (4)In at least one: (3), for detection of primer and the probe of HPV31, its base sequence is respectively SEQIDNO:7,9 and 14; (4) for detection of primer and the probe of HPV45, its base sequence is respectively SEQIDNO:15,16 and 19.
8. the kit as shown in claim 6, is characterized in that, described oligonucleotides also comprises (5)～(16)In at least one: (5), for detection of primer and the probe of HPV35, its base sequence is respectively SEQIDNO:8,9 and 14; (6) for detection of primer and the probe of HPV33, its base sequence is respectively SEQIDNO:10,13 and 14; (7) for detection of primer and the probe of HPV52, its base sequence is respectively SEQIDNO:11,13 and 14; (8) for detection of primer and the probe of HPV58, its base sequence is respectively SEQIDNO:12～14; (9) for detection of primer and the probe of HPV59, its base sequence be respectively SEQIDNO:17～19; (10) for detection of primer and the probe of HPV39 and HPV68, its base sequence is respectively SEQIDNO:20～22; (11) for detection of primer and the probe of HPV53, its base sequence be respectively SEQIDNO:23,24 and 28; (12) for detection of primer and the probe of HPV56, its base sequence be respectively SEQIDNO:25,26 and 28; (13) for detection of primer and the probe of HPV66, its base sequence be respectively SEQIDNO:25,27 and 28; (14) for detection of primer and the probe of HPV26, its base sequence be respectively SEQIDNO:29,31 and 32; (15) for detection of primer and the probe of HPV51 and HPV82, its base sequence is respectively SEQIDNO:30～32; (16) for detection of primer and the probe of HPV73, its base sequence is respectively SEQIDNO:33～35。
9. the kit as shown in one of claim 6-8, is characterized in that, described oligonucleotides also comprises: (17)For detection of interior target primer and probe, its base sequence is respectively SEQIDNO:36～38.
10. the kit as shown in one of claim 6-8, described Fluorescence PCR liquid also comprises TaqDNAPolymerase and anti-TaqDNA polymerase antibody.
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