CN105603121B - For detecting method, oligonucleotides and the kit of high-risk HPV - Google Patents
For detecting method, oligonucleotides and the kit of high-risk HPV Download PDFInfo
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Abstract
The present invention provides a kind of method for detecting common high-risk HPV, oligonucleotides and kit, using Fluorescence PCR assay, Preliminary detection is carried out to HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and 82 that may be present in sample and type is identified.The present invention can detect 18 kinds of high-risk types simultaneously, and carry out Genotyping to HPV16,18 simultaneously.The present invention is based on the affinity of 18 kinds of high-risk HPVs and other types on chadogram, design high-risk HPV primer and probe, while guaranteeing detection accuracy, sensitivity and specificity, background fluorescence activity is significantly reduced, and dramatically increases detection flux and greatly reduces testing cost.
Description
Technical field
The invention belongs to bioscience and field of biotechnology, in particular to a kind of 18 kinds of common high-risk HPVs of detection
Method, oligonucleotides and kit, using Fluorescence PCR assay, to HPV16 that may be present in clinical sample, 18,31,33,
35,39,45,51,52,56,58,59,68,26,53,66,73 and 82 Preliminary detection and type identification are carried out.
Background technique
Human papilloma virus (human papillomavirus, HPV) is that one kind can infect on people's epidermis and mucous membrane squamous
The small DNA virus of skin can cause the diseases such as condyloma acuminatum and cervical carcinoma.The mankind are unique hosts of HPV.HPV genome is
Double-stranded cyclic DNA is about 7900 pairs of bases (bp), contains 8 open reading frame (open reading frames, ORFs), by 3
A gene regions composition, including early stage area (area E), late region (area L) and noncoding region or upstream regulatory region.In order may be used in the area E
It is divided into E6, E7, E1, E2, E3, E4 and E5 totally 7 genes, wherein E6 and E7 is the Analyses of major carcinogens in mainstream gene of HPV, the cell with HPV
Transformation function is related to carcinogenicity.Mainly there are two genes of L1 and L2 in the area L.
Scientists have found 120 kinds or more of HPV hypotype at present.Different HPV Subtypes lead to different lesions, root
Whether can cause cancer according to it, HPV hypotype can be divided into: high-risk HPV, with cervical carcinoma and Cervical intraepitheliaI neoplasia (CIN I/
II/III generation) is related, common are the hypotypes such as HPV16,18,31,33,35,45,51,52,56,58,29,68；Low risk
HPV, it is generally related to condyloma acuminatum or low squamous intraepithelial lesion, seldom cause infiltrating carcinoma, common are as HPV6,11,
42, the hypotypes such as 43,44.About the division of high-risk HPV and low risk HPV, many mechanisms all give reference proposition in the world,
According to the research achievement 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 be classified as high-risk HPV, and by the totally 5 kinds of hypotypes column of HPV26,53,66,73 and 82
For medium risk HPV.When actually detected, medium risk HPV hypotype is also usually attributed to high-risk HPV.
80% women can infect HPV viruse in life, usually virus in 6~8 months can nature remove, only 25%
Precancerous lesion can occur.High-risk HPV repeatedly or persistent infection is considered as the necessary condition of nearly all uterine neck carcinogenesis.It is existing
There is result of study to show, cervical lesions degree is heavier, and high-risk HPV infection rate is higher；In CINI, CINII and CINIII patient
In, high-risk HPV infection rate is respectively 30%, 55% and 65%, and can be detected in 99.8% or more cervical cancer patient body
Cervical carcinoma hardly occurs for high-risk HPV infection, HPV negative patient.In these high-risk HPVs, most commonly seen is
Secondly HPV16 is HPV18, the infection rate highest of both high-risk HPV hypotypes, up to 70%；If along with HPV45 and
HPV31, the infection rate of this 4 kinds of high-risk HPVs is up to 80% or so.One, China is across 7 cervical cancer pathogenesis rate different regions
19 hospitals, be included in the cervix cancer in China correlation high-risk HPV subtype distribution (based on hospital) of 1244 cases altogether
Multicenter study confirms that the cervical carcinoma and the high of cervical cell pathological changes of Chinese different zones are all to infect based on HPV16/18, about
85% cervical carcinoma confirmed cases belong to both hypotypes, and the infection rate of HPV16/18 and carcinogenic rate are all apparently higher than other height
Danger type HPV (Chen W et al.Human papillomavirus type-distribution in cervical
cancer in China:the importance of HPV 16and 18.Cancer Causes and Control,
2009,20:1705-1713)。
It in addition, there will be research shows that infect the women of HPV16/18, it is whether in the recent period or at a specified future date, progress to high-grade cervical
The risk of lesion is all significantly larger than other high-risk HPVs.Based on this, U.S.'s vaginoscopy in 2013 and the meeting of uterine neck pathology
(ASCCP) especially set out will be to cytology ASC-US (nothing on the cytology found by uterine neck Liquid-based thin-layer cytology test
The change of the atypical squamous cell of meaning) feminine gender, 30 years old or more women of HPV positive progress HPV16/18 Genotyping
Detection.Due to pathogenic and prognosis the difference of different HPV hypotypes, parting is carried out to HPV16/18, it will help preferably right
High risk group carries out risk stratification management, finds the people at highest risk and ASC-US for suffering from CIN in cytology normal outcome in time
The crowd of closer follow-up is needed in positive findings.
Cervical carcinoma early symptom is not obvious, and there are longer, the reversible precancerous lesion phases in development process.From general
Precancerous lesions of uterine cervix development be cervical carcinoma took around for 10 years.As can carrying out medical intervention, palace in precancerous stage
Neck cancer cure rate is up to 98%.Therefore, the key that screening is still current prevention cervical carcinoma is carried out to high-risk HPV.
High-risk HPV detection method common at present has fluorescent PCR method, hybrid capture and PCR- revert dot blot hybridization
Deng.The shortcomings that hybrid capture is no internal standard, and detection time is long (generally requiring 5-6 hours), sensitivity it is low (3.5 ×
105Copies/ml), complicated for operation, detection genotype is on the low side, and false positive rate is higher.Although PCR- reverse hybridization is able to detect
More HPV hypotype, but the disadvantage is that detection time is long (at least needing 4 hours), testing cost is high, detection sensitivity is low
(104Copies/ml), complicated for operation, it is not able to satisfy the needs of clinical high flux examination.
Fluorescent PCR method sensitivity and specificity with higher, and real-time online detection, energy can be carried out to amplified production
Detection time is enough greatly reduced, while also avoiding the generation of residual contamination.Common fluorescent PCR method has SYBR Green I
Dye method, double probe hybrid methods and Taqman sonde method etc..Wherein SYBR Green I is due to being non-saturable dye, specificity
Not as good as double probe hybrid methods and Taqman sonde method, it is necessary to judge its specificity by observation solubility curve；And double probes
Method hybrid method cost again costly.
On domestic market, have the product that many producers are developed based on fluorescent PCR method detection high-risk HPV, and
It is to detect 13,14 or 15 kind of high-risk HPV simultaneously mostly, only a small number of producers develop while detecting using fluorescent PCR method
Common 18 kinds of high-risk HPVs (and of HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73
82) product, however, having plenty of in these products and designing a pair of of type-special primer and one for every kind of high-risk HPV
Type specificity probe, this can sharply increase testing cost, while the presence of so multiprobe can also dramatically increase background fluorescence letter
Number；Some or progress multitube detection, this can dramatically increase the dosage of sample and detection reagent, increase testing cost, and also not
Parting can be carried out to HPV16 and HPV18；What is more, primer and/or the probe limitation designed for different high-risk HPVs
On the same gene region, base sequence difference is too small, will lead to the amplified reaction of intercrossing, generates false positive, Bu Nengzhun
Really distinguish its specific type.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of for detecting high-risk HPV (especially common 18
Kind of high-risk HPV, i.e. HPV16,18,31,33,35,39,45,51,52,56,58,59,68,26,53,66,73 and side 82)
Method, oligonucleotides and kit, primer and probe involved in the method, oligonucleotides and kit are high-risk by constructing
The chadogram of type HPV, the affinity based on these high-risk HPVs on chadogram, allow some high-risk HPVs share upstream or under
Trip primer and probe dramatically increases detection flux, and greatly while guaranteeing detection accuracy, sensitivity and specificity
Reduce testing cost.
If the base sequence of primer and/or probe in the present invention contains alphabetical " Y ", " S ", " R " or " M ", show this
Primer and/or probe are degenerate primer or degeneracy probe, and wherein Y represents C or T, and S represents G or C, and R represents A or G, M represent A or
C。
The present invention is provided to the oligonucleotides of fluorescent PCR detection high-risk HPV, and the oligonucleotides includes: that (1) is used for
The primer and probe of HPV16 is detected, base sequence is respectively NO:1~3 SEQ ID；(2) for detect HPV18 primer and
Probe, base sequence are respectively NO:4~6 SEQ ID.
Further, the oligonucleotides further includes at least one of (3) and (4): (3) are for detecting drawing for HPV31
Object and probe, base sequence are respectively SEQ ID NO:7,9 and 14；(4) for detecting the primer and probe of HPV45, alkali
Basic sequence is respectively SEQ ID NO:15,16 and 19.
Further, the oligonucleotides further includes at least one of (5)~(16): (5) are for detecting drawing for HPV35
Object and probe, base sequence are respectively SEQ ID NO:8,9 and 14；(6) for detecting the primer and probe of HPV33, alkali
Basic sequence is respectively SEQ ID NO:10,13 and 14；(7) for detecting the primer and probe of HPV52, base sequence is respectively
SEQ ID NO:11,13 and 14；(8) for detecting the primer and probe of HPV58, base sequence is respectively SEQ ID NO:12
~14；(9) for detecting the primer and probe of HPV59, base sequence is respectively NO:17~19 SEQ ID；(10) for examining
The primer and probe of HPV39 and HPV68 is surveyed, base sequence is respectively NO:20~22 SEQ ID；(11) for detecting HPV53
Primer and probe, base sequence is respectively SEQ ID NO:23,24 and 28；(12) for detecting primer and the spy of HPV56
Needle, base sequence are respectively SEQ ID NO:25,26 and 28；(13) for detecting the primer and probe of HPV66, base sequence
Column are respectively SEQ ID NO:25,27 and 28；(14) for detecting the primer and probe of HPV26, base sequence is respectively SEQ
ID NO:29,31 and 32；(15) for detecting the primer and probe of HPV51 and HPV82, base sequence is respectively SEQ ID
NO:30~32；(16) for detecting the primer and probe of HPV73, base sequence is respectively NO:33~35 SEQ ID.
Further, the oligonucleotides further include: (17) for detecting interior target primer and probe, base sequence point
It Wei not NO:36~38 SEQ ID.
The present invention also provides a kind of methods for detecting 18 kinds of high-risk HPVs using fluorescent PCR, which comprises (1) mentions
Take the DNA in sample；(2) DNA in (1) is expanded in single tube using fluorescent PCR；(3) it determines whether there is described
At least one of 18 kinds of high-risk HPVs high-risk HPV；(4) if at least one high-risk HPV is only described 18 kinds high-risk
HPV16, HPV18 or HPV16 and HPV18 in type HPV are existed simultaneously, then detection terminates；If the high-risk HPV includes removing
At least one high-risk HPV except HPV16 and HPV18, then optionally, using it is described in addition to HPV16 and HPV18 at least
A kind of primer and probe of high-risk HPV specificity carries out fluorescent PCR amplification again, its specific type is determined, wherein in step (2)
Amplification be to be carried out in the presence of one group of primer and probe, the base sequence of one group of primer and probe is SEQ ID NO:
1~38.
The present invention also provides a kind of kits using fluorescent PCR detection high-risk HPV, and the kit includes sample core
Acid extracts reagent and Fluorescence PCR liquid, and the Fluorescence PCR liquid includes oligonucleotides, and the oligonucleotides includes: (1)
For detecting the primer and probe of HPV16, base sequence is respectively NO:1~3 SEQ ID；(2) for detecting drawing for HPV18
Object and probe, base sequence are respectively NO:4~6 SEQ ID.
Further, the oligonucleotides further includes at least one of (3) and (4): (3) are for detecting drawing for HPV31
Object and probe, base sequence are respectively SEQ ID NO:7,9 and 14；(4) for detecting the primer and probe of HPV45, alkali
Basic sequence is respectively SEQ ID NO:15,16 and 19.
Further, the oligonucleotides further includes at least one of (5)~(16): (5) are for detecting drawing for HPV35
Object and probe, base sequence are respectively SEQ ID NO:8,9 and 14；(6) for detecting the primer and probe of HPV33, alkali
Basic sequence is respectively SEQ ID NO:10,13 and 14；(7) for detecting the primer and probe of HPV52, base sequence is respectively
SEQ ID NO:11,13 and 14；(8) for detecting the primer and probe of HPV58, base sequence is respectively SEQ ID NO:12
~14；(9) for detecting the primer and probe of HPV59, base sequence is respectively NO:17~19 SEQ ID；(10) for examining
The primer and probe of HPV39 and HPV68 is surveyed, base sequence is respectively NO:20~22 SEQ ID；(11) for detecting HPV53
Primer and probe, base sequence is respectively SEQ ID NO:23,24 and 28；(12) for detecting primer and the spy of HPV56
Needle, base sequence are respectively SEQ ID NO:25,26 and 28；(13) for detecting the primer and probe of HPV66, base sequence
Column are respectively SEQ ID NO:25,27 and 28；(14) for detecting the primer and probe of HPV26, base sequence is respectively SEQ
ID NO:29,31 and 32；(15) for detecting the primer and probe of HPV51 and HPV82, base sequence is respectively SEQ ID
NO:30~32；(16) for detecting the primer and probe of HPV73, base sequence is respectively NO:33~35 SEQ ID.
Further, the oligonucleotides further include: (17) for detecting interior target primer and probe, base sequence point
It Wei not NO:36~38 SEQ ID.
Further, the Fluorescence PCR liquid further includes Taq archaeal dna polymerase and anti-Taq archaeal dna polymerase.
Further, the kit also contains positive control solution and negative controls.
The utility model has the advantages that the affinity based on 18 kinds of high-risk HPVs and other types on chadogram, present invention design is high-risk
Type HPV primer and probe allows some high-risk HPV hypotypes to share upstream/downstream primer and probe, thus significantly reduce primer and
The usage quantity of probe significantly reduces background fluorescence activity, and show while guaranteeing detection accuracy, sensitivity and specificity
It writes and increases detection flux and greatly reduce testing cost；In addition, the primer and probe that the present invention designs be directed to selection it is more
The high region of the tetraploid rice of a gene, without being limited in individual gene region, this help avoid cross reaction and
Further carry out type identification.
Detailed description of the invention
Fig. 1 is 18 kinds of common high-risk HPV evolution tree graphs.
Fig. 2 is the clinic channel uterine neck cotton swab subsample VIC (whether there is HPV16 in test sample) testing result figure.
Fig. 3 is the clinic channel uterine neck cotton swab subsample Texas Red (whether there is HPV18 in test sample) testing result
Figure.
Fig. 4 is the clinic channel uterine neck cotton swab sample F AM (with the presence or absence of in addition to HPV16 and HPV18 in test sample
At least one of other 16 kinds of high-risk HPVs) testing result figure.
Fig. 5 is the clinic channel uterine neck cotton swab subsample Cy5 (detection internal standard β-globin) testing result figure.
Fig. 6 is HPV16 positive reference product VIC Air conduct measurement result figure.
Fig. 7 is HPV18 positive reference product Texas Red Air conduct measurement result figure.
Fig. 8 is the positive reference of HPV26,31,33,35,45,56,58,59,66,73,82 product FAM Air conduct measurement result figure.
Fig. 9 is HPV National reference internal standard Cy5 Air conduct measurement result figure.
Figure 10 is 13 kinds of high-risk HPV positive reference product sensitivity technique result figures.
Figure 11 is the comparing result figure of background fluorescence signal.
Specific embodiment
18 kinds of common high-risk HPVs of detection (HPV16,18,31,33,35,39,45,51,52,56,58,59,68,
26,53,66,73 and 82) in, since the infection rate of HPV16/18 and carcinogenic rate are all apparently higher than other high-risk HPV hypotypes, because
This it is necessary in test sample with the presence or absence of at least one high-risk HPV hypotype there are while, while also reply HPV16 and
HPV18 carries out parting.
In invention, issuable non-specific amplification when in order to reduce fluorescent PCR amplification as much as possible, in fluorescence
Be added anti-Taq archaeal dna polymerase antibody in PCR reaction solution, the antibody in combination with Taq archaeal dna polymerase active site, to make
Taq archaeal dna polymerase cannot play a role, only in denaturation stage, anti-Taq archaeal dna polymerase antibody inactivate at high temperature and
It not can be incorporated on the active site of Taq archaeal dna polymerase, so that Taq archaeal dna polymerase can play catalytic action, close
At DNA chain new out.
Primer and probe design of the invention and its Genotyping thinking are as follows: being first depending on this 18 kinds of high-risk HPV Asias
The DNA sequence dna of type draws their chadogram (as shown in Figure 1), according to their affiliations on chadogram, allows some high
Danger type HPV hypotype shares upstream/downstream primer and probe, while to significantly reduce the quantity of primer and probe, not only significantly
Testing cost is reduced, while being significantly reduced background fluorescence signal, improves detection accuracy.
It is poor in view of the L1 genetic homology of HPV, it is usually used in parting, the present invention successfully selects some high-risk HPVs same
Property relatively high genome area in source designs some shared primer and probes as target, wherein the and of HPV31,33,35,52
58 have relatively high homology region on E1 gene；HPV45 and 59 has relatively high homology region on raq gene；
HPV39 and 68 has relatively high homology region on raq gene；HPV26,51 and 82 have relatively high on raq gene
Homology region；HPV53,56 and 66 have relatively high homology region on E7 gene.Based on this, in design primer and spy
When needle, the present invention allows HPV31 and HPV35 to share downstream primer (HPV31/35R), and HPV33, HPV52 and HPV58 share downstream and draw
Object (HPV33/52/58R), while this 5 kinds of high-risk HPVs share a probe (Probe3)；HPV45 and HPV59 shares one
Probe (Probe4)；HPV39 and HPV68 shares upstream primer (HPV39/68F), downstream primer (HPV39/68R) and a spy
Needle (Probe5)；HPV56 and HPV66 shares upstream primer (HPV56/66F), both high-risk HPV hypotypes are also total with HPV53
With a probe (Probe6)；HPV51 and HPV82 shares upstream primer (HPV51/82F), both high-risk HPV hypotypes are also
Downstream primer (HPV26/51/82R) and a probe (Probe7) are shared with HPV26, this is significantly reduced primer and probe
Quantity, while accordingly lower in view of the infection proportion of other 16 kinds of high-risk HPV hypotypes in addition to HPV16 and HPV18, allows spy
Needle Probe3, Probe4, Probe5, Probe6, Probe7 and Probe8 share identical fluorescent reporter group, these are all helped
Reagent development cost is reduced, convenient for the exploitation of product.
Importantly, the present invention, when detecting cervical carcinoma high risk clinical sample, each single tube detection just can determine every
Whether part sample contains HPV16 and HPV18, at the same also can determine whether there are in other 16 kinds of high-risk HPV hypotypes at least
One kind, at least one of other 16 kinds of high-risk HPV hypotypes, also may be selected in other 16 kinds of high-risk HPV hypotypes if it exists
The specific primer and probe of every kind of hypotype carry out fluorescent PCR detection again, further determine that its type.Based on HPV16 and
The infection rate of HPV18 is 70%, and using primer and probe provided by the invention, single tube detection can once exclude most of sample
In do not infect other 16 kinds of high-risk HPV hypotypes, without carrying out subsequent type detection again, this not only saves detection
Cost, while also increasing the detection flux an of batch.Furthermore the present invention is not as some products in the market, needle
To design primer and probe on the same gene of HPV (predominantly L1 gene), but set on 4 genes (L1, E1, E2 and E7)
Count primer and probe, can thus avoid as far as possible when designing different high-risk HPV primer and probes on the same gene because
The base sequence otherness of some type-special primers and probe is too small and cross reaction is caused to generate, thus cannot accurate area
Divide the type of high-risk HPV (especially HPV16 and HPV18).Finally, the present invention also introduces internal standard detection, this will increase detection
Accuracy and reliability.
The present invention uses 9 TaqMan probes, and 5 ' ends of every probe are fluorescent reporter group, 3 ' ends for issue fluorescence or
The quenching group of fluorescence is not issued, and middle probe Probe1 and Probe2 use different fluorescent reporter groups；Probe3,
Probe4, Probe5, Probe6, Probe7 and Probe8 use identical fluorescent reporter group；Probe1, Probe2 and
The fluorescent reporter group that Probe9 is used is different, and this three also with Probe3, Probe4, Probe5, Probe6,
The fluorescent reporter group that Probe7 and Probe8 are used is not also identical.The fluorescent reporter group of this 9 probes can be selected from FAM,
TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 and LC RED705
Deng；Quenching group can be selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and NFQ etc., the selection principle of quenching group
Being the fluorescent absorption spectrum of quenching group, there are Chong Die with the emission spectrum of fluorescent reporter group.
For ease of description, it is illustrated by taking the primer and probe that table 1 provides as an example.It is right in this way in single reaction pipe
Clinical sample carries out fluorescent PCR amplification, and point four colors detection HPV16 (channel VIC), removes HPV16 at HPV18 (channel TexasRed)
Other 16 kinds of high-risk HPVs (channel FAM), internal standard β-globin (channel Cy5) with except HPV18, not only can detecte sample
With the presence or absence of at least one high-risk HPV hypotype in product, and parting can also be carried out to HPV16 and HPV18.
1. primer and probe of table
Following embodiment further illustrates the present invention.These embodiments are not intended to limit the scope of the invention, and are to provide
A further understanding of the present invention.
Embodiment 1: sample DNA extracts
For ease of description, the present embodiment is contained with uterine neck cotton swab subsample and sample nucleic extraction reagent used and is split
It is illustrated for solution liquid, isopropanol and 1 × TE buffer:
(1) uterine neck cotton swab subsample is taken out, after concussion mixes, draws 1mL sample in 1.5mL centrifuge tube, 12000rpm
It is centrifuged 1min, careful inhale abandons supernatant, retain precipitating；
(2) 500 μ l lysates are added in the precipitating into (1), and concussion mixes, 100 DEG C of water-baths or dry bath 10min, wherein
Lysate be NaCl containing 0.2M, 10mM NaOH, 0.1%SDS, 0.5%NP-40 and 0.5%Tween-20 1 × TE buffering
Liquid；
(3) 500 μ l isopropanols are added, concussion mixes, and is placed at room temperature for 2min, and 12000rpm is centrifuged 5min, abandons supernatant, room
Temperature places 2min；
(4) 50 μ l 1 × TE buffers are added, sufficiently dissolve, are stored at room temperature 5min, 12000rpm is centrifuged 1min, supernatant
As DNA solution, it is spare.
Embodiment 2: Fluorescence PCR step
(1) high-risk type HPV fluorescence PCR reaction solution: 4 μ 10 × PCR of l Buffer (100mM Tris-HCl, 500mM is prepared
KCl)；0.08μl dATP(0.4mM),0.08μl dTTP(0.4mM),0.08μl dCTP(0.4mM),0.08μl dGTP
(0.4mM)；0.8μl BSA(10mg/mL)；6.4μl MgCl2(25mM)；0.05μl HPV16F(100μM),0.08μl
HPV16F(100μM),0.08μl Probe1(100μM)；0.05μl HPV18F(100μM),0.08μl HPV18F(100μM),
0.08μl Probe2(100μM)；0.08μl HPV31F(100μM),0.08μl HPV35F(100μM),0.08μl HPV31/
35R(100μM)、0.08μl HPV33F(100μM)、0.08μl HPV52F(100μM)、0.08μl HPV58F(100μM)、
0.08μl HPV33/52/58R(100μM),0.06μl Probe3(100μM)；0.08μl HPV45F(100μM),0.06μl
HPV45R(100μM),0.08μl HPV59F(100μM),0.06μl HPV59R(100μM),0.06μl Probe4(100μM)；
0.08μl HPV39/68F(100μM),0.08μl HPV39/68R(100μM),0.06μl Probe5(100μM)；0.08μl
HPV53F(100μM)、0.08μl HPV53R(100μM)、0.08μl HPV56/66F(100μM)、0.08μl HPV56R(100μ
M),0.08μl HPV66R(100μM),0.06μl Probe6(100μM)；0.08μl HPV26F(100μM),0.06μl
HPV51/82F(100μM),0.08μl HPV26/51/82R(100μM),0.06μl Probe7(100μM)；0.08μl
HPV73F(100μM),0.08μl HPV73R(100μM),0.05μl Probe8(100μM)；0.04μl IC-F(100μM),
0.04μl IC-R(100μM),0.03μl Probe9(100μM)；0.4 μ l Taq archaeal dna polymerase (purchase from Promega),
The anti-Taq archaeal dna polymerase antibody of 0.067 μ l；Remaining addition ddH2O makes 36 μ l of its volume, wherein the Taq DNA polymerize
Enzyme and anti-Taq archaeal dna polymerase antibody are preferably added in high-risk type HPV fluorescence PCR reaction solution before use；
(2) n parts of the sample number [+1 pipe negative controls of+1 pipe positive control solution of n=clinical sample number] that need to prepare is calculated,
The high-risk type HPV fluorescence PCR reaction solution prepared in 36 μ l (1) is added in different reaction tubes, then respectively toward different
The DNA solution extracted in 4 μ l embodiments 1,4 μ l negative control solutions and 4 μ l positive control solutions, middle-jiao yang, function of the spleen and stomach are added in reaction tube
Property comparison liquid be the solution containing high-risk HPV nucleic acid, negative controls be the solution without containing high-risk HPV nucleic acid.
(3) each reaction tube is put into certain sequence on fluorescent PCR instrument, carries out PCR amplification, amplification program by following procedure
It is as shown in table 2:
2 fluorescent PCR amplification program of table
(4) it after step (3), is detected according to the Ct value in the channel FAM, the channel VIC, the channel Texas Red, the channel Cy5
High-risk HPV whether there is, and testing result judgement is as shown in table 3:
The judgement of 3 testing result of table
Embodiment 3: clinical sample detection
The DNA in 140 HPV uterine neck cotton swab subsamples is extracted by the method in embodiment 1, then according in embodiment 2
Method respectively in this 140 HPV Cervical scrapes samples DNA carry out fluorescent PCR amplification.At the same time, for the ease of than
Compared with, using 48014 kinds of high-risk HPVs of Roche Combas (i.e. HPV16,18,31,33,35,39,45,51,52,56,58,
59,68 and 66) detection kit detects the DNA in this 140 HPV Cervical scrapes samples, testing result such as 4 institute of table
Show.
4 testing result of table
Result in table 4 the result shows that, in this 140 clinical samples, detection of the present invention to 134 clinical samples
As a result consistent in Roche kit, but there are also 6 clinical samples, the present invention is capable of detecting when the presence of high-risk HPV,
And Roche kit cannot then detect the presence of high-risk HPV.Confirmed by Sanger PCR sequencing PCR, this 6 samples contain respectively
There are HPV53, HPV82, HPV73, HPV53, HPV26 and HPV26.This further demonstrates detection accuracy of the invention.In addition,
The present invention is to carry out the inspection of four-way fluorescent PCR to 18 kinds of high-risk HPVs that may be present in clinical sample in single reaction tube
It surveys, wherein Fig. 2 is the channel VIC (whether there is HPV16 in detection clinical sample) testing result；Fig. 3 is the channel Texas Red
(whether there is HPV18 in detection clinical sample) testing result；Fig. 4 is that the channel FAM (whether there is in detection clinical sample and remove
At least one of other 16 kinds of high-risk HPVs except HPV16 and HPV18) testing result；Fig. 5 is the channel Cy5 (in detection
Mark β-globin) testing result.Determined in the amplification of first time fluorescent PCR preferably for those containing except HPV16 and
The clinical sample of at least one high-risk HPV except HPV18, its also recycling in addition to HPV16 and HPV18 of the present invention
The specific primer and probe of his 16 kinds of high-risk HPV hypotypes carry out fluorescent PCR amplification again, so that it is determined that its specific type.
In addition, 14 kinds of high-risk HPV detection kits relative to Roche, the present invention are able to detect more high-risk-types
HPV type, it is often more important that, for added high-risk HPV type, the present invention is not just simply every in them
The primer and probe of a type design specificity, and consider the affiliation of they and other types, allow HPV53 and
HPV56 and HPV66 shares same probe, and HPV82 and HPV51 is allowed to share upstream primer, makes HPV82, HPV51 and HPV26 total
With downstream primer and a probe, at the same also allow detection HPV53,73,82,26 and HPV31,33,35,39,45,51,52,56,
58,59,68 and 66 when used probe share the same sense channel, it is logical that this can be effectively reduced primer, probe and detection
The quantity in road, to be effectively reduced testing cost and background fluorescence signal.
Embodiment 4: specific detection result
The setting of HPV National reference is as follows:
5 kinds of negative reference product N1-N5: chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), neisseria gonorrhoeae are followed successively by
(NG), herpes simplex virus type II (HSV-2), HPV nucleic acid negative cervical cotton swab subsample；
20 kinds of positive reference product: 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 product.
Fluorescent PCR amplification is carried out to each HPV National reference according to the method in embodiment 2 to join each HPV country
It examines product and carries out fluorescent PCR detection, as a result as follows:
FAM, VIC of 5 kinds of positive reference product such as negative reference product N1-N5 and HPV6,11,61,67,69,71,81 and
Texas Red Air conduct measurement result is feminine gender；The VIC Air conduct measurement result of HPV16 positive reference product is the positive, such as Fig. 6 institute
Show；The Texas Red Air conduct measurement result of HPV18 positive reference product is the positive, as shown in Figure 7；HPV26,31,33,35,45,
56,58,59,66,73,82 positive reference product FAM Air conduct measurement results are the positive, as shown in Figure 8；Internal standard Cy5 Air conduct measurement knot
Fruit is the positive, as shown in Figure 9.
Embodiment 5: sensitivity technique result
Respectively to the positive reference product of HPV16,18,26,31,33,35,45,56,58,59,66,73 and 82 in embodiment 4
Carrying out gradient dilution to concentration is 1 × 103Copies/ml carries out each positive reference product according to the method in embodiment 2 glimmering
Light PCR amplification, test result are the positive, i.e., detection sensitivity of the invention is 1 × 103copies/ml.As a result such as Figure 10 institute
Show.
Embodiment 6: background fluorescence signal testing result
Respectively to the positive reference product of HPV16,18,26,31,33,35,45,56,58,59,66,73 and 82 in embodiment 4
Fluorescent PCR amplification is carried out to each positive reference product according to the method in the embodiment of the present invention 2；Simultaneously in order to be compareed, needle
To the present invention 18 kinds of common high-risk HPVs that can be detected, for the primer and one of a pair of of specificity of each high-risk HPV design
The probe of item specificity also carries out fluorescent PCR amplification to same positive reference product respectively under the same conditions, with the channel FAM
It is detected as example, carries out the comparison of background fluorescence signal, comparing result is as shown in figure 11.As shown in Figure 11, the present invention is due to making
Number of probes substantially reduces, and background fluorescence signal significantly reduces, and is convenient for result interpretation.
Claims (7)
1. the oligonucleotides for fluorescent PCR detection high-risk HPV, which is characterized in that the oligonucleotides includes:
(1) for detecting the primer and probe of HPV16, base sequence is respectively NO:1~3 SEQ ID；(2) for detecting
The primer and probe of HPV18, base sequence are respectively NO:4~6 SEQ ID；
(3) for detecting the primer and probe of HPV31, base sequence is respectively SEQ ID NO:7,9 and 14；
(4) for detecting the primer and probe of HPV45, base sequence is respectively SEQ ID NO:15,16 and 19；
(5) for detecting the primer and probe of HPV35, base sequence is respectively SEQ ID NO:8,9 and 14；
(6) for detecting the primer and probe of HPV33, base sequence is respectively SEQ ID NO:10,13 and 14；(7) it is used for
The primer and probe of HPV52 is detected, base sequence is respectively SEQ ID NO:11,13 and 14；(8) for detecting HPV58's
Primer and probe, base sequence are respectively NO:12~14 SEQ ID；(9) for detecting the primer and probe of HPV59, alkali
Basic sequence is respectively NO:17~19 SEQ ID；(10) for detecting the primer and probe of HPV39 and HPV68, base sequence
Respectively NO:20~22 SEQ ID；(11) for detecting the primer and probe of HPV53, base sequence is respectively SEQ ID
NO:23,24 and 28；(12) for detecting the primer and probe of HPV56, base sequence is respectively SEQ ID NO:25,26 and
28；(13) for detecting the primer and probe of HPV66, base sequence is respectively SEQ ID NO:25,27 and 28；(14) it is used for
The primer and probe of HPV26 is detected, base sequence is respectively SEQ ID NO:29,31 and 32；(15) for detect HPV51 and
The primer and probe of HPV82, base sequence are respectively NO:30~32 SEQ ID；(16) for detect HPV73 primer and
Probe, base sequence are respectively NO:33~35 SEQ ID；
(17) for detecting interior target primer and probe, base sequence is respectively NO:36~38 SEQ ID.
2. oligonucleotides as shown in claim 1, which is characterized in that the detection is carried out in single reaction pipe.
3. oligonucleotides as shown in claim 1, which is characterized in that (1) the fluorescence report base of every probe in~(17)
Group selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 or
LC RED705, the quenching group of every probe in (1)~(17) be selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl,
Eclipse or NFQ.
4. a kind of kit using fluorescent PCR detection high-risk HPV, the kit includes Fluorescence PCR liquid, described glimmering
Light PCR reaction solution includes oligonucleotides, which is characterized in that the oligonucleotides include: (1) be used for detect HPV16 primer and
Probe, base sequence are respectively NO:1~3 SEQ ID；(2) for detecting the primer and probe of HPV18, base sequence point
It Wei not NO:4~6 SEQ ID；
(3) for detecting the primer and probe of HPV31, base sequence is respectively SEQ ID NO:7,9 and 14；
(4) for detecting the primer and probe of HPV45, base sequence is respectively SEQ ID NO:15,16 and 19；
(5) for detecting the primer and probe of HPV35, base sequence is respectively SEQ ID NO:8,9 and 14；
(6) for detecting the primer and probe of HPV33, base sequence is respectively SEQ ID NO:10,13 and 14；(7) it is used for
The primer and probe of HPV52 is detected, base sequence is respectively SEQ ID NO:11,13 and 14；(8) for detecting HPV58's
Primer and probe, base sequence are respectively NO:12~14 SEQ ID；(9) for detecting the primer and probe of HPV59, alkali
Basic sequence is respectively NO:17~19 SEQ ID；(10) for detecting the primer and probe of HPV39 and HPV68, base sequence
Respectively NO:20~22 SEQ ID；(11) for detecting the primer and probe of HPV53, base sequence is respectively SEQ ID
NO:23,24 and 28；(12) for detecting the primer and probe of HPV56, base sequence is respectively SEQ ID NO:25,26 and
28；(13) for detecting the primer and probe of HPV66, base sequence is respectively SEQ ID NO:25,27 and 28；(14) it is used for
The primer and probe of HPV26 is detected, base sequence is respectively SEQ ID NO:29,31 and 32；(15) for detect HPV51 and
The primer and probe of HPV82, base sequence are respectively NO:30~32 SEQ ID；(16) for detect HPV73 primer and
Probe, base sequence are respectively NO:33~35 SEQ ID；
(17) for detecting interior target primer and probe, base sequence is respectively NO:36~38 SEQ ID.
5. the kit as shown in claim 4, which is characterized in that the Fluorescence PCR liquid further includes Taq archaeal dna polymerase
With anti-Taq archaeal dna polymerase antibody.
6. the kit as shown in claim 4, which is characterized in that the detection is carried out in single reaction pipe.
7. the kit as shown in claim 4, which is characterized in that (1) fluorescent reporter group of every probe in~(17)
Selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 or LC
The quenching group of RED705, every probe in (1)~(17) are selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse
Or NFQ.
Priority Applications (2)
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| CN105950788B (en) * | 2016-06-15 | 2019-07-30 | 亚能生物技术（深圳）有限公司 | Detect the primer, probe and kit of 18 kinds of high-risk HPV nucleic acid |
| CN106282413B (en) * | 2016-09-30 | 2019-11-22 | 广州易活生物科技有限公司 | Probe combinations, kit and the method for the high-risk strain Genotyping detection of HPV |
| CN107130058B (en) * | 2017-06-28 | 2018-08-03 | 广州市宝创生物技术有限公司 | A kind of novel 9 kinds of humam papillomavirus genotype parting kits and method |
| CN107130059B (en) * | 2017-06-28 | 2018-07-20 | 广州市宝创生物技术有限公司 | A kind of novel 21 kinds of humam papillomavirus genotype parting kits and method |
| CN108179226B (en) * | 2018-03-12 | 2020-10-02 | 江苏硕世生物科技股份有限公司 | Nucleic acid composition for detecting human papilloma virus, application thereof and kit |
| CN109402297A (en) * | 2018-11-05 | 2019-03-01 | 泰普生物科学（中国）有限公司 | Single tube detects the kit and method of 15 kinds of HPV genotype |
| CN110256566A (en) * | 2019-05-10 | 2019-09-20 | 江苏苏博生物医学科技南京有限公司 | Taq archaeal dna polymerase single-chain immunoglobulins IgG antibody, preparation method and its application in Genotyping detection |
| CN112961938B (en) * | 2019-12-13 | 2022-06-07 | 浙江我武生物科技股份有限公司 | Method and kit for detecting human papilloma virus |
| CN112981004A (en) * | 2019-12-17 | 2021-06-18 | 广东菲鹏生物有限公司 | Reagent for detecting HPV (human papillomavirus), kit and application thereof |
| CN112921033A (en) * | 2021-04-07 | 2021-06-08 | 深圳市刚竹医疗科技有限公司 | Nucleic acid composition and detection kit |
| CN114214463A (en) * | 2021-12-28 | 2022-03-22 | 广州安必平医药科技股份有限公司 | Primer probe composition for detecting HPV (human papillomavirus) and application thereof |
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